Lactic Acid Fermentation of Human Excreta For Agr-Wageningen University and Research 401835

Lactic Acid Fermentation
of Human Excreta for

Agricultural Application
Nadejda Andreev
LACTIC ACID FERMENTATION OF HUMAN
EXCRETA FOR AGRICULTURAL
APPLICATION
Nadejda Andreev
Thesis committee
Promotor
Prof. Dr P.N.L. Lens
Professor of Environment Biotechnology
UNESCO-IHE Institute for Water Education, Delft
Co-promotors
Prof. B. Boincean
Habil. Dr. of Agricultural Sciences, Research Professor
Research Institute of Field Crops “Selectia”
Balti, Republic of Moldova
Dr M. Ronteltap
Senior Lecturer in Sanitary Engineering
UNESCO-IHE Institute for Water Education, Delft
Other members
Prof. Dr G. Zeeman, Wageningen University & Research
Prof. Dr W.Verstraete, Ghent University, Belgium
Dr Tjaša Griessler Bulc, University of Ljubljana, Slovenia
Prof. Dr J.O. Drangert, Linkӧping University, Sweden
This research was conducted under the auspices of the SENSE Research School for Socio-
Economic and Natural Sciences of the Environment
Lactic acid fermentation of human excreta for agricultural application
Thesis
submitted in fulfilment of the requirements of
the Academic Board of Wageningen University and
the Academic Board of the UNESCO-IHE Institute for Water Education
for the degree of doctor
to be defended in public
on Friday, 29 September 2017at 1.30 p.m.
in Delft, the Netherlands
by Nadejda Andreev
Born in Nisporeni, Boldurești, Republic of Moldova
CRC Press/Balkema is an imprint of the Taylor & Francis Group, an informa business
© 2017, Nadejda Andreev
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DOI: https://doi.org/10.18174/401835
Dedication
This thesis is dedicated to the research community interested in resource oriented sanitation.
v
vi
Aknowledgements
I would like to express sincere gratitude to my promotor Prof. Dr. ir. Piet Lens
and to my co-promotors Dr. Mariska Ronteltap and Prof. Boris Boincean for scientific
support, guidance, encouragement and critical interpretation of the data, who enabled me to
earn an open minded research thinking and helped in a systematic way of finding answers to
questions.
I am also very grateful to prof. Elena Zubcov, Nina Boichenco and Natalia Borodin
from the Laboratory of Hydrobiology and Ecotoxicology, Institute of Zoology of Moldova;
Dr. Lidia Bulat from the Department of Sustainable Farming Systems of Crop Research
Insitute, Selectia, Baltsy, Moldova; Dr. Larisa Cremeneac from the National Insitute of
Zootechny and Veterinary Medicine, Moldova as well as Svetlana Prudnichonok and Olga
Coteț from the Laboratory of Sanitary Microbiology, National Centre of Public Health,
Moldova for the research advise and support during crucial parts of my field and laboratory
research experiments. Many thanks to Christopher Canaday, biologist at the Fundación
Omaere (Puyo, Ecuador) for proofreading the thesis and providing valuable comments for its
improvement.
Special thanks to my relatives Elena, Vladimir, Nadine, Vasile and Ana Vrabie,
Semion Varaniță as well as Vera and Boris Suciu who offerred valuable help during the field
work. I am also indebted to Sergiu Andreev who provided support in graphical design of the
thesis. I am also very thankful to my beloved children, Alina and Sandu for their patience and
understanding during my absence for field work or when being abroad. I give all the respect
to my departed mother and father, who gave me a lot of encouragement during the start of my
research.
Nadejda Andreev
Delft, 24 March, 2017
vii
viii
Table of Contents
Dedication ........................................................................................................................................ v
Aknowledgements ......................................................................................................................... vii
Table of Contents ........................................................................................................................... ix
Thesis summary.............................................................................................................................. xi
Samenvatting ................................................................................................................................ xiii
Chapter 1. General Introduction ............................................................................................ 1
1.1 Research justification................................................................................................................ 2
1.2 Scope of research ....................................................................................................................... 5
1.3 Thesis outline ............................................................................................................................. 6
Chapter 2. Increasing the agricultural value of human excreta by lactic acid
fermentation, composting and biochar addition (literature review) ................................... 9
Abstract .......................................................................................................................................... 10
2.1 Introduction ............................................................................................................................. 11
2.2 Nutrient and resource challenges in sanitation .................................................................... 14
2.3 Applying excreta and biochar to agriculture ........................................................................ 16
2.4 Challenges in agricultural reuse of excreta........................................................................... 23
2.4.2 Loss of nutrients, carbon matter and odour issues ..................................................... 25
2.5 Lactic acid fermentation of human excreta .......................................................................... 27
2.6 Post treatment of lacto-fermented human excreta ............................................................... 35
2.7 Agricultural effects of lacto-fermented excreta .................................................................... 40
2.8 Conclusions .............................................................................................................................. 45
Chapter 3. Treatment of source-separated human faeces via lactic acid fermentation
combined with thermophilic and vermi-composting for agricultural application ........... 47
Abstract .......................................................................................................................................... 48
3.1 Introduction ............................................................................................................................. 49
3.2 Materials and Methods ........................................................................................................... 52
3.3 Results ...................................................................................................................................... 56
3.4 Discussion ................................................................................................................................. 61
3.5 Conclusions .............................................................................................................................. 65
Chapter 4 The effect of lacto-fermented faeces, biowaste and addition of biochar soaked
in urine on soil quality, growth, yield and yield components of Zea mays L. ................... 67
ix
Abstract .......................................................................................................................................... 68
4.1 Introduction ............................................................................................................................. 69
4.2 Methods .................................................................................................................................... 73
4.3 Results ...................................................................................................................................... 84
4.4 Discussion ............................................................................................................................... 101
4.5 Conclusions ............................................................................................................................ 109
Chapter 5. Lactic acid fermentation of human urine for improving its fertilizing value
and reducing odour emissions in urine diverting dry toilets ............................................ 111
Abstract ........................................................................................................................................ 112
5.1 Introduction ........................................................................................................................... 113
5.2 Material and methods ........................................................................................................... 114
5.3 Results .................................................................................................................................... 117
5.4 Discussion ............................................................................................................................... 122
5.5 Conclusions ............................................................................................................................ 128
Chapter 6. General Discussion and Outlook ..................................................................... 129
6.1 Potential and limitations of the application of lactic acid fermentation and biochar in
excreta treatment ........................................................................................................................ 130
6.2 Product quality assurance for potential full-scale application .......................................... 135
6.3 Input materials required for lactic acid fermentation of excreta ..................................... 139
6.4 Agricultural application of lacto-fermented excreta and biochar .................................... 142
6.5 Application of lactic acid fermentation combined with composting and biochar in urine
diverting dry toilets in Moldova................................................................................................. 143
6.6 Potential entrepreneurial models ......................................................................................... 149
6.7 Conclusions ............................................................................................................................ 151
References .................................................................................................................................... 153
Appendixes ................................................................................................................................... 195
x
Thesis summary
Management of human excreta is one of the most pressing global issues. The application
of cost-effective, environmentally friendly treatment methods and agricultural reuse of excreta
are particularly important in the context of global nutrient and resource challenges, a rapid
depletion of soil nutrients and organic matter, eutrophication and scarcity of freshwater
resources, exacerbated by the population growth and climate change. This study focuses on the
use of lactic acid fermentation for the treatment of human faeces and urine for agricultural
reuse.
The efficiency of lactic acid fermentation and thermophilic composting versus lactic
acid fermentation and vermi-composting on pathogen removal and the post-effects on seed
germination (radish) and plant growth (tomatoes) were assessed in pot experiments. The main
effects of lacto-fermented faeces and bio-waste supplemented by biochar were also studied
during a two-year experiment under the conditions of a clay-loamy cernoziom in the Central
part of Moldova using corn, Zea mays L, as test crop. Urine lactic acid fermentation was also
investigated under laboratory conditions and real field conditions in a functional household
urine diverting dry toilet (UDDT) storage tank in the vicinity of Chisinau (Moldova).
The research results revealed that lactic acid fermentation combined with
thermophilic composting reduced concentration of the coliforms, Escherichia coli,
Enterococcus faecalis and Clostridium perfringens to below the detection limit, while lactic
acid fermentation combined with vermi-composting reduced the coliform concentration to 5
log CFU g-1 only. Lactic acid fermentation of the mix of faeces and bio-waste with the addition
of lactic acid bacteria and molasses followed by thermophilic composting led to a rapid increase
in sanitization temperature above 55° C, without the need of turning and aeration. The overall
composting period of lactic acid fermentation combined with thermophilic composting was 33
days compared to 130 days for combined lactic acid fermentation with vermi-composting. The
germination index of radish seeds fertilized by compost after lactic acid fermentation
xi
combined with thermophilic composting was higher than that obtained after lactic acid
fermentation combined with vermi-composting (90 versus 84 %, respectively). Moreover,
significantly bigger average fruit weight and total amount of biomass per tomato plant
(p<0.05) were obtained after compost amendment compared to vermi-compost and the
unamended control.
The difference of means by the Dunnet test showed that the mix of lacto-fermented
faeces and bio-waste, supplemented by urine charged biochar has increased significantly
(p<0.05) the corn growth, yield and yield components compared to the stored urine, faeces
and cattle manure. The yield of corn after fertilization of mineral fertilizer during the first year
was significantly lower than the investigated fertilizing mix, but during the second year, it
was not significantly different due to different weather (precipitation) conditions. The lacto-
fermented mix of faeces and bio-waste, supplemented by biochar also significantly improved
the soil bulk density, soil mobile potassium and soil nitrate content (the latter only in
comparison to the control and stored urine). The beneficial effects of lacto-fermented faeces
and bio-waste, supplemented by urine charged biochar on yield components might be
attributed to potential prevention of nitrate leaching by the biochar component in the root
zone.
The application of a lactobacilli solution of sauerkraut containing molasses and water
to fresh urine contributed to an efficient reduction of the pH to < 4-4.5 and of the ammonium
content by 1/3, while maintaining a high concentration of viable LAB (7.3 CFU ml-1)
compared to untreated, stored urine. Moreover, the odour strength was reduced twicely after
urine lactic acid fermentation. Also the seed germination was enhanced after application of
lacto-fermented urine, showing a potentially higher fertilizing value than the stored urine.
This study showed that lacto-fermented faeces and bio-waste, supplemented by urine
charged biochar can serve as a potential suitable soil conditioner. Besides, it showed that
lacto-fermented urine can be a potential bio-fertilizer for improving soil quality and crop
yield. The results of this thesis can be useful for improving the resource oriented potential of
urine diverting dry toilets.
xii
Samenvatting
De adequate behandeling van menselijke uitwerpselen is een van de meest uitdagende

mondiale kwesties. De toepassing van kosteneffectieve, milieuvriendelijke
behandelingsmethoden en agrarische hergebruik van uitscheidingsproducten zijn bijzonder
belangrijk in het kader van de globale uitdagingen op het gebied van nutriënten; uitputting
van de bodem met betrekking tot nutriënten en organische stof; vervuiling, eutrofiëring en
schaarste van zoetwaterreserves, welke versterkt worden door de bevolkingsgroei en
klimaatverandering. De huidige studie richt zich op de inzet van lacto-fermentatie voor de
behandeling van menselijke uitwerpselen en urine voor hergebruik in de landbouw.
De efficiëntie van lacto-fermentatie en thermofiele compostering versus lacto-
fermentatie en vermi-compostering op pathogeenverwijdering en vervolgens de effecten op
zaadontwikkeling (radijs) en plantgroei (tomaten) zijn beoordeeld aan de hand van een 24
maands onderzoek, zowel in de volle grond als in plantenbakken.
De belangrijkste effecten van lacto-gefermenteerde feces en groen afval aangevuld met
biochar werden ook bestudeerd tijdens een experiment van twee jaar in een leemklei-cernoziom
bodem in het centrale deel van Moldavië met behulp van maïs (Zea maïs L.) als testgewas. Urine
lacto-fermentatie werd eveneens onderzocht onder laboratoriumomstandigheden en echte
veldomstandigheden met urine opgevangen in een droog scheidingstoilet (urine diverting
dehydrating toilet, UDDT) in de nabijheid van Chisinau, Moldavië.
Resultaten van het onderzoek toonden aan dat middels lacto-fermentatie in combinatie met
thermofiele compostering het aantal coliforme bacteriën (Enterococcus faecalis, Escherichia coli en
Clostridium perfringens) kon worden teruggebracht tot onder de detectielimiet; middels lacto-
fermentatie in combinatie met vermi-compostering slechts tot 5 log CFUg-1. Lacto-fermentatie van
de mix van feces en bio-afval met de toevoeging van melkzuurbacteriën en melasse gevolgd door
thermofiele compostering leidt tot een verhoging van de temperatuur tot boven 55°C, zonder
mengen of beluchten. De algehele composteringsperiode van lacto-fermentatie gecombineerd met
thermofiele compostering was 33 dagen; voor gecombineerde lacto-vergisting met vermi-
xiii
compostering bedroeg dit 130 dagen. De kiemindex van radijszaden bemest met compost na lacto-
vergisting plus thermofiele compostering was hoger dan die na lacto-vergisting plus vermi-
compostering (90 tegenover 84%, respectievelijk). Bovendien werd er per tomatenplant een
significant grotere gemiddeld gewicht per vrucht en per plant verkregen (p < 0.05) dan na het
toedienen van vermicompost of geen compost (controle).
Het verschil van gemiddelden werd bepaald middels de Dunnet test. Hieruit kwam naar
voren dat groei, rendement en opbrengst van de maïs significant toenam (p < 0.05) na toevoeging
van lacto-gefermenteerde feces en bio-afval aangevuld met urine en biochar, ten opzichte van
gehydrolyseerde urine, feces en mest. De opbrengst van maïs na bemesting met minerale meststof
was aanzienlijk lager tijdens het eerste productiejaar maar niet significant anders dan de onderzochte
bemestingsmix tijdens het tweede productiejaar. Lacto-gefermenteerde mix van feces en bio-afval,
aangevuld met biochar, heeft de bulkdichtheid, mobiele kalium en het nitraatgehalte van de bodem
verbeterd (het nitraatgehalte in vergelijking met de controle en de opgeslagen urine). De gunstige
effecten van lacto-gefermenteerde feces en bio-afval aangevuld met urine en biochar op de
plantendelen kunnen worden toegeschreven aan potentiële preventie van nitraatuitspoeling in de
wortelzone door biochar component tijdens droge perioden, gevolgd door regen.
De toepassing van een oplossing van lactobacilli afkomstig van zuurkool, melasse en water
met verse urine droeg bij aan een verlaging van de pH naar minder dan 4-4,5 en van het
ammoniumgehalte met 30%, met behoud van een hoge concentratie van levensvatbare LAB (7,3 ml
CFU-1) in vergelijking met onbehandelde, gehydrolyseerde urine. Daarnaast droeg lacto-
gefermenteerde urine bij aan een verminderde waargenomen geur en had het een positieve invloed
op de kiemkracht van zaad.
Deze studie toonde aan dat lacto-gefermenteerde feces en bio-afval, aangevuld met urine
doordrenkte biochar, kunnen dienen als een potentiële bodemverbeteraar, en lacto-gefermenteerde
urine als potentiële bio-meststof voor de verbetering van de kwaliteit van de bodem en de opbrengst
van gewassen. De resultaten van deze thesis kunnen nuttig zijn voor verbetering van het
hergebruikspotentieel van de grondstoffen uit droge scheidingstoiletten (UDDTs).
xiv
Chapter 1. General Introduction
1
1.1 Research justification
Modern society is faced with several challenges regarding the disposal and
treatment of human excreta. Water and resource limitations in conventional sanitation
systems are becoming nowadays of a concern due to urbanization and population growth,
negative effects on freshwater and land resources as well as aggravating climate change. In
spite of technological development in the field of sanitation, still a high proportion of the
generated wastewater is discharged into the environment receiving a poor or even no
treatment at all (Sato et al., 2013). Many developed and developing countries bear significant
costs for maintaining and upgrading the sanitation infrastructure and treatment facilities
(Schertenleib, 2005a; Zimmer and Hofwegen, 2006; Kone, 2010).
The scarcity of water and limitations of financial resources or political willpower force
a majority of the population of Eastern and Central Europe, Africa, South, South-East and
East Asia as well as Central America who live in rural settlements to use unsewered,
environmentally polluting latrine types of sanitation (Bodik, 2007; Kone, 2010). If not
properly managed, these sanitation systems can lead to high risks for environmental and
public health in the crowded areas, those with a high water table, those that are periodically
flooded, hard to excavate or in sensitive coastal zones (Winblad and Simpson-Hebert, 2004).
Also modern agriculture is still based on intensive use of nonrenewable sources of
energy (oil, natural gases and coal) and mineral fertilizers, especially nitrogen. Recycling of
organic matter and nutrients to the soil is limited. While high amounts of nutrients, with a
fertilizing potential, are discharged via excreta or wastewater, the global phosphorous
resources are depleting (Cordell and White, 2011) and the soils are threatened due to a
decrease of their natural fertility through so-called "nutrient and organic matter mining". For
example, in many developing countries and those with economies in transition, where farmer
survival depends heavily on agriculture and land, the fertilizing input is very low due to high
prices or the absence of mineral fertilizers. Thus, soils are drained of organic matter and
nutrients making them more prone to degradation under natural conditions or by man made
factors. A similar phenomenon happens to many European soils, where approximately half of
2
General introduction
the soils have reached a low or very low organic matter content. In Southern Europe for
instance, a high proportion of intensively used agricultural soils reach a critical threshold
value for soil organic carbon (2%) (Rusco et al., 2001).
A "material-flow-oriented recycling sanitation", also called "resource oriented" or
"productive sanitation" approach is seen as an effective solution for recycling organic matter
and nutrients to the soil (Murray and Buckley, 2010). This new paradigm shifts the idea from
disposal of excreta to containment and recycling. This fits into the idea of a circular economy,
where environmental and economic gains are obtained from the recycling of nutrients and
organic matter. Recycling of nutrients from excreta to agriculture can reduce the costs from
the use and production of chemical fertilizers. Of a particular importance is the case of
phosphorus fertilizers, which prices are excalating at the global level due to shrinkage of rock
resources and raise in the costs for their extraction and transportation. For example, in 2008 a
steady increment by 800 percent was encountered for phophate rock due to boosting of oil
prices, cultivation of biofuels, more demand for fertilizers caused by higher meat
consumption and short term scarcity of phosphorus rock resources on the global market. That
led to rise in food prices that hit poor countries very hard (Cho, 2013).
Reuse of excreta is one of the potential ways to supplement the scarce phosphorus
resources. Estimations from USA show that recovery and recycling of phosphorus from
excreta, food waste and animal manure can return up to 37 % of annual crop requirements
(Carlson, 2016). The safe recycling of these nutrients, however, does require effective
sanitisation technologies. Working with source separated urine and faecal matter asks for
alternative methods to more conventional biological wastewater treatment technologies, for
which we need to reorient ourselves by either looking for innovative approaches or by
applying known technologies in a novel fashion.
One promising example from the past is the application of charred organic matter in
combination with other organic material, which was proven to be a viable strategy for
replenishing soil nutrients and organic matter deficiencies. In the past, highly fertile,
artificially created soils, "terra preta" or "black anthrosols" have been distributed over South
America, Australia, West Africa and Northern Europe (Downie et al., 2011; Frausin et al.,
3
2014; Wiedner et al., 2015a). These soils arose as a result of intentional or unintentional
deposition of organic (e.g. left-over cooking charcoal, human and animal excreta and plant
material) and inorganic waste (e.g. ceramic shreds) on the top of infertile soil (Glaser and
Birk, 2012a; Giani et al., 2014). After the discovery of terra preta soils and its potential was
realized, extended research was initiated on investigating the role of biochar in increasing soil
fertility as well as the re-application of ancient practices of waste management and soil
enrichment in the field of sanitation and agriculture (Kawa and Oyuela-Caycedo, 2008;
Lehmann and Joseph, 2009b; Novotny et al., 2009; Otterpohl and Buzie, 2011; Smetanova et
al., 2012; Otterpohl and Buzie, 2013; Windberg et al., 2013; Schuetze and Santiago-Fandino,
2014; Yemaneh, 2015).
Lacto-fermented and composted human excreta can provide a range of useful products
of natural origin for agriculture, e.g. bio-fertilizers such as lacto-fermented urine or soil
conditioners (compost obtained after lactic acid fermentation combined with thermophilic or
vermi-composting). The obtained end products are bio-degradable and have a lower detrimental
impact on humans and plants than chemical fertilizers. The microbial inocula applied can be
obtained from locally available resources such as sauerkraut, rice wash or milk products
(Aliyu & Muntari, 2011; Factura et al., 2010). Lactic acid fermentation is a low cost
technique that employs limited handling and makes use of simple equipment and limited
resources (Erickson et al., 2004).
Terra preta sanitation is an approach where human excreta is treated and recycled to
agriculture and horticulture in an original way via combined lactic acid fermentation as a
primary treatment step, followed by vermi-composting as a secondary post-treatment step.
Recently, thermophilic composting was proposed as a post-secondary step to efficiently recycle
the nutrients and organic matter as well as to reduce the pathogen contamination (Andreev et
al., 2017). Biochar supplementation at the end of the lactic acid fermentation or at the
thermophilic or vermi-composting stage increases the stability of carbon matter and reduces the
nitrogen loss via volatilization. Lactic acid fermentation of urine and faeces diminishes the
odour emissions and contributes to their stabilization. Faeces lactic acid fermentation after the
collection phase was combined with other types of bio-waste, such as high moisture and carbon
4
rich waste from the food industry, e.g. from diary or beverage industry or the kitchen scraps.
These wastes are easily degradable and can produce high emissions and pungent odours
(Kosseva, 2013). By combining lactic acid fermentation with thermophilic composting, the
health risks during faeces handling can be reduced as temperature increases occur in the
substrate with passive aeration only. Apart from faeces, also lactic acid fermentation of
human urine can be considered as it reduces the pH and decreases the ammonia volatilization,
thus potentially enhancing the fertilizing value of urine (effects on germination) and reducing
odour emissions. Moreover, lacto-fermented urine may have a better effect on germination
owing to the metabolites lactic acid bacteria (LAB) produce (e.g. organic acids and hydrogen
peroxide) and suppressing of a wide range of phytopathogens (Limanska et al., 2015), while
having a stimulatory effect on plant growth by triggering plant systemic resistance and
enhance production of plant growth regulators (Abel-Aziz et al., 2014).
1.2 Scope of research
This PhD thesis assessed the suitability of lactic acid fermentation for increasing the
fertilizing value of human excreta (Figure 1.1). The efficiency of lactic acid fermentation and
thermophilic composting versus lactic acid fermentation and vermi-composting on pathogen
removal and the post-effects on seed germination (radish) and plant growth (tomatoes) were
assessed in pot and field experiments. The main effects of lacto-fermented faeces and
biowaste supplemented by biochar on soil quality of a clay-loamy cernoziom in the central
part of Moldova as well as on the growth and yield of corn (Zea mays L.) were studied during
a two-year field experiment. The field research was carried out in randomized experimental
plots of 60 m2, with three replicates on a total area of 0.15 ha. Lactic acid fermentation of
faeces and the bio-waste mix was performed in fermentation pits and closed plastic barrels.
Thermophilic composting was carried out in a metallic insulated box and vermi-composting
in a windrow of 1 m2, for a period of four months. Earthworms (Eisenia foetida) were
acquired from the Institute of Biotechnologies in Zootechny and Veterinary Medicine
(Maximovca vg., Anenii Noi district, Moldova).
5
Faeces+bio-waste Urine, LAB,

LAB, molasses molasses
Field application (2 year experiment) Biochar

Corn productivity: yield, yield
LAF (10 days-4 weeks)
components, growth rate Germination test
Soil quality: humus content, bulk density,
soil moisture, P2O5, K2O, NO3-
TC (23-24 days) VC (4 months)
Quality assessment
SIB: Escherichia coli, Enterococcus faecalis,
Faecal coliforms, Clostridium perfringens
GI: as compared to the control (24 hours)
Plant growth test (pot experiment) – 4
months
Resource requirement
School UDDT
Apply cover material, innoculum,
collection stage
LAF+TC (faeces)
LAF (urine)
Figure 1.1 Main experimental stages for faeces and urine treatment via lacto-fermentation and
post-treatment. LAF - lacto-fermentation, LAB - lactic acid bacteria, TC - thermophilic
composting, VC - vermi-composting, SIB - sanitization indicator bacteria, GI - germination
index.
1.3 Thesis outline
This thesis is organized into 6 chapters: the introductory part, literature review and 3
research chapters as well as one discussion chapter. The introductory part gives background
information of the study topic, scope and outline of the research. The second chapter is a
literature review and describes the main nutrient and resource challenges faced by
conventional sanitation, the rate of soil degradation and the loss of organic matter in the soil
as well as the need for sustainable build-up of soil organic matter, including the application of
combined excreta and biochar, the fertilizing value of human excreta as well as advantages
6
and disadvantages of the use of biochar in agriculture. Furthermore, the chapter discusses the
challenges related to agricultural reuse of excreta such as hygienization aspects, loss of
nutrients, carbon matter and odour issues as well as the potential for lactic acid fermentation
to overcome these challenges. The chapter also addresses the limitations of lactic acid
fermentation, post-treatment of faeces via thermophilic composting and vermi-composting,
the role of biochar supplementation as well as agricultural effects of lacto-fermented excreta.
The third chapter evaluates the efficiency of lactic acid fermentation and thermophilic
composting versus lactic acid fermentation and vermi-composting for pathogen removal and
the effects on plant germination and growth. The results obtained from this chapter showed
that combined lactic acid fermentation with thermophilic composting are more efficient in
pathogen removal and plant growth than combined lactic acid fermentation with vermi-
composting. In the fourth chapter, the effects of lacto-fermented faeces and biowaste,
supplemented by biochar on soil quality (soil nitrate, phosphorus potassium and humus
content, moisture content as well as bulk density) and corn productivity (growth, yield and
yield components) was examined in a two year field experiment. A comparison was made
with no fertilization, lacto-fermented mix without biochar, stored human faeces, cattle
manure, urine as well as nitrogen, phosphorus and potassium mineral fertilizers. The lacto-
fermented mix of faeces and bio-waste, supplemented by biochar significantly improved plant
height (p < 0.05) compared to all fertilizers during the first production year and compared to
the control, stored faeces and vermi-compost during the second year. This fertilizer also
achieved a significantly higher corn yield compared to all other fertilizers during the first and
second production year, except for the lacto-fermented mix without biochar and the mineral
fertilizer, which showed no significant yield difference (p>0.05). It also reduced the bulk
density of the soil during both years and increased the soil potassium content during the first
production year. The yield components (rows per ear and kernels per ear) were improved
significantly when supplied with the lacto-fermented mix of faeces and bio-waste,
supplemented by biochar that might have attributed to potential prevention of nitrate leaching in
the root zone during dry spells, followed by rain flushes.
7
Chapter 5 discusses the potential of the improvement of the fertilizing value of urine
and the prevention of a pH increase and ammonia loss by urine lactic acid fermentation using
low cost LAB inoculum. The results showed that the urine lactic acid fermentation has led to
effective acidification to pH < 4 and a reduction by 1/3 of the ammonium content compared to
the stored urine. Moreover, lacto-fermented urine reduced twice the perceived odour strength
and improved seed germination, thus showing a potentially higher fertilizing effect than
untreated, stored urine.
Chapter 6 examines the potential applications of the research results in urine diverting
dry toilets (UDDT) in Moldova for the primary and secondary treatment of both the urine and
faeces fractions. The aspects on product quality requirements for potential full applications and
the input materials are given. Furthermore, an entrepreneurial model is proposed for the
application of compost obtained from lactic acid fermentation of faeces, kitchen and garden
waste. In this model, nutrient rich, lacto-fermented urine is used for the irrigation of a short
rotation copice (willow), grown for heating of schools. Faeces treated via combined lactic acid
fermentation and thermophilic composting is applied as growth medium for ornamental plants.
Potential revenue streams from sale of products (willow plantation and ornamental plants) are
highlighted.
8
Chapter 2. Increasing the agricultural value of human
excreta by lactic acid fermentation, composting and
biochar addition (literature review)
This chapter is based on:

Nadejda Andreev, Mariska Ronteltap, Boris Boincean, Piet N.L. Lens. Lactic acid
fermentation of human excreta for agricultural application (under review).
9
Abstract
Studies show that source separated excreta have a good fertilizing potential for
improving crop production and soil quality, the encountered effects are similar or even exceed
those of mineral fertilizers. The main challenges with agricultural application of excreta are
the high pathogenic risks (especially of the faeces fraction), increased loss of nutrients and
odour emissions. The use of lactic acid fermentation of excreta for improving its resource-
oriented value as well the post-treatment stages via vermi-composting and micro-aerobic
composting is reviewed. The review shows that lactic acid fermentation of excreta can
increase its agricultural value by reducing the amount of pathogens, minimizing the nutrient
loss and inhibiting the production of malodourous compounds. While pathogens as
Enterobateriacea, Staphylococcus and Clostridium can be reduced by 7 log CFU g-1 during 7-
10 days of fermentation, Ascaris may not be always efficiently removed. Direct application of
lacto-fermented faeces to agriculture may be constrained by incomplete decomposition, high
content of organic acids or insufficient hygienization. Post treatment by biochar addition,
vermi-composting or thermophilic composting will contribute to stabilization and sanitizing
of the material. The pot and field experiments on soil conditioners obtained by lactic acid
fermentation and post treatment steps by composting and biochar supplementation
demonstrate an increased crop yield and growth as well as improved soil quality in
comparison to the unfertilized control or other types of fertilizers (e.g. mineral fertilizer,
stored faeces, urine or cattle manure).
10
Increasing the agricultural value of human excreta by lactic acid
fermentation, composting and biochar addition (literature review)
2.1 Introduction
Global population growth, intense urbanization and economic development as well as

climate change increase competition over water, energy and land resources. While there is
recognition of the need to raise the food production for the growing population, there is also a
concern of the limitations of land and freshwater resources, which are undergoing worldwide
degradation (Hoff, 2011; Turral et al., 2011). Energy can be substituted from renewable
resources, while water and soil resources have no substitute. Therefore, it is important to
prevent their further degradation and where possible restore their quality.
Soil is lost at a 10-40 times faster rate than it is regenerated, approximately one quarter
of all cultivated land is undergoing degradation (Pimentel, 2006; Bai et al., 2008). For this
reason, farmers must spend more on energy, without a corresponding increase in crop yield,
thus negatively affecting the livelihood of poor populations, especially in the developing
countries of Africa, Asia and Latin America, whose economies depend on agriculture.
Erosion and desertification are increasing throughout the world, which declines agricultural
production in India, Pakistan, Nepal, Iran, Jordan, Lebanon and Israel (Pfeiffer, 2006).
Soil depletion and imbalances are a widespread problem in Africa and Asia (Tan et
al., 2005). The loss of nutrients and organic matter is also a concern in Europe, where
approximately 45% of soils have reached a low or very low organic matter content of 0-2%
(Rusco et al., 2001). This depletion is particularly intense in the Mediterranean region,
Southern and Eastern Europe, as well as in some countries in Western Europe (Jones et al.,
2012; Krupenikov et al., 2011; Virto et al., 2014).
The loss of soil organic matter leads to a reduction in soil fertility as well as its
structure, water holding capacity and biological activity of the soil. Soil organic matter is lost
when organic matter inputs are not replenished to the soil during cultivation (Vlek, 1997).
Along the history of crop fertilization, farmers gradually replaced organic fertilizers with
mineral fertilizers as the latter were more convenient to store, transport or spray (Schröder,
2014). In many parts of the world, increased application of nitrogen fertilizers was advocated
11
and subsidised (Mulvaney et al., 2009). Long term application of solely mineral fertilizers can
reduce soil organic carbon content (Boincean et al., 2014) or cause other adverse effects, such
as acidification (Guo et al., 2010).
Freshwater resources around the globe are also faced by degradation and scarcity
issues. For a number of countries in Africa and the Middle East, water is becoming physically
scarce, while for others (e.g. Latin America) it is economically scarce (Rijsberman, 2006).
Degradation of water bodies and water-related ecosystem services is a widespread global
problem (FAO, 2011). Sanitation is a major consumer and pollutant of water resources
(Gleick, 2003). Large volumes of drinking water are used to transport excreta, generating
enormous amounts of wastewater which cannot be fully cleaned with existing conventional
technology. This results in significant amounts of nutrients and organic matter being
discharged into surface waters, especially since 90% of the wastewater is released without
treatment. Reusing excreta in agriculture returns nutrients to the soil and reduces the pollution
of freshwater resources and, thus, the energy costs required for its treatment. Reusing excreta
to agriculture will allow replacing or complementing the mineral fertilizers and reduce the
pollution of freshwater resources and thus the energy costs required for its treatment.
Phosphorus reuse from sanitation is of particular interest, considering the projected
increase in the price and availability of phosphorous fertilizers due to depletion of mineral
reserves, the uneven geopolitical distribution and the increasing energy costs for their mining,
processing and extraction (Cordell and White, 2011). If only the phosphorous content from
urine and faeces could be recovered and reused, it would supply 22 % of the current global
phosphorous demand in agriculture (Mihelcic et al., 2011).
Prehistoric human societies have created long-lasting rich fertile soils by integrating
their excrement, biochar and other substances into the ground (Lehmann et al., 2003). This
occurred in the Amazon terra preta or Amazonian dark earths (Glaser and Birk, 2012; Woods,
2003), Northern Europe (Wiedner et al., 2015 a), Australia (Downie et al., 2011), and West
Africa (Frausin et al., 2014). This ancient practice of forming so-called terra preta soils
highlights the potential for application of excreta and biochar for improving the soil fertility.
12
This ancient practice of terra preta soils highlights the potential for modern application
of excreta and biochar for improving the soil fertility. A resource-oriented approach, named
"terra preta sanitation system" has recently been developed (Otterpohl and Buzie, 2011;
Otterpohl and Buzie, 2013; Schuetze and Santiago-Fandiño, 2014; Windberg et al., 2013),
which treats excreta by two combined processes: lactic acid fermentation (LAF), followed by
composting (usually worm composting). Biochar is also applied to reduce the nutrient losses
and obtain stable organic soil conditioners (Bettendorf et al., 2014; Glaser, 2015; Yemaneh et
al., 2014). Lactic acid fermentation contributed to controlling foul odour (Yemaneh et al.,
2014) and suppresses the growth of pathogenic bacteria (Scheinemann et al., 2015). It also
shortens the required stabilization time during the subsequent vermi-composting stage (with
earthworms), where further pathogen reduction, fragmentation and aeration occur in the
faeces (Otterpohl and Buzie, 2011; Otterpohl and Buzie, 2013)
Different aspects of terra preta sanitation have been investigated so far, e.g. integration
of terra preta sanitation in different sanitation systems (Schuetze and Santiago-Fandino, 2014;
Bettendorf et al., 2015), identification of effective LAB for excreta treatment; optimization of
carbohydrate sources for efficient hygienization and odour mitigation (Yemaneh et al., 2012;
Yemaneh et al., 2014; Yemaneh, 2015). Research on the effects of excreta treated via terra
preta sanitation on plant soil systems is rather scarce.
This chapter evaluates the efficiency of LAF, followed by vermi-composting and
microaerobic composting, in the treatment of excreta to improve its agricultural value, reduce
pathogens, control odour, and generate a long-lasting, stable product. The role of biochar for
avoiding nutrient loss and contributing to the formation of humus was highlighted and the
potential fertilizing effects of excreta treated via LAF combined with composting and biochar
addition were overviewed. Due to the scarcity of peer-reviewed publications on LAF of
excreta, articles on human food and animal feed were also reviewed. For a better appreciation
of the potential applications of this approach, a urine-diverting dry toilet was taken as an
example, in which the main fermentation conditions and additives required for their
optimization at different steps were analyzed separately for the urine and the faeces.
13
2.2 Nutrient and resource challenges in sanitation
Conventional water-based sanitation aims to increase hygiene, but it is not applicable

in all the societies of the world. Clean drinking water is used to transport excrement and is
then treated via an energy-intensive process, but is never 100% clean again (Figure 1, I). This
is inappropriate, especially in regions with scarcity of freshwater or energy resources, which
are growing due to droughts caused by global climate change (Hanjra and Qureshi, 2010). An
issue of concern is the pollution of freshwater resources that increases the costs of water
treatment.
Freshwater pollution increases the costs of potable water treatement, but the
wastewater that contaminates it contains numerous valuable resources, such as nutrients and
organic matter from the food people eat (Figure 2.1, II), in addition to constituents of concern,
e.g. pathogens and trace amounts of pharmaceuticals, hormones and pesticides (Figure 2.1,
III). Wastewater is collected and transported to the treatment facilities (Figure 2.1, IV), where
the pollutants are only partly removed (Muga and Mihelcic, 2008), in addition to getting
mixed with industrial wastewater and stormwater (Figure 2.1, V) (Tchobanoglous et al.,
2003). Even with advanced treatment such as microfiltration and reverse osmosis (Watkinson
et al., 2007), micro-pollutants (e.g. pesticides, phenol compounds, heavy metals,
pharmaceuticals and personal care products) cannot be fully removed during the treatment
processes (Tchobanoglous et al., 2003). They thus present a high risk to aquatic organisms
(Cirja et al., 2008) and people living downstream. At the same time, valuable resources like
nitrogen and phosphorus are not efficiently recycled (Katukiza et al., 2012; Rosemarin,
2010;). While part of them are recovered during the treatment process and reused in
agriculture (Figure 2.1, VI), another part is released into aquatic ecosystems, causing, along
with agricultural runoffs (Figure 2.1, VIII), eutrophication and impairment of water quality
(Figure 2.1, VII) (Tan et al., 2005; Jianyao et al., 2010; Syers et al., 2011).
14
Figure 2.1 Resource and nutrient challenges in conventional sanitation. A major

resource challenge is that treated water (I) is used as an excreta dilution and transportation
medium. Valuable resources (e.g. nutrients and organic matter) (II) enter our body through
food and are excreted (III), being flushed away into the wastewater collection systems (IV).
There, they are mixed with industrial wastewater (V) and treated at wastewater treatment
plants. Only a minor part of the nutrients and carbon matter is recycled to food production
systems (VI), while the major part is discharged into aquatic ecosystems (VII), causing
eutrophication and increasing the costs of water treatment. WTP - water treatment plant;
WWTP - wastewater treatment plant.
One limitation of conventional waterborne sanitation is the high maintenance and

operation cost (Schertenleib, 2005b). Most developed countries are already confronted with
significant costs for modernizing and upgrading their sanitation infrastructure, but in
15
developing countries, the scarcity of financial and human resources often renders large
wastewater treatment facilities dysfunctional only after a few years of operation. For example,
the government of Thailand has invested during the 1990s 1.5 billion USD in wastewater and
treatment facilities, but only 40 % were functioning properly shortly after their installation
(Schertenleib, 2005). Concern also rises over the limitations of the land suitable for landfilling
of sewage sludge resulting from wastewater treatment plants (Jiménez et al., 2010).
Most of the urban and peri-urban population of Africa, Asia and Central America as
well as rural populations from Eastern Europe are relying on pit latrines as primary means of
sanitation. This system is affordable for many people in water scarce areas and those with
limited financial resources (Kvarnström et al., 2006; Morella et al., 2009). They, however,
contribute to heavy groundwater pollution and contamination of the soil and water with
nutrients and pathogens, thus increasing the health risk of those using groundwater as a
drinking water source (Kimani-Murage and Ngindu, 2007; Graham and Polizzotto, 2013). In
addition, pit latrines are not suitable for crowded areas, sensitive coastal areas, soils with
rocky grounds or those with a high water table or periodically flooded (Winblad, 1997;
Hurtado, 2005). The potential increase in intensity of floods and rise of the groundwater level
as a result of climate change can make this type of sanitation less suitable for some areas,
especially in the coastal zones (Graham and Polizzotto, 2013).
2.3 Applying excreta and biochar to agriculture
2.3.1 Fertilizer value of human urine and faeces
If appropriately treated, human urine and faeces have a good potential for substitution
or complementing mineral fertilizers and soil conditioners. Resource oriented sanitation
systems are efficient for recycling nutrients and carbon matter of human excreta (Larsen et
al., 2009; Murray and Buckley, 2010). In some source separating sanitation systems, urine,
which the main source of plant available nutrients, is diverted by a separate outlet to a storage
tank and the faeces fraction is collected in special collection chambers or containers, to which
16
a bulking agent (e.g. sawdust or ash) is generally added for dehydration, smell reduction and
decomposition (Anand and Apul, 2014). After storage or other additional treatment for
hygienization and stabilization, urine and faeces can be applied to crop production systems,
thus ensuring a nutrient closed loop approach.
In human urine, nitrogen, phosphorous, potassium and sulphur are present in plant
accessible, ionic forms (Jönsson et al., 2004). Additional components with fertilizing value
contained in urine are calcium, magnesium and micro-elements, e.g. B, Cu, Zn, Mo, Fe, Co
and Mn (Kirchmann and Pettersson, 1994; Rodushkin and Ödman, 2001). The heavy metal
content is usually low in human urine (Kirchmann and Pettersson, 1994). It was calculated
that the application of urine can be economically more feasible than the production and use of
mineral fertilizers even when this is transported 100-200 km away (Johansson et al., 2000).
15 32
Pot experiments on barley with N and P labelling phosphorus and nitrogen crop uptake
from human urine which was comparable to the soluble nitrogen and phosphorus uptake from
mineral fertilizers (Kirchmann and Pettersson, 1994).
Faeces account for a smaller quantity than the urine (approx.1/10) and nutrients,
especially nitrogen, are organically bound (Jönsson, 2003; Winker et al., 2009). As the
amounts of nutrients, such as N, K and S (Rose et al., 2015) would probably cover only a part
of the crops' needs, the faecal derived compost could be used primarily as a P-rich organic
soil supplement and as a soil conditioner (Winker et al., 2009). For improving the fraction of
organic carbon and aeration (Strauss et al., 2003), mixing of faeces with organic waste and
carbon rich bulking material is recommended, which also enhances the thermophilic
composting process important for hygienization and reuse (Gotaas, 1956; Niwagaba, 2009;
Winker et al., 2009).
There have been many studies on the effect of source separated urine on different crops
and soils, but limited research exists on the effect of faeces or combined faeces and urine on
crops and soils (Table 2.1). Urine application had a beneficial influence on different crops such
as okra, cabbage, tomatoes and cucumber by increasing crop nutrient uptake, yield or pest
resistance, these effects being similar or even higher than those of mineral fertilizers
17
(Heinonen-Tanski et al., 2007; Pradhan et al., 2007; Pradhan et al., 2009; Akpan-Idiok et al.,
2012.
Table 2.1 Overview of the effects of source separated urine and faeces on crops and soil
quality
Length of
Experimental conditions Fertilizer Crop type Effects Ref.
experiment
Sandy loamy soil. Fertilizer rate: NPK and Okra1 Increase in the One growth 1
urine - 0,10, 15 and 20 m3L-1, control2, nutrient uptake, season
400 kg N ha-1, 2 weeks after UDT3 growth and yield
transplanting urine, at a similar rate to
Greeh. trial: plastic pots NPK mineral
field experim. - plots 20x20m fertilizer
50 plants/plot
Clay loamy soil. Seedlings UDT3 Cucumber2 The yield was not 2-3 months 2
transplanted from greenh. urine, significantly
outdoor and planted in banks of 1 mineral different than
m wide x 72 m long. Urine fertilizer mineral fertilizer
applied at 9.7 L/m2 10, 20, 30
and 40 days after planting.
Application rate: 233 kg N ha-1
(urine) and 34 kg N ha-1 (mineral
fertilizer)
Field experiments on an area of urine, Cabbage3 The yield was 89 days 3

63 m2 divided into plots of 4.5 mineral similar, pest
m2. Fertilizer application rate - fertilizer resistance higher
180 kg N ha-1. Urine - 1.4-2.0 than non-fertilized
L/m2 control and
mineral fertilizer
Greenhouse pot experiments of UDT Urine Tomato4 Urine and ash - 88 days 4
491 cm2. Fertilizer application urine and equal yield as
rate - 90 kg N ha-1 (min. fert.) ash, mineral mineral fertilizer
and 120 kg N ha-1 (urine). fertilizer and and 4 times higher
control yield than non-
fertilized plants.
Field plots experiments. UD6. Cabbage5 Higher yield, P One growth 5
Application rate: 50, 100, and Faeces, goat and K crop uptake season
200 kg N ha-1 manure and with goat manure,
Manual fertilizer application mineral but lower than in
incorporated by hoe, one week fertiliser mineral fertilizer
before seedling transplant. on a
18
pre-irrigated soil. Irrigation: 2

times or 3 hours per week.
Cabbage seedlings transplanted
to 3x3 m plots in cambisol at 36
plants per plot
7
Pot experim. with 2 month old Mixed Jatropha The use of 40-60% 12 weeks 6
seedlings faeces and of CTR
(CTR8) (stored for 122 days) urine incorporated into
allowed to dry (30 days) then (CTR) the soil
mixed at 0, 20, 40 and 60 % (wet Compared significantly
weight) with latosol. to mineral increased leaf
NPK fertilizer at 0 and 2 g per fertiliser and number, leaf area
pot was disolved in 200 ml of control and stem diameter,
water and mixed with latosol. the growth being
similar to mineral
fertilizers or
higher.
Water retention in
biotoilet residue
was 10 times
higher than in
control (latosol
only). Also, the N,
P, K content was
35, 7 and 9 times
higher than in the
soil
9
Field experiment on volunteer Combined Corn Positive effects on One growth 7
farming plots urine and corn yield and season
Plants planted in 10x10m blocks. faeces, water use
Fertilizer application after 4 control and efficiency, higher
weeks of planting at 200 ml/crop mineral effects compared
fertilizer to mineral
fertilizers and non-
fertilized control
1
Ambelmoschus esculentum, 2 control - no urine or mineral fertilizer was applied, 3Cucumber sativus ,
4
Brassica oleracea 5Lycopersicum esculentum 6Brassica oleracea 7UDT- urine diverting toilet 8,
9
CTR - composting toilet residue, 10Jatropha curcas 11Zea mays. References: 1 - Akpan-Idiok et. al,
2012; 2 - Heinonen-Tanski and Sjöblom, 2007; 3- Pradhan, 2007; 4 - Pradhan, 2009; 5- Mkeni and
Austin, 2009; 6- Triastuti et al., 2009; 7 - Guzha et al., 2005.
The use of faeces or combined faeces and urine has also contributed to a significantly
higher soil water retention and use efficiency in maize compared to mineral fertilizer (Guzha
19
et al., 2005). It also resulted in a higher yield of cabbage compared to goat manure (Mkeni
and Austin, 2009), due to higher available phosphorus and potassium concentration. The
compilation of these research findings (Table 2.1), clearly ilustrates the fertilizing value of
human excreta.
2.3.2 Advantages and disadvantages of biochar application to agriculture
Application of biochar to soils with or without compost and fertilizers has been
recognized among viable strategies for efficient restoration of soil organic carbon (Topoliantz
et al., 2005; Lehmann et al., 2006; Lal, 2009). This is related to its longevity in the soil, thus
its capacity to maintain stable soil organic matter for a longer period of time than application
of other organic matter, e.g. compost or manure (Lehmann et al., 2006). Biochars are
produced by pyrolyzation of biomass in an oxygen depleted atmosphere at a high temperature
of 400-800◦C (Verheijen et al., 2010). Hydrothermal carbonization produces another type of
biochar, called hydrochar, from wet biomass at low temperature (between 180 and 300 ◦C)
and high pressure (2 – 2.5 MPa). Application of biochar has a number of positive effects on
crops and soils, including increasing the water retention and cation exchange capacity (CEC)
(Abel et al., 2013) and reduction of nitrous oxide (N2O) by facilitating the transfer of
electrons to soil denitrifying microorganisms (Cayuela et al., 2013). As biochar contains
organic and inorganic compounds which are reduced under oxygen limiting conditions, these
act as electron donors under suboxic conditions for denitrifying microorganisms (Cayuela et
al., 2013). In paddy soils, biochar can absorb organic carbon onto its surface, thus reducing
the substrate availability to methanogens and generating less CH4 (Han et al., 2016). Biochar
can retain plant available nutrients, improve plant disease resistance (Elad et al., 2010) and
soil microbial biomass (Biederman and Harpole, 2013). It also enhances crop productivity
(Liang et al., 2006a; Lehmann and Joseph, 2009).
At high application rates, biochar may negatively affect plants (Buss and Mašek,
2014). Application of hydrochars or biochar produced from swine faeces caused negative
effects on soil and crops, including decreases in plant-available nitrogen and reduction in
20
plant growth (Gajić and Koch, 2012; Rillig et al., 2010) and biomass compared to the control
(George et al., 2012) or excessive increases in soil extractable phosphorus (Novak et al.,
2014). One of the causes is the volatile and leachable compounds produced during pyrolysis,
e.g. low molecular alcohols, ketones, phenols or polyaromatic hydrocarbons (Buss and
Mašek, 2014; Lievens et al., 2015). Furthermore, agricultural application of freshly produced
biochar may have no or negative effects on soil and plants. This is caused by highly labile
fractions of carbon that can lead to an increased mineralization of the organic matter already
present in the soil (Singh and Cowie, 2014) or nitrogen immobilization and, therefore, to a
reduction in crop growth and yield (DeLuca et al., 2009; Rillig et al., 2010). To make use of
freshly produced biochars productively, co-composting and enrichment with mineral or
organic fertilizers is recommended (Alburquerque et al., 2013; Schmidt et al., 2014). The
subsequent incorporation of some aromatic fractions into the humic substances increases the
humification indice of the final compost (Dias et al., 2010; Jindo et al., 2012). Moreover,
oxidation of biochar will improve its capacity to absorb nutrients and dissolved organic matter
(Cheng et al., 2006; Lehmann et al., 2003; Steiner et al., 2010).
2.3.3 Anthrosols: land application of human excreta and biochar
The reuse of animal or human excreta in combination with biochar was used
successfully in the past to transform easily weatherable sandy soils, poor in organic matter
and plant available nutrients into long lasting, highly fertile lands (Pape, 1970; Lehmann et
al., 2003). An example is the terra preta soil type, created as a result of surface deposition,
slush and burn cultivation or soil enrichment practices, with waste materials such as plant
residues, human excreta and charred biomass in permanent pre-Columbian Indian settlements
(Erickson, 2003; Woods, 2003). These soil types are distributed in Brazil, Columbia, southern
Venezuela, Peru and the Guianas (Sombroek et al., 2002; Kämpf et al., 2004). Analysis of
faecal specific steroids revealed a high input of human excreta into the terra preta soils
(Glaser, 2007; Glaser and Birk, 2012). The biochar component contributed to soil stability
and to an increased microbial biomass growth (Glaser, 2007; Glaser and Birk, 2012).
21
Recent studies have indicated the presence of terra preta analogous soils in Northern
Europe, Australia, Asia (e.g. China and Japan) and Africa (Downie et al., 2011; Frausin et al.,
2014; Wiedner and Glaser, 2015; Wiedner et al., 2015a), where the local indigenous
population had similar waste management and soil enrichment practices as those in South
America. In Europe, the anthrosols were formed from heath and grass, mixed with animal
manure, human faeces and charcoal there were deposited on sandy soils (Figure 2.2). The
total elemental content was highly enriched compared to the reference soil, also the biochar
content (30 tons ha-1) was of the same magnitude as in terra preta soils (Wiedner et al.,
2015b).
Figure 2.2 An example of plaggen soil (grey plaggic anthrosols) from Germany (source: Giani
et al., 2014). The thickness of the plaggic horizon is 70-120 cm, very dark in colour and
contains artefacts such as charcoal and bricks.
In China, the farmers mixed the vegetable waste with straw, turf, weeds, faeces and
soil and burned it, after which they applied it to the soil (Wiedner and Glaser, 2015). From
ancient times in Japan, farmers have mixed human and animal excreta with rice husk biochar
and wood ash for fertilization and conditioning of the soil (Ogawa and Okimori, 2010). In
Australia, the anthrosols containing charcoal were developed as a result of the disposal of
22
waste materials from cooking to the soil. The addition of charcoal improved the soil's physical
and chemical properties, including its nutrient status, cation exchange capacity and water
holding capacity. In West Africa, anthrosols are still under production and use (Frausin et al.,
2014). The waste materials added to the soil are charcoal from cooking, residues from palm-
oil soap production as well as organic materials from food preparation, crop processing, house
construction and agroforestry. All these studies suggest that charcoal in combination with
excreta created favourable conditions for maintaining soil fertility over long periods of time
during ancient times and this practice can be applied today to restore soils around the world.
2.4 Challenges in agricultural reuse of excreta
2.4.1 Pathogen reduction
One of the main challenges for excreta application in agriculture is the risk of
infecting people with disease via contaminated food crops. In human urine the number of
pathogens is usually low, but potentially dangerous pathogens may still be present in endemic
regions, due to urinary excretion, e.g. Leptospira, Salmonella, Ascaris and Schistosoma
(Drangert, 1998; WHO, 2006). In urine-diverting toilets, cross-contamination is difficult to
avoid. This also leads to an increase in the number of pathogens in urine: a study carried out
in Sweden showed that 22% of the samples collected from urine tanks were contaminated
with faeces (Höglund et al., 2002).
In contrast to urine, human faeces contain a much higher number of bacteria, viruses,
parasitic protozoa and helminths. Due to the potentially high amounts of pathogens, faeces have
always been considered contagious and handled accordingly, since poor excreta sanitation may
lead to the spread of disease (WHO, 2006). For example, in Vietnam, fertilization of farmlands
with fresh or partially composted faecal waste from latrines caused up to 30% of hookworm
infection among the local population and high indices of parasites like Ascaris, Trichurus and
Taenia (Jensen et al., 2005).
23
Different treatment procedures have been proposed to reduce the amount of pathogens
in excreta, among which storage and composting (thermophilic composting and vermi-
composting) are considered among the simplest in operation. Depending on the local climate,
storage during at least 1-6 months for urine and 1-2 years for faeces is recommended for
pathogen removal (WHO, 2006). Desiccation along with high pH was proven to be efficient in
pathogen destruction during the storage of faeces (Niwagaba et al., 2009). According to the
WHO, in order to reduce the pathogens in human excreta to a safe level, a moisture content
below 25% and a pH > 9 shall be ensured. In real life situations, such conditions of low
moisture content and high pH can rarely be achieved. For example, wood ash with the potential
to increase the pH above 9 does not always provide sufficient dehydration and thus pathogen
reduction (Kaiser, 2006). Moreover, moisture levels below 25% limits the activity of
decomposing bacteria and fungi (Anderson et al., 1979) and therefore the faecal material during
storage is more dehydrated than decomposed.
Composting is an effective method for sanitizing source-separated faeces. It does,
however, need to be mixed with organic waste, like vegetable scraps, at different ratios (Koné et
al., 2007; Niwagaba et al., 2009). A sanitizing temperature above 50 °C can be achieved and
maintained for a sufficient number of days in stored faeces, if equal amounts of faeces and food
waste are mixed: an E.coli and Enterococcus reduction of 3 log CFUg-1 and 4 log CFU g-1,
respectively, was achieved (Niwagaba et al., 2009). Also, the addition of a carbon source, such
as sawdust or straw is required to adjust the low C/N ratio of faeces to 5-10 (Gotaas, 1956). In
addition, good insulation (e.g., 25-75 mm styrofoam), frequent turning and moisture levels
below 65% are required.
Another way to reduce the pathogens present in faeces is via vermi-composting. The
mechanisms by which earthworms contribute to the reduction of pathogens are not clearly
described. Some studies have shown that the coelomocytes (a type of leukocyte) of earthworms
reduce the number of pathogens, through phagocytosis, encapsulation and secreting proteins
that adhere to the bacterial cells and inhibit their activities (Pablo, 2001; Popović et al., 2005)
during the passage of faeces through the guts of earthworms. Coliform bacteria were
significantly reduced in comparison to the control only at low doses of pig slurry
24
(approximately 1 g of slurry to 1.5 g of earthworms per day), while at a higher dose (3 g slurry :
1.5 g of earthworms per day) it did not reduce their number significantly (Monroy et al., 2009).
Studies also indicate that the chitinase enzyme of the earthworms can destroy the middle
protective shell of helminth eggs (Hill et al., 2013b), but several passages through the
earthworm gut are required. However, experiments on the reduction of Ascaris suum egg
viability did not prove efficient in comparison to the control soil without earthworms (Bowman
et al., 2006). Moreover, the number of viable eggs could even recover after 6 months of
vermicomposting (Bowman et al., 2006). An efficient reduction of E.coli, Salmonella and
Ascaris was achieved with a high stocking density, i.e. an earthworm biomass to biosolid ratio
of approximately 1 : 1.5 (Eastman et al., 2001). This is as much as 40 kg of earthworms to 60
kg of biosolid, an option which is not economically feasible in all cases (Mupondi et al., 2010).
Vermicomposting requires an extended time of 3-5 months (Bajsa et al., 2005; Sinha et al.,
2009), during which a significant amount of nitrogen can be lost.
2.4.2 Loss of nutrients, carbon matter and odour issues
Long term storage along with the use of alkaline covering material in faeces leads to
nutrient losses via volatilization (Nordin, 2010). During extended storage and aerobic
decomposition of faeces, up to 94% of the nitrogen and a significant loss of the carbon can be
released to the atmosphere (Lopez Zavala et al., 2002; Hotta and Funamizu, 2007).
Composting also leads to nitrogen losses through volatilization, leaching and denitrification
(Hao et al., 2001). Up to 92% of the ammonia content can be released during composting
(Eghball et al., 1997), the highest ammonia volatilization being encountered during the
thermophilic phase of composting, when intensive mineralization takes place (Bernal et al.,
1996; Hao et al., 2011). Part of the nitrogen is also emitted as N2O during the maturation
phase of composting (He et al., 2001). Also a significant portion of organic matter (e.g. up to
62%) is lost as CO2 during the bio-oxidative stage of composting (Bernal et al., 1996). During
vermi-composting, most of the nitrogen is emitted as N2O due to incomplete denitrification
processes occurring in the guts of the earthworms (Frederickson and Howell, 2003). The
25
variations in the nitrogen and organic matter loss depend on the time required for
decomposition of the organic waste.
Urine storage also leads to nutrient loss. Under the influence of urease positive
bacteria and free urease, urea is degraded to ammonia (NH4+) (Udert et al., 2003; Udert et al.,
2006), part of which can be lost during storage, transportation and field application (Sherlock
and Goh, 1984; Rodhe et al., 2004). The release of ammoniacal nitrogen increases at pH
values above 8 (Williams et al., 2011). Urine hydrolysis also facilitates the precipitation of
phosphates and carbonates, which are deposited at the bottom of the collection tanks (Udert et
al., 2003). Repeated application of stored urine to the soil can cause salinization, due to its
ionic composition (Ca2+, Mg2+ and SO42-) and a major source of soluble salts as NaCl (Neina
and Dowuona, 2014). Ammonia and other malodourous compounds (e.g. VFA, indolic and
phenolic compounds) formed during organic decomposition cause an undesirable odour of the
stored urine (Troccaz et al., 2013; Zhang et al., 2013), which might be of considerable
concern during agricultural applications.
Chemical acidification prevents volatilization of nitrogen, thus increasing the
fertilizing value of excreta. For example, acidified cattle slurry had a 26 % higher nitrogen
content than the non-acidified one (Kosmalska, 2012). Besides reducing the NH3 loss,
acidification of the slurry substantially reduces CH4 emissions (Petersen et al., 2012).
However, this method is costly compared to the values of increased nitrogen retention
(Kirchmann, 1994). Acidification with sulphuric acid can cause H2S emissions and may
negatively impact the soil by raising its electric conductivity (Frost et al., 1990). In addition,
acid treatment may cause an increase in the concentrations of volatile fatty acids and volatile
sulphurous compounds that may add to an overall increase in odour emissions (Ottosen et al.,
2009; Petersen et al., 2012). Therefore, lowering the pH value can be a promising possibility
for reducing gaseous emissions in excreta. However, chemical acidification may be
undesirable due to potential environmental concerns and odour intensification. Alternative
possibilities that use environmentally friendly bio-based products shall be considered.
26
2.5 Lactic acid fermentation of human excreta
2.5.1 Transformations occurring during lactic acid fermentation
Lactic acid fermentation has been widely applied in food and silage preservation,
treatment of kitchen and agricultural waste as well as animal manure (Kamra et al., 1984;
Andersson et al., 1988; Wang et al., 2001; Schroeder, 2004; Murphy et al., 2007). The use of
lactic acid fermentation for treatment of human excreta has been extensively studied within
the terra preta sanitation approach (Factura et al., 2010; Otterpohl and Buzie, 2011; Yemaneh
et al., 2012; Otterpohl and Buzie, 2013; Schuetze and Santiago-Fandino, 2014; Yemaneh et
al., 2014; Bettendorf et al., 2015).
Lactic acid fermentation consists of two major stages: an aerobic and an anaerobic
one. During the first stage, oxygen is consumed by a mixed culture of aerobic microorganisms
(Woolford, 1984). During the second stage, the soluble carbohydrates are transformed to
organic acids, ethanol, acetaldehyde and carbon dioxide by homofermentative and
heterofermentative lactic acid bacteria (LAB) (Murphy et al., 2007). The accumulation of
high amounts of organic acids results in a pH decline to a level that inhibits further microbial
growth. The lacto-fermented material thus becomes stabilized and can be preserved for a long
time.
The extracellular enzymes produced by fermentative bacteria contribute to the
breakdown of the polysaccharides, proteins and lipids into smaller monomers, which are
further used for synthesis of organic acids and microbial exopolysaccharides or for bacterial
growth (De Vuyst and Degeest, 1999; Sánchez, 2009). LAB produce a wide range of enzymes
such as hydrolytic, carbohydrate degrading enzymes, proteolytic, lypolytic and starch-
modifying enzymes (Patel et al., 2013; Petrova et al., 2013), which solubilise the solid
substrate. Thus, a better substrate utilization by bacterial and fungal consortia is possible
when additional treatment, such as composting, is performed after the lactic acid fermentation
(Xavier and Lonsane, 1994; Yang et al., 2006). As the excreted faeces contain cellulolytic
bacteria such as Clostridium and Bacteroides, also non-digestible compounds like
27
lignocellulotic materials (e.g. sawdust from cover material or toilet paper) can be partly
decomposed during lactic acid fermentation (Bryant, 1978; Wedekind et al., 1988;
Kalantzopoulos, 1997).
With a sufficient amount of organic acids, there is a pH decrease, which prevents the
degradation of proteins; does not significantly change the nutrient contents; and lowers the
organic matter loss. For example, the protein content did not change significantly after 10
days of lactic acid fermentation of poultry manure with 10 % of molasses and 3 % of LAB
inoculum at pH 4 (El-Jalil et al., 2008). In another study on a corn-broiler litter mixture, the
total nitrogen content as well as crude protein content changed insignificantly even after 80
days of ensiling (Caswell et al., 1977). Also, treatment of manure with acidic liquid bio-waste
from the milk and citrus industry decreased the pH to 5, reducing the CH4, N2O and NH3
emissions by 76-78%, 36-37 % and 84-86 %, respectively (Clemens et al., 2002; Samer et al.,
2014). After treatment of manure with lactic acid, no N2O emission was recorded anymore
and a 75-90% reduction of CH4 emission was observed (Berg et al., 2006).
Lactic acid fermentation can contribute to the reduction of pathogens in excreta
(Scheinemann et al., 2015; Yemaneh, 2015). However, its efficiency in the destruction of
resistant pathogens is still insufficiently investigated. An effective pathogen reduction was
attained with the addition of molasses and wheat bran as source of carbohydrates, which
contributed to the formation of lactic acid and pH reduction. Lactic acid fermentation of
faecal sludge and manure contributed to effective pathogen reduction with the addition of
molasses and wheat bran (10-12 %) as sources of soluble carbohydrates (McCaskey and
Wang, 1985; Scheinemann et al., 2015). Lactic acid fermentation of swine manure has
decreased the concentration of the Enterobacteriacea, Staphylococcus and Clostridium
populations by 7 log CFUg-1 after 5 - 10 days, when the pH was reduced to 4 (El-Jalil et al.,
2008). In another study on swine manure with corn, a complete destruction of faecal
coliforms, Salmonella derby and Treponema hyodysenteriae was obtained during 3 weeks of
ensilage at pH 4 (Weiner, 1984). The pathogeneity of Ascaris suum was lost completely after
56 days its viability was already reduced after 21 days (Scheinemann et al., 2015) at pH 5.0-
5.2. In a study on swine manure, Ascaris suum eggs remained infective even after 56 days of
28
fermentation processing at pH 3.9-4.4, even though their viability was considerably reduced
(Caballero-Hernández et al., 2004). Further investigations are needed on the efficacy of
combined lactic acid fermentation and thermophilic composting or vermi-composting in the
treatment of faeces.
The hygienization of faeces by lactic acid fermentation is caused by pH reduction and
the production of lactic acid and other compounds with antagonistic activities to fungi,
protozoa and a wide range of gram positive and gram negative bacteria (Rattanachaikunsopon
and Phumkhachorn, 2010). Protein complexes like bacteriocins and other compounds, e.g.
glucose oxidase, hydrogen peroxide and exopolysaccharides have also a suppressive effect on
pathogenic microorganisms (Patel et al., 2012; Saranraj, 2014). The mechanisms of the
reduction of helminths eggs are poorly described. One of the factors is the increased
temperature (36-37 °C) during fermentation (Scheinemann et al., 2015). As lactic acid
fermentation does not produce a temperature increase and the process usually takes place at
lower temperatures than those indicated above, it might be challenging to achieve this.
Lactic acid fermentation also deodorizes the offensive odour of excreta, as LAB
inhibit microorganisms producing malodourous ingredients, e.g. S-compounds (H2S), N-
compounds (NH3, indole and scatole) and C-compounds (lower fatty acids) (Hata, 1982; Zhu,
2000; Wang et al., 2001). Formation of volatile fatty acids, responsible for the odour
production in faeces, is inhibited during the lactic acid fermentation process (Kamra et al.,
1984; Yemaneh et al., 2014). After a few days of fermentation, the foul faecal odour is
changed to a sour-silage-like one. Urine lacto-fermentation leads to odour reduction by a
decrease in malodourous compounds (Yemaneh et al., 2012; Zhang et al., 2013). For
example, it was found that a range of urease positive bacteria such as Escherichia fergusonii,
Enterococcus faecalis, Citrobacter kaseri, Streptococcus agalacticae and Morganella
morganii are responsible for the representative stale urine odour due to emission of phenol,
indol and sulphide (Troccaz et al., 2013). LAB inhibit the activities of these organisms by
producing a wide range of secondary metabolites (Savadogo et al., 2004).
Considering the potential of lactic acid fermentation for hygienization as well as
reduction of nutrient loss and odour emissions, this treatment technique can be applied in
29
mixed and separately collecting sanitation systems (Windberg et al., 2013; Anderson et al.,
2015). In UDDTs, the addition of LAB and accompanying sugar-containing substrates can be
done both at the collection and post-collection stages (Figure 2.3).
Figure 2.3 Overview of the potential steps and additives used during lacto-fermentation of the
urine and faeces fraction of urine diverting dry toilet systems
LAB: lactic acid bacteria, SC: soluble carbohydrates.
30
2.5.2 Lactic acid fermentation of urine
At the collection stage, urine can be treated with a solution of LAB and soluble
carbohydrates, e.g. molasses or whey (Figure 2.3). A limited number of studies (Reckin,
2010; Beriso and Otterpohl, 2013; Wolf, 2013) report about urine treatment via lactic acid
fermentation. Furthermore, there is only scarce information regarding the added agricultural
or hygienic value. Some medical studies indicate that LAB strains such as Lactobacillus
casei, L. acidophilus, L. gasseri and L. plantarum produce substances that inhibit the activity
of urease positive bacteria, for example in the pathogenic bacterium Helicobacter pylori
(Michetti et al., 1999; Servin, 2004; Sgouras et al., 2004). Among the potential inhibitory
mechanisms, the production of bacteriocins is mentioned (Tabak et al., 2012). Similarly to
this, during lactic acid fermentation of urine, LAB can inhibit urease positive bacteria, thus
preventing urea hydrolysis.
During storage, the urine pH increases rapidly up to 8-9, when most urea is
hydrolyzed (Kirchmann and Pettersson, 1994; Udert et al., 2003). The hydrolyzed urine has a
high buffering capacity, it would be more appropriate if the soluble carbohydrate source and
the LAB are added to storage tanks prior to urine starts to accumulate there. Non-hydrolyzed
urine could be a good growth medium for LAB as it contain urea, amino acids and minerals
such as K+, Na+, Mg2+, PO43-, SO42- and Cl- which are important for the growth of LAB
(MacLeod and Snell, 1947; Udert et al., 2006). Experiments show good acidification effects
of the sauerkraut juice microbial inoculum to the fresh urine with a ratio of 1:6 by decreasing
the pH to 4 after 1.5 month treatment (Wernli, 2014). During the urine lactic acid
fermentation, the LAB inhibit the bacterial urease, thus preventing urea hydrolysis and
consequently reduce ammonia volatilization. For example, studies have shown that the urease
activity is diminished at pH < 5 (Larson and Kallio, 1954; Schneider and Kaltwasser, 1984). The
highest release of NH3 takes place between pH 7 and 10; below pH 7, NH3 volatilization
decreases and at pH 4.5 free ammonia is completely unavailable (Hartung and Phillips, 1994;
Williams et al., 2011) (Hartung and Phillips, 1994; Williams et al., 2011).
31
Further research on the application of lactic acid fermentation of urine is important

considering the potential for reducing the pH, thus minimizing the ammonia loss and
phosphorous precipitation as well as the associated consequences such as smell and pipe
clogging. It is also important to investigate the effects of lactic bacteria on malodourous
components in urine. Lactic acid fermentation can further add to a reduction of pathogens that
may result from faecal cross-contamination. The capacities of LAB to degrade
micropollutants such as hormones, pharmaceuticals and pesticides which may be excreted via
urine and diffuse into the aquatic environment after urine application in agriculture need
further investigation. Ensilage of corn grain mixed with broiler litter for a period of 80 days
reduced significantly (p < 0.01) the concentration of sulfoquinoxaline, a veterinary medicine
to treat coccidiosis compared to before ensilage (Caswell et al., 1977). Some LAB strains are
effective in the decomposition of organophosphorus pesticides, using them as a source of
carbon and phosphorus for the synthesis of the enzyme phosphatase (Cho et al., 2009; Zhang
et al., 2014b).
Lacto-fermented urine can also be soaked on biochar for nutrient capturing.
Application of wood biochar could reduce ammonia volatilization from ruminant urine by 45
%, while retaining its bioavailability to plants (Taghizadeh-Toosi et al., 2011). Acidified urine
may be more effective for biochar charging because sorption of ammonia and phosphate onto
biochar is higher at lower pH (Yao et al., 2011; Spokas et al., 2012). For example, phosphate
adsorption was maximal at pH 2 to 4.1 and decreased at pH above 6 (Yao et al., 2011). The
ions and organic molecules in the urine can enter the pore system of the biochar particles
(Schmidt et al., 2015). Besides, the organic compounds in the urine are adsorbed on the
biochar surface, forming an organic coating onto which anions and cations from urine (e.g.
phosphates and ammonium) are bound (Schmidt et al., 2015).
2.5.3 Lactic acid fermentation of faeces
Fresh human faeces contain a low number of microbial species facilitating lactic acid
fermentation, e.g. Lactobacillus, Pediococcus and Leuconostoc (Sghir et al., 2000; Dal Bello
32
et al., 2003). Thus, the addition of lactic acid-producing bacteria as well as substrates
favouring their growth is required (Otterpohl and Buzie, 2013). The use of lactobacilli strains
such as Lactobacillus plantarum, Lactobacillus casei and Pediococcus acidilactici as
additives promotes effective lactic acid fermentation of faeces (Yemaneh et al., 2012). Low
cost sources of LAB are waste products of milk, fish, meat and grains (Frazier, 1967; Park
and DuPonte, 2008; Windberg et al., 2013; Bulbo et al., 2014). The outer layers of grain,
especially rice, contain essential nutrients, carbohydrates, vitamins as well as microorganisms
from the families of Lactobacilacea, Pseudomonacea, Micrococcacea, and Bacilacea
(Frazier, 1967; Saman et al., 2011). Therefore, rice wash water can serve both as a growth
medium and inoculum.
The excreted carbohydrate fraction in faeces, which is an important substrate for lactic
acid bacteria, is usually composed of undigested cellulose, vegetable fibre, oligosaccharides
and polysaccharides, not hydrolyzed by the intestinal secretions of humans (Canfield and
Goldner, 1964; Southgate and Durnin, 1970). Soluble carbohydrates can be found in
molasses, whey or waste from starchy materials such as potato, wheat, manioc or barley
(Anuradha et al., 1999; Pandey et al., 2000). A potential cheap source of soluble
carbohydrates that can be added to the cover material for faeces dehydration is press mud, a
non-value waste from the sugar industry which contains 5-15 % sugar (Xavier and Lonsane,
1994; Solaimalai et al., 2001; Partha and Sivasubramanian, 2006) and is produced in large
amounts. For example, the sugar industry in India produces annually 5.2 million tonnes of
press mud (Rakkyappan et al.2001). Studies have shown that this material was successfully
used as a mixing agent in silages (van der Poel et al., 1998). Press mud addition is important
if lactic acid fermentation is followed by vermi-composting, as it is easily processed by
earthworms (Prakash and Karmegam, 2010). Additional components in the covering material
can be chopped corn stover, clay and rock flour (Snyman et al., 1986; Bottcher et al., 2010;
Bagar and Kavčič, 2013). Grinded corn stover was efficiently lacto-fermented with cattle
manure (Murphy et al., 2007). The rock flour stimulates microorganisms by providing them a
habitat and the necessary micronutrients required for the breakdown of organic matter. Clay
33
and rock flour will add to the formation of organo-mineral chelated complexes, thus
increasing organic matter stability (Stevenson, 1994; Bagar and Kavčič, 2013).
Some authors indicate that a mixture of ground biochar can also be added to the
covering material at the collection stage of faeces (Factura et al., 2010; Windberg et al.,
2013). The application of biochar as a matrix in composting toilets showed a 2.3 and 1.7
times lower carbon loss and a 1.9 and 1.3 lower nitrogen loss than rice husk and corn stalk,
respectively (Hijikata et al., 2015). Since the pH of the biochar is mostly alkaline (Verheijen
et al., 2010) it may hinder the fermentation process. Therefore, its addition at the end of the
lactic acid fermentation (Yemaneh et al., 2014) or at the post-treatment stage (thermophilic or
vermi-composting) is more appropriate (Bettendorf et al., 2014).
The quantity of source separated faeces and toilet paper generated per capita per year
is relatively low, approximately 70 kg and 6 kg (wet weight) per year, respectively (Rose et
al., 2015). After the collection stage, faeces and toilet paper can be treated via extended lacto-
fermentation along with bio-waste such as kitchen/food industrial waste (e.g. fruit, milk or
sugar industry wastes) or animal manure (Figure 2.3). Vegetable waste is rich in LAB,
enzymes (e.g. cellulolytic, lignolytic and pectinolytic) and carbohydrates (Jawad et al., 2013)
and can increase the efficiency of lacto-fermentation of faeces and cover material as well as of
the thermophilic stage of composting, thus shortening the overall time required for the
treatment of faeces (Ong et al., 2001). Pre-fermenting (simple storage in closed vessels) of
kitchen and fruit waste prior to being mixed with the faeces will release the sugars under the
influence of extracellular enzymes and facilitate a better fermentation of faeces. Addition of
cattle or swine manure to faeces during secondary faeces treatment is beneficial owing to a
higher number of lactic, cellulolytic and hemicellulolytic bacteria, that are capable of
fermenting a range of monosaccharides, oligosaccharides, polysaccharides and lignocelluloses
(Cotta et al., 2003; Dowd et al., 2008). These cellulolytic and hemicellulolytic bacteria, e.g.
Clostridia and Ruminococcus (Sijpesteijn, 1951; Wang et al., 2007; Dowd et al., 2008), are
important for an effective decomposition of lignine and cellulose from toilet paper and
sawdust in the cover material, before soil application. Micro-aerobic conditions occur in
compost when the oxygen concentration is less than 2.0% throughout the whole composting
34
process (Wang et al., 2001). Under these conditions, lignocellulose degradation takes place at
the initial composting stage as the bacteria which decompose celluloses are more effective.
2.5.4 Limitations of lactic acid fermentation
Among the main limitations of lactic acid fermentation is the need for creating an
enabling environment for the optimal growth of LAB, promoting acidification and
stabilization of excreta, e.g. provision of LAB strains and nutritional additives like a carbon
source (Saeed and Salam, 2013). The nature of added components (e.g. kitchen, waste from
food industry or cattle manure) as well as the interactions between different LAB strains and
fungi during the storage and the fermentation process may also influence the efficiency of the
lactic acid fermentation or combined lactic acid fermentation and composting (aerobic
thermophilic composting or vermi-composting) processes. These affect the structure of the
substrate, oxygen permeability, moisture content and pH value.
Another limitation is that an effective acidification of faeces is obtained only with the
addition of sources of easily fermentable carbohydrates such as molasses and wheat bran
(Yemaneh et al., 2012; Scheinemann et al., 2013; Böttger et al., 2014). These may have other
potential competitive uses, such as in the fermentation industry, production of renewable fuels
or food and feed additives (Leng, 1984; Noike and Mizuno, 2000; Dumbrepatil et al., 2008;
Siqueira et al., 2008; Javed et al., 2012). Therefore, it is important for revealing non-value
waste materials rich in sugar or starch, such as kitchen waste and food industry waste, to be
used in lactic acid fermentation (Samer et al., 2014; Yemaneh et al., 2014).
2.6 Post treatment of lacto-fermented human excreta
Lactic acid fermentation of faeces contributes to hygienization, pre-decomposition of

the digestate and odour reduction. However, direct application of lacto-feremented faeces to
the soil may create some problems. There are a number of constrains for soil application
(Figure 2.4): the lacto-fermented faeces may not always be fully sanitized (Factura et al.,
35
2010; Buzie and Körner, 2014), the lacto-fermented faeces is anaerobic and rich in organic
acids, its addition to the soil may cause incomplete denitrification or intensification of the
mineralization of soil organic matter (Hamer and Marschner, 2005; Green and Popa, 2011).
In poorly drained soils, an accumulation of organic acids may occur with a phyto-toxic
effect on plant roots (Fu, 1989). As the lacto-fermented material may be incompletely
mineralized, its direct application to the soil may cause a rapid decomposition, leading to a
decrease in oxygen concentration in the root zone and potentially to an increase in the
solubility of heavy metals (Jiménez and Garcia, 1989). Therefore, before being applied to the
soil, the lacto-fermented faeces can be either mixed with biochar or processed further via
vermi-composting or thermophilic composting.
2.6.1 Vermi-composting
Vermi-composted material has a better structure as the earthworms are mixing and
grinding the substrate, thus increasing nutrient availability and the content of humic
substances (Figure 2.3). The mineralization activity of bacteria, fungi, and actinomycetes
populating the earthworm gut release plant available nutrients, for example N, P K and trace
minerals (Zn, Mn and Fe) (Yadav and Garg, 2011). One limitation of this treatment option is
that it needs to be pre-conditioned, e.g. ventilated for at least 72 hours or mixed with bulking
material such as vermi-compost, cattle manure or biochar prior to inoculation with
earthworms (Buzie and Körner, 2014). Limited oxygen supply in the lacto-fermermented
material and low pH may be detrimental to earthworms as they die very quickly in anaerobic
conditions (Munroe, 2007). The lacto-fermented mix is also rich in organics (lactic and acetic
36
Figure 2.4 Effects of lacto-fermentation on human faeces, constraints for soil application and
post-treatment via combined lacto-fermentation with vermi-composting or thermophilic
composting. Human faeces mixed with bio-waste can be lacto-fermented under conditions of
low oxygen supply (e.g. pit earths covered with plastic foil) during which pathogens and
odour are reduced. Due to incomplete decomposition and insufficient pathogen removal,
additional treatment of excreta needs to be performed via a composting stage (this could be
either themophilic or vermi-composting.
37
acids) and has a low pH that might have a deleterious effect on earthworms (Frederickson and
Knight, 1988). For example, earthworms introduced in lacto-fermented faeces died within
24 hours (Factura et al., 2010).
Vermi-composting and earthworm activities generate high N2O emissions from the
worms themselves and their casts (Horn et al., 2003). Treatment of faeces and bio-waste via
lactic acid fermentation results in a high content of organic acids and amino acids, which
decrease the pH. The sugar and organic acids can serve as electron donors for nitrate
reduction in the gut of earthworms and thus can lead to more intense N2O emissions in the
course of incomplete denitrification (Karsten and Drake, 1997; Asuming-Brempong and
Nyalemegbe, 2014). Combining lactic acid fermentation with thermophilic composting is thus
a more preferred option than lactic acid fermentation with vermi-composting.
2.6.2 Thermophilic composting
Another way to improve the structure of the lacto-fermented faeces is through

composting. Compost maturation can be achieved with passive aeration and limited turning
only (micro-aerobic composting), with an oxygen supply of less than 2 % or by turning and
forced aeration (aerobic composting) (Wang et al., 2001). For example, the soluble sugar
content was significantly lower, while the rate of cellulose degradation was significantly
higher after 15 days of composting under micro-aerobic composting compared to aerobic
composting. Furthermore, based on the C:N ratio, the compost was mature at 45 days under
micro-aerobic conditions and at 60 days under aerobic conditions (Wang et al., 2001). Micro-
aerobic composting with limited oxygen supply might be more efficient for the degradation of
lacto-fermented substrates. The presence of labile forms of carbon in the initial material
(caused for example, by the addition of molasses) contributes to a reduction of ammonia
volatilization due to immobilization of the NH4+-N by the microbial biomass. Provision of
extensive air supply and turning is counterproductive as it leads to an intense release of
nitrogen (NH3-N) from the composting material (Liang et al., 2006b).
38
During the thermophilic phase of micro-aerobic composting, along with labile carbon,
slower degradable materials such as celluloses and hemicelluloses are also degraded by
facultative thermophile anaerobes, e.g., Clostridia, whose cellulolytic enzyme system works
optimally in micro-aerobic conditions, with O2 concentrations below 2 % and temperatures of
50-60 °C (Schwarz, 2001; Wang et al., 2007). In contrast, lignocellulolytic degradation will
be inhibited in aerobic composting and fungi and actinomycetes will perform degradation of
these materials at the end of the composting period. Therefore, the aerobic composting
process is taking longer to stabilize, it also requires turning and aeration (Wang et al., 2007).
With shorter turnover rates, micro-aerobic composting can recycle carbon matter to the soil
more efficiently than aerobic composting or vermi-composting. For example, during micro-
aerobic composting only 2% of carbon is lost as CO2, compared to 41% and 63%,
respectively, during aerobic composting and vermi-composting (Green and Popa, 2011;
Fornes et al., 2012).
2.6.3 Addition of biochar
Biochar has a high surface area (Jindo et al., 2012) and adsorbs NH3, NH4+ and NO3-,
thus preventing their loss via volatilization or leaching. By creating a favourable
microenvironment for nitrifying bacteria (Chowdhury et al., 2014; Jindo et al., 2016; López-
Cano et al., 2016), it also promotes nitrification. Biochar particles further improve the texture
of the lacto-fermented material thus improving aeration (Jindo et al., 2012). Biochar addition
at the vermi-composting stage is important for increasing the stability of the compost,
reducing the nutrient loss and improving the compost structure. Earthworms ingest biochar
particles, grind and excrete them in the vermicasts, thus mixing them with the compost
(Asuming-Brempong and Nyalemegbe, 2014; Eckmeier et al., 2007).
Adding biochar during micro-aerobic composting will improve the reduction of
pathogens in faecal matter by increasing the peak thermophilic temperature and shortening the
time for reaching the sanitizing temperature above 55 °C (Steiner et al., 2010). The high
temperature (± 70°C) generated by aerobic thermophilic composting further adds to surface
39
oxidation and an increase in the acidic functional groups, thus enhancing the biochar capacity
to absorb nutrients and dissolved organic matter (Cheng et al., 2006; Dias et al., 2010; Jindo
et al., 2012). It also accelerates humus formation due to the incorporation of biochar aromatic
fractions into the humic substances of the composted material (Dias et al., 2010; López-Cano
et al., 2016; Steiner et al., 2010). A high degree of humus formation was observed upon the
addition of only 2% dry weight of biochar to compost (Jindo et al., 2012).
2.7 Agricultural effects of lacto-fermented excreta
2.7.1 The effects of lacto-fermented excreta on soil and plants
Research on the agricultural effects of lacto-fermented human excreta is rather scarce.

The benefits reported are mainly traced from related studies on bokashi (anaerobically
fermented animal manure or vegetable waste with rice bran and microbial inocula) and also
on LAB. Studies have evaluated the improved growth, yield and quality of crops, increased
nutrient availability (e.g. phosphorus), enhanced soil biological activity and physical soil
properties as well as suppression of plant pathogens. Table 2.2 presents an overview of the
main effects on soils and crops of lacto-fermented human and animal excreta and vegetable
waste.
Application of lacto-fermented faeces and biochar to mung bean (25:75 residue : clay
soil ratio) contributed to a higher plant growth compared to the unfertilized control (Prabhu et
al., 2014). In a two year field experiment, lacto-fermented faeces and biowaste, supplemented
by urine charged biochar, significantly improved (p < 0.05) the yield of corn on a clay-loamy
chernoziom in Moldova compared to the unfertilized control, stored cattle manure, faeces,
urine and vermicomposted lacto-fermented mix. In contrast, the mineral fertilizer gave a
significantly lower corn yield during the first production year, but not significantly different
yield during the second production year. This fertilizing mix has also significantly reduced the
soil bulk density and increased the mobile potassium content in comparison to the control and
other fertilizers (Andreev et al., 2016).
40
Table 2.2 Overview of the main effects on soil and plants of the application of lacto-
fermented faeces, lacto-fermented and composted organic waste, and biochar
Experimental Soil type, Plants used Effects on soil and crops Reference
conditions geographical
location and
scale of the
experiment
Sewage sludge, Acidic clay Mung bean Plants with 25 % lacto- Prabhu et
charcoal (20%), and soil (pH=5.5- (Vigna fermented sludge had the al., 2014 a,
lactobacilli (2%) were 6.5), low in P radiata) highest leaf length and b
lacto-fermented for 28 and Mg. leaf area compared to
days in enclosed other treatments.
buckets, followed by Goa, India
vermin-composting
Pot
with Eudrillus eugenia
experiments
earthworms.
Source-separated feces Clay loamy Corn (Zea Yield was significantly Andreev et
and biowaste (manure, chernoziom mays) higher than the control, al., 2016
kitchen waste) was Eastern stored cattle manure,
lacto-fermented with Europe, feces, urine and
sugar beet molasses Moldova vermicomposted lacto-
and bacterial inoculum Two-year fermented mix. In relation
from sauerkraut in pits field to mineral fertilizer, a
in the ground lined and experiments significantly higher yield
covered with plastic for was produced during first
8 weeks. Before field year but not the second.
application, biochar Lacto-fermented mix with
that had been soaked in biochar had lowered soil
urine was mixed in. For bulk density and enhanced
the second production soil potassium content in
year, this mix was also relation to the control and
vermicomposted. other fertilizers.
Bokashi 1 and mixed Field Rice EM bokashi and Xiaohou et

culture of LAB, yiest experiments (Oryza subdrainage had al., 2008
and photosynthetic China, loam sativa) significantly lowered bulk
bacteria - EM2 clay saline density, increased organic
compared to NP3 soil matter content, available
fertilizer. Different P, cation exhange capacity
treatments included: and microbial biomass
drainage and without compared to all other
drainage; bokashi; and treatments. Also EM
41
mineral fertilizers bokashi and drainage has

significantly increased the
yield and quality of rice.
Bokashi (Rice bran, Field Peanut, Fresh weight of nodules Pei-Sheng

rice husk, rapeseed oil experiments (Arachis on lateral plants, the and Hui-
mill sludge, fish meal 42 m2 each hypogaea) number of pods with two Lian, 2002
anaerobically experimental seeds, and yield in plants
fermented with plot fertilized by EM bokashi
Lactobacillus and was significantly higher
yeast), application rate than in those fertilized by
3000 kg/ha compared chemical fertilizer
to NPK fertilizer
(15:15:15) at a rate of
600 kg/ha
Bokashi was obtained Field Corn Plants fertilized with EM Alattar et

by fermenting fruit experiments (Zea mays) bokashi were two times al., 2016
2
waste, grass weeds on a 197.4 m taller and had 14% more
with molasses and area leaves
effective
microorganisms in a
closed bucket for a
period of 10 days.
Comparison was made
with bokashi without
EM and control of
farmer’s normal
practice.
1
rice bran and cattle manure (90.4%), water (9.0%) with molasses and effective microorganisms
2
EM - mixed culture of lactic acid bacteria, yeast and photosynthetic bacteria (0.6%).
3
nitrogen and phosphorus fertilizer
Fermented cattle manure with molasses and effective microorganisms (mixed culture
of LAB, yeast and photosynthetic bacteria) has a decreased bulk density, but an increased
organic matter content, available phosphorous, cation exchange capacity, microbial biomass
as well as yield and quality of rice compared to mineral fertilizer (Xiaohou et al., 2008).
Anaerobically fermented rice bran, rice husk, rapeseed oil mill sludge and fish meal
fermented with Lactobacillus and yeast has significantly increased the fresh weight of
nodules, pod numbers and yield of peanut compared to chemical fertilizer (Alattar et al.,
2016). The application of lacto-fermented kitchen scraps produced twice taller corn plants (p
42
< 0.05) and more leaves than the control (Alattar et al., 2016). Fermented organic waste with
wheat bran and effective microorganisms improved nutrient availability, but produced a 29 %
lower tomato yield than mineral fertilizer mixed with effective microorganisms (Hui-Lian et
al., 2001). The quality of the fruits, e.g. sugar, organic acid and vitamin C concentration was,
however, higher in plants amended with fermented organic waste.
The addition of lacto-fermented material affects different biological, physical and
chemical soil components caused by the LAB themselves as well as by the compounds they
produce. The application of lacto-fermented vegetable waste and effective microorganisms
led to a 35 %, 23 % and 12 % increase of soluble N, P and K, respectively, compared to the
unfertilized control (Lim et al., 1999). LAB are able to solubilize water insoluble phosphates
(Zlotnikov et al., 2013) from compost and also from the soil, through mechanisms similar in
phosphorus solubilizing bacteria, e.g. production of organic acids, pH lowering and
phosphatase enzymes (Park et al., 2010), thus increasing its availability to plants. In addition,
some of the organic acids (e.g. lactic or acetic acid) produced by LAB can be used as carbon
source by other useful microorganisms, such as photosynthetic bacteria, able to fix N2
(Kantha et al., 2012).
LAB can beneficially influence the growth and yield of crops. For example, tomato
amended with LAB had 2-4 fold more fresh weight of fruits than the unamended control
plants (Hoda et al., 2011). Lactococcus lactis showed significantly higher growth than the
control, in cabbage grown under greenhouse conditions (Somers et al., 2007). Different
compounds such as carbohydrates, amino acids and organic acids and other metabolites
produced by the LAB may stimulate soil beneficial microorganisms and suppress phyto-
pathogens (Hoda et al., 2011). In a pot experiment, LAB have suppressed by 60 % the growth
of plant pathogen Phytium (Hoda et al., 2011). LAB have reduced by 63 % the disease
incidence of bacteria wilt Ralstonia solanacerum in tomato (Murthy et al., 2012) and have
caused significantly fewer damages upon a bacterial infection by Pseudomonas (number of
lesions per leaf and percentage of dead leafs; p < 0.05) in beans compared to the control
(Visser et al., 1986).
43
2.7.2 Effect of post-treatment by composting and biochar addition
Compost obtained from combined lactic acid fermentation and vermi-composting is

enriched with humic acids and phytohormones (Arancon et al., 2010; Zhang et al., 2014a).
Moreover, it has improved physical characteristics such as porosity and water holding
capacity. The quality of the obtained faecal compost treated via combined lactic acid
fermentation and vermi-composting had a higher total nitrogen, phosphorus and total organic
carbon content than certified compost (Bettendorf et al., 2014). A pathogenically safe
compost was obtained from combined lactic acid fermentation and thermophilic composting,
which beneficially influenced the germination of radish. Therefore, seeds treated with this
type of compost had a germination index of 90 % of the control, which was distilled water
(Andreev et al., 2015).
The biochar component may also provide beneficial effects on different soil
characteristics such as the structure, texture, porosity, particle size distribution, density,
oxygen content, water storage capacity, microbial and nutrient status in the root zone
(Atkinson et al., 2010). The main value of mixing biochar with excreta is the reduction of
nitrogen loss. NO3- is captured and retained on the biochar during the composting process,
thus will not be readily leached which makes it available for plants. For example, in a
lysimeter study with pasture soil, pine forestry plantation soil and soil amended with biosolids
in combination with biochar have contributed to a decrease in nitrate leaching compared to
the control soil (no biochar, no biosolids) and soil amended with biosolids only (Knowles et
al., 2011). Pot experiments with soil and gravel and addition of composted faeces from
compost toilets, where biochar was used as a matrix during the collection stage, increased
significantly the growth of mustard spinach Brassica rapa compared to sandy soil, without
nutrient addition (Hijikata et al., 2015). The authors related this growth increase to the
biochar contribution to NO3- retention, leading to increased nitrification rates as well as
preventing its loss via leaching. Biochar can absorb NO3- onto its surface, however,
nitrification could not be measured in this study (Clough et al., 2013). The fact that biochar
adds with improved aeration can reduce the activity of denitrifying bacteria which act under
44
anoxic conditions, thus preventing NO3- loss via denitrification (Wang et al., 2013; Zhang et
al., 2010).
Compost supplemented by biochar (2% wet weight) improved up to 5 times the
growth of Chenopodium quinoa (Kammann et al., 2015). In other studies, for example in pot
experiments conducted with a ferrasol with biochar amended compost and biochar as a
substitute for peat growth medium, biochar contributed to increased water retention, nutrient
uptake by plants and crop yield (Johnson et al., 2012; Agegnehu et al., 2015).
Additional research is still needed for clarifying the role of biochar in capturing the
nutrients from urine and faeces and their release to the soil and plants. For example, pumpkin
fertilized with biochar soaked in cow urine had a threefold higher yield compared to urine-
only fertilized plants (Schmidt et al., 2015). In addition, in a field experiment on a tropical
Andosol, a fertilizer composed of co-composed pasteurized faeces, mineral additives (e.g. ash
and brick particles), kitchen waste, urine and biochar gave a 16 % increase in grain corn yield
compared to an unamended control. It also increased the soil available phosphorous content in
a soil suspension from 0.5 to 4.4 mg kg-1 and the soil pH from 5.3 to 5.9, thus reducing
acidification (Krause et al., 2015b).
2.8 Conclusions
This chapter discussed the potential of the application of a low cost technique to treat
excreta for safe and efficient recycling of nutrients and carbon matter back to the soil. It
contributes to the overall approach of sustainable sanitation in which "safe disposal"
transitions to safe reuse of excreta and human excreta is valued as an important fertilizer to
help mitigate the current rapid global degradation of soils, while also reducing contamination
of aquatic ecosystems. Lactic acid fermentation can contribute to pathogen reduction, prevent
nutrient loss and control odour, thus enhancing the value of excreta as fertilizer. Lactic acid
fermentation can enhance excreta hygienization, prevent nutrient losses and reduce odour,
thus enhancing the fertilizing value of excreta.
45
The combination of lactic acid fermentation and composting (thermophilic

composting or vermi-composting) and biochar addition can improve the pathogen reduction,
availability of nutrients and stability of organic matter. Within this process, biochar enhances
the thermophilic composting, preserves more nutrients in the substrate and increases the
humus formation. The limited agricultural studies on the effects of applying lacto-fermented
excreta demonstrate ample benefits on both crop production and soil quality. Among the main
limitations of excreta lactic acid fermentation is the need to provide an enabling environment
for LAB growth by inoculating them, e.g. as sauerkraut, and adding easily fermentable
carbohydrate sources such as molasses and wheat bran. Identification of non-value carbon
sources is an important consideration.
46
Chapter 3. Treatment of source-separated human faeces
via lactic acid fermentation combined with thermophilic
and vermi-composting
composting for agricultural application

Nadejda Andreev, Mariska Ronteltap, Boris Boincean, Piet N.L. Lens. Treatment of source-
source
separated human faeces via lactic acid fermentation combined with thermophilic and vermi-
composting for agricultural application. Compost Science & Utilization (2017,
( in press,
10.1080/1065657X.2016.1277809
10.1080/1065657X.2016.1277809).
47
Abstract
Human faeces from urine diverting dry toilets can serve as valuable soil conditioners.
For a successful agricultural application, an efficient pathogen reduction needs to be ensured,
with no negative effects on the plants. This study assessed the efficiency of lactic acid
fermentation combined with thermophilic composting on pathogen removal from human
faeces and the post-treatment effects on germination and growth of radish (Raphanus sativus)
and tomatoes (Lycopersicum esculentum) compared to lactic acid fermentation combined with
vermi-composting and the control. The NH4+-N/NO3--N ratio of 2.99 and 3.6, respectively,
indicated the obtained compost and vermi-compost was not yet mature. A complete reduction
in the concentration of all investigated bacterial indicators (i.e. coliforms, Escherichia coli,
Enterococcus faecalis and Clostridium perfringens) from 5-7 log CFU g-1 to below the
detection limit (< 3 log CFU g-1) was achieved after lactic acid fermentation combined with
thermophilic composting. Lactic acid fermentation combined with vermi-composting also
contributed to pathogen die-off, but coliform bacteria were reduced to only 5 log CFU g-1.
Compost obtained after lactic acid fermentation combined with thermophilic composting to
seeds of radish had a higher germination index than vermi-compost obtained after lactic acid
fermentation combined with vermi-composting (90 versus 84%). Moreover, significantly
bigger average fruit weight and total biomass per tomato plant (P<0.05) were obtained after
compost amendment compared to vermicast or the unamended control.
48
Treatment of source-separated human faeces via lactic acid fermentation
combined with thermophilic and vermi-composting for agricultural application
3.1 Introduction
Urine diverting dry toilets (UDDTs) offer many environmental benefits among which
reduced waste volume as a result of waterless operation, simplicity of technological treatment
and the possibility of recycling nutrients and organic matter to the soil (Langergraber and
Muellegger, 2005; Rieck et al., 2012). In UDDTs, urine and faeces are collected and
contained separately, with a possibility of co-treatment with other types of organic wastes,
e.g. kitchen waste or cattle manure. The separation toilet technology was successfully
implemented in communities with high water tables, rocky areas or areas with no possibility
to connect to the sewerage network, e.g. in regions as Central, Eastern and Northern Europe,
Caucasus and Central Asia, Africa, China and Central America (Wendland et al., 2011; Rieck
et al., 2012). During the last years, there is a growing interest towards this technology due to
increasing pressures from population growth, decline of freshwater resources and soil
depletion of nutrients and carbon matter. In spite of its high potential, UDDTs still need
further improvements, for example on faeces sanitization and stabilization, reduction of odour
as well as prevention of nutrient and carbon matter loss.
The WHO guidelines (WHO, 2006) recommend long-term storage as the simplest
method of faeces sanitization. However, conditions required for pathogens or parasites die-off
such as moisture reduction below 25%, pH raise above 9 or temperature increase over 55°C
are seldom or never achieved in UDDTs faeces chambers in real situations (Redlinger et al.,
2001). Moreover, the heterogeneity of the mixed faeces and cover material can lead to a re-
growth of some pathogens (Niwagaba et al., 2009a). Extended storage can also cause
significant loss of nitrogen and organic matter. For example, in composting toilets up to
66-80 % of nitrogen and 75% of carbon can be lost from stored excreta (Zavala et al., 2002;
Zavala et al., 2005; Hotta and Funamizu, 2007).
Thermophilic composting and vermi-composting contribute to faecal matter sanitizing
and stabilization (Eastman et al., 2001; Vinneras et al., 2003; Niwagaba et al., 2009c; Yadav
et al., 2010; Juarez et al., 2011; Hill and Baldwin, 2012). The main problems with
thermophilic composting are the long-duration and the need to provide frequent turning for
49
oxygen supply (Niwagaba et al., 2009 b). In order to hygienize effectively the mix of source
separated faeces and food waste, a thorough mixing and prolonged exposure to high
temperatures is necessary during thermophilic composting (Niwagaba et al., 2009c). Vermi-
composting results in a homogenous end-product with high nutrient quality (Mupondi et al.,
2010). However, it does not always lead to a complete destruction of pathogens due to
insufficient heating. This technique contributed to pathogen reduction in sewage sludge and
source separated faeces (Eastman et al., 2001; Buzie-Fru, 2010; Yadav et al., 2010), but not
always sufficiently effective for decay of resistant pathogens such as Ascaris (Bowman et al.,
2006; Hill et al., 2013). Vermi-composting is also a lengthy process, e.g. 4-6 months (Alidadi
and Shamansouri, 2005; Bowman et al., 2006; Yadav and Garg, 2011), which leads to high
losses of nitrogen and carbon matter as NH3, N2O and CO2 (Frederickson and Howell, 2003;
Nasiru et al., 2014).
Lactic acid fermentation leads to deactivation of different types of pathogens such as
faecal coliforms, Salmonella, Staphylococcus or Clostridium due to pH reduction as well as
production of lactic acid and other compounds with suppressive effects to pathogens, e.g.
bacteriocins, glucose oxidase or hydrogen peroxide (Rattanachaikunsopon and
Phumkhachorn, 2010; Saranraj, 2014). A number of studies (Juris et al., 1997; Papajova et
al., 2000; Caballero-Hernández et al., 2004) showed that lactic acid fermentation may not
allow a complete hygienization. For example, Ascaris survived after 1-6 months of lactic acid
fermentation of swine faeces or silage. Another concern, besides insufficient hygienization, is
the incomplete organic matter decomposition. The low pH and lactic acid formation during
lactic acid fermentation inhibits the growth of microorganisms, including the growth of LAB
themselves, thus promoting preservation of faecal material and inhibiting further
decomposition (Murphy et al., 2007). An insufficiently decomposed material applied to the
soil will still have high microbial activity and may potentially deplete soil oxygen and
nitrogen (Gómez-Brandón et al., 2008) causing its deficiency to plant growth.
Combined lactic acid fermentation and vermi-composting has recently been
considered within the terra preta sanitation approach aimed at improving faeces stabilization
(Factura et al., 2010; Scheinemann et al., 2015). One limitation for combined lactic acid
50
fermentation and vermi-composting is the need to pre-condition the lacto-fermented material

as this is highly anaerobic and rich in organic acids, potentially negatively affecting
earthworms (Buzie and Körner, 2014). In addition, studies have shown that combined lacto-
fermentation and vermi-composting of human faeces was not efficient in the elimination of
Salmonella sp. (Stoeckl et al., 2013).
One possibility for better hygienization would be to combine lactic acid fermentation
with thermophilic composting. The effect of integrated lactic acid fermentation and
thermophilic composting on source separated faeces has thus far not been investigated.
Application of lactic acid fermentation at the stage of faeces collection contributes to odour
reduction and pre-processing of waste (Windberg et al., 2013; Yemaneh et al., 2013). Biochar
is also added at the end of the lactic acid fermentation in order to reduce the nutrient loss and
increase the stability of the carbon matter (Windberg et al., 2013; Glaser, 2015). The enzymes
and metabolites released by cellolytic and hydrolytic activities of LAB contribute to substrate
solubilization and transformation, which facilitate the activity of bacterial and fungal
consortia in the thermophilic composting stage (Morrison, 1988; Xavier and Lonsane, 1994;
Rasool et al., 1996; Yang et al., 2006). The use of LAB or other effective microorganisms in
combination with easily soluble carbohydrates (e.g. molasses) contributes to an increase in
sanitizing temperature above 55°C and maintain it for an adequate number of days for
elimination of most of the pathogens (Ong et al., 2001; Ma et al., 2010).
The goal of this research was, therefore, to assess the effects of combined lactic acid
fermentation and thermophilic composting on pathogen reduction in faecal matter and
biological effects of compost on plants compared to lactic acid fermentation and vermi-
composting as well as the control. The maturity of the obtained compost and vermicompost
was assessed by analysing the ammonium and nitrate ratio and the toxicological effects on
radish (Raphanus sativus) germination and tomato (Lycopersicum esculentum) growth.
51
3.2 Materials and Methods
3.2.1. Experimental set-up
The faeces used in the experiments were collected over a three month period from a
household UDDT in Chisinau (Moldova). Toilet paper was collected separately and stored
together with kitchen waste in an enclosed 60 L barrel at ambient temperature (27-30 °C
during August 2013 and 28-31 °C during August 2014). The experimental conditions are
described in Table 3.1. After collection, the faeces were mixed with kitchen waste and cattle
manure, molasses and microbial inocula. This mixture was lacto-fermented in an enclosed
barrel for a period of 10 days at ambient temperature, the pH at the end of the experimental
period was 5.5. After this pre-treatment, 10 % of biochar (wet weight) was added to the lacto-
fermented material and further processed via either thermophilic composting or vermi-
composting. Biochar was added as a bulking agent to stabilize the lacto-fermented material
and increase its aeration. The experiments were repeated in two consecutive years (2013 and
2014) and during 2015 with some modifications. The changes consisted in replacing molasses
by pre-fermented kitchen waste and no addition of LAB inoculum.
Chemical and microbiological analysis was done in duplicates, germination tests and
growth tests were performed in triplicates. Samples for microbiological and chemical analysis
were collected from 3 points and mixed together with a plastic shovel to form a representative
sample. Out of this, subsamples (in duplicates) of approximately 100 g were taken, placed
into plastic bags with zippers and transported to the laboratory for analysis. The composition
of the mixture is presented in Table 3.1.
52
Table 3.1 Description of the experimental conditions and measured parameters

Time of Cover material Composition (based Processes Parameters
experiment on wet weight, %) investigated
2013 Sawdust Source separated LAF+TC T (°C),
faeces (40 %), cattle microbiological
manure (30 %), fruit indicators
waste (17 %),
molasses (8 %), lactic LAF+VC
bacteria inoculum (5
Microbiological
%)
indicators
Seed germination
test
2014 Sawdust:pressmud As the 2013 LAF+TC T (°C),

3:1 by volume experiment microbiological
indicators, plant
LAF+VC growth test; NH4+-
N/ NO3--N
Microbiological
Faeces (40%), cattle
2015 Sawdust: pressmud: indicators, plant
manure (30%),
biochar 1:1:1 by LAF+TC growth test NH4+-N/
kitchen waste (30%)
volume NO3- -N, T (°C)
LAF - lactic acid fermentation and thermophilic composting, TC - thermophilic composting, VC -

vermi-composting.
53
Table 3.2 The description of the composition of the fertilizers added
Fertilizer Composition description

applied
LAF Source separated faeces with sawdust as cover material (40 %), 6 month
stored cattle manure (30 %), fruit waste from fresh juice boutiques (17%),
molasses (8%), microbial inoculum from sauerkraut juice (3 %).
LAF+TC As above, but the addition of biochar soaked in urine for a period of two
weeks
LAF+VC Lacto-fermented mix and biochar soaked in urine were vermi-composted for
4 months with Eisenia foetida
LAF- lactic acid fermentation, TC- thermophilic composting, VC- vermi-composting,
3.2.2 Composting
Composting was carried out during August (2013), August-September (2014) and
April-May (2015).
3.2.2.1 Thermophilic Composting
The thermophilic composting reactor was an insulated metallic box made from an old
fridge (95x58x45 cm). It was positioned aslope so that the leachate could be collected during
the composting period. The leachate was removed as it was accumulated and discarded. No
microbiological analysis or biological test on tomato plants was performed on the leachate.
Temperature was recorded once a day as an average of measurements taken from four points
of the composting reactor, using a portable digital thermometer. The average outdoor
temperature at the time of measurement was the following: a) 2013 - 27.8 °C (with a range of
20-33 °C), 2014 - 26.5°C (with a range of 21-30 °C) and 2015 - 22.8 °C (with a range of 18-29
°C).
54
3.2.2.2 Vermi-composting
Vermi-composting was carried out outdoor, in a windrow of one meter wide by one
meter long and 40 cm high, using Eisenia foetida, obtained from the Institute of
Biotechnology in Zootechnology and Veterinary Medicine (Moldova) with an inoculation
density of 5,000 worms per investigated windrow and the whole experimental period lasted
130 days. Considering that the lacto-fermented material was highly anaerobic and rich in
organic acids (lactic and acetic acids), with a potential deleterious effect on earthworms
(Frederickson and Knight, 1988), the lacto-fermented mix was kept for one week for aeration
and volatilization of toxic compounds, before being offered to the earthworms. Wet shredded
newspaper was used as bedding material.
3.2.2.3 Germination and plant growth experiments
Aqueous compost extracts were prepared by mixing the compost samples with
distilled water at 1:10 w/w, shaking for 1 hour and then filtered via Whatman pads (Zucconi
et al., 1981). For germination tests, 20 radish seeds were placed on a Whatman paper in Petri
dishes and treated with aqueous extract and dechlorinated water as control. After 72 hours,
germination was stopped by adding 1 ml alcohol (50%) to each of the Petri dishes. The
germination index was calculated according to Tiqua et al. (1996).
Tomatoes were sown in plastic pots, three seeds per pot. The seedlings were
transplanted after germination, one seedling per pot, into a similar soil and compost mix as
they were germinating in. The total amount of nitrogen corresponded to 90 kg ha-1, the
quantity recommended for tomatoes growth (Andries et al., 2013), the necessary quantity
being calculated in accordance with the total nitrogen (N) content as g per pot, according to
the methodology on pot experiments (Johnson, 2005). The compost was mixed with
cernoziom clay loamy soil, the control was without any compost amendment.
The tomato plants were cultivated for a period of approximately 3 months (09.06.2014
to 13.09.2014). Watering of plants was done daily. The plant height and stem diameter were
55
measured with a ruler, after 1.5 months from sowing and at the end of the growing period. In
addition, the wet and dry weight of the root, stem, leaves and fruits as well as total biomass were
weighed. The dry weight was measured by drying the material in the oven at 105° C until a
constant weight was attained. Any significant difference between the means was assessed by
using Dunnet multiple comparisons and one-way ANOVA with the Minitab 17 Statistical
software.
3.2.3 Microbiological analysis and analytical methods
Total coliforms, Escherichia coli, Enterococcus faecalis and Clostridium perfringens

were determined using serial dilutions of the compost and subsequent incubation on selective
and differential nutrient media according to the standard protocols for pathogen and indicator
microorganisms for soil and compost samples (MHRF, 2005): a) the coliform bacteria on
Tergitol-7 Agar at 37°C for 24 h, b) Escherichia coli - on HiChrome Coliform agar at 43°C
for 24 h, c) Enteroccocus faecalis - on Slanetz Bartley agar TTC at 37°C for 48 h and d) -
Clostridium perfringens on iron sulphite agar at 44 C° for 16-18 h. For Clostridium, the
samples were preliminarily heated to 75 °C for 25 minutes to eliminate the vegetative forms,
after which they were sequentially heated to 70 °C and cooled in water under anaerobic
conditions. The NH4+-N concentration was determined photocolourimetrically according to
GOST 26489-85 and the NO3--N concentration ionmetrically according to GOST 27894.4-88
(1989).
3.3 Results
3.3.1 Temperature development during thermophilic composting
Pre-processing of the faeces and biowaste mix via lactic acid fermentation promoted a
rapid increase in temperature during thermophilic composting. Figure 3.1 shows that only a
very short mesophilic period was encountered during the 1st year (2013). The thermophilic
56
phase, with temperature rises above 50°C started already on the second and third day. During
the second year, a temperature increase above 40 °C was achieved during the 5th day and
above 55 °C during the 6th day. The sanitization temperature of 56-65°C was reached at the
3rd-6th day and was maintained for a period of 9 days during 2013 and for 6 days during 2014.
The sanitization temperature during 2013 and 2014 was attained without any turning or
mixing, despite the relatively compacted, mostly anaerobic starting material obtained by the
lactic-acid fermentation of the faeces. In contrast, during the year 2015, the sanitizing
temperature was not attained: the overall active stage of composting occurred mainly at a
mesophilic temperature (below 40 °C) (Figure 3.1). The temperature increase above 50 °C
took place only after the addition of a small proportion of molasses (1%), but it was
maintained for three days only, thus showing a quick depletion of this available carbon
source. It might be also that the outdoor temperature had an influence on the compost
temperature.
80
70
60
Temperature (°C)
50
40
30
20
10
2015 2014 2013 SanT
0
1 6 11 16 21 26 31
Time (days)
Figure 3.1 Temperature variations during thermophilic composting after pre-processing of

faecal material and biowaste via lactic acid fermentation. SanT - Sanitization temperature
57
The decrease in temperature below 50 °C and transition to the curing phase took place after
12 days and lasted for 11 days during 2013. In 2014, the transition to the curing phase
occurred after 14 days of thermophilic composting and lasted for 11 days as well. At the end
of this stage, the temperature became similar to the ambient temperature, i.e. 25-27°C. The
overall composting period during 2013 and 2014 lasted for 23 days, but when also
considering 10 days from the lactic acid fermentation, it lasted for 33 days. During 2015, with
no pre-treatment via lactic acid fermentation, the overall composting period lasted for 33
days. The values of the NH4+-N/NO3--N ratio was 2.99 for the thermophilic compost and 3.6
for the vermi-compost, indicating that the compost was not yet mature (Bernal et al., 2009)
(Table 3.3). Additional research is required to assess the maturation and stabilization time
required.
Table 3.3 Nitrate and ammonium content in the compost (Mean ± SD, n = 2)
Sample type NO3--N, mg kg-1 NH4+-N, mg kg-1 NH4+-N/NO3--N pH
LAF+TC 334 ± 4.2 1000 ± 14.8 2.99 7.78. ± 0.2
LAF+VC 277.6 ± 17.7 1000 ± 14.1 3.6 7.97 ± 0.1
Even though no measurements were made on the decomposition of lignocellulotic

materials, visual observations showed that at the end of the thermophilic composting period,
there were almost no signs of toilet paper in the compost anymore; the compost was covered
by white mould.
3.3.2 Hygienization by lactic acid fermentation combined with

thermophilic composting or vermi-composting
Table 3.4 shows that the lactic acid fermentation stage contributed to a reduction of all
pathogen indicator organisms by 2-4 log10 CFU g-1. According to EPA pathogen reduction
requirements for class A biosolids, the level of coliform bacteria should be reduced
to < 3 log CFU g-1 for a safe agricultural application (Pepper et al., 2006). Lactic acid
fermentation alone did not reduce the pathogen indicators to this level. The concentrations of
58
microbiological indicators during this process were reduced as follows: coliforms from 7 to
3.9-4.5 log CFU g-1, E.coli from 4.6-6.7 to 3.2-4.5 log CFU g-1, E. faecalis from 6.7-7.2 to 5-
5.5 log CFU g-1 and Clostridium perfringens from 5 to 2.5-4.5 log CFU g-1 (Table 3.4).
Instead, lacto-fermentation combined with thermophilic composting decreased the
number of all four pathogen indicator organisms from 5-7 log CFU g-1 to the recommended
safe level below 3 log CFU g-1. Lactic acid fermentation combined with vermi-composting
also led to pathogen reduction, however, not to the safe level of 3 log CFUg-1 for coliform
bacteria both during 2013 and 2014.
Table 3.4 - Density of pathogen indicator organisms in faeces: biowaste mix before
treatment, after lactic acid fermentation and combined lactic acid
fermentation/thermophilic composting or vermi-composting (average values of the
samples analysed during 2013-2014).
Bacterial density log10 CFUg-1

Pathogen indicators
Raw material LAF1 LAF+TC2 LAF+VC3
(10 days)
2013
coliforms 7.09±0.5 3.9±2.6 2.7±0.6 5.2±1.0
E. coli 6.7±1.2 3.2±1.6 2.3±0.6 2.0±0.0
E. faecalis 7.2±0.4 5.0±1.7 2.0±0.0 3.7±0.0
C. perfringens 5.0±0.0 2.5±0.7 2.0±0.0 2.0±0.2
2014
coliforms 5.3±0.5 4.5±2.1 3.0±0.0 4.9±0.5
E. coli 4.6±0.0 4.5±2.1 3.0±0.0 3.0±0.0
E. faecalis 6.7±0.0 5.5±0.2 2.0±0.0 2.0±0.0
C. perfringens 1.0±0.0 4.5±0.7 1.5±0.7 2.3±1.0
1 2
LAF - lactic acid fermentation; LAF+TC - combined lactic acid fermentation and
thermophilic composting; 3LAF+ VC - combined lactic acid fermentation and vermi-
composting.
59
3.3.3 Effects on plant germination and growth
Both applied fertilizing mixes had beneficial effects on radish germination (GI >
80%). However, radish seeds treated with aqueous extract of compost obtained after lactic
acid fermentation combined with thermophilic composting showed a higher GI than that after
lactic acid fermentation combined with vermi-composting (90 % versus 84 %). The tomatoes
grown in soil supplemented with both compost and vermicast were more vigorous than in the
control (Figure 3.2 A and B).
Figure 3.2 Tomato plants treated with compost obtained after lacto-fermentation with
thermophilic composting (front row in photo A) and lacto-fermentation with vermi-composting
(front row photo B) in comparison to the control (back row photos A and B). Three most
representative pots of each treatment are presented in this picture.
There were 18% and 7 % taller tomato plants in pots fertilized by compost obtained
after lacto-fermention combined with thermophilic composting and lacto-fermentation
combined with vermi-compost respectively, compared to the control. After 1.5 months from
sowing, the plant height of tomatoes fertilized by compost and vermicast was significantly
higher than the control (Table 3.4).
Even though the root, leaf and stem weight (dry and wet weight) was not significantly
different than the unfertilized control, the fruits of tomato fertilized by compost obtained after
60
lactic acid fermentation combined with thermophilic composting were significantly bigger
than those of the control. In addition, this type of compost produced the highest amount of
tomato biomass (wet and dry weight) compared to vermicast or the control (Table 3.5).
Table 3.5 Effect of compost versus vermicast on growth and weight of tomatoes
Sampling Parameter Control LAF/VC LAF/TC

period
1.5 months after PH1 (cm) 4.6±0.8 A 6.63±1.0 B 8.2±1.0AB
sowing
End of experiment PH (cm) 39.6±0.5 A 37.6±3.5 A 41.8±1.04 A
1.5 month after SD2 (cm) 0.84±0.14 A 0.90±0.0 A 1.07±0.15 A
sowing.
End of experiment SD (cm) 1.45±0.02A 1.72±0.04A 1.96±0.02 A
End of experiment Rww3 (g) 1.74±0.9 A 1.4±0.15 A 2.29±0.2 A
End of experiment Rdw4 (g) 0.67±0.1 A 1.14±0.6 A 1.24±0.5 A
End of experiment Lww5 (g) 8.03±1.6 A 7.25±2.1 A 8.04±1.8 A
End of experiment Ldw6 (g) 2.24±0.6 A 2.4±0.1 A 2.73±0.7 A
End of experiment Sww7 (g) 11.73±0.4 A 11.8±1.7 A 15.2±2.1 A
End of experiment Sdw8 (g) 2.37±0.5 A 2.5±0.4 A 3.2±0.4 A
At harvest Fww9/plant (g) 31.±3.3 A 41.4 ±1.9AB 55.1±12.5 B
At harvest Fdw10 (g) 2.37±0.5 A 2.5±0.49A 3.17±0.4 A
End of experiment TBww11 (g) 52.5±2.5 B 62.6±4.9 B 78.91±11.3 A
End of experiment TBdw12 (g) 7.97±0.84 B 11.45±1.23 B 8.9±0.9 A
1
-plant height; 2- stem diameter; 3 - root wet weight; 4- root dry weight; 5- leaf wet weight; 6- leaf
dry weight; 7- stem wet weight; 8 - stem dry weight; 9 - fruit wet weight; 10 - fruit dry weight; 11-
total biomass wet weight (per plant), 12 - total biomass dry weight (per plant). The means were
compared for significance using Fisher Pairwise comparison (p<0.05). Means that do not share a
letter are significantly different.
3.4 Discussion
3.4.1 Combined lactic acid fermentation and thermophilic composting
This study showed that lactic acid fermentation combined with thermophilic
composting during a two year run experiment might contribute to an overall shorter
composting period (33 days) compared to the usual aerobic composting (45-180 days) (Wang
61
et al., 2007; Fornes et al., 2012). Thermophilic temperatures in the current study were
achieved during the second-fifth day without any manual turning with passive aeration only,
such type of compost being described as of the "micro-aerobic type" (Wang et al., 2007;
Green and Popa, 2011; Alattar et al., 2012), however to prove this determination of the
oxygen concentration in the compost pile is required.
The overall composting period of 33 days (together with the lactic acid fermentation
period) is similar to other studies (Wang et al., 2007). It would be interesting to investigate
how thermophilic and vermi-composting processes alone differ in their rate of decomposition
and stabilization of organic matter and nutrient content compared to the integrated processes of
LAF and thermophilic or vermi-composting. The fast temperature increase without any turning
was probable related to the addition of molasses, a source of labile carbon, which boosted the
number of thermophilic bacteria (Hayes and Randle, 1968). In addition, the use of lactic acid
fermentation as pre-processing stage most probable contributed to the partial decomposition
and solubilization of excreta, thus facilitating more efficient substrate utilization during
thermophilic composting. This might have been caused by the abundance of enzymes released
by the LAB (Morrison, 1988; Rasool et al., 1996; Ong et al., 2001; Yang et al., 2006). It is
important to understand the contribution of lactic acid bacteria and molasses (or other sources
of water soluble carbohydrates) to the transformation of faecal substrate prior to thermophilic
composting.
Based on the assessment of the NH4+-N/NO3--N ratio, the obtained compost was not
yet mature, higher than the indicator value for compost maturity of > 0.16 (Bernal et al.,
1998). For a more complete picture of compost maturity, other parameters should be
investigated, e.g. C/N ratio, respiration rate (Barrena et al., 2014), or the level of elemental
and functional composition of organic matter and the humification level (Bernal et al., 2008)
using electrophoresis, chromatography-mass spectrometry or UV-spectroscopy (Domeizel et
al., 2004; Bernal et al., 2009).
The overall aerobic composting takes longer than in micro-aerobic composting, e.g.
45-50 days versus 35 days. With the exclusion of turning and mixing, it is also possible to
reduce carbon and nitrogen losses as CO2 and NH3 (Liang et al., 2006). As the process was
62
micro-aerobic, thermophilic bacteria probably contributed to the partial decomposition of the

toilet paper during the thermophilic stage. In addition, enzymatic systems of lactic acid
fermentation bacteria might have added to the decomposition of celluloses and hemicelluloses
into simpler compounds during the lactic acid fermentation stage (Morrison, 1988; Rasool et
al., 1996).
3.4.2 Faeces sanitizing by combined lactic acid fermentation with

composting
This study showed that integrating the processes of lactic acid fermentation with
thermophilic composting was more efficient in the reduction of coliform bacteria and E.
faecalis than combining lactic acid fermentation with vermi-composting (Table 3.3). As the
pH was 5.5 only at the end of lactic acid fermentation, that was probably not the single factor
contributing to pathogen reduction. Also antimicrobial substances produced by LAB during
the fermentation might have contributed to pathogen reduction. For example, Lactobacillus
plantarum, which was applied in this study, produces bacteriocins, that are thermoresistant
and inhibit a wide range of pathogens. The bacteriocins, might have been broken down by
proteases produced in higher amounts in vermi-composting than in thermophilic composting
(Abo-Amer, 2007; Devi et al., 2009). The main mechanisms of pathogen reduction during
thermophilic composting are: competition between indigenous microorganisms and
pathogens, the antibiotic activity of excretions of fungi or actinomycetes, natural die-off and
thermal destruction (Burge et al., 1978; Haug, 1993). The most important factor in pathogen
destruction is, however, the increased temperature (Vinnerås et al., 2003). According to the
sanitization standards for biosolids (Noble et al., 2004; Schönning and Stenström, 2004), a
minimum temperature of 55°C for a period of 3-14 days is required for achieving a safe
product for agricultural application, depending on the heterogeneity of the compost material.
During this study, hygienization temperatures above 55 °C were maintained for 6-9 days,
which was likely sufficient to reduce the bacterial contamination to a safe level of below the
detection limit of < 3 log CFU g-1.
63
The mechanism of pathogen reduction in vermi-composting is caused by the direct

destruction of the pathogenic bacteria after passage via earthworm guts and under the effect of
coelomic fluids or glycoproteins excreted by the earthworms (Edwards et al., 1984; Popović
et al., 2005). The insufficient hygienization during vermi-composting was also mentioned by
other studies (Alidadi et al., 2007), which showed that vermi-composting reduced pathogens
to a level which satisfies EPA requirements for class B biosolids, but not for class A. In this
study during lactic acid fermentation combined with vermi-composting, at a density of
5000 earthworms/m2 there was insufficient reduction of coliforms and in some cases of
Enterococcus faecalis. Other studies reported a complete pathogen removal at a density
6-9 times higher than that used in this study (Eastman et al., 2001). Such a high density, if
applied at large scale, might not be economically feasible (Mupondi et al., 2010).
It would be interesting to study the effect of lactic acid fermentation combined with
thermophilic composting on resistant pathogens such as Ascaris. Lactic acid fermentation can
reduce the viability of Ascaris suum in swine faeces; however, it did not destroy them
completely even after 56 days of lactic acid fermentation (Caballero-Hernandez et al., 2004).
Scheinemann et al. (2015), has achieved, however, a succesful Ascaris elimination after 56
days of lactic acid fermentation, but only at temperatures above 36 °C.
3.4.3 The value of faecal compost obtained via combined lactic acid
fermentation with thermophilic composting as soil amendment
The germination index (>80%) of both the thermophilic compost and vermi-composts
showed good potential for improving plant growth when used as soil amendments. Studies
have reported that vermi-composts can be efficient soil amenders, with a higher quality than
normal compost (Chaoui et al., 2003; Tognetti et al., 2005). For example, while a total
replacement of the peat growth media with vermi-compost was possible, an increase in
compost dose above 50 % caused plant mortality (Lazcano et al., 2009). One of the
explanations could be that the nitrogen is released in the form of ammonium during
64
composting, which may be toxic to plants, while in the vermi-compost it was in the nitrate
form, which is plant available (Atiyeh et al., 2000).
This study showed that lactic acid fermentation with thermophilic composting had an
overall higher effect on germination of radish Raphanus sativus and growth of tomatoes
Lycopersicum esculentum than lactic acid fermentation with vermi-composting (Table 3.5).
Combined lactic acid fermentation with thermophilic composting can be a faster turnover rate
of human faeces than lactic acid fermentation with vermi-composting (33 versus 130 days),
therefore with potential to recycle more nutrients and organic matter. Most probably these
integrated processes produced a more stabilized end-product, with a higher nutrient content
than the lactic acid fermentation and vermi-composting. However, it did not have any
negative effects on germination, the NH4+-N/NO3--N ratio indicated that the compost was not
yet mature. Table 3 shows that the nitrate content in the compost was higher than in the
vermi-compost. This may be related to the fact the biochar added during the thermophilic
stage contributed to ammonia and nitrate retention via sorption (Knowles et al., 2011; Jindo et
al., 2012; Hijikata et al., 2015), thus reducing the potential toxic effects of ammonia to the
plants.
3.5 Conclusions
Lactic acid fermentation combined with thermophilic composting can be a cost-

effective method for the processing of source separated faeces from UDDTs for agricultural
reuse that could be performed without turning but with only passive aeration. This integrated
process was more efficient in the reduction of pathogen indicator bacteria and had a better
stimulatory effect on the germination of radish and growth of tomatoes and occurs at a shorter
turnover rate (33 days) than lactic acid fermentation combined with vermi-composting (130
days).
65
66
Chapter 4 The effect of lacto-fermented faeces, biowaste
and addition of biochar soaked in urine on soil quality,
growth, yield and yield components of Zea mays L.

Nadejda Andreev, Mariska Ronteltap, Piet N.L. Lens, Boris Boincean, Lidia Bulat, Elena
Zubcov (2016) Lacto-fermented mix of faeces and bio-waste supplemented by biochar
improves the growth and yield of corn (Zea mays L.) Agriculture, Ecosystems and
Environment, 232, 263-272
67
Abstract
Resource oriented sanitation has emerged as a need to improve the efficacy of excreta
treatment schemes, to reduce the environmental pollution from their disposal and to improve
soil fertility. In urine diverting dry toilets, storage alone is inefficient for faeces treatment due
to poor hygienization, incomplete decomposition as well as high losses of organic matter and
nutrients. The purpose of this study was to assess the effect of stored faeces and bio-waste
treated via lactic acid fermentation supplemented with biochar soaked in urine on the growth,
yield and yield components of corn in a two year field experiment. Also the soil quality was
assessed. Lactic acid fermentation of faeces and bio-waste was performed under limited
oxygen conditions in earth pits covered with plastic foil by the addition of microbial inoculum
and molasses. The effect of fertilizer was compared to an unfertilized control; a lacto-
fermented mix (faeces, bio-waste, molasses and microbial inoculum) without biochar; stored
faeces; cattle manure; urine and mineral fertilizer. In addition, during the second year, a
vermi-composted lacto-fermented mix supplemented with biochar was applied. Differences
among the treatments concerning corn growth parameters, yield, yield components and soil
quality were evaluated using the Dunnet test of multiple comparisons. The lacto-fermented
mix supplemented by biochar significantly improved plant height (p < 0.05 and confidence
interval CI with two negative values) compared to all fertilizers during the first production
year and compared to the control, stored faeces and vermicompost during the second year.
This fertilizer also achieved a significantly higher corn yield compared to all other fertilizers
during the first and second production year, except for the lacto-fermented mix without
biochar and the mineral fertilizer, which showed no significant yield and yield component
difference (p > 0.05, CI with both positive and negative values). With regard to the soil
quality, bulk density was reduced during both years, while the soil potassium content
increased during the first production year. The nitrate concentration significantly improved in
comparison to the control, stored urine and stored faeces amended plots.
68
The effect of lacto-fermented faeces, biowaste and addition of biochar soaked
in urine on soil quality, growth, yield and yield components of Zea mays L.
4.1 Introduction
Adequately treated human faeces from urine diverting dry toilets (UDDTs) can serve
as an important soil conditioner. Studies have demonstrated a number of benefits from stored
human faeces such as improving the soil structure, water holding capacity, soil microbial
activity, soil buffering capacity and nutrient use efficiency (Guzha et al., 2005). In UDDTs,
faeces have to be maintained dry and stored for one to two years, without addition of new
fresh faeces, in order to eliminate or substantially reduce the number of pathogenic organisms
(WHO, 2006). The number of infective vectors in the stored faeces decreases as a result of
natural die-off or by the influence of environmental factors such as low moisture content and
pH increase (Redlinger et al., 2001; Kaiser, 2006). However, reduced moisture content
(below 40 %) leads to an incomplete decomposition, whereas a high pH contributes to intense
nitrogen volatilization (Nordin, 2010). Storage can result in the loss of about 66-94% of the
nitrogen and a significant proportion of the carbon (Zavala et al., 2002; Zavala et al., 2005).
In addition, storage alone may not contribute to a complete sanitization of the faeces, as there
is a potential re-growth of some pathogens (Gibbs et al., 1997). This method may thus
provide a product with an incomplete decomposition, still unsafe to be applied in agriculture
and with a relatively low nitrogen and carbon content (Nordin, 2010).
Application of safely treated human excreta to agriculture, rather than land disposal,
supports the reduction of environmental pollution and allows for recycling of valuable
components such as nitrogen, phosphorus and organic matter to the soil (Murray and Buckley,
2010b; Lu et al., 2012). Fertilizing crops, by source separated human faeces, alone or in
combination with urine contributed positively to soil quality, growth and yield of different
crops, similar to or even better than manure or mineral fertilizers (Guzha et al., 2005b;
Mnkeni and Austin, 2009; Triastuti et al., 2009). Adding combined human faeces and urine
resulted in a higher water use efficiency by corn than urine or mineral fertilizer only (Guzha
et al., 2005).
The application of mineral fertilizer or urine alone might be less efficient due to
nitrogen leaching. For example, a lysimeter study with 15N-labeled cow urine has shown that
69
in a temperate climate, approximately 60 % of the nitrogen was leached within one year
following urine application (Wachendorf et al., 2005). Supplementation with urine and faeces
does not only increase the soil nutrient content but also the soil organic matter content, which
in turn raises the water holding capacity (Hatfield et al., 2014). For example, field
experiments showed that for every 1 % increase in soil organic matter, there is a 3 time
increase in soil water holding capacity (Lawton, 2014) and an accumulation of 24 kg ha-1 of
mineral nitrogen (Andries, 2007).
Studies have shown that human excreta in combination with biochar can improve soil
quality by increasing nutrient availability and reducing soil acidification (Krause et al., 2015a;
Krause et al., 2015b). Such studies have emerged after the discovery of highly fertile soils of
anthropogenic origin such as terra preta or Amazonian dark earths (Woods, 2003; Glaser and
Birk, 2012) and their analogues, e.g. in Northern Europe (Wiedner et al., 2015a), Australia
(Downie et al., 2011) and West Africa (Frausin et al., 2014).
An important prerequisite for agricultural application of human faeces is their
hygienization. Among different treatment techniques, lactic acid fermentation combined with
vermi-composting has been considered during the last years in the context of the so-called
"terra preta sanitation" approach (Windberg et al., 2013). At different stages, biochar can be
added to reduce excessive nutrient loss and stabilize organic matter (Schuetze and Santiago-
Fandiño, 2014). To restore soil fertility, soil management practices which maximize recycling
of nutrients and build up of soil organic matter need to be adopted (Roy et al., 2002; Andries,
2007). Lactic acid fermentation, also called ensilage, is a widely used technique for the
preservation of food and animal feed (Kamra et al., 1984; Andersson et al., 1988; Schroeder,
2004). Lacto-fermenting human faeces together with a microbial inoculum and other types of
organic waste such as cattle manure, molasses or vegetable residues can return a larger
amount of carbon and nitrogen to the soil than stored faeces or cattle manure alone because
their losses are reduced. For example, 58 % of organic matter is lost in the common compost
process, compared to only 2.5% in fermented material (Hitman et al., 2013).
Lactic acid fermentation of faeces eliminates the foul odour and suppresses the growth
of putrefactive bacteria (Hata, 1982; Freitag and Meihoefer, 2000; Wang et al., 2001). This
70
type of treatment proved to be an efficient method in reducing the number of pathogens in

manure and sewage sludge (Scheinemann et al., 2015). Reduction of pH during lactic acid
fermentation reduces nitrogen losses as NH3 (Berg et al., 2006) because the activity of lactic
acid bacteria inhibits urea hydrolysis, thus preventing the volatilization of ammonia (Larson
and Kallio, 1954; Schneider and Kaltwasser, 1984). Direct application of lacto-fermented
faeces may raise some problems for the soil. This is because the end-product of lactic acid
fermentation is anaerobic and rich in available carbon, thus its addition may intensify the
activity of soil decomposers and mineralization of soil organic matter (Hamer and Marschner,
2005; Green and Popa, 2011). As a consequence also the oxygen concentration in the root
zone may decrease (Jiménez and Garcia, 1989).
Mixing of lacto-fermented organic waste with biochar may contribute to synergistic
positive effects on nutrient level, plant available water retention and an increase in stable
carbon content (Dias et al., 2010; Liu et al., 2012). The labile carbon diffuses into the biochar
pores and is adsorbed onto the biochar surface, thus preventing abiotic and biotic organic
matter decomposition (Zimmerman et al., 2011). The organic matter added with the lacto-
fermented substrate as well as the biochar increases the cation exchange capacity of the soil
and, therefore, the adsorption and retention of NO3-, NH4+-N and NH3 (Jindo et al., 2012).
Studies have shown that biochar supplemented to compost adsorbed nitrate and released it to
the plants, in this way preventing nitrate leaching and thus acting as a slow release fertilizer
(Kammann et al., 2015).
Biochar can be enriched with nutrients from human urine (Andreev et al., 2016). The
small organic particles present in urine form a coating on the biochar onto which anions such
as nitrates as well as cations like ammonium can bind, which favourably influence the crop
yield (Kammann et al., 2015; Schmidt et al., 2015). For example, the addition of a mix of
cow urine and biochar significantly increased the yield of pumpkin compared to the urine or
biochar only treatments (Schmidt et al., 2015).
Rational nutrient and organic carbon fertilizing management schemes from sanitation
waste are particularly important for subsistence farmers whose agricultural benefits are
exacerbated by the increase in intensity and frequency of droughts. Limited financial
71
resources and lack of knowledge on the soil fertilization practices force farmers to use limited
or no mineral and organic fertilizers (Andries et al., 2014). Crop yield mainly relies on natural
fertility, thus leading to loss of humus and further impoverishing the soil (Krupenikov et al.,
2011).
In Moldova, the decrease in precipitation and frequent droughts occurring during the
last decades lead to severe declines in crop yields (Potop, 2011; Vronskih, 2014). Land
privatization has led to the formation of many small plots distributed to rural farmers that are
only enough for subsistence farming. While crops extract up to 150-180 kg ha-1 NPK, the
supplementation from fertilizers is almost 10 times less, contributing to a negative nutrient
balance (Andries et al., 2014). Hence, a permanently available and affordable nitrogen
fertilizer, such as co-treated human faeces and bio-waste, has a large potential in the
agricultural future of countries like Moldova. At the moment, several urine diverting dry
toilets are available all over the country. The faeces quantity is relatively small and it takes
several years until the faeces compartment is filled. Faeces from these urine diverting dry
toilets could be lacto-fermented together with other types of organic waste such as animal
manure or waste from the food and beverage industry.
The purpose of this study was to determine whether a lacto-fermented mix obtained by
combined treatment of faeces from UDDT and bio-waste, such as manure, fruit residues and
waste from sugar industry (molasses) by lactic acid fermentation with the addition of biochar
soaked in urine has a beneficial effect on the productivity of corn (Zea mays L.) and the soil
quality. The influence of the applied fertilizer on the growth rate, yield and yield components
of corn, nitrate, phosphorus, potassium and humus content, soil available water as well as soil
bulk density, were examined during two production years. The lacto-fermented mix
supplemented by biochar was compared to an unfertilized control, a lacto-fermented mix
without biochar, mineral fertilizer, stored faeces, urine and cattle manure. An additional
treatment step via vermi-composting after lactic acid fermentation was performed as well, but
this material was only applied on the test plots during the second production year.
72
4.2 Methods
4.2.1 Site description
The research was carried out during two growth seasons (2012-2013 and 2013-2014) on a
private farm located in the Bolduresti Village (Nisporeni District, Figure 4.1) at a latitude of
47° 8′6.72″ N and longitude of 28°2′5.85″E, 90 km North-West of Chisinau (Moldova). The
annual average minimum air temperature for Nisporeni during the 2012-2013 production
period was 5.52 °C and during the 2013-2014 production period it was 5.92 °C. The average
annual maximum temperature was 15.75 and 16.38 °C for the first and second production
period, respectively (Table 4.1). The total rainfall for the first production period was higher
than the second production period (580 mm versus 547 mm, Table 4.1). The fall and spring
were more favourable for the second production year, with higher precipitation during these
Figure 4.1 Location of the experimental plot in the village Bolduresti (Nisporeni district,
Moldova)
73
seasonal periods (Figure 4.1).
Table 4.1 Average temperature and precipitation data in the Nisporeni district during
the production periods
2012 2013
Aver. Aver. Aver. Aver.
max min Precip. maxt° min Precip
Month t°C t°C mm Month C t°C . mm
IX 16.74 12.22 43.9 IX 20.50 9.50 93.7
X 19.00 6.20 33.8 X 16.83 6.29 1.9
XI 14.17 3.06 16.9 XI 12.80 4.60 23.6
XII -0.58 -8.92 40.3 XII 4.88 -3.04 5.2
2013 2014
I 0.32 -6.13 46.3 I 1.77 -4.32 44.8
II 4.00 -1.79 21.7 II 2.48 -4.10 7.2
III 7.58 -1.74 32.9 III 14.26 2.81 15.6
IV 18.97 6.26 35.5 IV 17.20 5.66 76.2
V 25.48 11.96 84.8 V 23.03 10.22 138.4
VI 27.13 15.30 172.7 VI 25.33 13.20 29.4
VII 27.62 15.32 102.6 VII 28.00 15.81 96.4
VIII 28.55 14.52 49.9 VIII 29.48 15.06 14.7
Prod Prod
period 15.75 5.52 681.3 period 16.38 5.97 547.1
Source: Anonymous, 2015.
The means of precipitation for the investigated production years were compared to the
average multiannual precipitation, representing data from long term monitoring (1981-2010)
collected across the country (Nedealcov et al., 2013) (Table 4.2). The annual precipitation for
both investigated years was higher than the average long term (30 years, 1981-2010)
precipitation value for the Nisporeni district, 681 and 547 mm versus 538.9 mm, respectively
(Nedealkov et al., 2013) (Table 4.2).
The investigated soil was a clay-loamy cernoziom, slightly eroded, with a low humus
content of approximately 2.83 % and a bonity of 66 (Baluhatîi et al., 2002). Soil bonity
represents a comparative qualitative assessment, based on a scale from 0 to 100 points of soil
price or productive capacity and is calculated for each individual soil and compared to a
74
reference soil (Ursu et al., 2009). According to its humus content, the soil belongs to the IIIrd
bonity class, which is an average value characteristic for the current soil status of Moldova
(Leah and Andries, 2012). This type of soil bonity allows for an annual crop productivity of
winter wheat of 2.5 t ha-1 (National Bureau of Statistics of the Republic of Moldova, 2014).
The humus value is, however, quite low compared to the average humus content in the topsoil
for cernozioms in Moldova, which ranges from 3.5-4 to 5-6 % (Andries, 2006).
Table 4.2 Classification of the investigated production years (2013 and 2014) according
to the amount of precipitation (mm) during spring and summer
N/o Period Years Precipitation (mm) Precipitation (mm)
for the average value
investigated during 1981-2010
production year
1 Spring Ist production 51** 44
year
2 Spring IInd 77 **
production
year
3 Summe Ist production 108 ** 67.5
r year
4 Summe IInd 47 *
r production
year
1
- The average precipitation value is based on data for the spring and summer periods of the
investigated district (Nedealkov et al., 2013). * - significantly lower, **- significantly higher.
The pH of the soil varies between neutral to slightly alkaline. The investigated area
was previously used mainly for corn production for several years. According to the land
owner, no fertilizers were applied to the area since the land privatization (1991-1992) and the
yield was mainly based on the natural fertility of cernoziom soils.
4.2.2 Fertilizers applied in this study
This study is focused mainly on a lacto-fermented stored faeces from UDDT and bio-
waste combined with urine charged biochar. The combined fertilizer was compared to a lacto-
fermented mix without biochar, an unfertilized control, stored faeces, cattle manure and urine
75
as well as an NPK mineral fertilizer. In addition, during the second production year, the lacto-
fermented mix supplemented by biochar was also vermi-composted, using Eisenia foetida
earthworms. Since the entire vermi-composting process required four months for completion,
a sufficient quantity for field application was only obtained and applied during the second
year.
The lacto-fermented mix consisted of stored faeces, cattle manure, fruit residues, sugar
beet molasses and microbial inoculum from sauerkraut brine, which were added at a
proportion of 40:30:17:8:5 percent by wet weight. The one-year stored faeces were obtained
from UDDTs of three schools in the Nisporeni district (Moldova). The faeces were
homogenized and separated from any foreign material (for example toilet paper and hygienic
pads). The cattle manure that was previously stored at ambient temperature for a period of 6
months was obtained from a farm within the village of the experimental area. The fruit
residues were purchased from a fresh juice boutique in Chisinau and chopped in a grinder
prior to use. Molasses was obtained from a sugar factory in Faleshti (Moldova). The
sauerkraut brine was activated by mixing with sugar beet molasses and water at a ratio of
5:5:90 percent by volume and fermented for five days, which allowed microbial growth
(Jusoh et al., 2013). After a thorough mixing, the waste components were placed in an earth
pit and fermented for a period of eight weeks. The material was tightly compacted to ensure
micro-aerobic conditions, and plastic foil was used to line and cover the pit (Figure 4.2).
Lactic acid fermentation in earth pits is quite a laborious process for practical applications at
large scale, windrow compaction by tractor might be preferred for the latter.
Biochar residues (dust and small pieces) from a local enterprise producing wood
biochar are presented in Table 4.3. The biochar proximate analysis was carried out according
to reported methodologies (Ronsse et al., 2013).
76
Figure 4.2 The earth pit where the waste material was fermented. In order to ensure
microaerobic conditions, the following actions were undertaken: A) the earth pit was lined
with plastic foil and the material was tightly compacted by feet and B) the pit was covered
with plastic foil and soil.
Biochar samples (1 g) were crushed, placed in porcelain crucibles and oven dried at
105°C for 2 hours to determine the moisture content. The volatile matter was determined as
the weight loss of biochar by heating the samples at 950 °C for 11 min in covered crucibles.
The ash content was determined by heating for a minimum of 2 hours (uncovered crucibles)
at 750°C. The stable carbon fraction (fixed carbon) was calculated by subtracting its moisture
content, ash content and volatile matter content. The surface area and pore volume was
measured by nitrogen gas absorption using a quanta chrome instrument Autosorb 1 MP (Greg
and Sing, 1984).
The specific surface area of the biochar investigated (Table 4.3) was lower than that of
other reported wood biochars (Ronsse et al., 2013). The investigated biochar had a moisture,
ash and fixed carbon content within the limits for previously reported wood biochar (Brewer
et al., 2011). Also the volatile matter was within the limit of variation of 20-60 % (33%),
characteristic for wood biochar (Enders et al., 2012).
77
Table 4.3 Characteristics of the quality of wood biochar used as fertilizer supplement
Moisture Volatile Ash Fixed C Specific Pore volume
(%) matter (%) content (%) surface area (cm3/g)
(%) (cm²/g)
2.35 ± 1.9 32.65 ± 1 7 ± 2.82 60.35 ± 13.24 73.79 ± 8.57 0.063 ± 0.036
Prior to the application, the charcoal was charged with nutrients from human urine by
storing a mixture of the urine and charcoal (3:1) in closed plastic barrels of 35 L for a period
of two weeks. Analysis of the urine solution before and after soaking showed a retention of 29
% of the ammonia and 70% of the phosphate onto the biochar (Table 4.4).
Table 4.4 Ammonia and phosphate concentration in urine before and after biochar
soaking (mean ± SD, n = 3)
Urine solution N-NH4+ (mg/l) PO43--P (mg/l)
Before biochar charging 9600 ± 20 520 ± 10
After biochar charging 6900 ± 10.5 150 ± 6
As mineral fertilizer, a complex nitrogen and phosphorus (amofos) fertilizer was used
with a N and P content of 12% and 52 %, respectively, supplemented by ammonium nitrate
(NH4NO3) and potassium chloride (KCl with K content of 40%). The mineral fertilizer was
offerred by the Selectia Institute of Crop Research (Moldova).
4.2.3 Sample collection and frequency
The soil samples were collected one time per growth season, four weeks after
planting, using a spade and a scoop. A composite sample from each plot was obtained by
collecting random subsamples at depths of 0-20 and 20-40 cm. In order to ensure
representativeness of the sampling depth, a ruler was used and the same collection method
was applied along the whole experimental area. In each plot, three subsamples from both soil
depths were collected from the middle, these being mixed in a common sample of
approximately 1 kg, out of which a small subsample of 300 g was drawn for analysis.
78
4.2.4 Physico-chemical analysis
Bulk density was assessed by core sampling (taken in 3 points of the sampling plot)
and calculated as the ratio of oven-dried mass to its volume (Stadnik, 2010). Before the
chemical analysis, the soil was air-dried, crushed and sieved.
All the elemental analysis (total N, P, K and C) was done from the same soil sample.
Samples for nitrate analysis were collected separately and analysed during the following day
after collection. The soil was collected four weeks after planting, at a depth of 0-20 and
20-40 cm, as described in Andreev et al. (2016). For measuring nitrate concentrations, an ion
meter I-160 M (Belarus) and an electrode ELIS 121-NO3- were used. The calibration of the
electrode was done by using known N-NO3- standard solutions, made by serial dilution of a
1000 ppm solution.
The soil available water storage capacity is the difference between water at field
capacity and the permanent wilting point (Eq. 1):
= − ∗ ∗ ∗ 0.1 (Eq. 1)
Where Wa - soil available water storage capacity (mm), Wfc - soil gravimetric moisture (soil
water at field capacity, % Cw - permanent wilting point, % H - soil bulk density (g/cm3), d - soil
depth (cm) (Verigo and Razumova, 1973). Water at field capacity is the water remaining in the
soil one or two days after rainfall. A permanent wilting point is the moisture content of the soil
at which plants wilt and fail to recover when they are supplied again with sufficient moisture
(Verigo and Razumova, 1973). In this study, the calculated value for clay soils (Verigo and
Razumova, 1973) was taken into consideration.
The soil gravimetric moisture was determined by drying the soil at 105 °C to a
constant weight and measuring the soil sample weight before and after drying. Eq. 2 was used
for calculating soil gravimetric water (Stadnic, 2010):
!" ! #$%& !" !
% = ∗ 100 (Eq. 2)
$%& !" !
The humus content was determined according to the Tiurin method, based on the
oxidation of soil humus in the solution by potassium dichromate and sulphuric acid, followed
79
by the photocolourimetric determination of the trivalent chromium concentration (Mineev et

al., 2001). The mobile forms of phosphorus and exchangeable potassium were extracted with
0.5 M acetic acid and determined photocolourimetrically for phosphorous and
photometrically for potassium as described by Sokolov and Askinazi (1965). Total N and P
were determined by the Ginsburg method (Ginsburg, 1985) from the same soil samples.
4.2.5 Field layouts and application rates
Table 4.5 summarizes the treatment variants that were applied to the field. The
nutrient requirement for corn of 60 kg of nitrogen per ha was based on local recommendations
on the application of fertilizers for different crops (Andries et al., 2013). The quantity of the
fertilizer was calculated according to the amount of total nitrogen.
Table 4.5 Description of different fertilizer treatments applied to the corn field plots;
data calculated from own measurements
Fertilizer applied Total N Total P C content Quantity of Quantity of product
kg tonne-1 kg tonne-1 kg tonne-1 N applied applied
kg ha-1 kg ha-1
Lactofermented mix and 18.7 2.8 280 60 3200
biochar
Lactofermented mix 18.7 2.4 240 60 3200
Vermicomposted lacto- 15.5 N/a 350* 80 5172
fermented mix
and biochar
(vermicast)
Mineral fertilizers 12 5.2 - 60 115 NP fertilizer +
(N12%, P52%), 111.7 NH4NO3 and
N60P60K60 kg 150 KCl
active
ingredients
ha-1
Stored faeces 13.1 n.d 280 60 4580
Stored urine 9.6 n.d - 60 6250
Stored cattle manure 17.8 n.d 260 60 3370
n.d - not determined.
80
The composition of the mixtures is given in Table 4.6. The application of all fertilizers
was done manually, including the mineral fertilizer. Fertilization was carried out during the
fall of both 2012 and 2013, except for the stored urine which was applied during spring,
before sowing.
The experimental plots were 60 m2 (5 x 12 m), distributed over a total experimental
area of 1500 m2. At harvesting, in order to avoid edge effects, one outer side row was omitted
at each side. Moldroad ploughing was performed in the fall using a Belarusi 82 tractor, at 22
cm depth. Seeding of corn was done in April by a seeding-machine at 45000 plants/ha.
Weeding, application of fertilizers and corn harvesting was done manually. The experimental
field plots were positioned in a randomized block design with eight variants and three
replications (Figure 4.3). The plots were fixed, so that each plot was treated twice. The plots
where vermi-compost was applied were left unfertilized during the first year and fertilized
only during the second year. All the applied fertilizers were distributed as uniformly as
possible. The incorporation into the soil was done by tillage on the same day or the day after
the application. Weed control was performed by hoeing two times during the entire growth
season.
Table 4.6 The description of the composition of the fertilizers added

Fertilizer applied Composition description
Lactofermented mix with biochar Source separated faeces with sawdust as cover material (40
addition %), 6 month stored cattle manure (30 %), fruit waste from
fresh juice boutiques (17%), molasses (8%), microbial
inoculum from sauerkraut juice (3 %). After fermentation for 3
weeks, prior application, biochar preliminarily charged with
nutrients from human urine was added.
Lacto-fermented mix As above, but without biochar
Vermicast Lacto-fermented mix which was vermicomposted for 4
months with Eisenia foetida
Mineral fertilizer (amofos) As complex nitrogen and phosphorous NP mineral fertilizer,
N60P60K60 kg active ingredients ha-1, N-12%, P 52%,
supplemented by balanced amount of amonium nitrate
(N34%) and potassium chloride (K40%).
Stored urine 6 months stored urine from UDDTs
Stored cattle manure 6 months cattle manure from a local farmer stored at ambient
temperature.
81
Figure 4.3 Random distribution of the experimental plots layout to investigate the effect of
fertilizers on corn.
Fertilizers applied: 1-control with no fertilization; 2- mineral fertilizer (amofos); 3 - stored
human faeces; 4 - stored cattle manure; 5 - lacto-fermented mix with no biochar; 6 - lacto-
fermented mix with biochar; stored urine; vermicomposted lacto-femented mix and biochar.
4.2.6 Assessment of the effects of lacto-fermented mix and biochar on the

growth, yield and yield components of corn
The corn was planted in April and harvested in September during the two consecutive
years (2013 and 2014). Growth measurements were done at 7-10 leaves, during intensive
growth (40 days from germination). These measurements were made on 10 randomly selected
plants in each plot, for them the plant height (PH), stem diameter, the maximum leaf length
(LL), and the maximum leaf width (LW) were recorded using a measuring tape (Figure 4.4)
and a Vernier calliper.
The yield of corn was calculated by weighing the harvested corn cobs from each plot.
Out of each sack, 10 randomly selected corn cobs were chosen and hand shelled; grain was
weighed for evaluation of the ratio of grain to cob according to the requirements specified by
the state standard for corn (GOST UJCN 13634-90, 2010). The following formula was used to
calculate the yield (Dospehov, 1985):
( 100 − )
'=
8600
where Y - yield of corn at 14 % moisture (kg ha-1), which is the standard storage corn
moisture, according to the corn requirements for state purchases and deliveries (GOST UJCN
13634-90, 2010)
C - yield of cobs (weight of harvested corn cobs from each plot and estimated at kg/ha)
G - ratio of grain (%) (GOST UJCN 13634-90, 2010)
M - corn moisture (%)
8600 - coefficient of conversion of cob yield to grain yield at 14 % moisture.
82
Figure 4.4 Measuring the corn growth parameters in the field.
After the corn was harvested by standard methodology (Verhults et al., 2013), ten corn
ears were randomly chosen from the bags, for which the following yield component
parameters were recorded: number of rows per ear, number of kernels per row, number of
kernels per ear and average kernel weight. The number of rows per ear and the number of
kernels per row were obtained by actual counting of the rows and kernels, while the number
of kernels per ear was estimated by multiplying the number of rows per ear with the number
of kernels per row. The average kernel weight per ear was obtained after hand shelling and
weighing of kernels.
4.2.7 Statistical analysis
The experimental data were statistically processed via a Dunnet test of multiple
comparisons within Anova one-way analysis of variance and using Minitab 17 statistical
software. The means of all fertilizers were compared to the lacto-fermented mix with the
83
biochar supplement for significant differences (at 95 % significance, based on p values and
confidence interval CI).
4.3 Results
4.3.1 Influence of lacto-fermented mix and biochar on corn growth, yield

and yield components
4.3.1.1 Growth of corn
Table 4.7 shows that application of a lacto-fermented mix supplemented by biochar during
2013 produced significantly taller plants compared to the unfertilized control and all other applied
fertilizers (p < 0.05; CI contains only negative values) (Appendix A). The stem diameter was
significantly bigger only in relation to the control. The leaf length was larger compared to the
control and stored urine. No significant difference was observed in leaf width in comparison to the
other applied fertilizers (p > 0.05 and CI contains both negative and positive values).
During 2014, the lacto-fermented mix supplemented by biochar again contributed to
significantly taller plants, but this time only in relation to the control, stored faeces and
vermicomposted lacto-fermented mix and biochar. The mineral fertilizer produced significantly
taller plants than the lacto-fermented mix and biochar (p > 0.05, CI with both positive values). The
stem diameter was not significantly different in comparison to the other fertilizers investigated. The
leaf length was significantly longer only in comparison to the control and to the vermi-composted
lacto-fermented mix and biochar, but significantly smaller than those fertilized by mineral fertilizers
and stored faeces. The leaf width was significantly broader only in relation to the vermicomposted
lacto-fermented mix and biochar.
84
Table 4.7 The effect of application of the lacto-fermented mix and biochar compared to
other applied fertilizers on corn growth rate (mean ± SD, number of samples, n=3 per
each plot) during 2013-2014.
Applied fertilizer Plant height, Stem Leaf length, cm Leaf width,
cm diameter, cm cm
First production period, 2013
Lacto-ferm mix and 174.93 ± 1.01 A 2.67 ± 0.34 A 62.20 ± 1.80 A 9.66 ± 0.40 A
biochar
Lacto-ferm. mix only 160.56 ± 0.51* 2.53 ± 0.08 A 59.21 ± 1.71 A 9.20 ± 0.20 A
Mineral fertilizers 162.57 ± 1.16 * 2.31 ± 0.12 A 60.37 ± 1.27 A 9.07 ± 0.49 A
Stored faeces 157.03 ± 2.70 * 2.26 ± 0.21 A 59.43 ± 1.88 A 8.50 ± 0.61 A
Stored cattle manure 166.83 ± 1.02 * 2.41 ± 0.12 A 62.54 ± 1.35 A 8.97 ± 0.14 A
Stored urine 157.03 ± 2.70 * 2.22 ± 0.13 A 56.43 ± 1.19 * 7.88 ± 0.58 A
Control 145.37 ± 1.52 * 1.99 ± 0.22 * 56.29 ± 1.15 * 9.20 ± 1.57 A
Second production period, 2014
Lacto-ferm mix and 177.90 ± 0.80 A 2.67 ± 0.18 A 67.03 ± 1.42 A 9.53 ± 0.21 A
biochar
Lacto-fermented mix 180.13 ± 1.03 A 2.62 ± 0.14 A 69.50 ± 0.66 A 8.95 ± 0.35 A
only
Mineral fertilizers 182.67 ± 0.35 ** 2.75 ± 0.36 A 76.10 ± 0.85 ** 9.48 ± 0.16 A
Stored faeces 170.73 ± 1.10 * 2.36 ± 0.01 A 72.33 ± 0.81 ** 9.43 ± 0.13 A
Stored cattle manure 176.13 ± 0.81 A 2.43 ± 0.11 A 64.17 ± 1.26 A 9.15 ± 0.47 A
Stored urine 179.17 ± 1.26 A 2.56 ± 0.16 A 69.70 ± 4.45 A 8.84 ± 0.24 A
Control 168.00 ± 1.00 * 2.18 ± 0.15 A 59.97 ± 1.68 * 8.94 ± 0.37 A
Vermic lacto-ferm 174.07 ± 3.59 * 2.21 ± 0.48 A 61.20 ± 1.39 * 8.60 ± 0.36 *
mix + biochar
The comparison was made by Dunnett multiple comparisons, where lacto-fermented mix and biochar
was compared to all other fertilizers. Means labeled with letter A are not significantly different (p >
0.05, CI contains both positive and negative values), those not labeled with letter A are significantly
different, those marked with * have significantly lower values p > 0.05 and CI contains both negative values),
those with ** have significantly higher values (p < 0.05 and CI contains both positive values).
85
4.3.1.2 Grain yield and yield components
The lacto-fermented mix and biochar also contributed to a significantly higher grain
yield during 2013 compared to all other applied fertilizers, with the exception of only the
lacto-fermented mix, which showed no significant difference (Table 4.8, Appendix B).
Table 4.8 The effect of lacto-fermented mix and biochar compared to other applied
fertilizers on corn yield during two different production periods (2013-2014) (mean ±
SD, number of samples, n=3 per each plot).
APPLIED FERTILIZER CORN YIELD, kg ha -1
First production period
Lacto-fermented mix and biochar 2368 ± 32 A
Lacto-fermented mix only 2352 ± 106 A
Mineral fertilizers 1556 *± 58
Stored faeces 2010 * ± 129
Stored cattle manure 2098 * ± 88
Stored urine 2015* ± 102
Control 1578 * ± 62
Second production period
Lacto-fermented mix and biochar 3295 ± 118 A
Lacto-fermented mix only 2602 * ± 67
Mineral fertilizers 3404 ± 29 A
Stored faeces 2021 * ± 72
Stored cattle manure 2437* ± 118
Stored urine 2647* ± 67
Control 2136 * ± 72.4
Vermi-composted lacto-fermented mix 2416* ± 87
supplemented by biochar
* - Parameters marked with a star have significantly lower value than the lacto-fermented mix and
biochar.
The yield increase was in the following order compared to the unfertilized control:
lacto-fermented mix and biochar > lacto-fermented mix > stored urine > stored faeces >
stored cattle manure, which was 58 % > 45 % > 37 % > 32 % > 13 % > 9 % higher than the
control. During 2014, the lacto-fermented mix and biochar again contributed to a significantly
higher yield compared to the control and all other treatments, except for the mineral fertilizer,
86
which was not significantly different (Table 4.7). The grain yield increased in the following
order in relation to the control: mineral fertilizers > lacto-fermented mix and biochar > stored
urine > lacto-fermented mix > stored cattle manure > vermi-composted lacto-fermented mix
with a yield of 37 % ˃ 35 % ˃ 19 % ˃ 18 % ˃ 12 % ˃ 12 % higher than the control. The lacto-
fermented mix with biochar addition positively influenced the yield components of corn
during both production years (Table 4.9).
Table 4.9 Influence of the lacto-fermented mix and biochar on yield components of corn
in comparison to the unfertilized control and other types of fertilizers
1st Production year
Yield Fertilizer Means Diff of Diff of 95 % CI P-
component s applied levels means value
-1
Nr rows ear C 13.23 A C - LFB -0.367 (-1.996; 1.263) 0.964
SU 12.93 A SU -LFB -0.667 (-2.296; 0.963) 0.698
LF 13.9 A LF - LFB 0.300 (-1.330; 1.963) 0.986
SCM 12.63 A SCM - LFB -0.967 (-2.596; 0.663) 0.367
MF 13.47 A MF -LFB -0.133 (-1.763; 1.496) 1.000
SF 13.33 A SF - LFB -0.267 (-1.896; 1.363) 0.992
LFB 13.6 A
-
Nr kernels row C 24.23 C - LFB -7.53 (-12.54; -2.53) 0.003
1
SU 27.10 A SU -LFB -4.67 (-9.67; 0.34) 0.072
LF 32.73 A LF - LFB 0.97 (-4.04; 5.97) 0.982
SCM 24.33 SCM - LFB -7.43 (-12.44; -2.43) 0.003
MF 25.73 MF -LFB -6.03 (-11.04; -1.03) 0.016
SF 26.60 SF - LFB -5.17 (-10.17; -0.16) 0.042
LFB 31.77 A
Nr kernels C 327.07 C - LFB -106.4 (-150.3; -62,5) 0.000
-1
ear SU 355.1 SU -LFB -78.4 (-122.3; -34.5) 0.001
LF 447.7 A LF - LFB 14.2 (-29.7; 58.1) 0.850
SCM 317.8 SCM - LFB -115.7 (-159.6; -71.8) 0.000
MF 346.2 MF -LFB -87.3 (-131.2; -43.4) 0.000
SF 352.3 SF - LFB -81.2 (-125.1; -37.3) 0.000
87
LFB 433.5 A
Average kernel C 119.9 C - LFB -76.7 (-105.19; -8.21) 0.000
-1
weight ear SU 143.33 SU -LFB -53.33 (-81.83; -4.84) 0.000
LF 180 A LF - LFB -16.67 (-45.16; 11.83) 0.379
SCM 121 SCM - LFB -75.67 (-35.10; -9.23) 0.000
MF 188.33 A MF -LFB -8.33 (-36.83 20.16) 0.895
SF 153.33 -43.33 (-71.83; -14.84) 0.003
LFB 196.7 A
2nd Production Year
Yield Variants Means Difference Differenc 95% CI P-
component of levels e of value
means
Nr rows*ear-1 C 10.77 C - LFB -2.900 (-4,517; -1.283) 0.001
SU 12.47 A SU -LFB -1.200 (-2.817; 0.417) 0.188
LF 11.83 LF - LFB -1.833 (-3.450; -0,216) 0.024
SCM 11.17 SCM - LFB -2.500 (-4.117; -0.883) 0.002
MF 14.00 A MF -LFB 0.333 (-1.284; 1.950) 0.975
SF 12.8 A SF - LFB -0.867 (-2.484; 0.750) 0.460
LFB 13.67 A
Nr C 28.47 C - LFB -8.87 (-12.32; -5.41) 0.000
-1
kernels*row SU 29.73 SU -LFB -7.60 (-11.06; -4.14) 0.000
LF 33.07 LF - LFB -4.27 (-7.72; -0.81) 0.014
SCM 24.33 SCM - LFB -13.00 (-16.46; -9.54) 0.000
MF 37.97 A MF -LFB 0.63 (-2.82; 4.09) 0.986
SF 30.63 SF - LFB -6.70 (-10.17; -3.24) 0.000
LFB 37.33 A
Nr kernels*ear-1 C 315.8 C - LFB -180.2 (-273.5; -86.9) 0.000
SU 425.6 A SU -LFB -70.4 (-163.6; 22.9) 0.177
LF 395.2 LF - LFB -100.8 (-194.1; -7.6) 0.032
SCM 317.8 SCM - LFB -178.2 (-271,4 -84.9) 0.000
MF 528.17 A MF -LFB 32.2 (-61.1; 125.4) 0.815
SF 407.7 A SF - LFB -88.3 (-181.6; 4.9) 0.066
LFB 496 A
88
Average kernel C 119.97 C - LFB -76.7 (-105.19; 8.21) 0.000

-1
weight*ear SU 143.33 SU -LFB -53.33 (-81.83; -24.84) 0.000
LF 180.00 A LF - LFB -16.67 (-45.16; 11.83) 0.379
SCM 121.00 SCM - LFB -75.67 (-104.16; -7.17) 0.000
MF 188.33 A MF -LFB -8.33 (-36.83; 20.16) 0.895
SF 153.33 SF - LFB -43.33 (-38.84; -17.16) 0.000
LFB 196.7 A
C- control; LFB - lacto-fermented mix and biochar; LF - lacto-fermented mix; SCM - stored cattle
manure; MF- mineral fertilizer; SF - stored faeces. Means were compared by Dunnet multiple
comparison, those not labeled with letter A are significantly different than LFB.
During the first year, the number of kernels per row, number of kernels per ear and
average kernel weight per ear were significantly higher than the control, stored cattle manure,
mineral fertilizer, stored faeces and stored urine (p < 0.05; CI with both negative values)
(Table 4.9). The only exception came from the number of rows per ear, which was not
significantly different than the other applied fertilizers and the control during the first
production year (p > 0.05, confidence interval CI with both positive and negative values).
During the second production year, the lacto-fermented faeces and bio-waste supplemented
by biochar produced a higher number of rows per ear than the control, lacto-fermented mix
only and stored cattle manure (Table 4.9). Mineral fertilizer produced significantly lower
yield components and yield during the first year, but not significantly different yield and yield
components during the second year (Table 4.9). Stored faeces yielded a significantly lower
number of kernels per ear and average kernel weight per ear than the biochar supplemented
lacto-fermented faeces and bio-waste mix.
Addition of biochar had an added value to the yield components. The effect was
observed during the second year only when the lacto-fermented mix without biochar had a
significantly lower number of rows per ear, kernels per row and kernels per ear. It, however,
did not have any influence on the average kernel weight (Table 4.9). As in the case of yield
components, the yield of corn plots fertilized by stored cattle manure, faeces and urine as well
as the unfertilized control had significantly lower yields than the plots fertilized by lacto-
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fermented faeces and bio-waste, supplemented by biochar during both production years
(Figure 4.5).
3500
3000
Yield, kg/ha
2500
2000
1500
Treatment 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Yield 2013 Yield 2014
Figure 4.5 Yield of corn at 14 % moisture (kg ha-1) during the 1st production period (2013) and
2nd production period (2014).
1- Control; 2- stored urine; 3- lacto-fermented mix and biochar; 4 - lacto-fermented mix;
5 - stored cattle manure; 6 - mineral fertilizer; 7 - stored faeces
90
The variations on ear length and grain filling are shown in Figure 4.6, the results
presented are from the second production year.
Figure 4.6. Corn ear length and grain filling in corn fertilized by the lacto-fermented mix of
faeces and bio-waste supplemented by biochar in comparison to other applied fertilizers (each
column represents one of the triplicates of the applied fertilizers).
91
The corn fertilized by the lacto-fermented mix of faeces and bio-waste, supplemented
by biochar as well as mineral fertilizer produced the longest ears (Figure 4.6) compared to the
control and other fertilizing mixes. The shortest ears and the highest number of ears with
reduced number of kernels were observed in the unfertilized control. A large number of corn
plants with short ears were also observed in corn fertilized by the stored faeces, cattle manure
and urine alone, compared to the investigated mix of lacto-fermented faeces and bio-waste
supplemented by biochar. During the first production year, the yield of all applied fertilizers
was significantly lower than the average yield for Moldova during 2013 (Table 4.10).
Table 4.10 Corn yield of the applied fertilizers in comparison to the average value
(based on spring and summer precipitation data) and lacto-fermented mix
supplemented by biochar
Fertilizers applied Corn yield kg ha-1 Difference of yield of applied fertilizers
(mean ± SD)1 compared to the average yield of
Moldova during 2013 and 2014
1st production year
Control 1578 ± 62 -49 *
Stored urine 2015 ± 30 -35 *
Lacto-fermented mix 2352 ± 106 -24 *
Stored cattle manure 2098 ± 88 -32 *
Mineral fertilizer 1556 ± 58 -50 *
Stored faeces 2010 ± 129 -35 *
Lacto-fermented mix and 2368 ± 32 -23 *
biochar
2nd production year
Control 2136 ± 87 -36 *
Stored urine 2647 ± 67 -21 *
Lacto-fermented mix 2602 ± 67 -22 *
Stored cattle manure 2098 ± 88 -37 *
Mineral fertilizer 3404 ± 29 +2
Stored faeces 2021 ± 129 -40 *
Lacto-fermented mix and 3295 ± 118 -2
biochar
*significantly lower, no mark means not significantly different.
During the second production year, only lacto-fermented faeces and bio-waste,
supplemented by biochar as well as the mineral fertilizer produced yields that are not
significantly different from the average yield for Moldova during 2014 (Table 4.10). It should
92
be noted that two years of application is a rather restricted period to draw conclusions on the
effect of fertilizer use efficiency on yield in comparison to the overall corn yield at the
country level.
4.3.2 Influence of the lacto-fermented mix and biochar on soil quality
4.3.2.1 Soil available water storage capacity
During the first production year, fertilization by the lacto-fermented mix and biochar
had contributed to a significantly higher soil available water storage capacity in the soil layer
0-20 cm compared to the control. This value was, however, significantly lower than the
fertilization by cattle manure. During the second production year, no significant difference of
the available water storage capacity in the soil layer 0-20 cm was observed in comparison to
the control and the other applied fertilizers. In the deeper soil layer of 20-40 cm, stored cattle
manure contributed again with a significantly higher soil available water storage capacity
(Table 4.11), whose values (26-86 mm) were in conformity with the norm of productive
moisture stocks for soil required for corn (Bugaeva and Mironova, 2009). Cattle manure
increased the humus content during both production years and both soil layers (Table 4.12). In
spite of the increasing soil available water storage capacity, cattle manure produced a
significantly lower yield than lacto-fermented faeces and bio-waste, supplemented by biochar,
probably due to a higher loss of nitrogen as NH3 via volatilization or via leaching.
Data on average precipitation during spring and summer for the first and second
production periods (Table 4.1) show that for both spring and summer, the precipitation was
significantly higher than the average precipitation for the Nisporeni district (Nedealkov et al.,
2013). Apparently, the first production period received a more favourable amount of
precipitation in spring and summer compared to the second production period. However, there
was a much more uniform distribution of the rainfall during the key corn growing periods of
the second production year (i.e. June and July). This is likely to have positively influenced the
yield. During both production years (Table 4.2), the seasonal rainfall was less than 81 mm, a
93
below average value during summer or spring. Such conditions may lead to a corn yield
decreased by 15% (Vronskih, 2014).
4.3.2.2 Soil available nitrate content
The C:N ratio in the lacto-fermented mix supplemented by biochar, lacto-fermented

mix alone and cattle manure was < 15 (Andreev et al., 2016). Such a C:N ratio should have
accelerated the organic matter decomposition and increased the availability of mineral
nitrogen (Qian and Schoenau, 2002); however, probably higher losses of N have occurred
after application of cattle manure. In the case of stored faeces, which contained sawdust,
added during the collection stage, might not have been fully decomposed, the C:N ratio was >
15 (Andreev et al., 2016). With this range of C:N ratio, part of the nitrogen from the applied
stored faeces was probably immobilized by microbial consortia that continued to decompose
the material into the soil (Qian and Schoenau, 2002).
The mineral fertilizer gave the highest nitrate content in both soil layers during the
first production year. The lacto-fermented mix of faeces and bio-waste, supplemented by
biochar, has increased significantly the soil nitrate concentration in the 0-20 cm soil layer
during the first production year, but only compared to the control and stored urine (Table
4.11). During the second year, this particular fertilizer has increased significantly the soil
nitrate content in relation to the control, stored urine and faeces only in the 20-40 cm soil
layer, while for the 0-20 cm soil layer there was no significant difference compared to all
fertilizers applied (Table 4.11).
94
Table 4.11 Influence of the lacto-fermented mix and biochar on soil moisture and nitrate
content in comparison to the unfertilized control and other types of fertilizers
Soil 1st Production year
compon.
Variant Mean Difference Diff of 95% CI P-value
of levels means
Soil C 20.9 C - LFB -5.48 (-10.37; -0.58) 0.026
available SU 28.8 A SU -LFB 2.38 (-2.51; 7.28) 0.549
water LF 27.5 A LF - LFB 1.11 (-3.79; 6.01) 0.962
storage SCM 25.5 A SCM - -0.89 (-5.79; 4.00) 0.367
capacity, LFB
mm,
MF 28.9 A MF -LFB 2.49 (-2.40; 7.39) 0.507
0-20 cm
SF 28.0 A SF - LFB 1.59 (-3.30; 6.49) 0.847
LFB 26.4 A
C 64.4 A C - LFB 8.19 (-16.50; 32.87) 0.837
SU 29.2 SU -LFB -27.00 (-51.68; -2.31) 0.030
LF 64.3 A LF - LFB 8.07 (-16.61; 32.75) 0.844
SCM 86.2 SCM - 29.95 (-12.44; -2.43) 0.021
LFB
MF 61.1 A MF -LFB 4.88 (2.63; 35.93) 0.016
SF 71.6 A SF - LFB 15.35 (-3.38; 29.91) 0.063
LFB 56.2 A
Soil nitrate C 39.9 C - LFB -21.40 (-33.52; -9.28) 0.001
content, kg SU 38.9 SU -LFB -22.43 (-34.55;-10.31) 0.000
ha-1 LF 48.8 A LF - LFB -12.47 (-24.59; -0.35) 0.043
0-20 cm SCM 67.7 A SCM - 6.40 (-5.72; 18.52) 0.473
LFB
MF 81.9 MF -LFB 20.60 (8.48; 32.72) 0.001
SF 53.0 A SF - LFB -8.27 (-20.39; 3.85) 0.249
LFB 61.3 A
Soil nitrate C 115.1 A C - LFB -28.5 (-62.2; 5.1) 0.112
content, kg SU 173.2 A SU -LFB 29.6 (-4.1; 28.8) 0.095
ha-1 LF 138.8 A LF - LFB -4.8 (-38.5; 11.83) 0.996
20-40 cm SCM 160.6 A SCM - 17.0 (-16.7; 50.7) 0.515
LFB
MF 191.6 MF -LFB -26.4 (14.4; 81.7) 0.005
SF 117.2 A SF - LFB -26.4 (-60.1; 7.3) 0.153
LFB 143.3 A
95
2nd Production Year

Soil Variant Mean, Differerence Differ. of 95% CI P-value
component mm of levels means
Soil C 29.2 A C - LFB -5.64 (-12.28; 1.00) 0.111
available SU 26.9 A SU -LFB A -2.28 (-8.92; 4.36) 0.817
water LF 26.1 A LF - LFB A -3.05 (-9.69; 3.59) 0.600
storage SCM 27.7 A SCM - LFB A -1.52 (-8.16; 5.12) 0.961
capacity, MF 30.71 A MF -LFB A 1.52 (-5.12; 8.16) 0.961
mm, 0-20 SF 29.2 A SF - LFB A 0.01 (-6.63; 6.65) 1.000
cm LFB 29.2A
Soil C 59.8 A C - LFB 1.72 (-14.92; 18.37) 0.999
productive SU 72.3 A SU -LFB 14.19 (-2.45; 30.84) 0.109
moisture, LF 55.4 A LF - LFB -2.69 (-19.34; 13.95) 0.992
mm, 20-40 SCM 77.3 SCM - LFB 19.28 (2.63; 35.93) 0.021
cm MF 74.0 A MF -LFB 15.92 (-0.72; 32.57) 0.063
SF 71.3 A SF - LFB 13.27 (-3.38; 29.91) 0.144
LFB 58.0 A
Soil nitrate C 60 A C - LFB -19.55 (-44.02; 4.93) 0.143
content, kg SU 73 A SU -LFB -22.30 ( -46.77; 2.17) 0.177
ha-1, 0-20 LF 72.4 A LF - LFB 1.71 (-22.76; 6.19) 1.000
cm SCM 77.2 A SCM - LFB 6.50 (-17.97; 0.97) 0.927
MF 78 A MF -LFB -7.21 (-31.68; 7.27) 0.89
SF 49.5 A SF - LFB -21.16 (-45.63; 3.31) 0.102
LFB 58.0 A
Soil nitrate C 119.9 C - LFB -73.1 (-115.7; -30.4) 0.001
content, kg SU 142.1 SU -LFB -50.8 (-93.4; -8.2) 0.017
ha-1, 20-40 LF 190.3 A LF - LFB -2.6 (-45.2; 40.0) 1.000
cm SCM 210.2 A SCM - LFB 17.2 (-25.4; 59.9) 0.706
MF 187.9 A MF -LFB -5.0 (-47.6; 37.6) 0.999
SF 112.4 SF - LFB -80.6 (-126.2; -37.9) 0.000
LFB 192.9 A
The values labeled with letter A are not significantly different than lacto-fermented mix and
biochar. C - control, SU - stored urine, LF - lacto-fermented mix, without biochar, SCM - stored
cattle manure, MF - mineral fertilizer, SF - stored faeces, LFB - lacto-fermented mix and biochar.
Under such dry periods, corn intensively consumes nitrogen and water for vegetative
growth and reproductive development. Under the conditions of reduced precipitation, the
analysed spring soil moisture may not fully reflect the complete influence of the applied
fertilizers on soil moisture. In spite of the increasing soil available water storage capacity,
cattle manure produced a significantly lower yield than the lacto-fermented mix and biochar.
96
The mineral fertilizer contributed to the highest nitrate content in both soil layers
during the first production year. The lacto-fermented mix of faeces and bio-waste,
supplemented by biochar increased significantly the soil nitrate concentration in the soil layer
0-20 cm during the first production year, but only compared to the control and stored urine
(Table 4.11). During the second year, this particular fertilizer has increased significantly the
soil nitrate content in relation to the control, stored urine and faeces only in the soil layer 20-
40 cm, while for the 0-20 cm soil layer there was no significant difference compared to all
applied fertilizers (Table 4.11).
4.3.2.3 Mobile phosphorus and exchangeable potassium
The content of mobile phosphorous and exchangeable potassium of the investigated

soil refers to a medium nutrient content (Sokolov and Askinazi, 1965). During the first year of
application, the available phosphorus in both soil layers did not differ significantly in
comparison to the other applied fertilizers and the unfertilized control in any of the production
years investigated. An accumulation of mobile phosphorus and exchangeable potassium was
observed during the second production year for all treatments compared to the control (Table
4.12, Appendices D and E). The lacto-fermented mix supplemented by biochar contributed to
significantly higher values of phosphorus in the soil layer 0-20 cm compared to the control,
stored cattle manure and the vermi-composted lacto-fermented mix supplemented by biochar.
For the deeper soil layer (20-40 cm), significantly higher phosphorus values were found only
in relation to the control. The values were, however, significantly lower than in the plots
fertilized by the stored urine. The potassium content increased significantly during the first
production year after application of the lacto-fermented mix supplemented by biochar, but
only in the soil layer 20-40 cm (Table 4.12).
97
Table 4.12 Mobile P1 and exchangeable K2 content in the soil with different fertilizer
treatments during 2013-2014 (number of samples, n = 3 per each plot)
Applied fertilizer P2O5 (mg kg-1) K2O (mg kg-1)
First production period 0-20 cm 20-40 cm 0-20 cm 20-40 cm

Lacto-fermented mix
and biochar 110.6 ± 4.6 A 101.7 ± 4.8 A 72.0 ± 2.5 A 75.3 ± 3.0 A
Lacto-fermented mix 103.0 ± 6.7 A 101.0 ± 2.9 A 80.0 ± 1.6 ** 64.5 ± 1.8 *
Mineral fertilizers 108.8 ± 2.1 A 105.2 ± 5.9 A 73.3 ± 1.4 A 78.5 ± 1.9 A
Stored cattle manure 107.8 ± 10.6 A 102.3 ± 5.9 A 68.1 ± 2.32 A 62.5 ± 1.3 *
Stored faeces 105.6 ± 4.9 A 96.7 ± 13.3 A 69.7 ± 0.87 A 63.6 ± 1.9 *
Stored urine 107.8 ± 10.6 A 101.9 ± 6.9 A 75.0 ± 2.5 A 64.1 ± 1.9 *
Control 99.9 ± 3.4 A 103.0 ± 6.4 A 69.1 ± 0.8 A 61.7 ± 1.3 *
Second production
period 0-20 cm 20-40 cm 0-20 cm 20-40 cm
Lacto-fermented mix
and biochar 132.6 ± 12.7 A 113.0 ± 3.6 A 81.8 ± 5.5 A 78.6 ± 22.7 A
Lacto-fermented mix 127.9 ± 8.9 A 116.4 ± 2.3 A 80.7 ± 4 A 83.0 ± 7.6 **
Mineral fertilizers 137.8 ± 1.4 A 110.5 ± 2.9 A 100.7 ± 7.8 A 105.1 ± 4.6 **
Stored faeces 139.7 ± 0.6 A 118.7 ± 2.8 A 92.4 ± 1.2 A 90.7 ± 1.8 **
Stored cattle manure 112.2 ± 3.0 * 114.5 ± 1.03 A 96.1 ± 7.6 A 98.4 ± 7.6 **
Stored urine 134.4 ± 0.5 A 130.2 ± 4.10 ** 81.4 ± 3.3 A 97.3 ± 22.7 **
Control 101.3 ± 3.1 * 99.5 ± 0.00 * 78.7 ± 0.2 A 65.5 ± 0.00 A
Vermi-composted lacto-
fermented mix and
biochar 117.2 ± 1.6 * 111.2±8.0 A 83.0±7.6 A 67.6 ± 3.56 A
The values labeled with letter A are not significantly different than lacto-fermented mix and
biochar. *values marked with one star are significantly lower than the lacto-fermented mix and
biochar and those marked with two stars ** are significantly higher than lacto-fermented mix and
biochar. 1,2 - expressed as K and P respectively, see Appendix F.
In the soil layer 0-20 cm, a significant increase was observed only for the lacto-
fermented mix without biochar. During the second production year, in the soil layer of 0-20
cm, there was no significant difference of the potassium content in comparison to the other
fertilizers investigated. Nevertheless, in the soil layer 20-40 cm, the potassium content was
significantly lower compared to the other applied fertilizers, except for the control, where no
significant difference was found.
98
4.3.2.4 Humus content
No significant difference in the humus content was observed after fertilization with the
lacto-fermented mix supplemented by biochar in comparison to the control plots and other
fertilizers applied in either of the two production years investigated (Table 4.13, Appendix C).
Table 4.13 Humus content in the soil layers 0-20, 20-40 cm during the first and second
production years (number of samples, n = 3 per each plot)
Applied fertilizer Humus content, %
0-20 cm 20-40 cm
First production period
Lacto-fermented mix and biochar 2.80 ± 0.09 A 1.92 ± 0.08 A
Lacto-fermented mix only 2.98 ± 0.20 A 1.92 ± 0.05 A
Mineral fertilizers 2.59 ± 0.11 A 1.57 ± 0.04 A
Stored faeces 2.59 ± 0.38 A 1.90 ± 0.12 A
Stored cattle manure 2.81 ± 0.15 A 2.16 ± 0.28 A
Stored urine 2.57 ± 0.07 A 1.81 ± 0.05 A
Control 2.69 ± 0.18 A 1.91 ± 0.18 A
Second production period
Lacto-fermented mix and biochar 2.68 ± 0.08 A 2.71 ± 0.13 A
Lacto-fermented mix only 2.76 ± 0.12 A 2.79 ± 0.12 A
Vermicomposted lacto-fermented 2.75 ± 0.37 A 2.64 ± 0.32 A
mix and biochar
Mineral fertilizers 2.46 ± 0.35 A 2.39 ± 0.47 A
Stored faeces 2.53 ± 0.37 A 2.33 ± 0.35 A
Stored cattle manure 2.85 ± 0.06 A 2.81 ± 0.06 A
Stored urine 2.48 ± 0.17 A 2.42 ± 0.16 A
Control 2.54 ± 0.32 A 2.44 ± 0.29 A
The values labeled with letter A are not significantly different from the lacto-fermented mix and
biochar.
For the layer 20-40 cm in the first year, the data showed a big difference with the
humus content compared to that of the second production year. This might have been
influenced by a different degree of oxidation of the organic material during laboratory
99
analysis or non-uniformity of organic material incorporation in the soil. The total amount of
carbon applied with the lacto-fermented mix supplemented by biochar and lacto-fermented
mix without biochar was lower compared to the stored faeces and vermi-composted lacto-
fermented mix supplemented by biochar. However, a higher (but not significantly higher)
humus content was encountered for these fertilizers during the first production year. A slight
decrease in the humus content was, nevertheless, observed during the second year (Table
4.12).
4.3.2.4 Bulk density
During the first production year, the lacto-fermented mix supplemented by biochar has
contributed to a significantly lower bulk density in the superior layer of the soil (0-20 cm)
compared to the mineral fertilizer and stored faeces (Table 4.14, Appendix F).
Table 4.14 Bulk density (g/cm3) in the soil layers 0-20 and 20-40 cm during spring of
2013 and 2014 (number of samples, n = 3 per each plot)
Applied fertilizer 1st production period 2nd production period
0-20 cm 20-40 cm 0-20 cm 20-40 cm
Lacto-fermented mix
and biochar 1.05±0.05 A 1.28±0.02 A 1.04±0.03 A 1.27±0.02 A
Lacto-fermented mix 1.14±0.04 A 1.23±0.03 A 1.08±0.03 A 1.25±0.03 A
Mineral fertilizer 1.38±0.02 ** 1.40±0.05 A 1.23±0.01 ** 1.47±0.07 **
Stored faeces 1.23±0.02 ** 1.27±0.03 A 1.07±0.02 A 1.41±0.05 **
Stored cattle manure 1.14±0.03 A 1.13±0.03 * 1.14±0.06 A 1.41±0.03 **
Stored urine 1.11±0.03 A 1.27±0.06 A 1.21±0.07 ** 1.42±0.06 **
Control 1.14±0.08 A 1.25±0.02 A 1.08±0.03 A 1.33±0.07 A
Lacto-feremented mix
and biochar +V1 1.04±0.03 A 1.27±0.02 A
The values labeled with letter A are not significantly different than the lacto-fermented mix and
biochar. *values marked with one star are significantly lower than the lacto-fermented mix and
biochar and those marked with two stars ** are significantly higher than the lacto-fermented mix
and biochar. 1 lacto-fermented mix and biochar was supplementary vermi-composted.
100
For the deeper soil layer (20-40 cm), no significant difference in the bulk density was
found compared to other fertilizers, except for cattle manure which had a significantly lower
bulk density. During 2014, similar to 2013, a significantly lower bulk density was observed
after application of the lacto-fermented mix supplemented by biochar compared to mineral
fertilizer and stored urine. For the deeper soil layer (20-40 cm), the bulk density was
significantly lower than in the mineral fertilizer, stored cattle manure, stored faeces and urine.
The application of mineral fertilizer (layers 0-20 and 20-40 during 2013 and layer 20-40 cm
during 2014) contributed to a bulk density exceeding 1.35 g/cm3 (Table 4.14), such values
might show a tendency of soil compaction (Andries et al., 2014). The same tendency was also
observed during 2014 in the 20-40 cm soil layer for the plots fertilized by cattle manure,
stored faeces, and urine.
4.4 Discussion
4.4.1 Corn growth
This study demonstrated that the lacto-fermented mix and biochar had a beneficial
effect on the growth of corn. Although some of the growth parameters were not consistent for
both production years, a significant increase in the key growth parameters such as corn height
and leaf length was observed. In spite of the fact that during 2014, the mineral fertilizer had
significantly better effects on the corn height and leaf length than the lacto-fermented mix
supplemented by biochar, it did not have a significantly higher yield during that specific year
(Tables 4.7 and 4.8).
4.4.2 Corn yield and yield components
The corn yield fertilized by the lacto-fermented mix supplemented by biochar was
very similar to the average for Moldova (Table 4.10) during 2006-2013 (National Bureau of
Statistics of the Republic of Moldova, 2014). Considering the low humus content of the soil
101
investigated and also the lack of application of any fertilizers to the area, a beneficial effect
from the fertilization with the lacto-fermented mix and biochar was observed even at a
relatively low application rate (60 kg N active ingredients ha-1) for a crop like corn, which
requires a high nitrogen fertilization rate, e.g. 120-180 kg ha-1 (Andries, 2007).
During the two year field application, lacto-fermented faeces and bio-waste,
supplemented by biochar had also an overall significantly higher effect on yield and yield
components of corn than the unfertilized control, lacto-fermented mix of faeces and bio-waste
without biochar, mineral fertilizer, stored cattle manure, faeces and urine (Table 4.9). The
corn plants had bigger kernels, longer ears, and fewer ears with reduced numbers of kernels
(Figure 4.6). The organic matter from the lacto-fermented faeces and bio-waste, which was
stabilized by the supplemented biochar and the biochar properties themselves (Andreev et al.,
2016) might have provided a more efficient use of moisture during flowering and grain
production. Organic matter adds to an improvement of pore volume and water conductivity as
well as water retention (Fischer and Glaser, 2012). Biochar also improves soil physical
properties such as aggregate stability, pore size distribution and, therefore, water use
efficiency (Obia et al., 2016). Such conditions probably allowed the corn fertilized by the
lacto-fermented mix of faeces and bio-waste, supplemented by urine charged biochar to
develop a better root system than the other applied fertilizers and a subsequent more intensive
growth (Andreev et al., 2016), even during the conditions of reduced precipitation, and
ultimately produced a higher yield. Also the yield was enhanced as the investigated fertilizing
mix has beneficially affected several soil parameters, such as soil potassium and humus
content as well as soil bulk density (Andreev et al., 2016).
4.4.3 Effect of precipitation on yield and yield components
The corn fertilized by the mineral fertilizer had an increased yield compared to the
control and all other fertilizers only during favourable precipitation conditions, while the
lacto-fermented mix supplemented by biochar favoured a yield increase during both years,
regardless of the weather conditions (Table 4.8). This is probably related to the capacity of the
102
fertilizer, especially its biochar component, to retain nitrates from leaching and potentially
improving the soil water holding capacity (Kammann et al., 2015).
The difference in yield during the first and second years of the application of mineral
fertilizer can be explained by variations in intensity and quantity of annual precipitation and
temperature during 2013 and 2014 (Table 4.1), that might have influenced nutrient
availability and leaching (Udawatta et al., 2006). Indeed, Table 4.1 shows that during the first
production period, there was a wetter fall season which might have contributed to
accumulation of soil moisture stocks during spring. Also, abundant precipitation during April
and May, twice as much as the previous year, occurred during the second production period
(Figure 4.7).
120.00
Average monthly precipitations, mm
110.00 1st production

period
100.00
90.00
80.00 2nd
70.00 production
60.00 period
50.00
40.00
30.00
20.00
Autumn Winter spring summer
Annual distribution of precipitation
Figure 4.7 Difference in the seasonal distribution of precipitation during first and second
production years.
In addition for this second production year, the whole month of July had a uniform
precipitation distribution, with a maximum precipitation after 20 of July (Anonymous, 2015),
the period of corn intensive growth and use of soil moisture and nitrogen. This probably
favoured both more efficient mineral nitrogen uptake and intense accumulation by the stem
and leaves in the plots fertilized by the lacto-fermented mix of faeces and bio-waste,
supplemented by biochar. Moreover, such conditions during the second production year most
103
probably also allowed a more efficient translocation from the vegetative parts to the grain
during the dry spells of August and September than during the first production year.
In contrast, during the first production year, heavy rains were encountered during the
end of June and beginning of July, while the rest of the month was dry with high temperatures
encountered. The thunderstorms during June-July (Anonymous, 2015) coupled with a wetter
autumn-winter-early spring period during 2012 (Table 4.1), possibly contributed to nitrate
losses via leaching from the plots fertilized by mineral fertilizer, stored cattle manure, stored
faeces and urine, thus negatively affecting the pre-anthesis period and kernel setting (Runge,
1968). In addition, the temperature at the end of June and beginning of July was more
favourable during 2014 than 2013, thus positively influencing the grain yield.
Application of such fertilizers to crops with a high soil fertility requirement as corn
(Paponov and Engels, 2005) can thus be an alternative to mineral fertilizer, stored cattle
manure, faeces or urine alone, particularly under the conditions of decreased organic matter
content, frequent drought or heavy rains as those occurring in Moldova (Potop and Soukup,
2009; Potop, 2011). Therefore, building up of the soil organic matter through the application
of lacto-fermented faeces and bio-waste in combination with biochar could be more
favourable than the application of mineral fertilizers alone.
More research is needed on the corn nitrogen uptake and nitrogen distribution within
different vegetative parts of the corn at anthesis and grain filling. There is a big difference in
the nitrogen uptake at the different development stages of corn (Spiertz and De Vos, 1983).
During the growing period, nitrogen uptake exceeds that of mineralization and the nitrogen
reserves in the soil are very quickly depleted. During grain filling (e.g. August-September),
the nitrogen supply from the soil becomes less important, as nitrogen is being relocated from
the vegetative parts (Spiertz and De Vos, 1983). Additional knowledge is required on the
proportion of nitrogen accumulated in the beginning of the growing period and that translocated
from vegetative parts to the grain during the grain filling period. Such research will offer
information on the amount of nitrogen to be applied to the soil with a lacto-fermented mix of
faeces and bio-waste, supplemented by biochar and the timing of the fertilizer application so as
to maximize grain yield.
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4.4.4 Effect of soil available water and nitrate content on yield

components
An increase in the soil available water storage capacity might be related to an

enhancement in the soil organic matter, which raised the water holding capacity of the soil
(Diacono and Montemurro, 2010). The lacto-fermented mix of faeces and bio-waste
supplemented with biochar or without biochar have both contributed to a high humus content
during the first year, which slightly decreased during the second year (Andreev et al., 2016).
Additional research is important on the changes induced by the lacto-fermented mix
supplemented by biochar on the soil available water storage capacity along the whole soil profile
(0 to 100 cm) and for the whole corn development stage.
The average precipitation during spring of the first production year was significantly
higher than the average multiannual precipitation for the Nisporeni district (Nedealcov et al.,
2013). However, the precipitation value during the studied period was still far below the average
rainfall for Moldova, such an amount of spring precipitation is more close to the conditions
characterized by a dry spring (Vronskih, 2014). Under dry conditions, nitrate accumulation in the
soil may take place (Lucci et al., 2013). During June and the first half of July, corn requires a high
soil nitrogen content, since it is the period of intensive vegetative growth, when the root system,
corn panicle and primordia of the ears are formed. However, spring dry spells during the first
production year might have decreased the plant capacity to take up nitrogen (Lucci et al., 2013).
Rapid rewetting, for example during flushes from heavy rains after the dry period may cause
intense leaching of nitrates. Therefore, at the end of June, in the first production year, 51% of all
monthly precipitation has fallen during 2 days only and in the beginning of July 69% during one
day only (Anonymous, 2015). Studies have shown that during June, up to 20 to 76% of NO3-
leaching may occur (Corrêa et al., 2005).
Another period which might have also contributed to a decrease in the corn yield
components and yield for the unfertilized control or corn fertilized by mineral fertilizer, stored
faeces, urine and cattle manure during the first production year, is the second half of July and the
whole month of August to beginning of September. During this time, corn has a high water and
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nitrogen demand for internode elongation, tasseling, silking, pollination, kernel setting and filling
(Pandrea, 2012). The two weeks following pollination up to maturity (October) are crucial in
yield formation as the final kernel weight is determined in that period. Thus, the soil moisture
stress or nitrogen deficiency during this period of time may result in reduced kernel size and
consequently in reduced yields (Hollinger and Changnon, 1991) as in our case happened for the
unfertilized control and plots fertilized by stored faeces, cattle manure or urine alone (Table 4.9,
Figure 4.5).
Lack of nitrogen during June might have contributed to ovule abortion, thus resulting in
fewer kernels (Gallais and Hirel, 2004), which was indeed observed in Figure 4.6. Nitrate is a
mobile anion, not absorbed by soil organic matter particles. It is highly available following
application of mineral fertilizer or biosolids and can thus easily be leached below the root zone,
leading to groundwater pollution (FAO, 1996). For example, in a study on pheosem soil (Esteller
et al., 2009), among the applied fertilizers, mineral fertilizer had the highest proportion of nitrate
leached (37 % and 24 % during the first and second year, respectively), followed by composted
(17 % and 14 %) and untreated (11 %) biosolids. Under the conditions of insufficient soil nitrogen
as those caused by nitrate leaching as a result of heavy summer rains, corn can remobilize
nitrogen from other parts such as the stem or leaves. However, the dry period which continued
over August and early September (Anonymous, 2015), coupled with high temperatures, might
have caused leaf senescence and thus loss of accumulated nitrogen. As a consequence, a decrease
in the number of filled kernels leading to an overall yield decrease might have occurred in the
plots on which mineral fertilizer, stored cattle manure, stored faeces or urine were applied.
An integrated view on the efficiency of nitrogen use from the lacto-fermented mix of
faeces and bio-waste, supplemented by biochar could have been obtained if all mineral nitrogen
species would have been measured, including N-NO3- and NH4+-N. Field experiments could
identify the level of mineralization or immobilization of nitrogen in the applied fertilizers. In such
experiments, soil can be placed in polyethylene bags and buried in the 0-60 cm horizon and the
mineral nitrogen content in the bag can be compared to that in the root zone at selected time
intervals. The bags are retrieved and the nitrogen content measured (Westermann and Crothers,
106
1980; Lentz et al., 2012), the net N mineralization being calculated by subtracting the inorganic N
concentration in the initial soil from that in the soil of the retrieved bag.
4.4.5 Humus content
Although mineral fertilizers contribute to the availability of phosphorus and exchangeable

potassium in the soil profile, a loss of soil organic carbon may also occur. For example, long term
experimental research carried out on different crops on chernoziom soils demonstrated a carbon
loss of up to 28-34% in the soil profile of 0-40 cm and 66-72 % in the 40-100 cm profile in the
plots treated with mineral fertilizers compared to the unfertilized control (Boincean et al., 2014).
In the absence of a supplementary carbon source, mineral fertilizers may lead to intensification of
humus decomposition and degradation of soil fertility. At the same time, even though the lowest
amounts of organic matter were added with the lacto-fermented mix supplemented by biochar as
well as the lacto-fermented mix without biochar, these fertilizers contributed to the highest humus
content during the first year. The slight decrease observed during the second production year may
be the cause of the priming effect, an increase in the decomposition of soil organic matter due
high microbial activity after application of fresh organic matter. Incubation experiments or C-
labelling in the applied substrates would help to trace the decomposition of the added organic
matter and the portion that remains stable in the soil and its effect on mineralization of the native
soil organic matter (Perelo and Munch, 2005).
In the first production year, there were variations in the humus content in the 20-40 cm
layer compared to the second year (Table 4.11). Such differences might be caused by a non-
uniform distribution of organic matter (during application or ploughing). Two years is a rather
short period for the evaluation of the changes in the soil organic matter content and long time
research (e.g. 5-7 years) is required to validate these findings.
4.4.6 Potassium content
The potassium content increased significantly during the first production year after
application of the lacto-fermented mix supplemented by biochar, but only in the soil layer
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20-40 cm. In the soil layer 0-20 cm, a significant increase was observed only for the lacto-
fermented mix without biochar. During the second production year, in the soil layer of 0-20 cm,
there was no significant difference in the potassium content in comparison to the other
investigated fertilizers. Nevertheless, in the soil layer 20-40 cm, the potassium content was
significantly lower compared to the other applied fertilizers, except for the control, where no
significant difference was found.
The lacto-fermented mix supplemented by biochar also had a positive influence on the
soil mobile potassium content during the first year of application (soil layer 20-40 cm) compared
to all applied fertilizers, except for the mineral fertilizer (Table 4.12). That was probably related to
an increase in the soil cation exchange capacity favoured by the biochar component (Liang et al.,
2006b). The lower potassium content in the soil compared to other applied fertilizers during the
second production year could be related to a more efficient uptake of this element by plants during
the first year (Haynes and Williams, 1993). Additional research is required on the fate of
potassium in the soil after application of the lacto-fermented mix supplemented by biochar.
4.4.7 Soil bulk density
The soil bulk density was positively affected by the application of the lacto-fermented mix
supplemented by biochar compared to mineral fertilizer and stored faeces (first year, 0-20 cm),
mineral fertilizer and stored urine (second year, 0-20 cm) as well as mineral fertilizer, stored,
faeces, urine and cattle manure during the second year (20-40 cm). Research indicates that around
40 % of the crop yield depends on the soil physical conditions (Krupenikov, 1967; Scerbacov and
Vassenev, 2000; Berezin and Gudima, 2002). This could be related to an improvement in the soil
organic matter and its dilution effect on the soil bulk density, as organic matter is mixed with the
denser mineral fraction of the soil. A compilation of different studies by Khaleel et al. (1981)
demonstrated a linear regression (r = 0.69) between the increase of soil carbon and the percentage
reduction of the soil bulk density.
Studies have shown that only slight changes in bulk density can already contribute to better
root development and indirect beneficial effects for root penetration, increased soil infiltration
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capacity and decreased runoff volumes (Mbah and Onweremadu, 2009; Lawal and Girei, 2013). An
improvement of the root penetration increases the capacity of the plants to take up water and
necessary nutrients for plant growth and development, which in turn stimulates plant biomass and
yield (Mbah and Onweremadu, 2009), as might have happened for the applied lacto-fermented mix
supplemented with biochar and the lacto-fermented mix alone. The results are in line with other
studies on the application of bokashi soil conditioner (a mixture of cattle manure with water,
molasses, effective microorganisms and rice bran), which contributed to a reduction in the soil bulk
density, and an increase in the soil permeability and porosity as well as nutrient availability
(Xiaohou et al., 2008). Increased soil bulk density and compaction, for example as in the case of
mineral fertilizer, could have led to a number of negative effects, e.g. decreasing the soil respiration
and worsening of the soil borne diseases in the root zone during drier or wetter conditions (Batey
and McKenzie, 2006).
4.4.8 Effect of supplementary treatment by vermi-composting
In spite of the beneficial effects of the vermi-composted lacto-fermented mix and biochar on
the germination index and root length (e.g. the root length exceeded by 35% that of the control
under laboratory conditions, Andreev et al., 2014), the current study did not show the additional
treatment via vermi-composting was advantageous compared to the lacto-fermented mix
supplemented by biochar, since most of the growth parameters and the corn yield after application
of this fertilizer were significantly lower (Table 4.7). It had a positive effect on the soil humus
content, but this was not significant as reflected by Table 4.11. However, a single year field
application is insufficient for a generalization.
4.5 Conclusions
Two years of application of a lacto-fermented mix of faeces and bio-waste supplemented by

biochar on corn growing on a clay-loamy cernoziom showed beneficial effects on corn growth,
especially on plant height compared to the control, stored faeces, urine, cattle manure.
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During the first year, the nitrate content of plots fertilized by lacto-fermented faeces and bio-
waste supplemented by biochar was significantly higher than that of a control and plots fertilized by
stored urine, but significantly lower than the applied mineral fertilizer. The higher corn yield and
yield components produced might be attributed to the potential of the biochar to prevent nitrate
leaching as well to the improvement of soil quality parameters such as organic matter and soil bulk
density. Soil conditioners obtained by lacto-fermenting faeces and bio-waste with biochar
supplementation might thus be an appropriate strategy for improving soil fertility and crop yield as
well as preventing nitrate losses, particularly under unfavourable weather conditions, such as
extended droughts followed by rainfall flushes.
The corn fertilized by this soil improver had an increased number of kernels per row and
kernels per ear compared to the unfertilized control, stored cattle manure, faeces and urine, which
also improved the yield. Compared to mineral fertilizer, the yield was significantly higher during the
first production year, but not significantly different during the second production year. Potassium
availability was significantly increased during the first production year in the soil layer 20-40 cm
and the soil bulk density was lowered during both production years. In low productive soils and
those poor in organic matter content, the lacto-fermented mix supplemented by biochar may serve
as an important component in restoring the productivity by improving the soil organic matter and
physical soil properties (e.g. lowering bulk density) as well as by preventing nutrient losses. Long
term field research is required to fully understand the sustainability of this application, especially on
the changes occurring after application of this fertilizer on the soil organic matter and nutrient
availability.
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Chapter 5. Lactic acid fermentation of human urine for
improving its fertilizing value and reducing odour
emissions in urine diverting dry toilets

N. Andreev, M. Ronteltap, P.N.L. Lens, B. Boincean, M. Wernli, E.Zubcov, N. Bagrin, N.
Borodin Lactic acid fermentation of human urine for improving its fertilizing value, Journal of
Environmental Management,198, 63-69.
111
Abstract
During storage of urine, urea is biologically decomposed to ammonia, which can be

lost via volatilization and cause malodor. Lactic acid fermentation of urine can decrease
nitrogen volatilization and reduce odour emissions. Fresh urine (pH = 5.2 - 5.3 and NH4+-N =
1.2 - 1.3 g L-1) was lacto-fermented with bacterial inoculum from sauerkraut and compared to
urine stored for 36 days in glass jars. In the lacto-fermented urine, the pH was reduced to 3.8 -
4.5 and the ammonium content by 22 - 30 %, while in the untreated urine the pH rose to 6.1
and its ammonium content increased by 32 %. The concentration of lactic acid bacteria (LAB)
in lacto-fermented urine was 7.3 CFU ml-1, suggesting that urine is a suitable medium for
LAB growth.
The perceived odour was twice stronger in the untreated urine than in the lacto-
fermented one. Lacto-fermented urine induced higher radish germination than stored urine.
Adding LAB inoculum to storage tanks in a urine diverting dry toilet reduced the pH from 8.9
to 7.7, while the ammonium content increased by 35 %, probably due to the high initial pH of
the urine. As the hydrolyzed (stored) urine has a high buffering capacity for an efficient urine
lacto-fermentation, the LAB inoculum should be added to urine storage tanks before urine
starts to accumulate there.
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Lactic acid fermentation of human urine for improving its fertilizing
value and reducing odour emissions in urine diverting dry toilets
5.1 Introduction
During the last decade, a global concern has risen over a more efficient utilization of
nitrogen from the application of nitrogen-based fertilizers without producing adverse
environmental impacts. Alternative fertilizers capable to replace or complement mineral
fertilizers need to be considered. Human urine is recognised as a potentially good fertilizer
owing to its high nutrient content and low hygienic risk (Höglund et al., 2002). In fresh
human urine, the main part of nitrogen (75-90%) is in the form of urea [CO(NH2)2], but also
as uric acid and amino acids. Only a small proportion (up to 7 %) is in the form of ammonia
(Kirchmann and Pettersson, 1994). Besides nitrogen, urine also contains phosphorous (H2PO4-
and HPO42-) and potassium (K+) in ionic forms, calcium (Ca2+), sulphate (SO42-) and soluble
organic matter (Lind et al., 2001; Maurer et al., 2006), which potentially have a fertilizing
value as well.
Urine diverting dry toilets (UDDT) are ideal systems for harvesting urine for
fertilizing applications. However, misuse of UDDT systems may cause faecal cross-
contamination of the urine. In order to reduce the pathogens to a safe level, urine has to be
stored for 1-6 months (WHO, 2006; Jaatinen et al., 2016). During storage, under the influence
of bacterial activity and particularly the urease enzyme, urea is degraded to ammonia, which
can volatilise (Mobley and Hausinger, 1989; Udert et al., 2006). Urine hydrolysis and urea
decomposition are pH dependent. Urease activity is optimal at pH 7-8 (Schneider and
Kaltwasser, 1984; Krajewska et al., 2012), while it is inhibited at low pH < 5 (Larson and
Kallio, 1954; Schneider and Kaltwasser, 1984). At pH 8.9-9.0, 95% of the nitrogen in the
stored urine is in the form of ammoniacal nitrogen (Kirchmann and Pettersson, 1994). As a
result of alkalinisation and an increase in the concentration of compounds such as bicarbonate
and ammonia, the urine buffering capacity increases as well (Udert et al., 2006).
Besides impacting the efficiency of nitrogen recovery, ammonia volatilization causes
undesirable odour emissions in separate collection sanitation systems. The odour is intensified
by other malodourous components such as volatile fatty acids, formed under the influence of
113
bacterial activity (Zhang et al., 2013). The odour emissions are annoying for the toilet users as
well as for the residential areas in the vicinity where urine is applied onto agricultural fields.
Different methods have been proposed to reduce ammonia volatilization and inhibit
urea decomposition in the urine. A successful result of the inhibition of urea decomposition
by maintaining a low pH < 4 was obtained by adding strong acetic and sulphuric acids (2.9 g
L-1) (Hellström et al., 1999). A limitation of this method is the economical aspect and the
health risks during handling of the acids (Maurer et al., 2006). There is also a lack of research
on the impacts of acidified urine on soil and crops after its application.
An alternative method for stabilizing nitrogen is biological nitrification with the use of
ammonia and nitrite oxidizing bacteria (Udert and Wächter, 2012). However, maintaining
bacterial activity in high strength ammonia solutions as urine is challenging and requires
skilled handling (Udert and Wächter, 2012). Thus, there is a need to develop a cost effective
method to acidify urine that does not affect its fertilizer value and land applicability.
This study, therefore, focused on the efficiency of lactic acid fermentation of source
separated urine for increasing its resource oriented potential. The change in pH, ammonium
content, buffering capacity, odour reduction, chemical oxygen demand (COD) and potential
biological effects on plants of lacto-fermented urine was compared to that of untreated, stored
urine. This expands the knowledge regarding such applications in sanitation which is
currently limited.
5.2 Material and methods
5.2.1 Experimental set-up
Storage and lactic acid fermentation of fresh urine was performed under laboratory
conditions in two trials, each with three replicates, over two periods of 36 days each, during
December 2015 - January 2016 and April - May 2016. Urine samples were collected from 2
donors (a female, aged 44 and a child boy, aged 7) for a period of three days and stored in 1 L
glass jars tightly closed with a plastic lid. At the end of the collection period, all urine of both
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donors was thoroughly mixed, chemical analysis was performed and then separated into two
parts. The first part was mixed with a lactic acid bacteria (LAB) solution (1:1) and lacto-
fermented in glass containers for a period of 36 days. The second part was stored in parallel in
tightly closed glass containers for the same period of time as the lacto-fermented urine.
For obtaining the LAB solution, cabbage was fermented over a one month period,
after which juice was collected, mixed with sugar beet molasses and water at a proportion of
1:1:9 and kept in a closed plastic jar until the pH dropped to below 4.
After the treatments, chemical analysis was performed in both the lacto-fermented and
stored urine samples. In addition, the LAB solution as well as the lacto-fermented urine was
analysed for their E.coli and lactobacilli concentration. No analysis of E.coli was performed
in the untreated, simple stored urine.
The efficiency of lactic acid fermentation was also evaluated under real field
conditions in a functional household urine diverting dry toilet (UDDT) storage tank in the
vicinity of Chisinau (Moldova), used by a family of two adults (male 45 years and female 44
years) and one boy child (7 years). In this test, the 300 L plastic urine storage tank and the
urine pipes were thoroughly washed and rinsed with vinegar prior to the experiments. Then,
urine was collected in the tank for a period of one week (for pH and ammonia analysis) after
which LAB bacterial inoculum and molasses were added in the tank (2.5 kg of molasses was
added to 2.5 L of sauerkraut juice and mixed with 10 L of water). This was applied to 130 L of
urine accumulated from the 3 people during 36 days. This ratio was considered taking into
consideration the optimal ratio obtained from the laboratory experiments capable to reduce
the pH below 4. During each toilet use, the urinal and urine compartment were sprinkled with
a LAB solution, which was approximately 70-100 ml per day.
5.2.2 Odour evaluation
The odour intensity of lacto-fermented and stored urine was evaluated by a panel of
four people (2 males and 2 females) independent from each other. Perception of the strength
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of the perceived odour was evaluated according to a rank scale from 0 (no odour) to 6
(extremely strong odour) as described in Table 5.1 (Misselbrook et al., 1993).
Table 5.1 Rank scale for different perceived urine odour strengths
Perceived odour strength Rank scale
No odour 0
Very faint odour 1
Faint odour 2
Distinct odour 3
Strong odour 4
Very strong odour 5
Extremely strong odour 6
5.2.3 Germination tests
The treated urine samples were diluted 1:10 with distilled water. Twenty seeds of
radish Raphanus sativus were placed on Petri dishes lined with Whatman filter pads and an
amount of 3 ml of 1:10 urine:water mix was added to each replicate. As control 3 ml of
distilled water was used. The urine dilution rate (1:10) was obtained after preliminary
germination testing and was required because of the low pH of the lacto-fermented urine.
After 72 hours, germination was stopped by adding 3 ml of 50% alcohol to each of the Petri
dishes. The germination index (GI) was calculated according to the following formula (Eq.1):
(, = ( ∗ --( ∗ 100 (1)

where G - number of seeds germinated in the sample/number of seeds germinated in the
control and RRG - relative root growth (mean root length in the treated sample/mean root
length in the control) (Tiqua et al., 1996; Mitelut and Popa, 2011).
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5.2.4 Microbiological analysis
The LAB solution and lacto-fermented urine were thoroughly mixed and serially
diluted 8 times. Subsequently, 1 ml of the solution was incubated by inoculation on agar
plates. E.coli incubation was done on HiChrome Coliform agar at 43°C for 24 h, while
lactobacilli incubation was done on M.R.S agar (CMO361) at 36 °C for three days under
anaerobic conditions (ISO, 1998; MHRF, 2005). The analysis was performed in duplicates.
5.2.5 Chemical analysis
The ammonium (NH4+-N) concentration was determined by a UV-VIS Analytik Jena

Specord 210 spectrophotometer at 400 nm using cuvettes of 10 mm according to the standard
SM-SR-ISO7150-1:2005 (Anon, 2005). Urine was diluted 1000 times (0.5 ml to 500 ml of
distilled water) from which 50 ml was taken and added with Seignette salt (C4H4KNaO6) and
Nessler's reagent.
Chemical oxygen demand (COD) was analysed by dichromate oxidation, using the
closed reflux method (Aliokin et al., 1973). Buffer capacity was determined by measuring the
initial pH in urine using a Hanna portable EC/pH meter and titrating with 0.1 mol NaOH until
the pH changed by one unit (Kirchmann and Pettersson, 1994). All chemical analyses were
conducted in triplicates.
5.3 Results
5.3.1 Concentration of Lactobacillus and E.coli in LAB solution and

lacto-fermented urine
The microbiological analysis indicated high bacterial counts of Lactobacillus both in

the LAB solution added to the urine as well as in the lacto-fermented urine (Figure 5.1). The
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Figure 5.1. Plate counts of LAB (10-6 dilution) in the LAB solution (A) and lacto-fermented
urine (B).
Lactobacillus concentrations were, respectively, 7.5 and 7.3 log CFU ml-1 showing
that non-hydrolyzed urine offered favourable growth conditions for LAB. E.coli was not
detected in the LAB solution nor in the lacto-fermented urine. E.coli may appear in urine in
the case of urinary tract infection or potential faecal contamination during urine collection
(Kunin et al., 1992; Höglund et al., 2002). Under the influence of LAB, E. coli growth is
inhibited because of the low pH and excretion of inhibitory substances, such as bacteriocins,
lactic acid, hydrogen peroxide, glucose oxidase and other compounds (Saranraj, 2014).
5.3.2 Changes in chemical composition of urine by lactic acid

fermentation
Lactic acid fermentation did not change the buffer capacity of the urine; however,
urine storage increased the buffer capacity approximately two times (Table 5.2). Immediately
after the addition of the LAB solution to the urine, the ammonium content was slightly
reduced, probably by the dilution (1:1). The pH of the urine during lactic acid fermentation
decreased to 4.5-3.8 due to the formation of lactic acid. At this pH value, the bacterial urease
is normally inhibited and urea hydrolysation stops (Larson and Kallio, 1954; Schneider and
Kaltwasser, 1984). It is important to note that this is only effective with fresh urine; the
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hydrolysis process is non-reversible, so the lactic acid fermentation needs to take place before
ureolysis sets in.
The ammonium content in lacto-fermented urine decreased by approximately 22-30%
compared to the fresh urine (Table 5.2). In the untreated urine, the hydrolysis was not
inhibited, hence the ammonium content increased by 32% compared to the fresh urine and by
44-53% compared to the lacto-fermented urine. The pH changed only slightly compared to
the fresh urine (5.94-6.02 compared to 5.2-5.3, respectively).
Table 5.2 Effect of lactic acid fermentation on pH, buffer capacity and ammonium
concentration of urine (mean ± SD).
Sample type Buffer NH4+-N NH4+-N pH pH GI GI
-1 -1
capacity, gL gL I II I II
mmol L-1 I II (%) (%)
Fresh urine 0.9±0.01 1.3±0.05 1.2±0.02 5.3 5.2 - -
Urine+LAB 0.9±0.01 - 1.1±0.03 4.5 4.4 - -
Lacto-fermented urine 0.9±0.01 0.9±0.05 0.9±0.1 4.7 3.8 86 74
Stored urine (glass jar) 1.5±0.01 1.9±0.05 1.6±0.1 6.0 5.9 2.2 31.2
Stored urine (urine tank)1 - - 2.9±0.08 - 8.9 - -
2
LAF urine (urine tank) - - 4.6±0.09 - 7.7 - 0.17
Stored urine (one month, urine 9.6±0.02
tank)
1-
before lactic acid fermentation, 2 - after lactic acid fermentation, GI - germination index, I -
first experimental run; II - second experimental run.
In the urine tank urea hydrolysation occurred at a much faster rate compared to that in
the tightly closed glass containers, since the pH increased rapidly to 8.9 during one week
only. The addition of the LAB inoculum and molasses in the urine tank of a UDDT
contributed to a reduction of the urine pH by 1.25 units; however, it did not stop the
hydrolysation process. Even though the pH was reduced to 7.7 after 36 days of lactic acid
fermentation, the ammonium content continued to increase and was 1.5 times higher than the
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initial value and 3 times higher than in the stored urine (Table 5.2). However, compared to
previous one-month stored urine, the amount of ammonia was almost twice lower.
The addition of LAB to urine contributed to a change in the COD concentration (Table
5.3). During lactic acid fermentation, the soluble carbohydrates were converted to lactic acid
leading to a pH decline and inhibition of decomposition of organic compounds (Murphy et
al., 2007). Therefore, the COD value in lacto-fermented urine was expected to remain similar
to that of fresh urine. In contrast, it was 15 % higher (Table 5.3). Also in the untreated, simple
stored urine there was no COD reduction, but a slight increase of approximately 7 %
compared to the fresh urine (Table 5.3).
Table 5.3 Effect of lactic acid fermentation on COD of urine

Sample COD, g O2 L-1
Fresh urine 21.1 ± 0.9
1
LAB+urine 21.5 ± 1.6
Lacto-fermented urine (glass jar) 25.4 ± 3.0
Stored urine (glass jar) 22.8 ± 0.8
1
measurement was done immediately after the addition of the LAB solution
5.3.3 The effect of lacto-fermented urine on seed germination
Lacto-fermented urine had beneficial effects on the germination of seeds of Raphanus

sativus relative to the control, with a GI of 74-86 %. The stored urine with an ammonium
content of 1.6 g L-1 had a beneficial effect (GI =31.2%) on the germination (Figure 5.2 A). In
contrast, the stored urine from the glass jars with a higher ammonium content (1.9 g L-1) had
an inhibitory effect on germination and the GI was only 2.3% (Figure 5.2 B). In the urine
tank, with an ammonium content of 4.6 g L-1, the germination was also inhibited: the GI was
only 0.008 % of that of the control (Table 5.2), probably related to the toxicity of ammonia
and ammonium.
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Another factor that might have influenced the GI is the pH. Lacto-fermented urine had
a higher GI in the first experimental run (pH = 4.7) than in the second run (pH=3.8). The pH
of the lacto-fermented urine with a GI of 74% after the dilution of lacto-fermented urine with
water (1:10) was increased to only 4, such low pH values are usually unfavourable to
germination, while the pH of the stored urine was 6.3 (1st run), which favoured germination.
Figure 5.2. Seed germination in control (A), lacto-fermented urine (B) and stored urine (C)
(one out of 3 replicates per each sample).
5.3.4 Odour reduction during urine lactic acid fermentation
The odour of fresh urine was perceived as faint to distinct, that of the lacto-fermented
urine distinct to faint and that of the stored urine from very strong to extremely strong (Table
6.4). In the toilet room, the urine odour was reduced compared to when no LAB solution was
used for rinsing the urine compartments. In addition to this evaluation by different odour
panelists, the odour strength in the fresh, lacto-fermented and stored urine could also be
perceived during the laboratory analysis. When diluted with distilled water, the fresh and lacto-
fermented urine did not produce any nuisance, the odour almost dissapeared after dilution.
Instead, the odour of stored urine, particularly in the urine tank, was stronger and smelled even
after dilution. Additionally, it was observed that after 36 days of lactic acid fermentation, the pH
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of lacto-fermented urine had decreased to 3.5 and the urine odour was replaced by a
medicinal/ester odour type.
Table 5.4 Strength in the perceived odour of stored and lacto-fermented urine
Sample type Perceived odour strength Rank scale
Fresh urine Faint odour - distinct odour 2.2 ± 0.5

LAF urine Distinct-faint odour 2.6 ± 0.5
Stored urine (glass jar) Very strong - extremely strong 5.6 ± 0.5
odour
Stored urine (urine tank) Extremely strong odour 6.0 ± 0.0
5.4 Discussion
5.4.1 Urine as a suitable growth medium for LAB
This study showed that fresh urine can serve as a suitable growth medium for LAB as
their number in both the bacterial inoculum and lacto-fermented urine was higher than 7 log
CFU after 36 days of incubation. The complex proteolytic system of LAB (Savijoki et al.,
2006) probably allowed them to adapt to grow in the urine. Non-hydrolyzed urine is rich in
important components such as peptides, urea, hippuric acid, amino acids, citric acid and
minerals such as K+, Na+, Mg+, PO43-, SO42- and Cl-, which support or can stimulate the
growth of LAB (MacLeod and Snell, 1947; Strong et al., 2005; Udert et al., 2006). Other
components in non-hydrolyzed urine detected in small quantities in healthy people are
carbohydrates such as glucose, lactose, galactose, lactose, xylose and arabinose (White and
Hess, 1956; Date, 1958).
As LAB are not able to synthesize all amino acids by themselves, they need additional
amino acids and peptides in the growth medium (Niamsiru and Batt, 2000). Both non-
hydrolyzed urine and molasses, added as a carbohydrate source for LAB, contain most of the
essential amino acids required for the growth of the LAB and the production of lactic acid
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(Dunn et al., 1947; Stein and Carey, 1953; Mee et al., 1979) (Table 5.5). Amino acids and
peptides can also serve as a nitrogen source, since LAB cannot catabolise mineral nitrogen
(Saeed and Salam, 2013). During the current study, the NH4+-N concentration has decreased
as a result of lactic acid fermentation, one explanation could be that some free NH3 was
immobilized into the bacterial cell wall or precipitated as ammonium lactate (Ikawa and Snell,
1960; Kuromiya et al., 2010), thus decreasing the NH4+- N concentration.
Table 5.5 Amino acids excreted in human urine and present in molasses and those
essential for lactic acid bacteria (LAB) growth.
Amino acid Range value Composition Essential for
excreted in ratio (%) in cane LAB growth
urine, mg/day molasses
Histidine 113 - 320 0.57 +
Methyl histidine 17 - 384 - -
Taurine 86 - 294 - -
Glycine 110 - 199 3.33 +
Aspargine 34 - 92 - -
Serine 27 - 73 3.33 +
Alanine 21 - 71 13.3 +
Threonine 22 - 53 2.19 +
Tyrosine 15 - 49 3.33 +
Lysine 7 - 48 0.21 +
Phenylalanine 9 - 31 0.65 +
Leucine 13 - 28 2.19 -
Isoleucine 13 - 26 1.19 +
Glutamic acid 8 - 40 17.73 +
Cystine 10-21 2.19 +
Arginine < 10 - 18 1.09 +
Aspartic acid < 10 - 17 38.76 +
Methionin < 5 - < 10 0.57 +
Valine 4 - 10 6.62 -
Sources: Duun et al., 1947; Mee et al., 1979; Stein & Carey, 1953.
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5.4.2 Benefits of urine lactic acid fermentation
5.4.2.1 Urine lactic acid fermentation and its potential fertilizing value
Ammonia and bicarbonate formed during the process of urea hydrolyzation under the
influence of urease positive bacteria contribute to an increase in the buffering capacity (Udert
et al., 2006). Due to its high buffering capacity, it is not economical to treat urine with acids
to lower NH3 volatilization (Udert et al., 2006) and thus increase its fertilizing value. A
combination of high buffering capacity, increased pH and high NH3 (NH4+-N) concentration
might also have a negative impact on soil bacterial nitrification processes. This is caused by
inhibition of nitrite oxidation and facilitating its accumulation in the soil (Burns et al., 1995).
Nitrite accumulation is undesirable due to its potential phytotoxicity (Beauchamp, 1988).
Another negative effect could be the loss of ammonia from the soil following urine
application, particularly in soils with high pH and high buffering capacity as well as low
cation exchange capacity (Sherlock, 1984). For example, a lower soil cation exchange
capacity will allow a smaller percentage of NH4+-N cations to bind to the exchange sites.
Urine lactic acid fermentation in this study reduced the buffer capacity, pH and
ammonium content. A drop of the pH below 5 usually decreases the urease activity (Larson
and Kallio, 1954; Schneider and Kaltwasser, 1984). The formation of free NH3 and its loss via
volatilization is also pH dependent. The highest NH3 concentration is formed and subject to
loss at a pH range 7 to 10 (Hartung and Phillips, 1994) as was obtained in the UDDT urine
tank, while at pH 4.5 and below as obtained with urine lactic acid fermentation in the bottles
(Table 5.2), no free ammonia is formed (Hartung and Phillips, 1994; Williams et al., 2011).
Even though the addition of the LAB inoculum in the urine tank of a UDDT did contribute to
a decrease in pH from 8.9 to 7.7, it has not stopped urea hydrolysis and thus the increase of
the NH4+-N/NH3 concentration (Table 5.2). However, if comparing to a previous analysis of
the one month stored urine, the concentration was almost twice lower. Additional research is
required to test if the lactic acid fermentation technique can be effective in preventing urea
hydrolysis before any urine starts to accumulate there.
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Protein type compounds such as amino acids and peptides from urine and molasses
are more efficiently used by the proteolytic system of LAB (Niamsiru and Batt, 2000) than
urea (Carvalho et al., 2011). Therefore, nitrogen in urine, after its lactic acid fermentation,
will remain available mainly as urea. Urea based fertilizers are among the most frequently
used nitrogen fertilizers with more than 50 % of all nitrogen usage at a global level (Glibert et
al., 2006), owing to low production cost, high nitrogen content and widespread availability
(Hawke and Baldock, 2010). However, concerns are arising over the efficiency of nitrogen in
the soil system, due to loss of NH3 immediately following urea fertilizer application (Hawke
and Baldock, 2010). Urea applied to the soil undergoes hydrolysis to NH4+-N under the
influence of soil urease and is subsequently partly lost as NH3. Ammonia might also have
adverse effects on seed germination (Bremner and Krogmeier, 1989). We assume that
applying lacto-fermented urine will not cause increased ammonia volatilization since LAB
and their associated pH decrease may act as urease inhibitors in the soil, where urea
hydrolysis was inhibited (Table 5.2).
This study showed that the urine with the highest ammonium content completely
inhibited the germination (Table 5.2). This is probably related to the toxicity of the increased
NH3 and NH4+-N concentrations. The stored urine kept in the glass jars with increased
ammonium content (1.9 g L-1) inhibited germination (Table 5.2), thus suggesting a potential
threshold value of ammonium phytotoxicity. Uptake of high concentrations of NH4+-N by
plants may cause intracellular pH disturbance as well as reduction of synthesis of organic
acids and plant growth hormones such as cytokinin (Britto and Kronzucker, 2002). In contrast
to untreated stored urine, lacto-fermented urine showed more beneficial effects on
germination (Figure 5.2 A and B). The action of LAB from lacto-fermented urine and the
metabolites they produce may bring additional benefits to the plants and soil. For instance,
LAB contributed to a 2-4 fold increase in tomato fresh weight of fruits than the unamended
control plants (Hoda et al., 2011) and significantly higher growth of cabbage (Somers et al.,
2007). LAB may also exert a range of beneficial effects on soil such as solubilization of the
water insoluble phosphate compounds present in the soil, thus increasing their availability to
plants (Zlotnikov et al., 2013). Additionally, LAB can suppress soil pathogens, e.g. bacteria
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and fungi (Visser et al., 1986; Hoda et al., 2011; Murthy et al., 2012) by producing different
compounds with antagonistic activity such as organic acids, hydrogen peroxide, cyclic
peptides, and phenolic or proteinaceus compounds (Hoda et al., 2011; Fhoula et al., 2013). A
better understanding of the mechanisms of urine lactic acid fermentation on nitrogen
volatilization, plant uptake and the benefits of LAB on biological, physical and chemical soil
components can be achieved in long term field studies that are currently lacking.
An aspect that needs further investigation is the use of lacto-fermented urine for
nutrient charging of biochar to form a slow release fertilizer (Schmidt et al., 2015). Studies
have shown that ammonia and phosphate absorption onto biochar is more effective at a lower
pH, with a maximal phosphate absorption at pH 2.0-4.1, but minimal at pH higher than 6
(Yao et al., 2011; Spokas et al., 2012). Also the adsorption mechamism requires further
research. Besides direct adsorption on the carbon structure of biochar, the organic compounds
present in urine can form an organic coating to which anions and cations such as phosphorous
and ammonium can bind (Schmidt et al., 2015).
5.4.2.2 Urine lactic acid fermentation for odour reduction
Offensive odours in urine formed over time are produced as a result of bacterial
metabolism or thermal reactions (Troccaz et al., 2013). Ammonia volatilized during urine
storage and urea hydrolysis is a major malodourous compound (Zhang et al., 2013). However,
other organic compounds with lower odour threshold values than ammonia play also a
significant role in the formation of the typical stale odour of urine after storage (Troccaz et
al., 2013). A range of volatile fatty acids are formed as a result of anaerobic degradation of
carbohydrates, proteins, peptides and amino acids from urine by (facultative) anaerobes.
Volatile fatty acids are among the key factors contributing to odour emissions. Among them,
acetic, propionic and butyric acid have the strongest offensive odours (Zygmunt and Bannel,
2008). Other main odour contributors in the stored urine are dimethyldisufide, methyl
mercaptan, ethyl mercaptan, thrimethylamine, phenol and indol (Troccaz et al., 2013; Zhang
et al., 2013; Liu et al., 2016). Oxidation of methionine to methional and methanethiol leads to
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the formation of dimethyldisulphide and methyl mercaptan (Wagenstaller and Buettner, 2013;
Liu et al., 2016). Dimethyldisulphide was recognised as one of the most offensive odourous
compounds emitted from human urine (Liu et al., 2016). Lactic acid fermentation inhibited
the activity of bacteria (Wang et al., 2001) like Enterococcus faecalis, Streptococcus
agalactiae, Morganella morganii and Escherichia fergusonii that generate key odourous
compounds such as dimethyldisulphide, thrimethylamine, phenol or indol in stored human
urine (Troccaz et al., 2013).
During lactic acid fermentation, LAB can metabolise amino acids via their enzymatic
system into flavour compounds such as alcohols, aldehydes, esters and sulphur compounds
(Savijoki et al., 2006). Therefore, the reduction of odour emissions during urine lactic acid
fermentation was probable caused by the decrease in ammonia emissions as well as by the
synthesis of flavour compounds by LAB. For example, LAB can metabolize citrate from
urine into diacetil, acetoin and butanediol, which are important flavour compounds (Hassan et
al., 2012). Also, hippuric acid, present in urine, is decomposed completely during lactic acid
fermentation, this being the precursor for the synthesis of benzoic acid, which is another
flavour compound (Güzel-Seydim et al., 2000).
In contrast to the glass containers, where odour was considerable reduced after lactic
acid fermentation, the odour was stronger in the UDDT urine tank (Table 5.4). This was
probable caused by the fact that free urease and urease positive bacteria prevailed in the pipes
and urine tank. Their complete elimination is impossible, therefore urea hydrolysis and
anaerobic decomposition of organic matter did take place and the LAB could not dominate
the processes in the urine tank, even though they contributed to a pH reduction (Table 5.2).
Another factor that might have hindered urine lactic acid fermentation was that the urine tank
was not completely anaerobic, thus other microorganisms besides LAB could compete for
organic matter and nutrients from the urine.
This study showed that COD values do not reflect the decomposition of organic
compounds from urine (Table 5.3). The increasing, instead of decreasing, COD concentration
in the stored and lacto-fermented urine was probably due to the nitrogen organic compound
urea, which has no COD value on a molar basis, thus the results being underestimated and
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explaining the increased value in stored urine where anaerobic bioconversion into organic
compounds occurred. The increased COD concentration in lacto-fermented urine might also
have been caused by lactic acid, which is not completely oxidized during bichromate
oxidation in the COD analysis (Elizarova, 2000). An additional explanation might be that
hydrogen peroxide generated by LAB (Kang et al., 2005) consumes the oxidation agent
potassium dichromate, leading to an overestimation of the COD concentration. For example,
hydrogen peroxide present in anaerobically digested livestock wastewater led to a 9 - 14 %
overestimation of the theoretical COD values (Lee et al., 2011).
5.5 Conclusions
Urine lactic acid fermentation might be an effective low-cost technique that may lower
ammonia volatilization and reduce odour emissions from UDDT systems. The application of
a LAB solution of sauerkraut containing molasses and water to fresh urine led to effective
acidification to pH < 4 - 4.5 and a reduction by 1/3 of the ammonium content, while
maintaining a high concentration of viable LAB (7.3 CFU ml-1) compared to the stored urine.
In addition, lacto-fermented urine reduced by two times the perceived odour strength and
beneficially affected seed germination, showing a potentially higher fertilizing value than
untreated, stored urine. Applying this technique to an urine tank of a UDDT reduced the pH,
however did not stop urea hydrolyzis. Compared to previous studies on the concentration of
NH4+-N in one month stored urine the ammonium content was twice lower after urine lactic
acid fermentation, thus suggesting that ammonia reduction might be possible. However,
additional research is required to transfer these research findings to practical application in
UDDT systems.
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Chapter 6. General Discussion and Outlook
129
6.1 Potential and limitations of the application of lactic acid

fermentation and biochar in excreta treatment
During the last few decades, the growing threats such as depletion of soil nitrogen,
phosphorus and organic matter, eutrophication of rivers, competition for land between
farmers and other interests (Blum, 2013), loss of soil fertility due to erosion and
overexploitation and the challenge of establishing sustainable sanitation call for alternative
approaches in sanitation and agriculture. The productive use of excreta from UDDTs can
bring many economic and social benefits and contribute to an improvement in the soil quality
by substituting or complementing chemical fertilizers. The recycling of nutrients and organic
matter from excreta to the soil is becoming extremely important, given the current rate of loss
in soil organic carbon stocks (FAO, 2015), combined with the depletion of non-renewable
sources of nitrogen and phosphorus fertilizers (Dawson and Hilton, 2011) and the uneven
distribution of potassium resources in nature (Ciari et al., 2015). The application of only
chemical fertilizers to the soil has proven to be unsustainable, especially for countries that
face financial limitations and ever increasing prices of the fertilizers. Long term experiments
(Boincean et al., 2014) have shown that the application of increased amounts of chemical
fertilizers leads to rapid decomposition of humus and even decreased yields (Mulvaney et al.,
2009). Supplementation with organic matter is important for maintaining soil fertility and
increasing soil resistance to erosion (Lal, 2002).
Access to safe water and sanitation is a great challenge for the rural and peri-urban
areas of Eastern European regions. A significant part of the population lives in rural areas and
the connection to sewerage is either unfeasible or unaffordable in the immediate future. The
abundant existing pit latrines in these regions affect the quality of groundwater sources and
the environment (Bodik, 2007). Therefore, dry sanitation, such as urine-diverting dry toilets
(UDDTs), can be a sustainable alternative to latrines, as they provide valuable fertilizer and
reduce groundwater pollution. Also, in order to avert the water crisis in the future, it is crucial
130
General Discussion and Outlook
to adopt management techniques for more efficient use and protection of water resources that
are applicable under economic limitations as well as weather or hydrological extremes (e.g.
droughts).
In Moldova, a significant proportion of the rural population depends on shallow wells
for their drinking water, and these get polluted by onsite sanitation systems (pit latrines and
septic tanks) and the disposal of animal manure (Andreev and Andreev, 2010). Even though
there is connection to sewerage systems in some rural areas, the treatment facilities are either
dysfunctional or not existent (The World Bank, 2014), with the wastewater infiltrating into
the soil or discharged into the surface waters without treatment. The predictions from the
impact of climate change indicate that water supply will be threatened due to scarcity in
surface and groundwater resources. Currently a significant proportion of the rural population
is already confronted with the issues of insufficient water availability during extended
droughts (OECD, 2013).
UDDTs are quite well promoted at the national level (ApaSan, n.d.), are supported by
the government (e.g. National Centre of Public Health, Ministry of Environment) and are
socially accepted at the local level. The Swiss Development and Cooperation Agency'
Programme ApaSan has so far built 52 school UDDTs and more are planned in the future.
Also, several pilots of such facilities were built at the household level. UDDTs need further
improvement for reducing the required long-term storage of 1-2 years for faeces and 6 months
for urine (WHO, 2006) in order to recycle more nutrients and organic matter to the soil and
control the odour from the urine compartments.
Soils in the Republic of Moldova are an important economic asset. Maintenance of its
productive capacity over the long term is a prerequisite for sustainable development of the
country. Once having rich soil resources, Moldova is currently faced with serious
dehumification and erosion of soil resources, which greatly affect the economy. The main
soils of Moldova (in Russian: chernozioms) are naturally resilient, thanks to their unique
physical and mineralogical properties, the thickness of the humus in the topsoil, and their
restoration capacity. Nevertheless, having been intensively exploited in the past, these soils
have become susceptible to the loss of humus and nutrients, through erosion and compaction.
131
During the last 30 years, an acceleration of soil erosion and continuous loss of soil organic
matter and nutrients has occurred in Moldova (Krupenikov et al., 2011c). In contrast to the
past overuse of chemicals, currently there is a limited application of chemical and organic
fertilizers, such as cow manure particularly in subsistence farming systems. The use of
fertilizers in Moldova has declined substantially compared to the 1980s due to land
privatization (Spoor and Izman, 2009), when land was distributed from centrally controlled
big collective farms to small plots for farmers without a transfer of knowledge on soil
management practices. Twenty times less mineral fertilizer is currently being applied
compared to 30 years ago and the application of farmyard manure has declined to only a few
hundred kg ha-1 (National Bureau of Statistics of the Republic of Moldova, 2016 a). Due to a
lack of fertilization, particularly of replenishment of soil organic matter, areas affected by
erosion and dehumification continue to expand (Leah, 2012; Andries et al., 2014).
Soil fertility can be restored by enhancing the factors that form soil. One of the
important factors is the enrichment of soils with organic matter, which helps to improve the
soil quality and to increase the yields. Soil conditioners obtained from a lacto-fermented mix
of faeces and bio-waste, supplemented with biochar and lacto-fermented urine would be
inexpensive alternatives to chemical fertilizers. Co-composting faeces with waste from the
food and beverage industry is an interesting option worth exploring. For example, waste from
the food and beverage industry accounted for approximately 8% of the industrial waste. The
amount of waste generated during 2015 was 309,000 tons, out of which only 19% is used; 16
% of this waste was landfilled and the rest mainly remained on the properties of these
businesses (National Bureau of Statistics of the Republic of Moldova, 2016 b).
This thesis highlights the possibilities and limitations of the application of lactic acid
fermentation in excreta treatment (Chapter 2), including the effect of combined lactic acid
fermentation and thermophilic composting on pathogen removal (Chapter 3), the effects of
co-treated faeces, bio-waste and biochar on corn growth, yield, yield components and soil
quality (Chapter 4), and the treatment of urine via lactic acid fermentation for ammonia
reduction and odour control (Chapter 5). Based on the results of the current research, an
132
overview is made of the application of lactic acid fermentation combined with composting
(thermophilic composting or vermi-composting) and biochar addition for the treatment of
human excreta to improve resource-oriented sanitation using UDDT and its potential reuse in
agriculture or horticulture. The main advantages and limitations of lactic acid fermentation of
human excreta are explained in Chapter 2, which is a useful guide to help practitioners to
better understand the use of this microbiological process for improving the functionality of
UDDTs, together with better nutrient recycling.
The findings reported in Chapter 3 outline the safe treatment of human faeces through
the use of lactic acid fermentation combined with thermophilic composting for pathogen
removal. The effect of this compost on the germination and growth of plants is also explored
in this chapter to understand the potential for the recycling of the nutrients present in the
human faeces that are collected in UDDTs. The pathogen removal in lacto-fermented excreta
(Table 3.3) did not comply with the recommendations for soil amendments that are obtained
from biological treatment (Hogg et al., 2002), aside from improved hygienization through
supplementary treatment via thermophilic composting (Andreev et al., 2017).
An interesting fact is that the sanitizing temperature of 56-65°C during the post-
treatment composting stage, after the lactic acid fermentation, was achieved without turning
or mixing (Andreev et al., 2017). This may be related to the types of microorganisms that are
involved in this process under low oxygen conditions (Wang et al., 2007b), the enzymes
synthesized by lactic acid bacteria during fermentation (Muller, 2001), or the concentration of
easily degradable organic carbon in the substrate (Hayes and Randle, 1968). This research
suggests that the treatment of excreta via lactic acid fermentation is best carried out at the
post-collection stage, in combination with other types of waste such as cattle manure or fruit
waste. This will also help prevent other environmental problems since food waste can easily
get spoiled, generating offensive odour (Qu et al., 2015), contributing to gas emissions (Qu et
al., 2015) and attracting disease vectors (e.g. rodents or insects) (Drechsel et al., 2015). For
example, according to EPA estimations, during 2012 only 3 % of food waste was composted in
the USA, while the rest was landfilled, generating approximately 18 % of all US methane
emissions (Tim, 2015).
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Chapters 4 provides valuable insight on potential agricultural applications of lacto-

fermented faeces and bio-waste, supplemented by urine charged biochar. This research
highlights the effects of faecal based soil conditioner on corn growth, yield, yield components
and soil quality (Chapter 4, Tables 4.4-4.10). An emphasis is made on the role of such type of
soil conditioners under the conditions of decreased soil organic matter and increased drought
frequency. This research revealed that corn fertilized with lacto-fermented faeces, bio-waste
and biochar soaked in urine had a significantly higher yield (p < 0.05) than an unfertilized
control and plots fertilized by stored urine, faeces, cattle manure, and even chemical fertilizer
(during the first production year).
Higher nitrogen availability, coupled with the capacity of biochar to retain water, had
probably allowed for better root development of the corn and more efficient nitrogen uptake
during the period of intensive growth, thus making the corn more resilient to the following
drought during yield formation (Spiertz and De Vos, 1983). This aspect could be clarified
further with additional research on nitrate uptake by corn during the period of intensive
growth (June-July) and the translocation of nitrate from vegetative parts of the plant to the
grain, during the grain formation (August-September) following the application of lacto-
fermented faeces, bio-waste and biochar, and the chemical fertilizer (Chapter 4). These data
also need to be linked to soil nitrate content, soil available water storage capacity,
precipitation data (Vronskih, 2014), and estimations on nitrogen loss by leaching (Burgos et
al., 2006). The applied lactic acid fermented excreta and biochar has added to an improved
organic carbon content in the soil even though the increase was not significant (p>0.05, Table
4.13). The experimental length was too short to observe changes in the soil organic carbon
concentration. Such changes can only be observed by using C-labeled incubation experiments
(Perelo and Munch, 2005). Further, it is also required to explore the role of biochar in the process
of lacto-fermentation and composting and its role in nutrient retention. It is important to study
further how biochar influences ammonia retention from urine and organic carbon stability in
the compost (Glaser, 2015).
134
Chapter 5 explores lactic acid fermentation of urine. Data on this approach is very limited
and is important as it offers useful guidelines for practitioners, particularly those from countries
with scarce financial resources, on cost effective techniques for improving the fertilizing value of
urine and reducing odour emissions. This study's laboratory research on urine lactic acid
fermentation brings new perspectives for reducing the ammonia emissions and odour control in
UDDTs. Although further research is needed to further support these findings and to confirm
the efficiency in real-life conditions, the available evidence indicates that lactic acid bacteria
from common home-made products such as sauerkraut can survive in such a high strength
solution as urine, contributing to pH reduction to less than 4, an ammonium concentration
decrease and odour reduction (Chapter 5).
In this chapter, recommendations are given for the use of lactic acid fermentation
combined with composting and biochar in UDDTs for the treatment of both the urine and faeces
fractions for their reuse. Furthermore, practical recommendations on the application of the treated
urine and faeces for irrigating willow trees or growing ornamental plants are also given.
6.2 Product quality assurance for potential full-scale application
When there is a demand for large scale production and marketing of a product,
appropriate standards which stipulate the conditions for adequate pathogen reduction should
be applied (Hogg et al., 2002). In Europe, the application, testing and product quality
assurance of organic fertilizers, including compost, are based on statutory and voluntary
standards. The statutory standards cover most important characteristics for the protection of
the environment and human health (Hogg et al., 2002). Voluntary systems of quality
assurance support these standards by making recommendations regarding suitability of
products for different enduses. These standards refer to a number of requirements for
feedstock materials, the characteristics of the process, end-product quality and protection of
soil quality (Figure 6.1). Feedstock requirements categorize the input materials that are
allowed in this specific biological treatment. According to the existing regulations at the
European level, the input materials like bio-waste, sewage sludge, animal manure, yard waste
135
and agro-industrial by-products are included in the list of acceptable substrates for biological
treatment. Source-separated excreta are not listed as feedstocks for biological treatment and
there are currently no regulations at the European level (Hogg, 2002).
Product Quality
Soil quality
Feedstock
protection
Process End-product
Figure 6.1 Main components to be considered in the development of a standard lacto-fermented

fecal compost (Hogg et al., 2002)
Biochar is not included among the recommended feedstocks, but its quality shall be
assured according to the guidelines of the European Biochar Certificate (EBC, 2012). The
main quality criteria for biochar specified by the European Biochar Certificate versus those
investigated in this thesis are reflected in Table 6.1.
Table 6.1 Main quality parameters of biochar as specified by the European Biochar
Certificate (Source: ECB, 2012).
Quality Parameter Required as specified by EBC Results on wood charcoal used
in the current study
C content > 50 % 60 %
Nutrient content N, P, K, Mg, Ca Not investigated
Heavy metals Pb, Cd, Cu, N, Hg, Zn, Cr, As Not investigated
Volatile organic To be specified, threshold values Not investigated
compounds,
polychlorinated
biphenyls
Other parameters Bulk density, electric pH, moisture, ash content,
conductivity, pH, ash content, specific surface area, pore
volatile matter, water holding volume
capacity
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It refers to the carbon and nutrient content, the concentration of polychlorinated

biphenyls (PCB), volatile organic compounds (VOC), heavy metals and the water holding
capacity of biochar. The process specifications refer to the maximum acceptable limits for
pathogens, weed and plant propagules. These requirements vary across different countries. A
summary of these requirements for pathogen indicators, weed germination and plant tolerance
for compost maturity are indicated in Table 6.2.
Table 6.2 Standards for pathogens, weed seeds and propagules in compost.
Indicator Requirements specified Results from this research
by existing standardsa
Salmonella Absent Absent (Andreev et al., unpublished data)
Parasites (Ascaris) 0 in 100 g sample 0 in 100 g sample
3 -1
Enterobacteriacea < 10 CFU g LAF: E.coli - < 3 CFU g-1, Enterococcus
faecalis < 103CFU g-1
Enterococcus E.coli < 103CFU g-1 after LAF+TCb and
faecalis <103 CFU g-1 after LAF+VC c
Weed propagule test 5 viable plants L-1 Not investigated in this study
Plant tolerance 20 % below control LAF - 1-3% above the control, LAF+TC -
10 % below the control, 6 LAF+VC - 16 %
below the control
a
- Hogg et al., 2002
b
- LAF+TC - lactic acid fermented mix of faeces and bio-waste followed by
thermophilic composting.
c
- LAF+VC - lactic acid fermented mix of faeces and bio-waste followed by vermi-
composting.
Even though the lacto-fermented material was not sufficiently sanitized, it had
stimulatory effects on the growth of plants. This may be related to the substances synthesized
by lactic acid bacteria together with the effect of biochar onto which nutrients from urine were
adsorbed. The required time for curing and maturation is not specified in most compost
standards. The only exception is the Irish regulation, which stipulates that compost must be
cured for at least 21 days (Hogg et al., 2002). In this study, the curing phase of lactic acid
137
fermentation followed by thermophilic composting lasted for only 11 days (Chapter 3, Figure
3.1), which was not in accordance with the recommendations for a mature compost. In
addition, both germination tests were within the recommended limits and the tomato growth
exceeded that of the control (Chapter 3, Figure 3.2). Nonetheless, additional research on
compost maturity indicators, e.g. C:N ratio, respiration level, concentration of stable organic
carbon is required to enable safe use of human waste derived compost.
Depending on the type of use (agriculture, horticulture or other), standards on the
quality of the final compost specify the nutrient concentrations of N, P and K and the
maximum acceptable values for toxic substances and pathogens. Table 6.3 compares the
nutrient content of compost treated by aerobic composting, digestate treated by anaerobic
digestion (Hogg et al., 2002) and lacto-fermented mix of faeces, bio-waste and biochar (this
thesis). Both the nutrient content and the effect on plants growth and yield from radish
germination tests (Andreev et al., 2014) and two year field experiments on corn (Andreev et
al., 2016) indicated beneficial effects. However, the level of pathogens in the lacto-fermented
mix was not in accordance with the requirements of a compost (Table 6.2). In this case, it
would be appropriate that application of lacto-fermented faeces, bio-waste and biochar is used
for non-food (e.g. horticultural and ornamental) plant cultivation or additional treatment via
thermophilic composting is performed. The concentration of heavy metals such as Cd, Cr, Cu,
Hg, Ni, Pb, Zn, As, Co is required to be analysed to assess if the compost complies with the
certification.
Table 6.3 Nutrient content in compost obtained via aerobic composting, anaerobic
digestion and lactic acid fermentation with the addition of wood charcoal loaded with
nutrients from urine.
Nutrient Aerobic compost, kg/tonne-1 Digestate, kg tonne-1 LAF+C1, kg tonne-1
N 2-4 4-4.5 18.7
P 1-2 0.5-1.0 2.4
K 1-2 2.5-3 Not investigated
1
- without the content in charcoal
138
The soil quality determines the application rates of heavy metal and nutrients that can
be added the soil (mg kg-1 d.m.) with a compost, e.g. what are the admissible loads of heavy
metals (kg ha-1 year-1) and admissible loads for nutrients, such as nitrogen and phosphorous.
This thesis contributed with data on the nutrient content in the soil following application of lacto-
fermented excreta and biochar. Based on these data and additional research, it will be possible to
calculate the acceptable loading rates of lacto-fermented excreta and biochar for avoiding nutrient
pollution, while ensuring a beneficial agricultural effect.
6.3 Input materials required for lactic acid fermentation of

excreta
The main input materials (Figure 6.2) included in the lactic acid fermentation of excreta are
waste materials available in areas where UDDTs are located. The main limiting input materials are
the carbohydrate sources and the LAB inoculum. As lactic acid fermentation is widely used today,
with plenty of homemade fermented products (e.g. diary products and vegetables), these can serve
as sources of LAB to replace commercial bacterial inocula. Fermented foods are preferred by
consumers because of their taste, texture, nutritional values, antitoxin and antimicrobial effects
(Mokoena et al., 2016). Application of lactic acid fermentation in UDDTs at household level may
increase the need for more lacto-fermented products, thus contributing to a healthy, probiotic
nutrition.
Human faeces 5%
8%
Cattle manure
40%
17%
Fruit waste
Molasses
Lacto-bacilli 30%
Figure 6.2 Input materials added for lactic acid fermentation of faeces. Biochar soaked in urine
(10 % wet weight) was added after 8 weeks of lactic acid fermentation.
139
One of the limiting factors for scale application of lactic acid fermentation in UDDTs
is the need for the addition of sources of soluble carbohydrates or sugar containing substrates,
as sugar is the main growth substrate for LAB (Yemaneh et al., 2013). For an efficient lactic-
acid fermentation, sources of soluble carbohydrates need to be used that have other
competitive economic uses. For example, experiments on excreta lactic acid fermentation
were carried out with the application of wheat bran and mollases (Yemaneh et al., 2013;
Böttger et al., 2014; Scheinemann et al., 2015; Andreev et al., 2016; Andreev et al., 2017).
Low cost substitutes for the input materials have to be sought. Press mud, a sugar byproduct
(Figure 6.3) that contains 5-15% of sugar (Xavier and Lonsane, 1994) can be a potential
alternative.
Figure 6.3 Press mud - a by-product of the sugar industry and potential cost-effective
carbohydrate source for faeces lactic acid fermentation (scale: cm).
It has been shown that this by-product of the sugar industry can be an inexpensive
substrate for lactic acid production (Xavier and Lonsane, 1994). Its potential needs to be
studied for the use as an additive during the faeces collection stage in UDDTs or at the of
lactic acid fermentation stage. In Moldova, this waste product from the sugar industry is piled
on potentially agricultural land, thus its availability is not a limitation. Some waste products
such as e.g. fruit waste, grass clippings and kitchen scraps can be used as sources of water
soluble carbohydrates. Every day, a huge amount of fruit and vegetable waste is generated at
the household level, vegetable markets or fresh fruit boutiques as well as by the food
140
processing industry. Food waste can be a substitute for molasses; however, it requires pre-
processing, i.e. storage in an oxygen limiting environment to release sugars (Wang et al.,
2001). For example, a study carried out at the European level estimated that food waste
accounts for approximately 20 to 30% of the produced food (Stenmarck et al., 2016), many of
such wastes could be useful for lactic acid fermentation. It would also be interesting to test the
application of weed plants which contain elevated concentrations of glucose, fructose,
succrose or inulin, e.g. Cicorium intybus, Sonchus oleraceus, Lactuca seriola, Inula helenium,
Helianthus tuberosus (Van Laere and Ende, 2002; Petkova and Denev, 2012).
The availability of biochar can also be a limiting factor in scaling of excreta lactic acid
fermentation, when it is not produced at the local level. One of the options is to develop
portable, economically viable small scale biochar kilns (Nsamba et al., 2015) for the farmers
who practice integrated agricultural activities with agroforestry to produce their own biochar.
For example, a study carried out in Thailand showed that a farmer who grows maize, rice and
mixed agroforestry can produce up to 4.68 t of biochar from both field residues and
agroforestry prunings (Frogner, personal communication). Biochar selling to interested
compost producers can be an additional source of income to the farmers.
Another option can be the wood charcoal produced for griling, if this is not treated
with any additives to maintain ignition. For example, in Moldova there are few local
enterprises who produce wood charcoal for grilling in self-made metallic pyrolysers (Figure
6.4).
Figure 6.4 Metallic pyrolysers for wood charcoal production in Rezeni (Moldova)
141
These entreprises sell the charcoal, however the small pieces and dust, which remain
can be piled and used for agriculture. The quality of wood charcoal is in acceptable limits that
do not harm plants (Andreev et al., 2016). However, it is important to investigate other
quality parameters stipulated by the European Biochar Certificate to assess the applicability of
this material in agriculture.
6.4 Agricultural application of lacto-fermented excreta and

biochar
This research indicates that if even the agricultural effects of lacto-fermented faeces,
bio-waste and biochar are positive for both soil quality and hygiene, and organic carbon
stability, it is important that the lactic acid fermentation stage is combined with thermophilic
composting or biochar is added to the lacto-fermented mix alone in horticulture. Additional
research is needed to clarify the level of decomposition and stabilization of organic matter and
the degree of nutrient loss at both the lactic acid fermentation and the thermophilic
composting stage and how biochar can improve the stabilization of carbon and prevent
nutrient loss. For example, research has shown that the labile carbon diffuses into the biochar
pores and is adsorbed onto its surface, thus preventing its abiotic and biotic decomposition
(Zimmerman et al., 2011). The fertilizing effect of the combination of biochar with lacto-
fermented faeces and urine still needs to be investigated in long term field experiments,
especially with respect to the mechanisms of nutrient absorption from urine, nutrient
availability and nutrient uptake by the crops.
In separate collection systems, urine collection is also of particular importance, owing
to the abundance of nutrients and their availability to plants and its larger volume as
compared to faeces. The research carried out on the effect of lacto-fermented urine on plants
(Chapter 5) is yet at the incipient stage with only germination tests so far and should be
supplemented with field experiments. It is important to assess the potential of biochar to
142
absorb nutrients from acidified urine, considering the potentially more efficient nutrient
absorption onto biochar at a lower pH (Yao et al., 2011; Spokas et al., 2012).
The lacto-fermented faeces, bio-waste and biochar was found to be a superior fertilizer
compared to stored urine, faeces and cattle manure, even though further investigation is
required if the latter does improve the soil moisture and humus content. The fate of the mobile
phosphorus and potassium in the soil and plants, in addition to the level of nitrogen
mineralization in the soil after application of lacto-fermented faces, bio-waste and biochar.
The effect of chemical fertilizer differed between the two production years, with no increase
in yield during the first year and a significant one during the second year (Figure 4.5, Table
4.8), compared to the unfertilized control. This was probably related to the effect of heavy
rainfall on nitrate leaching during the first year (Chapter 4, Figure 4.5, Table 4.8). Research
on nitrate leaching and plant uptake after application of lacto-fermented excreta with biochar
compared to mineral fertilizers under different rainfall conditions can help to reveal if this soil
conditioner can be a fertilizer substitute under different climatic conditions.
6.5 Application of lactic acid fermentation combined with

composting and biochar in urine diverting dry toilets in Moldova
6.5.1 Site description
Moldova has a rich experience in implementation of ecological sanitation. During the

last 8 years, several pilot household and school urine diverting dry toilets (UDDTs) were
implemented by external donors and NGOs. UDDTs were recommended as appropriate
decentralized sanitation solutions for rural areas in the Country Document on Target Settings
for the Protocol on Water and Health (UNECE, 2011). Moreover, recently, the international
association Women in Europe for a Common Future (WECF) in cooperation with Women in
Sustainable Development of Moldova (WiSDOM) and local stakeholders, developed practical
guidelines on the construction of urine-diverting dry toilet facilities and guidelines for the use
of corresponding fertilizers are being prepared. Therefore, suitable conditions exist for real-
143
world testing, setting an example of ecological sanitation for similar countries, including the
resource required and technical issues for correctly implementing and operating of UDDTs.
However, many schools where UDDTs have been introduced are facing challenges,
particularly regarding the handling of urine and faeces as well as overcoming the odour
emissions. Such an example is the school Slobozia Mare, located in the Cahul district, South
of Moldova. Through an interview with the school director and the toilet care taker in this
school, it was seen that the administration of the school faces challenges, in particular the
rapid filling of the urine tank and the lack of experience regarding tank empting and the use of
urine as a fertilizer. During the visit, the door of the toilet was closed with a sign saying
"entering is strictly prohibited" (Figure 6.5 A), the pupils continuing to use the old pit latrine
outside of the school (Figure 6.5 B).
A B
Figure 6.5 A) The closed door of the UDDT in Slobozia Mare, (Cahul,
Moldova) and B) Pupils use again the pit latrine in the school yard.
144
6.5.2 Lactic acid fermentation of faeces followed by thermophilic

composting and biochar addition as post-treatment
At the collection stage of the faeces, cover material rich in carbohydrates is added,
including a sugar beet factory waste called press mud, chopped corn stalk and grass. This
helps to control the odour and promotes the growth of lactic acid bacteria already available in
the faeces. In order to reduce the pathogens and stabilize the faecal material, a secondary
treatment of faeces is carried out, in which the faeces are mixed with other kinds of bio-waste
(kitchen scraps, green refuse or animal manure), molasses and LAB for lacto-fermenting it for
a period of 10 days (Chapter 2 and 3). Kitchen and garden waste should be pre-fermented in
closed barrels before being mixed with faeces. Next, thermophilic micro-aerobic composting
has to be carried out for a period of approximately one month (Chapter 3). The compost
becomes mature when the temperature drops to its initial level. Biochar is added at a
concentration of 10-15% of the compost weight when the temperature raises above 50 °C to
lower the ammonia concentration. When temperature falls below 30-40 °C, at the maturation
stage, the compost becomes covered with a white mold (Figure 6.6).
Figure 6.6 The compost obtained via combined lactic acid fermentation and
thermophilic composting of faeces and other bio-waste together with biochar, at a
maturation phase of 30-40°C, which is characterized by the growth of white mold.
145
6.5.3 Lactic acid fermentation of urine
Acidification of urine is important for the reduction of ammonia volatilization and

odour emissions, effective hygienization and improving the overall agricultural value of urine.
The main suggested management steps applied in UDDTs are:
Acidification of urine in the storage tank. For succesful lactic acid fermentation in the
urine tank, a LAB solution has to be added before the urine starts to accumulate there, to
avoid abundance of urease positive bacteria and to ensure a cost effective lactic acid
fermentation. The urine tank should be closed to limit oxygen penetration since lactic acid
bacteria are facultative anaerobes. For reasons of hygiene, urine needs to be preserved for 1-2
months before its agricultural application. For the application of the LAB solution in those
emptied urine tanks, it is important that the pipes and urine tank are well rinsed with acetic
acid or a LAB solution. For example, sauerkraut juice that has been matured with sugar rich
liquids, e.g. sugar molasses or whey, depending on the availability. Another cheap alternative
for schools is the water remaining after the washing the rice or pots that contained dairy
products. This liquid may also be obtained by simply rinsing the spent milk packages. The
liquid obtained after rinsing the pipes and urine tanks is not toxic and can be poured on a
compost pile or used to water trees.
Acidification of urine within the toilet. After each UDD toilet use, the urinals and urine
section of the toilet seat should be sprayed with a LAB solution. Preferably, the LAB should
be activated before addition to the urine tank by mixing it with molasses and leaving it for
bacteria to grow in this solution for at least one week. The LAB solution shall be preserved in
a cool place and not stored longer than 1-3 months.
Potential post-treatment of urine after lactic acid fermentation. Acidified urine can be
used to soak on biochar, the latter can be mixed with compost. Alternatively, the acidified
urine can be diluted with water (at least 1:10, as suggested by the germination test with lacto-
fermented urine, see Chapter 5) and applied to the soil around the cultivated plants.
146
The costs of acidification of urine by lactic acid fermentation were three times lower
than chemical treatment by concentrated sulfuric acid (Hellström et al., 1999). As reflected in
Table 6.4, the overall costs of urine acidification via lactic acid fermentation can be at least 3
times cheaper than chemical acidification with sulfuric acid. Note that the cost for the LAB
solution includes sauerkraut, salt and molasses and the price of H2SO4 is the price of the
concentrated acid used in laboratory analysis, not of a highly purified quality. Aside from the
economic aspects of the treatment, potential risks and limitations have to be taken into
account. While for lactic acid fermentation, the availability of molasses could be the main
limitation, the negative side effects in the application of sulfuric acid include the health hazard
of handling and transporting the acid, increased H2S emissions that may negatively impact the
soil by raising its electric conductivity (Frost et al., 1990) and increased concentrations of
malodourous volatile fatty acids and volatile sulphurous compounds (Ottosen et al., 2009;
Petersen et al., 2012).
Table 6.4 Costs and potential risks/limitations for urine acidification by lactic acid
fermentation compared to the chemical acidification with concentrated sulfuric acid.
Process Quantity of additives* Cost/m3 of urine, € Potential risk or
required/m3 of urine limitation
Urine acidification 20 L LAB inoculum; 3 Molasses availability
by lactic acid 20 kg molasses; 200 L
fermentation water 1
Urine acidification 2.94 L concentrated 10 Health risks during
by sulphuric acid** H2SO4 handling and
addition treatment, potential
agricultural negative
impacts
*The additives used here are referred to a LAB solution obtained from sauerkraut, molasses and
water, the prices are in accordance to those available on the market (Sancom, 2016).
**This refers to concentrated sulphuric acid, of not a high quality available on the market in
Moldova (Kompas, 2017).
147
6.5.4 Potential for large scale sanitation and bio-waste management at

schools with nutrient recycling to agriculture
According to recent ApaSan monitoring data (2014), only about 1.5 l of urine acummulates
per capita per month and a school accumulates around 4 m3 per year. The quantity seems to be
insignificant, since 1.5 l is the quantity that one person produces per day. This means that the
UDDTs are not used at their full potential and school children continue to use their old pit
latrins as indicated for Slobozia Mare school (Figure 6.6 B). Estimations made during this
research showed that a family of 3 people, who were adding lactic acid bacteria after each
urination accumulated 600 l of urine per year or 0.55 l per person/day, including the LAB
solution added during the toilet use, but also considering that the family members are at home
only for a part of the day. These estimations indicate that a family of three people produce
monthly 9 kg of faeces and cover material, 2 kg of toilet paper and 5 kg of kitchen waste or
annually 108 kg, 24 kg and 57 kg respectively (Table 6.5).
Table 6.5. The quantity of different waste types estimated to be accumulated at

household level and extrapolated for schools with UDDTs (mean±SD).
Accumulation rate Faeces and cover Toilet Kitchen Urine (l)
material (kg) paper (kg) waste (kg)
Monthly 9 ± 3.6 2.0 ± 0.2 5 ± 0.1
Annual 108 ± 43.3 24 ± 1.8 57 ± 1.4 600 ± 100
Annual per capita 36 ± 14.4 8 ± 0.6 19 ± 0.5 200 ± 33
Potentially to be generated per 14400 1000 5200 80000
school (400 users, for 8 months)
Therefore, extrapolating these data for a school of 400 people, an amount of 14.4
tonnes of faeces and cover material, 1 tonne of toilet paper and 5 tons of kitchen waste can be
obtained that can be processed via combined lactic acid fermentation and thermophilic
composting (Table 6.5). The amount of the compost produced from 1 tonne of raw material is
150 kg. Considering the amount of faeces, toilet paper and bio-waste that could be generated
(Quora, 2016), approximately 3000 tonnes of compost can be obtained during one school
148
year. The price of potting soil in Moldova, depending of the package weight, ranges from 1.2
to 5 MDL l-1 (0.055-0.23 € l-1). Thus, the profit obtained would be between 2400-15000 MDL
(110-700 €), but it would be better if the schools use the compost for growing ornamental
plants.
In addition, a school of 400 children produces about 80 tons of urine that could be
lacto-fermented and used to fertilize a willow plantation. An urine tank is emptied usually
once a year or once per two year, during which time the fertilizing value of urine is reduced
due to ammonia volatilization during storage or application. However, only if the school
children use the UDDTs and not the pit latrines. Chapter 5 showed that lacto-fermented urine
is a better nitrogen source than stored urine and thus better maintaining its fertilizer value.
6.6 Potential entrepreneurial models
In a potential entrepreneurial model (Figure 6.8), excreta can be obtaind from UDDTs
by a service provider. The entrepreneur treats the urine on-site (at school where the urine
tanks are located) through the application of LAB supplemented with a source of
carbohydrate. The urine, together with LAB and carbohydrate, is accumulated in tanks, which
are emptied monthly and transported to a common short rotation copice (willow) plantation.
The entrepreneur also collects once a month the accumulated quantity of faeces and bio-waste
from the canteen and garden. Considering that willow requires 50-75 kgN/ha and that urine
contains about 9 kgN/m3 (Plămădeală et al., 2011), the amount of nitrogen in the urine that
accumulates in the UDDT during one school year (80 m3) can fertilize 9 to 13 ha of willow.
Taking into account the results from this research, in which 24-32 % of ammonia is prevented
from being lost via volatilization by lactic acid fermentation (Chapter 5), the surface of
irrigated willow could be increased by 20-30 %. However, this needs to be confirmed through
field research. The willow can be used for producing renewable energy products (wood chips
or pellets), in addition to the traditional Moldovan basket and furniture, which are highly
valued in many countries. Also, willow cuttings easily set roots when planted, so reforestation
with this tree species is simple (Figure 6.7).
149
Considering the price per ton of fresh willow woodchips for energy production, the
total benefit could be up to 9000 euros per year, although the investment costs for willow
planting, maintenance and harvesting along with the price of land would also need to be
calculated to make any conclusions about the cost-benefit of this new sanitation approach.
Schools can be encouraged to collect faeces, urine and vegetable waste through a discount
card system for the collection services or for buying ornamental plants. More responsible
schools can get small financial rewards that they could use to buy sanitary and school
supplies. Wood from tree pruning as well as woodchips from the willows can be also used for
local production of wood biochar. The compost can be used on site as growth medium for
ornamental shrubs and the yard pruning waste for biochar production (Figure 6.7).
Figure 6.7 A potential entrepreneurial model where excreta is collected and treated via lactic
acid fermentation to produce different revenue streams. LAB - lactic acid bacteria, SC - soluble
carbohydrate source.
150
Depending on the type of plants grown, an optimal ratio of compost mixes can be
determined. This research shows that a 1:3 compost:soil ratio was favourable for plant growth
(Chapter 3). The profits obtained from growing ornamental plants depend on the plants
species used as well as the demand for these plants. For example, decorative plants such as
the fast growing shrub, Hortensia, are highly appreciated by gardeners for outdoor
landscaping. Also Petunia is an annual plant, used widely in the gardens and for decoration of
balconies. However, a more comprehensive cost-benefit analysis is required taking into
account willow and ornamental plant growth, the price of compost at the local level, the
demand for ornamental potted plants and shrubs and the amount of decorative plants
produced.
6.7 Conclusions
The following main conclusions can be derived from this dissertation:
• The compost obtained via lactic acid fermentation of excreta, mixed with biochar is
beneficial for agriculture. This type of compost produces a similar or even higher corn
yield and improves soil quality compared to other fertilizers, such as NPK mineral
fertilizers or simple stored urine, faeces and cattle manure.
• When lactic acid fermentation is combined with thermophilic composting with addition
of biochar, it produces a safe soil conditioner with only passive aeration without the need
for turning, this compost enhances plant germination and growth.
• Use of urine as a fertilizer can generate foul odours. When applying lactic acid
fermentation, odour is largely controlled and there is less loss of nitrogen via ammonia
volatilization.
• A cost-benefit analysis of the application of this technique is required. Also, additional
field research on the effect on crops and soil is needed. Nonetheless, lactic acid
fermentation of excreta has shown to be a sustainable alternative to mineral fertilizers
which maintains soil fertility, where purchase of these fertilizers is not affordable.
151
152
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Appendixes
Appendix A
Corn growth rate as influenced by lacto-fermented mix and biochar in comparison to other
fertilizers applied
Dunnett Simultaneous Tests for Level Mean - Control Mean
1st production period
Plant height, cm
Difference Difference SE of 95 % CI T-value P-value
of levels of means difference
1-3 -29.57 1..20 (-33.06; -26.07) -21.63 0.000
2-3 -14.37 1.20 (-17.86; -10.87) -11.97 0.000
4-3 -8.10 1.20 (-11.60; -4.60) -6.75 0.000
5-3 -12.37 1.20 (-15.86; -8.87) -10.30 0.000
6-3 -17.17 1.20 (-20.66; -13.67) -14.30 0.000
7-3 -17.90 1.20 (-21.40;-14.40) -14.91 0.000
Stem diameter, cm
1-3 -677 0.155 (-1.129; -0.224) -4.35 0.003
2-3 -0.133 0.155 (-0.586; -0.319) -0.86 0.892
4-3 -0.257 0.155 (-709; 0.196) -1.65 0.407
5-3 -0.357 0.155 (-0.809; 0.096) -2.30 0.150
6-3 -0.410 0.155 (-0.863; 0.043) -2.64 0.082
7-3 -0.443 0.155 (-0.896; 0.009) -2.85 0.056
195
Leaf length. cm
1-3 -5.90 1.23 (-9.48; -2.32) -4.80 0.001
2-3 -3.00 1.23 (-6.58; 0.58) -2.44 0.117
4-3 -0.34 1.23 (-3.23; 3.92) 0.28 1.000
5-3 -1.83 1.23 (-5.41; 1.74) -1.49 0.501
6-3 -2.77 1.23 (-0.863; 0.043) -2.25 0.162
7-3 -5.77 1.23 (-9.34; -2.19) -4.69 0.002
Leaf width. cm
1-3 -0.467 0.619 (-2.271; 1,337) -0.75 0.934
2-3 -0.467 0.619 (-2.271; 1,337) -0.75 0.934
4-3 -0.697 0.619 (-2.501; 1.107) -1.12 0.741
5-3 -0.600 0.619 (-2.404; 1.204) -0.97 0.835
6-3 -1.167 0.619 (-2.971; 0.637) -1.88 0.292
7-3 -1.800 0.619 (-3.604; 0.004) -2.91 0.051
2nd production period
Plant height, cm
1-3 -9.90 1.24 (-13.54; -6.26) -7.96 0.000
2-3 2.23 1.24 (-1.40; 5.87) 1.80 0.356
4-3 -1.77 1.24 (-5.40; 1.87) -1.12 0.582
5-3 4.77 1.24 (1.13; 8.40) -1.42 0.008
6-3 -7.17 1.24 (-10.80; -3.53) -5.76 0.000
7-3 1.27 1.24 (-2.37; 4.90) 1.02 0.841
8-3 -3.83 1.24 (-7.47; -0.20) -3.08 0.037
Stem diameter, cm
1-3 -233 0.202 (-0.823; 0.356) -1.16 0.757
2-3 0.200 0.202 (-0.389; 0.789) 0.99 0.855
4-3 0.333 0.202 (-0.256; 0.923) 1.65 0.406
5-3 0.343 0.202 (-0.246; 0.933) 1.70 0.008
6-3 0.067 0.202 (-0. 523; 0.656) 0.33 1.000
7-3 0.100 0.202 (-0.489; 0.689) 0.50 0.995
8-3 -0.200 0.202 (-0.789; 0.389) -0.99 0.855
196
Leaf length, cm
1-3 -7.07 1.58 (-11.69; -2.44) -4.47 0.002
2-3 2.47 1.58 (-2.16; 7.09) 1.56 0.493
4-3 -2.87 1.58 (-7.49; 1.76) -1.81 0.348
5-3 9.07 1.58 (4.44; 13.69) 5.73 0.000
6-3 5.30 1.58 (-0.67; 9.93) 3.35 0.022
7-3 2.67 1.58 (-1.96; 7.29) 1.69 0.417
8-3 -5.83 1.58 (-10.46; -1.21) -3.69 0.011
Leaf width, cm
1-3 -0.593 0.250 (-1.324; 0.137) -2.37 0139
2-3 -0.567 0.250 (-1.297; 0.164) -2.27 0.167
4-3 -400 0.250 (-1.131; 0.331) -1.60 0.467
5-3 -0.067 0.250 (-0.797; 0.664) -0.27 1.000
6-3 -0.100 0.250 (-0.831; 0.631) -0.40 0.999
7-3 -0.667 0.250 (-1.397; 0.064) -2.67 0.081
8-3 -0.933 0.250 (-1.664; -0.203) -3.73 0.010
1- Control
2- Lacto-fermented mix
3- Lacto-fermented mix and biochar
4- Cattle manure
5- Mineral fertilizer
6- Stored faeces
7- Stored urine
8- Vermi-composted lacto-fermented mix and biochar
197
Appendix B
The yield of corn as influenced by lacto-fermented mix and biochar in comparison to other
fertilizers.
2013
1-3 -789.7 71.8 (-998.9; -580.4) -10.99 0.000
2-3 -16 71.8 (-225.3; 193.3) -0.22 1.000
4-3 -269.3 71.8 (-478.6; -60.1) -3.75 0.010
5-3 -811.3 71.8 (-1020.6; -602.1) -11.29 0.000
6-3 -357.3 71.8 (-566.6; -148.1) -4.97 0.001
7-3 -352.3 71.8 (--561.6; -143.1) -4.90 0.001
2014
1-3 -1159.3 64.7 (-1348.4; -970.3) -17.93 0.000
2-3 -693.0 64.7 (-882.1; -503.9) -10.72 0.000
4-3 -858.3 64.7 (-1047.4; -669.3) -13.27 0.000
5-3 109.3 64.7 (-79.7; 298.4) 1.69 0.000
6-3 -1274.0 64.7 (-1463.1; -1048.9) -19.70 0.413
7-3 -648 64.7 (-837.1; -458.9) -10.02 0.000
8-3 -878.7 64.7 (-1067.7; -689.6) -13.59 0.000
198
Appendix C
Soil humus content as influenced by the application of lactofermented mix and biochar in
comparison to other fertilizers
Humus content, 0-20 cm, 2013

Dunnett Simultaneous Tests for Level Mean - Control Mean
1-3 -0.113 0.157 (-0.570; 0.343) -0.72 0.945
2-3 0.177 0.157 (-280; 0.633) 1.13 0.740
4-3 0.013 0.157 (-0.443; 0.470) 0.09 1.000
5-3 -0.213 0.157 (-0.670; 0.243) -1.36 0.586
6-3 -0.047 0.157 (-0.503; -0.410) -0.30 0.999
7-3 -0.233 0.157 (-0.690; 0.223) -1.49 0.504

1-3 -0.0.017 0.128 (-0.390; 0.357) -0.13 1.000
2-3 -0.007 0.128 (-380; 0.367) -0.05 1.000
4-3 0,233 0.128 (-0.140; 0.607) 1.82 0.320
5-3 -0.350 0.128 (-0.723; 0.023) -2.73 0.070
6-3 -0.023 0.128 (-0.397; 0.350) -0.18 1.000
7-3 -0.113 0.128 (-0.487; 0.260) -0.88 0.879

1-3 -0.147 0.181 (-0.677; 0.383) -0.81 0.937
2-3 0.073 0.181 (-457; 0.603) 0.40 0.998
4-3 0.170 0.181 (-0.360; 0.700) 0.94 0.883
5-3 -0.227 0.181 (-0.757; 0.303) -1.25 0.697
6-3 0.010 0.181 (-0.520; 0.540) 0.06 1.000
7-3 -0.207 0.181 (-0.737; 0.323) -1.14 0.769
8-3 -0.040 0.181 (-0.570; 0.490) -0.22 1.000
199
1-3 -0.147 0.191 (-0.705; 0.411) -0.77 0.950
2-3 0.073 0.191 (-485; 0.631) 0.38 0.999
4-3 0.170 0.191 (-0.388; 0.728) 0.89 0.905
5-3 -0.227 0.191 (-0.785; 0.331) -1.19 0.738
6-3 0.010 0.191 (-0.548; 0.568) 0.05 1.000
7-3 -0.207 0.191 (-0.735; 0.351) -1.08 0.804
8-3 -0.090 0.191 (-0.468; 0.648) 0.47 0.996
200
Appendix D
Phosphorus content, mg/kg 0-20 cm, 1st production period, 2013

1-3 -10.70 5.24 (-25.96; 4.56) -2.04 0.228
2-3 -7.60 5.24 (-22.86; 7.66) -1.45 0.528
4-3 -2.83 5.24 (-18.10; 12.43) -0.54 0.985
5-3 -1.87 5.24 (-17.13; 13.40) -0.36 0.998
6-3 -5.00 5.24 (-20.26; 10.26) -0.95 0.843
7-3 -2.87 5.24 (-18.13; 12.40) -0.55 0.984
Phosphorus content, mg/kg 20-40 cm, 1st production period, 2013

1-3 1.93 5.68 (-14.61; 18.48) 0.34 0.999
2-3 -0.07 5.68 (-16.61; 16.48) -0.01 1.000
4-3 1.27 5.68 (-15.28; 17.81) 0.22 1.000
5-3 4.10 5.68 (-12.44; 20.64) 0.72 0.945
6-3 -4.33 5.68 (-20.88; 12.21) -0.76 0.931
7-3 0.83 5.68 (-15.71; 17.38) 0.15 1.000
Phosphorus content, mg/kg 0-20 cm, 2ndproduction period, 2014

1-3 -31.07 4.65 (-44.66; -17.47) -6.68 0.000
2-3 -4.40 4.65 (-17.99; 9.19) -0.95 0.879
4-3 -20.17 4.65 (-33.76; -6.57) -4.34 0.003
5-3 5.37 4.65 (-8.13; 19.06) 1.18 0.746
6-3 7.37 4.65 (-6.23; 20.96) 1.58 0.477
7-3 2.07 4.65 (-11.53; 15.66) 0.44 0.997
8-3 -15.17 4.65 (-28.76; -1.57) -3.26 0.026
201
Phosphorus content, mg/kg 20-40 cm, 2ndproduction period, 2014
1-3 -16.60 3.10 (-26.57; -7.58) -5.35 0.000
2-3 0.30 3.10 (-8.77; 9.37) 0.10 1.000
4-3 -8.43 3.10 (-17.50; 0.63) -2.72 0.074
5-3 -5.57 3.10 (-14.63; 3.50) -1.80 0.356
6-3 2.57 3.10 (6.50; 11.63) 0.83 0.930
7-3 14.13 3.10 (-11.53; 15.66) 4.56 0.002
8-3 -4.90 3.10 (-28.76; -1.57) -1.58 0.479
202
Appendix E
Potassium content, mg/kg, 0-20 cm 1st production period
1-3 -3.00 1.51 (-7.41; 1.41) -1.98 0.251
2-3 7.90 1.51 (3.49; 12.31) 5.22 0.001
4-3 -4.00 1.51 (-8.41; 0.41) -2.64 0.082
5-3 1.27 1.51 (-3.14; 5.67) 0.84 0.901
6-3 -2.37 1.51 (-6.77; 2.04) -1.56 0.458
7-3 2.93 1.51 (-1.47; 7.34) 1.94 0.268
Potassium content, mg/kg, 20-40

cm Difference SE of 95 % CI T-value P-value
Difference of means difference
of levels
1-3 -13.60 1.58 (-18.19; -9.01) -8.63 0.000
2-3 -10.83 1.58 (-15.43; -6.41) -6.87 0.000
4-3 -12.87 1.58 (-17.46; -8.27) -8.16 0.000
5-3 3.13 1.58 (-1.46; 7.73) 1.99 0.249
6-3 -11.77 1.58 (-16.36; -7.17) -7.46 0.000
7-3 -11.20 1.58 (-15.79; -6.61) -7.10 0.000
Potassium content, mg/kg, 0-20 cm 2nd production period

1-3 -0.37 4.39 (-13.21; 12.48) -0.008 1.000
2-3 -1.13 4.39 (-13.98; 11.71) -0.26 1.000
4-3 14.27 4.39 (1.42; 27.11) 3.25 0.026
5-3 18.93 4.39 6.09; 31.78) 4.31 0.003
6-3 10.60 4.39 (-2.24; 23.44) 2.41 0.130
7-3 -3.07 4.39 (5.07; 23.20) -0.70 0.968
8-3 1.17 4.39 (-28.76; -1.57) 0.27 1.000
Potassium content, mg/kg, 0-20 cm 2nd production period

1-3 0.27 3.90 (-11.14; 11.67) 0.07 1.000
2-3 17.70 3.90 (6.30; 29.10) 4.54 0.002
4-3 33.10 3.90 (21.70; 44.50) 8.49 0.000
5-3 39.83 3.90 (28.43; 51.24) 10.21 0.000
6-3 25.40 3.90 (14.0; 36.80) 6.51 0.000
7-3 32.07 3.90 (20.66; 43.47) 8.22 0.000
8-3 2.30 3.90 (-9.10; 13.70) 0.59 0.987
203
Appendix F
The mobile P2O5 and exchangeable K2O expressed as P and K
Fertilizer applied P (%) K (%)

Soil depth (cm)
1st Production year 0-20 20-40 0-20 20-40
Lactofermented mix and biochar 24.14 20.20 30.02 31.40
Lactofermented mix 22.48 22.05 33.36 26.90
Mineral fertilizer 23.75 22.97 30.57 32.73
Stored cattle manure 23.05 22.33 28.40 26.06
Stored faeces 23.05 21.11 29.06 26.50
Stored urine 23.53 29.33 31.30 26.73
Control 21.81 22.11 28.38 25.73
2nd Production year
Lactofermented mix and biochar 24.13 24.30 34.61 28.20
Lactofermented mix 22.50 24.70 28.95 32.78
Mineral fertilizer 23.75 25.41 30.08 43.83
Stored cattle manure 23.53 24.99 30.50 37.82
Stored faeces 23.05 25.91 24.49 41.03
Stored urine 23.53 28.42 26.73 40.57
Control 21.80 21.72 21.72 27.31
Vermicomposted lacto-fermented mix and biochar 25.60 24.30 34.61 28.18
For equivalence calculation the mobile P2O5 and exchangeable K2O are expressed as P and K; the proportion of
P in P2O5 is calculated from P2O5 (31x2+16x5=142) being equivalent to 100%, and a molecule of P as 21.83%;
for K2O (40+40=80+16=96), similarly, one molecule of K is 41.7%.
204
Appendix G
Soil bulk density, 1st production period
0-20 cm
1-3 0.0800 0.0355 (-0.0233; 0.1833) 2.26 0.161
2-3 0.0500 0.0355 (-0.0533; 0.1533) 1.41 0.553
4-3 0.0800 0.0355 (-0.0233; 0.1833) 2.26 0.161
5-3 0.0833 0.0355 (-0.0199; 0.1866) 2.35 0.137
6-3 0.3267 0.0355 (0.2234; 0.4299) 9.21 0.000
7-3 0.1700 0.0355 (0.0667; 0.2733) 4.79 0.001
20-40 cm
1-3 0.0867 0.0334 (-0.0106; 0.1839) 2.60 0.089
2-3 -0.0233 0.0334 (-0.1206; 0.0739) -0.70 0.952
4-3 -0.1233 0.0334 (-0.2206; -0.0261) -3.69 0.011
5-3 0.1267 0.0334 (-0.0294; 0.2239) 3.79 0.009
6-3 0.0000 0.0334 (-0.0972; 0.972) 0.00 1.000
7-3 -0.0067 0.0334 (-0.1039; 0.0906) -0.20 1.000
Soil bulk density, 2nd production period
0-20 cm
1-3 0.0433 0.0350 (-0.0589; 0.1456) 1.24 0.704
2-3 0.0433 0.0350 (-0.0589; 0.1456) 1.24 0.704
4-3 0.1000 0.0350 (-0.0022; 0.2022) 2.86 0.057
5-3 0.1967 0.0350 (0.0944; 0.2989) 5.63 0.000
6-3 0.0333 0.0350 (-0.0689; 0.1356) 0.95 0.875
7-3 0.1700 0.0350 (0.0678; 0.2722) 4.86 0.001
8-3 0.0067 0.0350 (0.0956; 0.1779) 1.37 0.619
205
20-40 cm

1-3 0.0567 0.415 (-0.0646; 0.1779) 1.37 0.619
2-3 0.0433 0.415 (-0.1466; 0.0976) 1.24 0.990
4-3 0.1000 0.415 (-0188; 0.2612) -0.56 0.020
5-3 0.1967 0.415 (0.0944; 0.2989) 5.63 0.002
6-3 0.0333 0.415 (-0.0689; 0.1356) 0.95 0.020
7-3 0.1700 0.415 (0.0678; 0.2722) 4.86 0.015
8-3 0.0067 0.415 (0.0956; 0.1779) 1.37 1.00
206
This work was performed at IHE, Delft Institute for Water Education (The Netherlands) and
the Research Institute for Field Crops, Selectia, Balti (Moldova). The field work was carried
out in village Boldurești, Nisporeni district (Moldova). This research was funded by the
Nuffic Fellowship Program (grant CF1080). The laboratory and field experimental work was
also partly covered by the Laboratory of Hydrobiology and Ecotoxicology of the Institute of
Zoology (Academy of Sciences of Moldova) and WiSDOM association.
207
Netherlands Research School for the
Socio‐Economic and Natural Sciences of the Environment

D I P L O M A
For specialised PhD training

The Netherlands Research School for the
Socio‐Economic and Natural Sciences of the Environment
(SENSE) declares that
Nadejda Andreev
born on 4 January 1972 in Nisporeni, Moldova

has successfully fulfilled all requirements of the
Educational Programme of SENSE.

Delft, 29 September 2017

the Chairman of the SENSE board the SENSE Director of Education

Prof. dr. Huub Rijnaarts Dr. Ad van Dommelen

The SENSE Research School has been accredited by the Royal Netherlands Academy of Arts and Sciences (KNAW)

The SENSE Research School declares that Ms Nadejda Andreev has successfully fulfilled all
requirements of the Educational PhD Programme of SENSE with a
work load of 35.3 EC, including the following activities:

SENSE PhD Courses
o Environmental research in context (2012)
o Research in context activity: Initiate and organize the workshop "Perspectives of the reuse of
human excreta from ecological sanitation for restoration of soil fertility in Moldova" (2014)

External training at a foreign research institute
o Training on soil sampling methods and analysis, Selectia Research Institute of Field Crops,
Balti, Republic of Moldova (2013‐2014)
o Online course Scientific and professional publishing on environment and sustainability, Open
University Heerlen (2014‐2015)
o Environmental problems: crossing boundaries between science and society, Open University
Heerlen (2014‐2015)

Management and Didactic Skills Training
o Supervising BSc student with thesis entitled ‘The potential for the use of combined
treatment of lacto‐fermentation and vermi‐composting of human waste for the application
in agriculture’ (2014)
o Co‐organising the international conference ‘Environmental Challenges in Lower Danube
Euroregion’, Galati, Romania (2015)

Oral Presentations
o A concept for a sustainable sanitation chain based on the semi centralised production of
terra preta for Moldova, 4th International Dry Toilet Conference, 22‐24 August 2012,
Tampere, Finland
o Production of terra preta soil improvers by lacto‐fermentation of organic residues,
International conference ‘Rational use of natural resources ‐ the basis for sustainable
development’, 10‐11 October 2013, Balti, Republic of Moldova
o The effect of a terra preta‐like soil improver on the germination and growth of radish and
parsley, 1st International Terra Preta Sanitation Conference, 28‐31 August 2013, Hamburg,
Germany
o Terra preta nova production for resource oriented excreta management in separately
collecting sanitation facilities, 3rd International Water Association (IWA) Development
Congress & Exhibition, 14‐17 October 2013, Nairobi, Kenya
o Processing of faeces from urine diverting dry toilets by combined biological processes: lactic
acid fermentation and vermi/thermal composting, International Conference on Terra Preta
Sanitation and Decentralised Waste Water Systems, 18‐21 November 2015, Goa, India
SENSE Coordinator PhD Education

Dr. ing. Monique Gulickx
Human excreta is a valuable fertilizer for A faeces treatment process by combined
improving soil quality and crop productivity, lacto-fermentation with thermophilic
with a potential to replace or complement composting and biochar supplementation
the mineral fertilizers. The main challenges had better reduction of coliforms, Escherichia
related to human excreta regarding coli, Enterococcus faecalis and Clostridium
agricultural applications are microbial perfringens, and higher germination of radish
contamination risks, loss of nutrients, and and growth of tomatoes than combined
odor issues. Fertilization by lacto-fermented lacto-fermentation with vermicomposting.
faeces supplemented by biochar has Urine lacto-fermentation contributed to a
benefits such as improved soil bulk density, pH reduction below 4, a decrease in the
nitrate and potassium concentrations as ammonium concentration and odor strength,
well as the yield and yield components of as well as an increase in the germination
corn, compared to untreated, simple stored rates compared to untreated stored urine.
faeces, urine, cattle manure, and unfertilized
controls. Even though the mineral fertilizer The results of this study provide important
produced corn with significantly higher height information that can set the basis for scaling
and leaf length, it did not add significantly up a sustainable technology for the treatment
higher yields than lacto-fermented faeces of source separated human excreta while
supplemented by biochar. improving its potential for resource recovery.
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Lactic Acid Fermentation

of Human Excreta for

© 2017, Nadejda Andreev

ISBN 978-1-138-04989-5 (Taylor & Francis Group)

De adequate behandeling van menselijke uitwerpselen is een van de meest uitdagende

1.1 Research justification

1.2 Scope of research

Faeces+bio-waste Urine, LAB,

Field application (2 year experiment) Biochar

TC (23-24 days) VC (4 months)

1.3 Thesis outline

This chapter is based on:

Global population growth, intense urbanization and economic development as well as

2.2 Nutrient and resource challenges in sanitation

Conventional water-based sanitation aims to increase hygiene, but it is not applicable

Figure 2.1 Resource and nutrient challenges in conventional sanitation. A major

One limitation of conventional waterborne sanitation is the high maintenance and

2.3 Applying excreta and biochar to agriculture

2.3.1 Fertilizer value of human urine and faeces

Field experiments on an area of urine, Cabbage3 The yield was 89 days 3

pre-irrigated soil. Irrigation: 2

2.3.2 Advantages and disadvantages of biochar application to agriculture

2.3.3 Anthrosols: land application of human excreta and biochar

2.4 Challenges in agricultural reuse of excreta

2.4.1 Pathogen reduction

2.4.2 Loss of nutrients, carbon matter and odour issues

2.5 Lactic acid fermentation of human excreta

2.5.1 Transformations occurring during lactic acid fermentation

2.5.2 Lactic acid fermentation of urine

Further research on the application of lactic acid fermentation of urine is important

2.5.3 Lactic acid fermentation of faeces

2.5.4 Limitations of lactic acid fermentation

2.6 Post treatment of lacto-fermented human excreta

Lactic acid fermentation of faeces contributes to hygienization, pre-decomposition of

2.6.2 Thermophilic composting

Another way to improve the structure of the lacto-fermented faeces is through

2.6.3 Addition of biochar

2.7 Agricultural effects of lacto-fermented excreta

2.7.1 The effects of lacto-fermented excreta on soil and plants

Research on the agricultural effects of lacto-fermented human excreta is rather scarce.

Bokashi 1 and mixed Field Rice EM bokashi and Xiaohou et

mineral fertilizers bokashi and drainage has

Bokashi (Rice bran, Field Peanut, Fresh weight of nodules Pei-Sheng

Bokashi was obtained Field Corn Plants fertilized with EM Alattar et

2.7.2 Effect of post-treatment by composting and biochar addition

Compost obtained from combined lactic acid fermentation and vermi-composting is

The combination of lactic acid fermentation and composting (thermophilic

This chapter is based on:

fermentation and vermi-composting is the need to pre-condition the lacto-fermented material

3.2 Materials and Methods

3.2.1. Experimental set-up

Table 3.1 Description of the experimental conditions and measured parameters

2014 Sawdust:pressmud As the 2013 LAF+TC T (°C),

LAF - lactic acid fermentation and thermophilic composting, TC - thermophilic composting, VC -

Table 3.2 The description of the composition of the fertilizers added

Fertilizer Composition description

3.2.2.1 Thermophilic Composting

3.2.2.3 Germination and plant growth experiments

3.2.3 Microbiological analysis and analytical methods

Total coliforms, Escherichia coli, Enterococcus faecalis and Clostridium perfringens

3.3.1 Temperature development during thermophilic composting

Figure 3.1 Temperature variations during thermophilic composting after pre-processing of

Even though no measurements were made on the decomposition of lignocellulotic