Professional Documents
Culture Documents
Review of Current and Future Bio Based Stabilisation Produ - 2021 - Transportati
Review of Current and Future Bio Based Stabilisation Produ - 2021 - Transportati
Transportation Geotechnics
journal homepage: www.elsevier.com/locate/trgeo
A R T I C L E I N F O A B S T R A C T
Keywords: In situ soil modification is required in order to improve the primary engineering properties of the material to meet
Bacillus species a road construction standard. Bio-stabilised soil is an environmentally friendly, cost-effective alternative to
Biopolymers imported granular fills, concrete, costly hauling of materials or export to a landfill. In-service soil performance
Soil stabiliser
and required maintenance is highly dependent on methods of stabilisation, ranging from expensive mechanical
Road construction
stabilisation to chemical processes. As such, many alternative materials originating from bio-based sources are
Soil stabilisation
Bio-additives being explored as potential stabilising additives to improve weak subgrade soils (i.e., dispersive, erodible and
collapsible soil, and soft or expansive clays). Some key solutions include the use of bio-derived enzymes, mi
crobes, and polymeric additives to avert road failure caused by water penetration and/or erosion. The role of
microbial substrate specialisation has been largely unexplored, since the level of research done on alternative
stabilisers consists mostly of small ad hoc studies. In addition, research has focused on a reduction in permeability
and an increase in compressive strength using enzymes and polymers, however, the complexity of these products
and their implementation for a wide range of soil types and structural applications remain limited. Currently
there is a need for more supporting research methodologies and systematic approaches on the implementation of
bio-based materials for infrastructure development. This also includes the simplification of bio-based products
for potential construction applications. This review provides (a) an overview of soil stabilisation techniques, (b)
the primary challenges that lay ahead for future research in bio-based stabilisation products application in the
road sector and (c) innovations to address the challenges of using modernised techniques in the road construction
industry (i.e., weak subgrade and the required maintenance thereof, as well as the development of potential bio-
based additives for unpaved road construction application).
* Corresponding author at: The CSIR Future Production, Chemicals, Meiring Naude Drive, Brummeria, PO Box 395, Pretoria 0001, South Africa. Tel.: +27 12 841
3780/+27 84 299 3794.
E-mail addresses: [email protected], [email protected] (V.M. Ramdas).
https://doi.org/10.1016/j.trgeo.2020.100458
Received 14 May 2020; Received in revised form 19 October 2020; Accepted 20 October 2020
Available online 22 October 2020
2214-3912/© 2020 Elsevier Ltd. All rights reserved.
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
emissions occurs throughout its lifecycle phases (i.e., construction and The challenge is to find low-cost, high-volume applications of bio-based
operation, albeit to lesser extent maintenance and rehabilitation) products for in-place soils, and to convert waste into value-added
(Alzard et al., 2019). As expected, the construction industry will products. This approach could be used for numerous geotechnical ap
continue to grow at a fast pace just to accommodate urban population plications such as dust suppression, dam control, wind soil-erosion
that will increase to 6.4 billion by 2050 (Pacheco-Torgal, 2016). Recent control, earthquake liquefaction mitigation, construction of reactive
estimates on urban expansion suggest that by 2030 urban land cover will barriers, and long-term stabilisation of contaminated soils. With respect
increase by 1.2 million km2 (Seto et al., 2012). Therefore, the demand to their mode of action, this presents an opportunity to discover novel
for alternative construction materials will also rise. micro-organisms through suitable assessment of their properties such as,
In developing countries, roads are expensive to develop and main secretion of one or more metabolic products, enzymes of interest, or
tain. There has been a concerted effort in industry to reduce construction biopolymers and investigate it as soil additives. It becomes clear that due
costs particularly in challenging ground conditions. However sustain to a lack of knowledge on bio-based soil stabilisation several areas of
able and environmentally friendly road development is a challenge to applied research need to be addressed in the biotechnology sector. Such
implement. According to the South African National Roads Agency as, appropriate upstream processes for biomass production, downstream
Limited (SANRAL), the total asset value of South African roads is esti processes to yield stable products (including the necessary in-vitro
mated at $200 billion (2014) with the value of the paved road network mechanisms and field testing), effectively formulated products and the
making up 80% of this (approximately $160 billion). New roads and appropriate application of each soil stabiliser under various environ
vital repairs of existing roads typically cost $300–500 thousand per mental conditions. This is discussed in more detail in the paper.
kilometre for a low volume paved rural road, while constructing and
maintaining a heavy freeway structure can cost millions per kilometre Challenges and conventional stabilisation methods used by the construction
(United States Dollar (USD)) (National Treasury, 2015; Siyepu, 2016). In industry
developing countries, soil roads are constructed for low traffic volume
areas such as access roads in rural areas or as estate roads. In South Soil stabilisation is defined as a process of improving the physical
Africa (SA) there is ample evidence pointing to lack of adequate main and engineering properties of a soil to achieve a predetermined target
tenance of current road infrastructure, as well as halted development of level of performance (Latifi et al., 2016). Stabilisation of in situ material
new roads. This is worsened by the persistent increase in traffic volumes is considered to be an important method for improving the strength and
as well as moisture damage caused by heavy rains. A lack of reliable road performance of the treated soil material, which contributes to prolong
infrastructure also undermines prospects for future development. ing the service life of the subsurface and results in a more economical
Unproclaimed roads, which are at best gravel roads that do not offer all- design (Mgangira, 2009a).
weather access is a prime example of sub-par infrastructure. According Bearing capacity of subsoil is one of the major site selection com
to Paige-Green (2008) these types of roads can have a significant ponents in geotechnical design criteria. Once the bearing capacity of soil
negative impact on the economic well-being of the affected communities is deemed poor, the following options are considered (a) change the
and also result in negative environmental impacts (i.e., soil lost due to design to suit the site condition, (b) remove and replace the in situ soil; or
erosion, dust clouds). (c) abandon the site. Abandoned sites linked to undesirable bearing
The construction industry is investigating new technologies such as, capacities have dramatically increased, and the outcome of this is the
deep mixing, electro kinetics, nano-materials for sensors and sensing, as scarcity of land and an increased demand for natural resources. The
well as the use of micro-organisms for groundwater remediation affected areas include those susceptible to liquefaction, those covered
(Wijeyesekera, 2014). These have effectively advanced the road con with high level clay material and organic soils which can be problematic
struction practice, resulting in a ‘new’ interdisciplinary approach to civil because of low shear strength and high compressibility, contaminated
and geotechnical engineering asset management by integrating concepts land, as well as areas in a landslide (Makusa, 2013). Problematic soil is
and techniques from other disciplines. The development of microbio recognised by its sudden volumetric reduction (i.e. collapsible soils),
logical processes for improvement of the soil properties is one of the after exposure by various environmental conditions such as, increasing
more recent manifestations of this trend. humidity, wind deposited sand or silt, or under water immersing con
Bio-based exploration for use in construction is a potentially cost- ditions, hence altering cementation bonds between soil particles
effective, and an environmentally sustainable engineering approach to (Ayeldeen et al., 2017).
recondition and strengthen the existing road base, sub-base, or subgrade Drainage problems are caused by a lack of diversion of rainwater,
materials for an extended lifespan (Pei et al., 2015). Biological stabil which can lead to deterioration of the road structure or pavement sur
isers such as Bacillus spp. produce a wide range of metabolites and eco- face. This is mostly due to moisture retention within the road prism and
friendly additives that offer cumulative nature of benefits for stabilisa in particular within the subgrade soils relating to typical subgrade
tion and compaction in structural applications (Samang et al., 2018), problems, as described in Table 1 (Paige-Green, 2008). The adverse ef
such as lowered cost, environmental and performance-based benefits. fects of increase in moisture content on the soil behaviour have also been
Several Bacillus spp. and their derivatives have been considered as po a major concern among geotechnical and pavement engineers. Soil
tential alternatives to conventional chemical soil stabilisers, particularly possesses excellent performance at the optimum moisture content or
in challenging ground conditions. This is clearly an excellent example of below the optimum moisture content (dry-side of optimum) however,
the application of multi-faceted green technologies promoting multi the strength of soils reduce significantly as the moisture content in
disciplinary research and collaboration between road/pavement engi creases beyond the optimum (wet-side of optimum). Sensitive soils tend
neering, green chemistry, biotechnology and geomicrobiology. It is to swell with moisture content increase, particularly in areas of higher
important to pursue these greener technologies, which necessitates rainfall, as a result subsurface soil becomes too weak to support the
research and development for new eco-friendly materials as an alter pavement or road loads resulting in collapsed road surfaces and potholes
native to conventional cement. The significance of harnessing geo (Stabnikov et al., 2011). The replacement of weak in situ soil is not al
microbiological research (using novel bioenzymes, biopolymers or ways a good option, especially in pavement or road developments due to
waste, such as fly ash, to catalyse various reactions that stabilise in situ the ongoing construction cost conundrum in cash strapped countries,
soil for road applications. This includes their mode/s of action) makes it which is the typical situation in most developing countries.
possible to devise engineering solutions for temporary and/or perma An opportunity exists to improve unpaved roads in both urban and
nent geotechnical engineering problems. rural areas. Unpaved roads are defined as those with a surface coarse of
A significant economic incentive exists for developing new and unbound aggregate (gravel), where no binder or chemical additive is
innovative, yet environmentally safe bio-based product applications. used (Netterberg and Paige-Green, 1988). Good wearing coarse on
2
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
3
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
Table 1 (continued ) and safety, as a result of the reduction in traffic generated dust from the
Soil type Key disadvantages Current methods Effectiveness of the treated roads.
& typical damage for recognition & standard practice For improvement of soil structure and engineering properties con
to roads counter measures for road ventional approaches include dynamic compaction, soil–cement stabi
(physical/ application lisation, soil–lime stabilisation, drainage of vegetation, geotextiles, as
chemical)
well as injecting synthetic materials such as micro-fine cement, epoxy,
leading to serious with waste rubble acrylamide, phenoplasts, silicates, and polyurethane into the pore space
failures (resulting or grouted.
to bind soil particles together (Saleh et al., 2019). This is accomplished
to loss of life &
costly using a variety of chemicals, jetting, and permeation grouting tech
amendments). niques. It must be noted that these approaches create serious environ
f. Soft clays Associated with Predominantly The materials are mental concerns as all chemical grouts apart from sodium silicate may
fluctuating water found on coastal associated with low be toxic and/or hazardous (Bryson and El Naggar, 2013).
tables causing low areas, making it shear strength,
shear strengths, impossible to walk high
In most civil engineering projects, it is difficult to obtain a con
high on (shear strength compressibility, struction site that will meet all design requirements without introducing
compressibility, of 10–40 kPa). low permeability, ground modification. Therefore, the current practice is to modify the
and low Embankments are also organic matter engineering properties of the native problematic soils to meet the design
permeability constructed contents make it
specifications (Ikeagwuani and Nwon 2019). This leads to using alter
resulting in time- slowly layer by difficult to predict
related settlement layer; or the use of their properties. native (non-traditional/biochemical) stabilisers which are one of several
problems. geosynthetic The long-term methods of soil improvement. In terms of viability, this may create a
products as differential viable substitute to cement but it is all dependent on cost. Non-
separation layers settlements require traditional additives are diverse in their composition and in the chem
and to facilitate ongoing
and accelerate maintenance to
ical and physical manner in which they interact with soil (Rajoria and
drainage. provide an Kaur, 2014). Unfortunately, little is known about their interaction with
The use of the adequate different geotechnical materials and their fundamental stabilisation
wide range of performance of the mechanisms. Despite the lack of research, an environmentally friendly
geosynthetic road.
solution is still advantageous, as the larger construction companies are
products as
separation layers very conscious of their environmental footprint, and thus eager to invest
and to facilitate in smart ‘green’ construction materials (Shehata and Poulos, 2018).
and accelerate
drainage has Discussion
contributed to
improved
construction over The use of bio-based innovations for potential road application
such areas.
4
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
Large scale laboratory testing and in situ demonstrations of bio-based cycle), for the OPC treated samples > 600 kPa (before FT cycle) and
additives usually causes serious damage, as expected with ~ 40% decrease on
strength. Shear strength investigated by Ng et al. (2012) using
Microbial biocement B. megaterium MICCP process found that the shear strength ratio of
One of the most studied methods is the precipitation of calcite uni treated to untreated increased from 1.40 to 2.64. Compressive strength
formly within soils through biological action to elevate the pH that of at least 300 kPa was reported by van Paassen et al. (2010) similar to
creates supersaturated conditions known as microbial induced calcium findings were reported by Nafisi (2019) which show that the tensile
carbonate precipitation (MICCP), commonly referred to as microbial strength varies between 210 kPa and 710 kPa depending on sand type
biocement (Oyediran and Ayeni, 2020). For example, Bacillus pasteuri is and mass of carbonate. The author van Paassen et al. (2010) also re
an alkalophilic soil bacterium with a highly active urease enzyme that ported a 60% reduction in the permeability of treated soils at ~ 100 kg/
consumes urea and decomposes it into carbon dioxide (CO2) and m3 CaCO3 precipitation however, with increasing concentration of the
ammonia (NH3). In the presence of water, NH3 is converted to NH+ 4 and solution calcite crystal clogged pore spaces which reduced permeability
CO2 equilibrates in a pH-dependent manner with carbonic acid, car between 50 and 99% using 1 M cementation solution (Ivanov et al.,
bonate, and bicarbonate ions. The increase in pH provides the alkaline 2010), as the crystals formed at the contact points can maintain the
environment and the carbonate required for the reaction with Ca2+ and connection of pores without restricting the pore water mobility. Even
precipitation of calcite (CaCO3), schematically shown in Fig. 2. though these tests characterise the strength properties at large scale,
Cheng et al. (2013) demonstrated bio-enzymatic mediated triaxial test is recommended to test the response of biocemented soils
improvement of soil using unconfined compressive strength (UCS) of toward the monotonic and cyclic loadings as it simulates the natural
MICCP treated samples before and after freeze–thaw (FT) cycling that behaviour of soil in the field for road applications.
showed<10% decrease on strength occurred from > 800 kPa (before FT Cheng et al. (2013) reports mechanical behaviour of the
Fig. 2. Biocement is two-step process a: Phase I, bacteria ingest nutrients such as, sugar, nitrogen, & proteins with the growth of the bacterial population and
enzymes produced from the bacterial species they hydrolyse urea components in the presence of water to form ammonium and carbonate ions which leads to b: Phase
II, again the addition of nutrients such as calcium chloride, in the presence of calcium ions and nucleation sites on the soil particles, the carbonate ions react
spontaneously with the calcium ions to form calcium carbonate, c-d: the calcite precipitates/the cementing agent (produced by the bacteria) used to bind the soil
particles together to increase strength and stiffness of the soil.
5
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
6
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
appear to be suitable candidates for improving durability aspects of have many advantages compared to conventional chemical stabilisers, it
concrete as no other potentially detrimental metabolic by-products, is unclear how these products work and under what conditions. The
such as ammoniums in the ureolytic pathway, are produced (Jonkers stabilising mechanisms of these products are limited in application, and
and Schlangen, 2007). Moreover, alkaliphillic species of the genus Ba their proprietary chemical composition makes it difficult to predict their
cillus are not only adapted to be metabolically active in highly alkaline long-term performance. According to Khan and Taha (2015) the re
environments, but are also able to produce spores, (i.e., specific robust ported formulation of Terrazyme is composed of ethoxylated alcohols,
‘dormant’ cells which survive being incorporated in the concrete matrix fermented vegetable extracts, and non-ionic surfactants (Khan and Taha,
for self-healing). Bacillus megaterium, is an example of ureolytic bacteria 2015). Permazyme is composed of multiple enzymes, proprietary in
that’s able to adapt to soil environments (Jiang et al., 2016). gredients, and unspecified organic material, according to the manufac
Bacillus subtilis, a common soil bacterium has been found to induce turer. Further laboratory investigation showed the presence of proteins
silica precipitation and other metals by binding it to the cell wall of the and non-ionic surfactants in both preparation (AbouKhadra et al., 2018).
bacteria (Urrutia and Beveridge, 1994). These studies show an exem The bioenzymes treatment of soil roads with a combination of different
plary approach of cell wall modification using Bacillus spp. via ethyl multi-enzyme formulation or in combination with other polymer com
enediamine to become electropositive to bind SiO2- 3 ion from the silicic pounds serves the purpose of waterproofing and hardening soil. There
acid. In line with this approach, cement induces calcium silicate hy fore, it becomes important to perform a research study that can give
drated (C-S-H) gel and calcium hydroxide in aqueous form after reacting objective scientific support for the use of novel enzymes as a product to
with water during the primary reaction, the secondary hydration will stabilise soils. Addition of enzymes to clayey soils provide a desirable
proceed with the presence of pozzolans (i.e. siliceous and aluminous reduction in the compaction effort necessary to reach target soil den
material) inside the concrete and produce an additional C-S-H gel that sities and improves resistance to freeze–thaw cycling (Rajoria and Kaur,
accelerate and enhance the strength of final concrete. In this case, micro- 2014). However, the recommended soil types are limited to 12–30%
organisms may be used as an alternative method to produce a precipi clay content, 18–30% cohesive fines and should have a plastic index
tation that possesses characteristics similar to that of the cement poz between 2 and 10, and a max liquid limit of 30% (Jayathileka and
zolans which can be derived naturally from the process of Adikari, 2007; Guthrie et al., 2015).
biomineralisation from micro-organisms - a process that forms precipi The idea of using enzyme stabilisation for roads was developed from
tation and fills the pores that enhanced the mechanical properties in the application of enzymes to products used to treat soil in order to
concrete materials, such as strength and durability. Therefore, evidence improve horticultural applications. A modification to the process pro
of bacterial modification in the presence of silicate by reacting aqueous duced a material which is suitable for stabilisation of poor ground for
calcium hydroxide to form C-S-H and enhancements made to the road traffic. When added to a soil, the enzymes increase the wetting and
compressive strength development in soil constitutes further investiga bonding capacity of the soil particles. It is suggested that the enzyme/s
tion in this field (Afifudin et al., 2011). react with soil molecules to form a cementing bond that stabilised the
soil structure. At higher application rates the treated soil can be stabi
Bioenzymes and their role in construction engineering lised to form a dense, firm, hard, and water-resistant bond layer that
Recent experimental progress focuses on applications of protein en may be used as a road surfacing (Lim et al., 2014).
gineering as a means for biochemical soil modification. More attention Preliminary laboratory investigation of various enzyme products as a
has been given to the use of bioenzymes as soil stabilising agents due to soil stabiliser originates from a common description: certain enzymes
expansion in manufacturing capacity, the low cost, and relatively wide are adsorbed by the clay lattice, initiating a catalytic reaction (i.e. metal
applicability compared to standard chemical stabilisers (i.e., hydrated cation exchange mechanism). Cation exchange refers to an immediate
lime, Portland cement, and bitumen), which are required in large reaction of the clay with the enzyme, within a few minutes of mixing,
amounts to stabilise soils (high associated cost). Innovative thought is resulting in improved soil texture. They have an important effect on the
provoked in observing the engineering prowess of the ‘termite engi clay lattice, initially causing them to expand and then to tighten. The
neers’ in the construction processes of their all-weather proof termite enzymes can also be absorbed by colloids enabling them to be trans
mounds (Wijeyesekera, 2014). Ant saliva, full of enzymes is used to ported through the soil electrolyte media. The enzymes aid the soil
build soil structures, which are rock hard and meters high. These bacteria to release hydrogen ions, resulting in pH gradients at the sur
structures are known to stand firm despite heavy tropical rain seasons faces of the clay particles, which assist in breaking down the lattice. The
(Assam et al., 2016). With research developing within this direction, change in the structure causes a decrease in the moisture sensitivity and
some of these biochemical solutions for soil stabilisation include en increases the workability and constructability of the soil (Marasteanu
zymes such as peroxidases, phospholipase, cellulase, phytase, lipase, et al., 2005; Rajoria and Kaur, 2014).
luciferase, and proteases (Ivanov et al., 2015; Das and Varma, 2010). Furthermore, an enzyme activity generally corresponds with element
A review of previous studies on enzyme-based soil stabilisation stabilisation. A number of enzymes require the presence of metal ions,
indicate enzymes have been used successfully to stabilise roads in such as calcium ions, for the maintenance of their stable and active
Malaysia, China, India, and the Western USA despite the heavy traffic structures. These ions are bound to specific binding sites on the surface
and high rainfall (Taha et al., 2013). Besides an increase in the strength of the molecules and restrict local flexibility and unfolding (Guncheva
and durability of the roads, a reduction in project cost has also been and Zhiryakova, 2011). Enzymes require trace amounts of specific
achieved. In Mendocino County, the California Department of Trans divalent metal ions such as Mn2+ for glucosylgydrolase, Zn2+ for phos
portation has conducted several tests of a compaction additive based on phohydrolase, and Ni2+ for amidohydrolase enzymes as functional co
enzymes. This natural product helped the road base to set very tightly, factors, which can be substituted by elements with similar ionic
reducing dust and improving chip-seal applications. With air quality and properties when present in excessive concentrations. In practice, the
water quality agencies requiring dust reduction, this is a potentially biochemical reactions are brought about largely through the catalytic
effective new product, cheaper than asphalt (Marasteanu et al., 2005). contribution of enzymes and variable substrates that serve as an energy
Products such as Permazyme and EcoBric are non-hazardous, and eco- source for micro-organisms. Enzymes containing Ca2+ binding domains
friendly, which use low energy techniques for soil stabilisation that have or other metal binding sites such as Co2+, Mg2+, and Zn2+ may act as a
weathered resistance approaching that of concrete. The advantages of soil stabiliser by ion exchange, thus replacing the monovalent cations
competitive bioenzyme products (i.e. Renolith, Fujibeton, and Terrazyme) surrounding the surface of expansive clay soil particles and may
are that it is rapid, cost-effective, convenient to use, and utilises eco- decrease bound water molecules. This results in a stronger bond/clay
friendly technology yielding a ‘value added’ product (Rajoria and lattice structure and particle cohesion (i.e. enabling clay particle floc
Kaur, 2014). Even though these enzyme-based soil stabilisers appear to culation) (Carvalho et al., 2013).
7
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
The utilisation of enzymes is unique in that it is one of the key According to AbouKhadra et al. (2018) enzymatic stabilisation
present-day strategies towards environmentally benign energy– and introduced a significant improvement in UCS and CBR for the fine
material saving biochemical processes. The need is to identify more grained soils. In this study, improvement of the enzyme product as soil
novel enzymes and to elucidate their modes of reactivity on various soil strength was normalised to that of untreated soil and represented by
geochemistry and/or waste, such as fly ash. The potential of using en (CBR)r. For fine grained soil, the improvement in (CBR)r ranges from
zymes to stabilise waste material raises the question of whether it can 2.75 to 4.5 times those values observed for non-treated soil and the best
work synergistically. However, enzymes such as amylase, arylsulpha concentration was 2.5 g/L. Also, the soil strength increased with in
tases, β-glucosidase, cellulose, chitinase, dehydrogenase, phosphatase, crease clay content therefore percentage fines important criteria. At
protease, lipase, and urease. have shown potential in related industrial 0.5% concentration Permazyme at 30–40% clay content achieved
applications (Ivanov et al., 2015; Das and Varma, 2010). maximum (CBR)r (4) also the fines material UCS was 800–1200 kN/m2.
Several reported stabilisation mechanisms termed enzyme induced It is inferred from the results that using the enzymatic preparations
calcite precipitation (EICP) are proposed (similar to the process micro significantly increased the strength of fine grained soil, the rate of
bial induced calcite precipitation (MICP)) such as bacterial lipases that improvement was found to be proportional to the clay content in the
was found to be activated by Ca2+, and other metal ions. These prop soil. Panchal et al. (2017) also found that using the correct dosages was
erties make immobilised lipase an attractive option for soil stabilisation fundamental such as, 700 mL dosage of Terrazyme enzyme solution
applications (Carvalho et al., 2013). These Ca2+ activators may increase which treated 1 m3 soil, showed consistent increment in the CBR values
adsorption of calcium on soil particles, thereby liberating metal ions to of the soil of 9.80% from an initial value of 8.31% (unsoaked) and 4.19%
bond with clay soil particles. Enzyme products containing high amounts (soaked) after the first week of curing, 13.43% after second week curing,
of silica (i.e. silica-immobilised lipase (Kumar et al., 2006) or calcium and 14.80% after fourth week curing. However, significant variation
and sodium) is hypothesised to make the enzyme product act more like was shown in CBR values with longer curing periods and increased
cement by forming hydrated calcium silicate when added to soil (Car dosages (900–1000 mL/m3). This classification of bio-catalyst namely,
valho et al., 2013). Bio-phosphate cements are known as microbial Terrazyme (comprised of water, ethoxylated alcohols and matured
induced deposition of phosphate. Alkaline phosphatase constantly hy vegetable concentrate) proved its efficacy in varying strength properties,
drolyses phosphate monoester in bacterial solutions, thus PO3- 4 ions are consistency limits and various other geotechnical properties of fine
obtained. MHPO4, MPO4 or M3(PO4)2 are prepared by the reaction of grained soils. However, to acquire the best performance from this bio
PO3-
4 ions with metal cations (alkaline earth element Ba) and adopted to enzyme additive, soil gradation ought to be fulfilled with minimum clay
cement loose particles. The cementing mechanism of barium hydrogen content required for the reaction, sufficient active elements to react with
phosphate particles by microbial precipitation can form large agglom metal cations resulting in a succession of breaking down of bonds as well
erates with each other and interact with quartz sand to produce van der as the advancement of a new system of links. The characteristics of the
Waals bonds in sandstones. Bio-phosphate cement can bind loose sand soil mixtures also decide the diversity in its breaking down process of the
particles into a bio-sandstone (Yu et al., 2015). Furthermore, the effi enzyme. In that study an essential element of the bio-catalyst is getting
ciency of soil enzymatic stabilisers for road applications highlights two disregarded inadvertently, due to decline in strength improvement after
prominent commercial preparations Terrazyme and Permazyme. Overall, longer curing periods. Therefore, as indicated by Thomas and Ran
the use of bio-enzymatic soil stabilisers reduces the amount of water gaswamy, 2019 low concentrations of enzymes are enough for the soil
required at the construction sites, minimise the need to haul materials improvement since they do not get attenuated in the regeneration of the
and are promising additives to reduce the application of OPC. Factors enzyme catalyst after the reactions completed. The authors demon
such as, non-toxic formulation, manufactured through natural fermen strated an ideal soil combination, clay soil with the presence of 1%
tation processes make enzyme products highly economical with cement added and 0.06 mL/kg of Terrazyme which exhibited higher
considerable cost savings of 3–5 times when compared with conven UCS. Initially, the strength of clay soil was 28 kPa, and improved to 127
tional road construction methods (Taha et al., 2013). kPa after introducing 1% cement and 0.06 mL/kg of the bioenzyme and
The paper presents efficiency and limitations from studies such as, it is about 354% higher than the UCS of untreated soil. This improved
soil type, curing time, and method for application which significantly strength could be sustained with extended curing periods. These changes
affect the effectiveness of the bio-enzymatic treatment. Peng et al. in the strength properties of soil matrix were supported by shifts of peaks
(2011) found under air-dried curing conditions UCS of enzyme treated in the FT-IR results, and the formation of additional C-S-H gel from the
soil were better in comparison to OPC because the cement particles SEM images (Thomas and Rangaswamy, 2019).
cannot be fully hydrated under the air-dry conditions, in comparison to Most of the enzyme stabilisers on the market are imported with
sealed curing conditions the UCS of the bio-stabilised soils were < 0.5 respect to SA, and have shown inconsistencies in performance, including
MPa. Marasteanu et al. (2005) conducted triaxial tests and determined frequent changes of names for the same product, which has led to their
the resilient modulus for two types of soils that were treated by two limited support in the local road construction industry (Moloisane and
different enzymatic preparations (1 cc of enzyme per 5 L of the water), Visser, 2014). Due to lack of innovative solutions using conventional
comprising of soil-1 96% of fines (75% of clay) and soil-2 60% of fines approaches, the construction industry is exploring the use of biological
(14.5% of clay). The triaxial tests were performed on two confinement methods to improve structural properties such as strength, volume sta
pressures (27.6 kPa and 55.16 kPa). The results showed that the soil bility, durability, and permeability in road construction applications.
treatment of enzyme-1 increased the shear strength of soil-1 and soil-2 Due to the lack of performance, a gap has been identified to produce
by 9% and by 23%, respectively. In case of enzyme-2 treatment, the improved bio-stabilisers for the local market.
increases in shear strength for soil-1 and soil-2 was 31% and 23%,
respectively. The resilient modulus tests with a deviatoric stress = 96.5 The role of biopolymers in construction engineering
kPa and confinement pressure = 13.8 kPa, and the resilient modulus for For more than half a century fossil fuels have provided the resources
the treated soil was between 86453.4 kPa and 5805.4 kPa which was for the polymer industry. However, the future scarcity of petroleum
greater than the mean of the untreated soil. Resilient modulus of soil is a resources (Sorrell et al., 2012) is a fact that must be faced. Sustainability
common parameter defining the stiffness of a soil. For instance, at 48.3 has now become important because of the significant depletion of fossil
kPa deviator stress, improvement in resilient modulus was noticed for resources and major scientific and political players have encouraged the
soil-1 with enzyme-2 whereas the increase in resilient modulus was 55% paradigm shift to ‘bio-based material’. The twentieth century became
to 85%. For soil-2, at the same deviator stress level the increase of the age of admixtures, the history of which started in the 1920 s with the
resilient modulus reached 51% to 61% with enzyme-1 and 57% to 137% introduction of lignosulfonate, a biopolymer, for OPC concrete plasti
with enzyme-2. cisation, the first functional polymer used in construction on a large
8
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
scale (Plank, 2004). Recent years have seen a tremendous increase in the soil inter-particle cohesion (Fatehi et al., 2018). Studies on xanthan and
number of publications on using bio-based polymers (Chang and Cho, guar gum by Ayeldeen et al. (2017) show remarkable soil strengthening
2019), however the fact is that these materials still constitute only a for road sector application. Whereas, Chang et al. (2015a) used two
small fraction of the polymer industry. Biopolymers include polymers thermos-gelation biopolymers (gellan gum and gaur gum), which enable
from agro-resources (polysaccharides, cellulose, starch, chitin, chitosan, hydrogen bonds to enhance the strength and durability of sandy and
and alginates), from micro-organisms by fermentation (poly clayey soils. While time was not a crucial parameter, temperature played
hydroxyalkanoates, such as, polyhydroxybutyrate) and from biotech an important role in soil treatment. Investigation is still required as the
nology via conventional synthesis (polylactides (PLA), polybutadiene soil improvement degree differs according to polymer types, soil types
succinate, biopolyethylene (PE), polytrimethylene terephalate, poly-p- and compositions, doses, and curing conditions.
phenylene) (Averous and Pollet, 2012). The reuse of agricultural and These studies provide an eco-friendly solution to improve the
biomass waste will contribute to the environmental advantages of bio geotechnical behaviour (strength) of different types of soil, so this may
polymers opposed to traditional petroleum-based polymers (Hottle resolve the need to provide complex conditions regarding microbial
et al., 2013). injection methods (Chang et al., 2016). The economic feasibility of
Biopolymers are naturally existing polymers that often have poly soil–biopolymer matrix interactions and related soil strengthening
meric carbohydrate structures. The types currently used in the con mechanisms, in the field, may be analysed in comparison to ordinary
struction industry include, xanthan, guar gum, welan, succinoglucan, Portland cement (OPC). According to Chang and Cho (2019), the cost of
curdlan, chitosan, sodium gluconate, and gellan gum. These are used in utilising biopolymers such as gellan gum for soil treatment remains
dry-mix mortars, wall plasters, self-levelling under layers, or injection relatively high compared to conventional soil treatment (OPC). They
grouts to improve viscosity, water retention, set retardation and flow showed that the total cost to treat one ton of soil with gellan gum via
ability (Plank, 2004). The biopolymer b-1,3/1,6-glucan has been shown thermal treatment is approximately $25–30 USD, in comparison to the
to aggregate soil particles and subsequently enhance the overall soil current cost ($10 USD) of 10% cement treatment. However, recent an
compressive strength. As well as, to have a positive effect on the com alyses of economic feasibility and prospects show that the use of bio
pactability, Atterberg limits and swelling index of treated soil and a polymers for construction purposes is becoming more feasible as a result
negative effect on the consolidation coefficient. A small amount of of the current rapid growth of the global biopolymer market. Findings
biopolymer has a significant effect in reducing soil erosion by enhancing also demonstrate that biopolymers that are of high-tensile strength (e.g.
Table 4
Bio-based in situ soil stabilisers cost benefit analysis.
Method of bio- Usage requirements Performance effect Main consumables Method for road application Price for soil treatment1
based (L/m3)/(concentration) (Compressive strength
stabilisation (MPa))
Ordinary 10% cement content to 2 2 (Chang et al., 2015b) Cement, water Deep mixing, injection, grouting, direct 60–100 USD/tonne (Chang
Portland the weight of soil. Mixing spray. et al., 2015b; Chang and Cho,
cement (OPC) 2012)
Chemical 10% content to soil 0.8 3 (Chang et al., Chemically synthesised Injection, grouting, Spray. 200 USD/tonne (Chang et al.,
stabiliser mass. 2016) 2016)
Enzymatic 0.033 L/m3 2.5 (Peng et al., 2011) Multienzyme Sprayed on the soil (water truck). 5–6 USD/tonne 4 (Specialised
stabiliser formulation, surfactants Protection Products, Gauteng,
product South Africa)
Polymeric 14–24 L/m3 2–4.3 (Chang et al., Biopolymers, water Direct mixing, injection, 285–250 6 USD/tonne (Chang
stabiliser 2015b; Chang and Cho, spraying exo-cultivated polymers. et al., 2015b; Chang and Cho,
2012) 2012)
Microbial 1 × 107 cells/mL 7
1.8 (shear strength) ( Micro-organisms, urea, Surface spraying (urea broth & Ca2+ 60–90 USD/100 L 8 (DeJong
biocement DeJong et al., 2006) starch, nutrient solution. solution); Subsurface injection; et al., 2006)
mechanically mixed in soil.
Benefits Industrial production Eco-friendly additives Biodegradable Soil competitiveness Sustainable (longevity for
bioreactor (Yes/No) unsealed roads)
9
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
bioplastic PLA) have strong potential to replace cement as a soil treat include the bio-stabilisation with microbes, as shown in a recent study
ment material within the context of environment-friendly construction by Samang et al. (2018), in which they demonstrated improvement of
and development (Babu et al., 2013; Muniyasamy et al., 2016). the bearing capacity of subgrade soil using B. subtilis. The Bacillus or
There is a growing need for utilisation of economical stabilisers on ganism class will form the first line of intervention towards a solution for
locally available soils (in situ) that are cost-effective and environmen the challenges mentioned. The advantages of the upstream and down
tally friendly. Great potential exists for the development of bio-based stream processing for biomass production is outlined in Fig. 3 (Maharajh
stabilisers, however further research is required for testing of the et al., 2008).
concept and development of the product prototypes for application on The proposed mechanism is that Bacillus spp. (i.e. B. subtilis,
the in situ construction materials. Table 4 provides a preliminary guide B. cereus) produce hydrolase enzymes, such as lipases, which work in
on cost and benefits of soil stabilisers. The data shows current products combination with added ionic stabilisers that may increase the strength
on the market and the potential development of an integrated and and the number of ionic bonds present on the surface of the soil-clay
validated process for the potential pilot manufacture of the desired particles. The result is the formation of a more highly weathered, less-
products to meet techno-economic requirements. Several factors may be expansive clay structure. Four major classes of polymers are produced
considered for further investigation into cost benefit ratios such as, by bacteria: polysaccharides, polyesters, polyamides and inorganic
material availability, equipment, technology viability, duration of con polyanhydrides (such as polyphosphates). The biopolymers and the
struction, cost of hauling, reduction in carbon footprint and manpower enzymatic stabilisers are proposed to form polymer-soil matrices due to
(Chang et al., 2016, 2015b; Chang and Cho, 2012; Larson et al., 2013; hydrogen and ionic bonds between the soil-clay particles. In this case the
DeJong et al., 2006; Maclaren and White, 2003; Velde and Kiekens, enzymes will have a dual role in polymer stabilisation, as they form both
2002; Stocks-Fischer et al., 1999; Bang et al., 2015). A comparative inorganic and organic catalysts with the natural polymers, to strengthen
analysis between the various stabilisers was limited at this stage, but a soil structural properties. The result is a soil material that is much less
techno-economic feasibility study may be performed to fully understand sensitive to moisture, more workable, and can be compacted to a better
the advantage of bio-stabilisers over conventional products. Moreover, particle-interlock state by equipment and traffic forces. Better particle-
cost versus performance is a requirement for upscaled technologies and interlock means higher internal friction and improved bearing capac
is also important for the determination of product feasibility or alter ity (Firoozi et al., 2017).
native applications. The importance of Bacillus spp. (particularly B. subtilis) has been
shown in their production of surfactants, such as glycolipids,
Micro-level analysis to understand the mechanism of interaction of soil polysaccharide-protein complexes, fatty acids, phospholipids as well as
stabilisers lipopeptides. These compounds enable greater surface and interfacial
Non-traditional additives are used to improve the engineering tension reduction, lower critical micelle concentration, emulsification
properties of subgrade soil. The assessment of these products, based on a properties, and tolerance to ionic strength. Recent trends in soil research
combination of in situ standard performance testing, determines the also suggest the stabilising activity of extracellular polymeric substances
macro-level strength characteristics. However, studies show limitations (EPS) produced by B. subtilis and B. licheniformis. EPSs can bind toxic
in predicting the expected performance on a macro-scale (Mgangira and metals, promote the dissolution of minerals and enhance aggregation
Ndibewu, 2010). Thus, there’s a need to identify more complementary associated with biofilms. When released into a solution, they can react
methods in the design. As such, using micro-structural details for the with other solutes and soil-particle surfaces and may contribute to the
determination of a mechanism of action may contribute to identifying stabilisation of microbial-derived organic carbon in soil. More generally,
the interaction between the product and the soil particles. such microbial products can be expected to enhance the binding capa
Physico-chemical bonding mechanisms of stabilised material bility of unstable/weak subsoil particles and is discussed in more detail.
induced by stabilisation processes are advantageous for geotechnical Fig. 4 shows fermentation components as potential material for testing.
engineering applications. Mechanistic tactics namely, bioclogging, bio Components of Bacillus spp. ferment that are well known is bacterial
aggregation and biocementation may be identified via micro-level
analysis. Latifi et al. (2016) show the mechanisms of pore filling and
particle bonding by cementitious products through the following tech
niques: X-ray powder diffraction (XRD), Field Emission Scanning Elec
tron Microscope (FESEM), Energy-Dispersive X-ray Spectroscopy (EDX),
Fourier Transform Infrared Spectroscopy (FTIR), Brunauer, Emmet and
Teller (BET) surface area analysis and particle size analysis (PSA), and
using a laser diffraction approach. In conjunction with these tests, UCS
tests show an improvement in strength with calcium based additives
such as, SH-85, a commercial product (Latifi et al., 2013). Micro-level
analysis such as, Field Emission Scanning Electron Microscopy
(FESEM) can be used to determine a morphological representation to
assess the prototype bio-additives for in situ soil stabilisation (Mgangira
and Ndibewu, 2010). Since the mode/s of action of biological stabilisers
are not mutually exclusive, and bio-additive/s may function with one
functional effect, or a combination thereof, it is thus important to
identify the level of laboratory performance testing necessary for
determination of soil-particle interaction, that complement large scale
tests.
10
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
11
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
fundamental research Mgangira (2009b), Moloisane and Visser (2014) (Visser and Hudson, 1983). The high levels of dust created from these
and Van Veelen and Visser (2007) have demonstrated a national unpaved roads reduces visibility and hinders traffic safety. The unpaved
standardised (ex situ) approach to the determination of strength char road network makes up a significant portion of the overall road network
acteristics of both polymer- and enzyme-based additives. This approach in SA, and in most developing countries and >90% of these roads are in
infers the modes of action and associated mechanisms may be attributed rural areas (Burrow et al., 2016). Thus, unpaved roads provide access for
or demonstrated by a pre-defined strength criterion and to the perfor the majority of the population and directly support social and economic
mance effect of key bio-molecules (i.e. resistance to: compression, growth. The problems associated with unpaved roads include, low
erosion, abrasion, and water absorption), as described below: standards, poor riding quality, increased erosion in wet weather, and
large quantities of dust that is generated by moving vehicles and the
• Bio-stabiliser effect: resistance to compression load wind. Contributing reasons, according to (Moloisane and Visser, 2014)
is that many unpaved roads do not have the ideal range and distribution
The capacity to resist applied stress is an indication of structural of particle sizes to give a good load-bearing capacity when wet (coarser
integrity and quality of a given material. Investigating the compression particles) or sufficient plastic capability (clay) when dry to prevent
load infers testing the material’s resistance to deformation and is material from breaking loose.
therefore an important assessment of material strength to ensure the Historic data shows that approximately 3 million tonnes of dust was
long-term durability and stability. For example, uniaxial compression generated on SA unpaved/unsealed roads per year in 2004. The estimate
test of biocemented sand (calcite) shows an increase in compressive was that two-thirds of this dust resettles on the road and the remaining
strength. According to recent finding by Gao et al. (2019) the million tonnes are permanently lost, leading to a negative environ
improvement in this material durability is sufficient to fulfil geotech mental impact and rapid deterioration due to loss of material (Jones and
nical applications. In addition, auxiliary additives such as, fly ash, Ventura, 2004). The lost material requires replacement and frequent
lignosulfonate, and biopolymers have all indicated improvement in maintenance, due to the development of potholes and ruts, contributing
compression strength (Chang et al., 2015a; Fatehi et al., 2018). Sup to a decrease in durability and strength properties of unpaved roads.
ported research at laboratory scale and field applications show that Replacement of gravel material is unsustainable as natural resources are
compression testing is a key design criterion for quantifiable assessment being depleted. A potential solution to this ever-increasing problem is
of the effect of a bio-based stabiliser/additive on performance. the use of soil stabilisers (additives). As previously discussed road ma
terials can be improved using traditional additives such as cement and
• Bio-stabiliser effect: resistance to erosion lime, however, studies on ionic additives (i.e. enzymes and sulfonated
products) suggests a cost-effective and practical solution to ongoing
Surface erosion occurs once the shear pressure applied by moving problems (Moloisane and Visser, 2014). An investigation into dust
fluids exceeds a critical value. This surface erosion often leads to ma suppressors, termed dust palliatives (e.g. SoilSeal, Aggrebind product)
terial failures of geo-structures. Several techniques have been used to which are agents applied to the road surface, to prevent the formation of
increase the soil erosion resistance, including chemical methods. Ham dust clouds and their associated environmental impact is therefore
et al. (2018) shows improvement of surface erosion resistance by using important.
microbial biopolymers, altering the mechanical and the hydrological
behaviour of soils, such as inter-particle bonding, stiffness, strength, and • Bio-stabiliser effect: resistance to water absorption
permeability by the insoluble biomaterials. Potential mechanisms of
biomaterials to enhance erosion resistance include, (a) gel like EPS Unpaved roads are more vulnerable to weather conditions, especially
(polymers – hydrophobic properties) that fill the pores of soils and cause during rainy seasons as soil material can expand as well as lose cohe
bio-clogging by reducing hydraulic conductivity; (b) grain coating and siveness, with increased porosity and possessing a higher permeability.
bridging by film-like biopolymers increases the apparent cohesion and Chemical/biochemical stabilised soils may have adequate compressive
erosion resistance, as reported in previous literature (Chang et al., strength under dry conditions; but they will lose their strength under
2015a); and (c) the biocement method is also suitable for the control of adverse moisture content. The amount of water absorbed by the stabi
soil erosion induced by hydraulic scour. These observations are also lised soil is therefore of importance in this case. A key factor in reducing
consistent with Alazigha et al. (2018), MacLaren and White (2003), soil permeability is the presence of additives (polymers – hydrophilic
Chang and Cho (2019) which use gellan gum, xanthan, lignosulfonate, properties) in soil which reduce the pore volume and increase the vis
and cement to increase resistance to erosion. Biopolymers and clays as cosity of pore water, and thereby decreasing soil permeability. Field
inclusion in sands play a similar role in erosion control behaviour, experiments have been conducted using a process of bioclogging and can
increasing the apparent cohesion of the soils and reducing the perme be utilised to tackle seepage problems such as the reduced leakage
ability and void ratio. The erosion test is one of several tests that are used volume of earth dikes and dams through the biofilm method (Blauw
to assess the material’s resistance to running water, simulating rainy et al., 2009). In another field experiment, the biocement grouting
conditions, a preliminary test necessary for further assessment (field method was applied for ground reinforcement and remediation of heavy
studies). metal-contaminated soil water seepage and wind forces (Cuthbert et al.,
2013). Therefore, water repellent agents are considered an effective
• Bio-stabiliser effect: resistance to abrasion method of preventing moisture damage in construction materials.
Recently, a field application of the bio-augmentation – enzyme
The dominating form of deterioration on unpaved or earth roads induced precipitation technology was investigated by Hodges and
depends on whether it is during the wet conditions or dry conditions. Lingwall (2020). They used three large scale field test sites which
Good wearing course on these roads should have the ability to resist focused on mitigation of road surface erosion and dust suppression. One
abrasive action of traffic and erosion by water and wind, freedom from of the test sites with weathered shale showed that the bio-stimulators
excessive dust during dry weather and freedom from excessive slipper possessed potential for mitigation of surface erosion prevention. This
iness during wet weather (Netterberg and Paige-Green, 1988). All un was due to the soil properties containing a clay content opposed to the
paved and earth roads, when dry, suffer from surface abrasion loss sandy soils. In their study the main ‘take-away’ was its several short
resulting in dust generation caused by a reduction in adhesion between comings in terms of field implementation, such as if the soil has large
the soil particles. The movement of loose material when there is insuf concentrations of salts, microbial biomineralisation may not occur at all
ficient binding power of the material to keep the surface intact, leads (i.e., large amounts of salt kill bacteria if chlorine levels exceed that to
into corrugations under traffic action, and ravelling of the road surface which the bacteria have tolerance). By analysis of this case study, two
12
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
research questions for future bio-stabiliser product development and • Testing of top prototypes in real world road subsurface studies.
performance was suggested, (1) will an alternative formulation (using Laboratory simulated tests are to be used to demonstrate perfor
bacterial carriers) for the installation of microbial based bio-stabiliser mance, alternatively the use of the Heavy Vehicle Simulator is
treatment on-site be ideal (opposed to live cultured bacterial suspen recommended.
sions on-site, or transported from laboratories); and (2) will longer
curing times such as 28-days be optimal as this is directly dependent on Limitations identified in current literature
strength gains and positively affects sorption, coating, and the aggre
gation effect of the ingredients by the stabiliser. Numerous experimental and demonstration formats have been con
The aim of this literature survey was to address challenges in road structed for performance testing by companies using their own addi
construction. CSIR (SA) has identified various mechanisms of action for tives. However, very little has been reported, especially with regards to
improving the strength properties of construction materials by using cost and benefits. This has led to suppliers of road additives not being
microbial components and produced metabolites (i.e., bio-enzymatic, able to provide sufficient information to road authorities and engineers
surfactants, ionic, and polymer soil stabilisation), refer to Table 5 for a to make a decision on the appropriate use of these products, which
summary of bio-mediated treatments. This allows for proper isolation would instead use conventional stabilisers rather than implementing a
and assessment of strains based on their ability to produce unique more expensive design (Jones and Ventura, 2004).
compounds of interests, for achieving a specific performance require The most widely available liquid stabilisers are enzyme based, usu
ment, such as workability, mix-ability, binding and compaction result ally verified by laboratory performance testing (Boutique et al., 2007).
ing in longer lasting road surfacing, improved road substructure and For example Taha et al. (2013), Rajoria and Kaur (2014) describes the
reduction of ongoing maintenance requirements. The research is improvement in the engineering properties of the treated soils using
important as it outlines important processes involved for the design and enzyme based products. Field tests reported by Velasquez et al. (2006)
development of soil stabilisation products from Bacillus spp. and related show good performance of enzyme treated soils and Van Veelen and
bio-additives for road construction application. These bio-stabiliser Visser (2007) have also reported significant improvement in CBR values
products are representative of whole cell bacterial suspension, fermen of enzyme-treated material in field test panels. Whereas, testing by
tation components and biopolymers which aim to address challenges in Mgangira (2009a) show variation in performance in the use of enzyme
road construction. At present research focusing on developing novel products for several soil types. The inference from these studies shows a
products from Bacillus spp. Ferment is limited. Therefore, the purpose of clear lack of significant, consistent improvements in the structural
this literature research was to obtain and evaluate the potential use of properties of treated material. Several of these studies also do not cover
fermentation material such as active whole cell, biopolymers and the microstructural details that may be responsible for the mechanisms
‘sticking’ agents produced by microbes in the soil stabilisation processes. that contribute to the interaction between the product and the soil
A summary of key focus areas for development of bio-stabiliser product particles, and the accompanying bio-geotechnical engineering proper
research: ties. As previously outlined, the characteristics of the treated materials
at a micro-scale can lead to an understanding of the expected perfor
• Development of medium- to high-throughput screening to assess key mance of the materials at the macro-scale. It also infers the potential of
structural characteristics of biological components/fractions and modifying the products in order to provide significant improvements in
hence to select the top performers (Ramdas et al., 2020). the stability of treated soils (Mgangira and Ndibewu, 2010) as well as
• Large scale demonstration of functional components and verified select the correct stabiliser for the intended purpose in a consistent
selection of candidate fractions. manner.
• Showing the mode of action (i.e. structural criterion including: A more controlled testing on the performance of bio-stabilisers is
resistance to abrasion, erosion, water absorption and compression needed for unpaved road application, prior to in situ field testing. The
load) of the candidate prototypes. initial testing programme should focus on demonstrating the effective
ness of the various microbial derived bio-additives in terms of bonding
Table 5
Microbial bio-treatment types related to construction-biotechnological processes.
Admixture Biochemical origin Mechanism Descriptive features
Whole cell Whole cell bacterial slurry; Bacillus spp. e.g. B. flexus, Process of metabolically mediated calcium Microbial pore filling material, termed
bacteria & Dry biomass or liquid B. pasteurii, B. licheniformis, carbonate precipitation; bioclogging and ‘Biocement’, water entering freshly formed
spores formulation. B. subtilis, B. cereus. biocementation process. concrete cracks that will activate the
bacteria which in turn will seal micro-
cracks.
Bacterial cell Bound extracellular Aerobic cultivation of Bacillus The cell wall of the Gram-positive B. subtilis Microstructural filler for concrete:
walls substances spp. such as B. subtilis. contains peptidoglycan as the major increasing compressive strength of
(polysaccharides & component, similar chemical structure to construction materials (i.e. concrete) &
proteins) to cells walls. polymers commonly used in construction. decreasing its porosity; reduce segregation &
bleeding of materials.
Bioenzymes Immobilised/liquid Bacillus spp. hydrolytic Involves cationic exchange capacity (CEC), a Reduction in the compaction effort
formulation. enzymes e.g. lipases, ureases, clay-water effect & an interaction of necessary to reach target soil densities &
proteases, phosphatases and enzymes; the electrical double layer of clay improves resistance to freeze–thaw cycling.
phytases particles neutralises the charge of the The enzyme regeneration property is an
particle & breaks down the clay lattice important consideration for long term
causing pore-water dissipation. stabilisation of soil (increasing strength over
time).
Extracellular Mixture of polysaccharides, B. pumus, B. megaterium, Polyelectrolytic EPS contain various The use of bacterial EPS may result in
polymeric amino sugars, proteins, B. cereus, B. flexus, B. subtilis, ionisable functionalities: carboxyl, flocculation & agglomeration, also as
substances teichoic & nucleic acids. B. licheniformis, B. subtilis, & phosphoryl, amino & hydroxyl groups, they hydrophobic agents to act as water erosion
(EPS) B. polymyxa may modify the charge & hydrophilicity of repellents. Due to their interplay of effects
the mineral surfaces, thereby conditioning direct use of microbial components/EPS
surfaces for bacterial attachment, (enzymatic, polymeric, protein) may
colonisation & biofilm formation. improve road construction materials.
13
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
performance at small scale. This test design should inform process Other envisioned applications – environmental
development of standard large scale product prototype production. The
mechanical properties of biocemented sand should be understood before World-wide there’s a strong drive to facilitate environmentally sus
large scale applications, as the stabilising mechanisms of these products tainable– low-carbon economy. The need is to identify ways that
are limited in application especially in predicting their long-term per contribute to this transition. Finding value in solid waste in the prepa
formance and maintenance. Thus, investigating various environmental ration of soil based construction material is one of the several technically
factors prior to field applications which affect the long-term durability feasible and cost-effective solutions for waste management. In South
and biodegradability of a product, such as investigating ground condi Africa, 54.425 tonnes of waste is generated daily with this amount being
tions with rapid fluid flow is important. The current biological approach the 15th highest world-wide. It is not surprising that South Africa’s
requires specific parameters to be addressed for acceptable performance landfill areas are rapidly running out of space (Chvatal and Smit, 2015).
of bio-based additives. These include, ideal temperature, pH, concen The development a novel bio-based technology includes exploring
tration, electron donor, electron acceptors, the rate of nutrients (Ca2+), alternative applications for bio-based products. One such application is
and metabolites. A study showed the in situ microbial biopolymer for the potential for improvement of the mechanical properties of weak soils
mation appears to be less effective in enhancing the erosion resistance, with the addition of by-products and waste material of industrial origin
compared to other biochemical agents, because the quantity of such as fly ash.
biopolymer produced by bacteria is fairly small (< 2% pore saturation). Numerous waste materials can be turned into valuable products and
Ham et al. (2018) suggests re-designing this bio-stimulation strategy. this is a proposed step in the right direction, as these techniques optimise
This review on biological based additives for structural applications the use of local materials. The utilisation of high energy waste material
identifies the significance of an entirely new value chain that is being such as ash, plastic, paper or agricultural waste (i.e. sugarcane bagasse)
established. This presents a challenge for previously unassimilated in as a binding agent can address the numerous challenges faced in soil
dustries like the construction industry. It is evident today that several stabilisation, by focusing on enhancing the particular physical and me
joint ventures have been formed between biotechnology, geochemistry chanical properties of a soil (i.e. improved resistance to erosion or
and civil engineering; this integration will leverage potential synergies. abrasion, hydraulic conductivity, and durability against repeating wet
The research strategy on bio-stabilisers is imperative to ensure that bio- ting and drying, as well as for environmental revitalisation). Knowledge
stabilised materials for the future construction and maintenance of un on the needs in the construction industry, and the symbiotic relationship
paved roads meet acceptable level of service for all road users, within between research and product development is paramount in creating
the limited budget available to road authorities, while protecting the these new opportunities for waste utilisation. The challenge is to find
environment in terms of level of dust generation and gravel loss. low cost but high-volume applications, and to convert waste into value-
added products.
Recommendations for future studies
Industrial wastes as proposed auxiliary additives for in situ soil
In this paper, several microbes and microbial consortia involved in stabilisation
the study of bio-based stabiliser techniques as well as, alternative
manufacturing methods for in situ soil stabilisation are presented. Ba • Ash waste
cillus spp. production of various components, in the fermentation and
dual functioning, makes it a superior candidate for an investigation into Bottom ash is collected from the boilers (i.e. a coarse, granular,
its structural properties. This indicates viable alternatives to chemical or incombustible by-product of coal combustion), and fly ash is obtained
traditional additives for many current uses of earthen construction from the gas washers (i.e. pulverised coal burned in the bottom of
materials. However, more supporting data is required to demonstrate boilers, most of these unburned material is caught in the flue gas) (Le
the superiority, even though the theoretical argument through mecha et al., 2018). Ash is currently a major problem, approximately 46 million
nism description/definition previously stated clearly demonstrates this. tonnes of fly ash is produced globally per annum (Gomes et al., 2020).
The development of bio-stabiliser soil additives/products for the Bottom ash is composed of both organic and inorganic fractions,
construction industry requires a systematic approach in the design of the whereas fly ash consists of more organic content. The heavy metals
experiments. These techniques include targeted isolation of micro- within fly ash are not biodegradable; they can only be transferred from
organisms, followed by medium- to high-throughput screening to a set one chemical state to another, which changes their mobility and
of pre-defined strength criteria that is associated with commercially toxicity. The technical utilisation of ash has potential for more advanced
relevant desirable characteristics. The use of large scale ex situ tests to uses (i.e. deep soil application), however gaps exist in terms of toxicity,
select stabilising additives from large numbers of isolates are expensive, and leaching into the environment (Kumpiene et al., 2008). One pro
time consuming, material limiting and not easily achievable. It is thus posed solution to counteract this is the use of micro-organisms. Micro-
critical to perform extensive in-vitro evaluation and selection processes organisms can influence metals in several ways, (a) some forms of metals
to reduce the number of isolates, biological fractions, and material costs. can be transformed either by a redox reaction or by alkylation; (b)
This pre-assessment in conjunction with large scale strength tests (i.e., metals can also be accumulated by micro-organisms by metabolism-
brush test, erosion test, and trial compression testing) has basis and will independent (passive) or by intracellular metabolism-dependent
provide an overall indication of the bio-stabiliser/s strength character (active) uptake; (c) micro-organisms can influence metal mobility
istics as previously proposed in Rauch et al. (2002). A step-wise process indirectly by affecting pH; and (d) by producing or releasing compounds
is provided for potentially establishing new bio-based stabilisers for road which change mobility of the metals (Igiri et al., 2018). The proposed
application: bio-molecules (i.e. EPS) derived from Bacillus spp. and/or in synergy
with other green composites (Fig. 4) are postulated as potential carriers/
• Development of a Bio-stabiliser for application on selected in situ stabilising agents of ash waste.
materials.
• Study of the effect of Bio-stabilisers on soil microstructure. • Sugarcane bagasse
• Elucidation of Bio-stabiliser-soil interaction mechanisms.
• Establish the application of Bio-stabiliser in road construction under Sugar cane bagasse is a solid waste with economic importance,
South African conditions in a field trial. produced as a by-product from the sugar manufacturing industry in
• Compare the technology – technical, economical and environmental enormous quantities (South African sugar mills produce 24.7 million
impact against other commercial benchmark products. tonnes of sugarcane annually, each 10 tonnes of sugarcane crushed
14
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
produces 3 tonnes of bagasse) (Muniyasamy et al., 2016). Bagasse is the hydroxyl, and hydrophobic groups consisting of carbon chains. The
fibrous residue remaining after extraction of the cane juice from sug stabilisation effect of the LS-treatment leads to reduction in Plasticity
arcane. Sugarcane bagasse consists of approximately 50% cellulose, Index (PI) of the soil, improved stiffness and UCS of the soil without any
25% hemicellulose, and 25% lignin. Whereas, sugarcane bagasse ash brittle nature. The increase in strength properties is attributed to the
consists of silica rich material that can play the role of an effective electrostatic reaction that occurs between the LS-water mixture and soil
pozzolans leading to enhanced pozzolanic reactions resulting in better particles resulting in the PI changes which could be due to the alteration
performing building materials (James and Pandian, 2017). The eco of the lattices of the soil minerals (i.e., decrease in crystalline size of the
nomic importance of these solid waste materials has been realised with double layer thickness of clay minerals) and better flocculation prop
several applications such as, adsorbents, filters, ceramics, briquettes, erties of the soil (i.e., formation of stable soil aggregation), this in effect
bricks, and soil modifiers. In addition, the use of bioplastics (i.e. PE), a improves the plasticity and the stabilised soil strength. Since LS is a by-
recent development from bagasse and maize stalks may have a future product of other processes (i.e., produced annually in large quantities
role in road construction due to their permeability properties, remedi (50 million tonnes) worldwide), they are relatively cheap and can be
ation of soil and high tensile strength (Muniyasamy et al., 2016). With competitive with other chemical stabilisers (Zhang et al., 2017).
regards to the biodegradable aspect of bio-based materials, the tests are
complex and subject to the environment in which the testing is Conclusion
completed, however standards are available, (i.e., American Society for
Testing and Materials (ASTM) and International Organization for Stan In summary, the construction industry is developing new opportu
dardization (ISO)) to adopt, and test green composite materials in soil nities involving the use of biological processes and products to modify
environments, to manage their end-life cycle. It is notable for con the structural properties of subpar soils (i.e., strength, volume stability,
struction purposes, that not all bioplastics are biodegradable but instead durability, and permeability) through innovative engineering tech
recyclable (see Fig. 5) (Muniyasamy et al., 2013). niques. The bio-based stabilisation technology such as bio-stabilisers has
the potential to reduce the use of conventional options such as cement
• Lignin-based waste and lime, whose production processes results in a higher environmental
load/GHG emissions and therefore preserve the environment and nat
Lignin is a by-product of paper and timber industry. Similarly, wood ural resources. Enzymes and polymers have been long introduced into
is composed of cellulose, hemicellulose, lignin, and extractives, while the market as soil stabilisers. However, the application of these imported
lignin is resistant to biodegradation in an anaerobic environment and eco-friendly products to improve the engineering properties of weak soil
can remain in a landfill for exceedingly long periods of time (Ramage is not readily adopted in countries such as, South Africa. Furthermore,
et al., 2017). Lignin is the most abundant organic polymer in existence novel biopolymers or ‘sticking agents’ of microbial origin are currently
on earth after cellulose. Sustainable reuse options for lignin in civil not manufactured in South Africa. In addition, these complexities and
engineering applications, such as road embankments and dam founda limitations can be circumvented by recognising that Bacillus spp. rep
tions or use of lignin as a stabilisation additive for soil may be viable resents unique and exclusive assemblages of diversity. The development
options for the reuse of this bio-based organic by-product (Zhang et al., of a bio-stabiliser product from an indigenous strain (Bacillus spp.) could
2017). Lignosulfonate (LS) (i.e. industrial by-product) is also being therefore address this gap. Microorganisms and their products such as
investigated as a soil stabiliser (Ta’negonbadi and Noorzad, 2017). In secondary metabolites, enzymes, endospores, and extracellular poly
this research by Ta’negonbadi and Noorzad (2017), improved soil meric substances have been considered as interesting alternatives to
properties was induced by a polymer with lignin-base that included conventional chemical soil stabilisers for the development of sustainably
hydrophilic groups, comprising of alcoholic hydroxyl sulfonate, phenyl road infrastructure. The primary challenges with this bio-based
15
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
approach is microorganism selection and bioprocess optimisation to Ayeldeen M, Negm A, El-Sawwaf M, Kitazume M. Enhancing mechanical behaviors of
collapsible soil using two biopolymers. JRMGE 2017;9:329–39. https://doi.org/
obtain maximum yields of the desired product for testing and use. The
10.1016/j.jrmge.2016.11.007.
extracellular components, consisting of EPS, enzymes, and biopolymers, Bang SC, Bang SS, Choi SR, Lee SJ, Lee JH and Kim JS. Echo Phile, Lotte Engineering &
are hypothesised to create a process of cross-linking with various soil Construction and South Dakota School of Mines, 2015. Method of manufacturing soil
particles via various interactions. This in essence, has the potential of reinforced by microbe-based bio-binders and soil produced by the method. U.S.
Patent 9,074,134.
improving the strength properties by improving shear strength and Babu RP, O’Connor K, Ramakrishna K. Current progress on bio-based polymers and their
compressibility of soils. It is therefore essential that the active ingredient future trends. Progr Biomater 2013;2. https://doi.org/10.1186/2194-0517-2-8.
contained in the product is produced at a high concentration during Burrow MPN, Evdorides H, Ghataora GS, Petts R, Snaith MS. The evidence for rural road
technology in low-income, countries. Thomas Telford Ltd.; 2016. p. 366–77.
fermentation for an efficacious product. Adoption of the bio-stabiliser Blauw M, Lambert J, Latil MN. Biosealing: A method for in situ sealing of leakages. In:
technology has the potential to improve road infrastructure delivery Proceedings of the international symposium on ground improvement technologies
and therefore contribute toward socio-economic development. By using and case histories; 2009. p. 125–30. 10.3850/GI132.
Boutique JP, Baeck A, Vanwyngaerden N, Stonehouse J, Gizaw Y and Rothgeb T. 2007.
bio-stabilisers, in situ waste utilisation, such as fly ash, may be possible in Enzyme stabilizer: Google Patents.
construction, which reduces waste to landfill. The application of the Bryson S, El Naggar H. Evaluation of the efficiency of different ground improvement
proposed technologies and products are envisioned to improve strength techniques. Proceeding of the 18th International Conference on Soil Mechanics and
Geotechnical Engineering. 2013.
properties of in situ materials and reduce permeability characteristics. Carvalho NB, Barbosa JMP, Oliveira MVS, Fricks AT, Lima ÁS, Soares CMF. Biochemical
This may result in improved subgrade quality and marginal materials for properties of Bacillus sp. ITP-001 lipase immobilized with a sol gel process. Quim
potential application in structural pavement layers and for wearing Nova 2013;36(52–58). https://doi.org/10.1590/S0100-40422013000100010.
Chang I, Cho GC. Shear strength behavior and parameters of microbial gellan gum-
coarse on unpaved roads. The stabilised soil may also be suitably used
treated soils: From sand to clay. Acta Geotech. 2019;14:361–75. https://doi.org/
for constructing pavement layers of low volume, rural (unpaved) roads, 10.1007/s11440-018-0641-x.
bicycle tracks, and pedestrian walkways. Of interest are the other areas Chang I, Im J, Cho GC. Introduction of microbial biopolymers in soil treatment for future
of opportunity beyond normal soil stabilisation such as, compressed environmentally-friendly and sustainable geotechnical engineering. Sustainability
2016;8:251. https://doi.org/10.3390/su8030251.
earth blocks (CEB blocks), promoting low technology manufacturing Chang I, Prasidhi AK, Im J, Cho GC. Soil strengthening using thermo-gelation
from waste material and soil. This will help in ensuring effective biopolymers. Constr Build Mater 2015a;77:430–8. https://doi.org/10.1016/j.
implementation and proper use of natural resources as well as, bring conbuildmat.2014.12.116.
Chang I, Im J, Prasidhi AK, Cho GC. Effects of Xanthan gum biopolymer on soil
about transparency against the intrusion of reinforcing physical pro strengthening. Constr Build Mater 2015b;74(65–72). https://doi.org/10.1016/j.
cesses or the use of hazardous chemicals in soil to create artificial conbuildmat.2014.10.026.
binding. Therefore, in view of the enormous challenges and rising de Chang I, Cho GC. Strengthening of Korean residual soil withβ-1,3/1,6-glucan
biopolymer. Constr Build Mater 2012;30:30–5. https://doi.org/10.1016/j.
mand for sustainable alternative solutions, novel biological in conbuildmat.2011.11.030.
terventions (i.e. bioenzymatic, and biopolymer soil stabilisation) is the Cheng L, Cord-Ruwisch R, Shahin MA. Cementation of sand soil by microbially induced
logical way forward to improve conventional soil stabilisation methods. calcite precipitation at various degrees of saturation. Can Geotech J 2013;50:81–90.
https://doi.org/10.1139/cgj-2012-0023.
Chu J, He J, Ivanov V. Mitigation of liquefaction of saturated sand using biogas.
Geotechnique 2013;63:267–75. https://doi.org/10.1680/geot.SIP13.P.004.
Declaration of Competing Interest Chvatal JA, Smit ADV. Waste management policy: Implications for landfill waste
salvagers in the Western Cape. IJEWM 2015;16:1–26. https://doi.org/10.1504/
IJEWM.2015.070480.
The authors declare that they have no known competing financial Costa F, Silva B and Tavares T. Biofilm Bioprocesses. In: Christian Larroche C, Sanromán
interests or personal relationships that could have appeared to influence MA, Du G, Pandey A, editors. Current Developments in Biotechnology and
the work reported in this paper. Bioengineering: Bioprocesses, Bioreactors and Controls. Elsevier, 2017, p. 143. Doi:
10.1016/B978-0-444-63663-8.00006-9.
Cuthbert MO, Mcmillan LA, Handley-Sidhu S, Riley MS, Tobler DJ, Phoenix VR. A field
Acknowledgements and modeling study of fractured rock permeability reduction using microbially
induced calcite precipitation. Environ Sci Technol 2013;47:13637–43. https://doi.
org/10.1021/es402601g.
Thanks to the Department of Science and Innovation (DSI), and the Das SK and Varma A. Role of enzymes in maintaining soil health, In: Shukla G and Varma
Council for Scientific and Industrial Research (South Africa, ZA) for the A, editors, Soil Enzymology. Berlin Heidelberg: Springer, 2010, p. 25-42. Doi:
funding support. 10.1007/978-3-642-14225-3_2.
Dejong J, Soga K, Kavazanjian E, Burns S, Van Paassen L, Al Qabany A, et al.
Biogeochemical processes and geotechnical applications: Progress, opportunities and
References challenges. Geotechnique 2013;63:287. https://doi.org/10.1680/geot.SIP13.P.017.
Dejong JT, Mortensen BM, Martinez BC, Nelson DC. Bio-mediated soil improvement. Ecol
Eng 2010;36:197–210. https://doi.org/10.1016/j.ecoleng.2008.12.029.
AbouKhadra A, Zidan AF, Gaber Y. Experimental evaluation of strength characteristics of
DeJong JT, Fritzges MB, Nusslein K. Microbially Induced Cementation to Control Sand
different Egyptian soils using enzymatic stabilizers. Cogent Eng 2018;5:1517577.
Response to Undrained Shear. J Geotech Geoenviron Eng 2006;132:1381–92.
https://doi.org/10.1080/23311916.2018.1517577.
Dhami NK, Reddy MS, Mukherjee A. Improvement in strength properties of ash bricks by
Achal V, Mukherjee A, Reddy MS. Effect of calcifying bacteria on permeation properties
bacterial calcite. Ecol Eng 2012;39:31–5. https://doi.org/10.1016/j.
of concrete structures. J Ind Microbiol Biotechnol 2011;38:1229–34. https://doi.
ecoleng.2011.11.011.
org/10.1007/s10295-010-0901-8.
Fatehi H, Abtahi SM, Hashemolhosseini H, Hejazi SM. A novel study on using protein
Afifudin H, Nadzarah W, Hamidah M, Hana HN. Microbial participation in the formation
based biopolymers in soil strengthening. Constr Build Mater 2018;167:813–21.
of calcium silicate hydrated (CSH) from Bacillus subtilis. Procedia Eng 2011;20:
https://doi.org/10.1016/j.conbuildmat.2018.02.028.
159–65. https://doi.org/10.1016/j.proeng.2011.11.151.
Firoozi AA, Olgun CG, Firoozi AA, Baghini MS. Fundamentals of soil stabilization. Int J
Alamgir M, Campbell MJ, Sloan S, Goosem M, Clements GR, Mahmoud MI, et al.
Geo-Eng 2017;8:26.
Economic, socio-political and environmental risks of road development in the
Gao Y, Hang L, He J, Chu J. Mechanical behaviour of biocemented sands at various
tropics. Curr Biol 2017;27:R1130–40. https://doi.org/10.1016/j.cub.2017.08.067.
treatment levels and relative densities. Acta Geotech 2019;14:697–707. https://doi.
Alazigha DP, Indraratna B, Vinod JS, Heitor A. Mechanisms of stabilization of expansive
org/10.1007/s11440-018-0729-3.
soil with lignosulfonate admixture. Transp Geotech 2018;14:81–92. https://doi.org/
Ghosh P, Mandal S, Chattopadhyay B, Pal S. Use of microorganism to improve the
10.1016/j.trgeo.2017.11.001.
strength of cement mortar. Cem Concr Res 2005;35:1980–3. https://doi.org/
Alzard MH, Maraqa MA, Chowdhury R, Khan Q, Albuquerque FD, Mauga TI, et al.
10.1016/j.cemconres.2005.03.005.
Estimation of greenhouse gas emissions produced by road projects in Abu Dhabi,
Gomes HI, Funari V, Ferrari R. Bioleaching for resource recovery from low-grade wastes
United Arab Emirates. Sustainability 2019;11:2367. https://doi.org/10.3390/
like fly and bottom ashes from municipal incinerators: A SWOT analysis. Sci Total
su11082367.
Environ 2020;715:136945.
Andrews RC. Surface longevity treatments for unsealed roads. Transport SA MTRD
Greening T. Quantifying the impact of vehicle-generated dust: A comprehensive
Report no. 97/PA/056 Adelaide, South Australia. 1999; 1-68.
approach. Report Prepared for Bank for Reconstruction and Development/The World
Assam S, Okafor F, Umoh U. Potentials of processed termite as a stabilizing agent in clay
Bank. 2011. http://siteresources.worldbank.org/.
soil. J Mech Civ Eng 2016;13:40–50. https://doi.org/10.9790/1684-1304014050.
Guncheva M, Zhiryakova D. Catalytic properties and potential application of Bacillus
Averous L and Pollet E. 2012. Biodegradable polymers. In: Avérous L, Pollet E, editors.
lipases. J Mol Catal B-Enzym 2011;68:1–21. https://doi.org/10.1016/j.
Environmental silicate nano-biocomposites. London: Springer; 2012; 13–39. Doi:
molcatb.2010.09.002.
10.1007/978-1-4471-4108-2.
16
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
Guthrie WS, Simmons DO, Eggett DL. Enzyme stabilization of low-volume gravel roads. Ma W, Peng D, Walker SL, Cao B, Gao CH, Huang Q, et al. Bacillus subtilis biofilm
Transp Res Rec 2015;2511:112–20. https://doi.org/10.3141/2511-13. development in the presence of soil clay minerals and iron oxides. NPJ Biofilms
Ham SM, Chang I, Noh DH, Kwon TH, Muhunthan B. Improvement of surface erosion Microbiomes 2017;3:1–9.
resistance of sand by microbial biopolymer formation. J Geotech Geoenviron Eng Maclaren DC, White MA. Cement: its chemistry and properties. J Chem Educ 2003;80:
2018;144:06018004. https://doi.org/10.1061/(ASCE)GT.1943-5606.0001900. 623. https://doi.org/10.1021/ed080p623.
Hodges TM and Lingwall BN. February. Laboratory Study in the Treatment of Burned Maharajh D, Roth R, Lalloo R, Simpson C, Mitra R, Görgens J, et al. Multi-copy
Soils with Microbial Augmentation for Erosion Control. In: Geo-Congress 2020: expression and fed-batch production of rhodotorula araucariae epoxide hydrolase in
Biogeotechnics. 2020: 20-28. Reston, VA: American Society of Civil Engineers. yarrowia lipolytica. Appl Micro Biotech 2008;79:235–44. https://doi.org/10.1007/
Hottle TA, Bilec MM, Landis AE. Sustainability assessments of bio-based polymers. Polym s00253-008-1420-7.
Degrad Stabil 2013;98:1898–907. https://doi.org/10.1016/j. Makusa GP. Soil stabilization methods and materials in engineering practice: State of the
polymdegradstab.2013.06.016. art review. Luleå Tekniska Universitet 2013.
Igiri BE, Okoduwa SI, Idoko GO, Akabuogu EP, Adeyi AO, Ejiogu IK. Toxicity and Marasteanu MO, Hozalski RM, Clyne TR, Velasquez R. Preliminary laboratory
bioremediation of heavy metals contaminated ecosystem from tannery wastewater: investigation of enzyme solutions as a soil stabiliser. Minnesota: University of
A review. J Toxicol 2018. https://doi.org/10.1155/2018/2568038. Minnesota Department of Civil Engineering; 2005.
Ikeagwuani CC, Nwonu DC. Emerging trends in expansive soil stabilisation: A review. Meyer F, Bang S, Min S, Stetler L, Bang S. Microbiologically-induced soil stabilization:
J Rock Mech Geotech Eng 2019;11:423–40. https://doi.org/10.1016/j. application of Sporosarcina pasteurii for fugitive dust control. In: Geo-frontiers 2011
jrmge.2018.08.013. Advances in geotechnical engineering; 2011. p. 4002–11.
Ivanov V, Chu J, Stabnikov V. Basics of construction microbial biotechnology. In: Mgangira M, 2009a. Evaluation of the effects of enzyme-based liquid chemical stabilizers
Pacheco Torgal F, Labrincha JA, Diamanti MV, Yu CP, Lee HK, editors. on subgrade soils. Paper presented at the Southern African Transport Conference
Biotechnologies and biomimetics for civil engineering biotechnologies and (SATC) South Africa. Available: https://researchspace.csir.co.za/dspace/handle/1
biomimetics for civil engineering. Switzerland: Springer; 2015. p. 21–56. 0204/3654 (Accessed 05.01.2018).
Ivanov V, Stabnikov V. Basic concepts on biopolymers and biotechnological admixtures Mgangira M. Properties controlling the resistance to abrasion and erosion of stabilized
for eco-efficient construction materials. In: Pacheco-Torgal F, Ivanov V, Karak N, sandy soils using non-traditional additives. In: Proceedings of Proceedings of the
Jonkers H, editors. Biopolymers and biotech admixtures for eco-efficient 17th International Conference on Soil Mechanics and Geotechnical Engineering: The
construction materials. first ed. England: Woodhead Publishing; 2016. p. 253. Academia and Practice of Geotechnical Engineering, Alexandria, Egypt, IOS Press,
James J, Pandian PK. A short review on the valorisation of sugarcane bagasse ash in the volume 1. IOS Press, 2009b; 213-216.
manufacture of stabilized/sintered earth blocks and tiles. Adv Mater Sci Eng 2017. Mgangira M and Ndibewu P. Identification of microscale characteristics of treated
https://doi.org/10.1155/2017/1706893. subgrade materials and how they relate to macroscopic properties. In: Proceedings of
Jayathileka S and Adikari A. Report on Determination on effect of natural Enzyme Proceedings of the 29th Southern African Transport Conference (SATC 2010), South
(Permazyme 11X) on Soil Stabilization. Report no. CP-2563, Industrial technology Africa, 2010.
Institute, Colombo, Sri Lanka. 2007. Mikutta R, Zang U, Chorover J, Haumaier L, Kalbitz K. Stabilization of extracellular
Jiang NJ, Soga K, Kuo M. Microbially induced carbonate precipitation for seepage- polymeric substances (Bacillus subtilis) by adsorption to and coprecipitation with Al
induced internal erosion control in sand–clay mixtures. J Geotech Geoenviron Eng forms. Geochim Cosmochim Acta 2011;75:3135–54. https://doi.org/10.1016/j.
2016;143:04016100. https://doi.org/10.17863/CAM.24. gca.2011.03.006 3136.
Jimenez-Lopez C, Jroundi F, Pascolini C, Rodriguez-Navarro C, Piñar-Larrubia G, Moloisane R, Visser AT. Evaluation of the strength behaviour of unpaved road material
Rodriguez-Gallego M, et al. Consolidation of quarry calcarenite by calcium treated with electrochemical-based non-traditional soil stabilisation additives. J S
carbonate precipitation induced by bacteria activated among the microbiota Afr Inst Civ Eng 2014;56:28–39.
inhabiting the stone. Int Biodeter Biodegr 2008;62:352–63. https://doi.org/ Morales L, Garzón E, Romero E, Sánchez-Soto PJ. Microbiological induced carbonate
10.1016/j.ibiod.2008.03.002. (CaCO3) precipitation using clay phyllites to replace chemical stabilizers (cement or
Jones D, Ventura D. A procedure for fit-for-purpose certification of non-traditional road lime). Appl Clay Sci 2019;174:15–28. https://doi.org/10.1016/j.clay.2019.03.018.
additives. Proceedings of the 24th SATC, South Africa. 2004. Muniyasamy S, Anstey A, Reddy MM, Misra M, Mohanty A. Biodegradability and
Jonkers H, Mors R, Sierra-Beltran M, Wiktor V. Biotech solutions for concrete repair with compostability of lignocellulosic based composite materials. J Renew Mater 2013;1:
enhanced durability. In: Pacheco-Torgal F, Ivanov V, Karak N, Jonkers H, editors. 253–72. https://doi.org/10.7569/JRM.2013.634117.
Biopolymers and biotech admixtures for eco-efficient construction materials. first ed. Muniyasamy S, Ofosu O, John MJ, Anandjiwala RD. Mineralization of poly (lactic acid)
England: Woodhead Publishing; 2016. p. 253. (PLA), poly (3-hydroxybutyrate-co-valerate)(PHBV) and PLA/PHBV blend in
Jonkers HM, Schlangen E. Crack repair by concrete-immobilized bacteria. In: compost and soil environments. J Renew Mater 2016;4:133–45. https://doi.org/
Proceedings of the first international conference on self healing materials; 2007. 10.7569/JRM.2016.634104.
p. 1–7. Nafisi A. Elucidating the Failure Behavior and Bond Mechanics of Bio-cemented Sands.
Joshi K, Kumthekar MB, Godhake VP. Bacillus subtillis bacteria impregnation in concrete North Carolina State University; 2019.
for enhancement in compressive strengh. IRJET 2016;3:1129–239. National Treasury. Provincial budgets and expenditure review 2010/11 – 2016/17,
Khan TA, Taha MR. Effect of three bioenzymes on compaction, consistency limits, and South Africa, National Treasury, Pretoria, South Africa. 2015.
strength characteristics of a sedimentary residual soil. Adv Mater Sci Eng 2015. Netterberg F and Paige-Green P. Wearing coarse for unpaved roads in southern Africa.
https://doi.org/10.1155/2015/798965. Review: Proceedings of the 8th Quinquennial Convention of South African institute
Katz L, Rauch A, Liljestrand H, Harmon J, Shaw K, Albers H. Mechanisms of soil for Civil Engineers and Annual Transportation Convention, Pretoria, 1988. Vol. 2D.
stabilization with liquid ionic stabilizer. Transp Res Rec 2001:50–7. 10.3141/1757- Ng W, Lee M, Hii S. An overview of the factors affecting microbial-induced calcite
06. precipitation and its potential application in soil improvement. Int J Civ Environ Eng
Kumar S, Pahujani S, Ola RP, Ola RP, Kanwar SS, Kanwar SS, et al. Enhanced 2012;6:188–94. https://doi.org/10.5281/zenodo.1084674.
thermostability of silica-immobilized lipase from Bacillus Coagulans BTS-3 and Oyediran IA, Ayeni OO. Comparative effect of microbial induced calcite precipitate,
synthesis of ethyl propionate. Acta Microbiol Imm H 2006;53:219–31. https://doi. cement and rice husk ash on the geotechnical properties of soils. SN Appl Sci 2020;2:
org/10.1556/amicr.53.2006.2.8. 1–12. https://doi.org/10.1007/s42452-020-2956-0.
Kumar CG, Joo HS, Choi JW, Koo YM, Chang CS. Purification and characterization of an Pacheco-Torgal F. Introduction to biopolymers and biotech admixtures for eco-efficient
extracellular polysaccharide from haloalkalophilic Bacillus sp. I-450. Enzyme Microb construction materials. In: Pacheco-Torgal F, Ivanov V, Karak N, Jonkers H, editors.
Technol 2004;34(673–681). https://doi.org/10.1016/j.enzmictec.2004.03.001. Biopolymers and Biotech admixtures for eco-efficient construction materials. first ed.
Kumpiene J, Lagerkvist A, Maurice C. Stabilization of As, Cr, Cu, Pb and Zn in soil using England: Woodhead Publishing; 2016. p. 1–5.
amendments – A Review. J Waste Manag 2008;28:215–25. https://doi.org/10.1016/ Pacheco-Torgal F, Labrincha J. Biotech cementitious materials: Some aspects of an
j.wasman.2006.12.012. innovative approach for concrete with enhanced durability. Constr Build Mater
Larson S, Newman JK, O Connor G, Griggs C, Martin A, Nijak G, Lord E, Duggar R, 2013;40:1136–41. https://doi.org/10.1016/j.conbuildmat.2012.09.080.
Leeson A. Biopolymer as an Alternative to Petroleum-based Polymers to Control Soil Pacwa-Płociniczak M, Płaza GA, Piotrowska-Seget Z, Cameotra SS. Environmental
Erosion: Iowa Army Ammunition Plant. Environmental security technology applications of biosurfactants: Recent advances. Int J Mol Sci 2011;12:633–54.
certification program office (DOD). Arlington Va 2013. https://doi.org/10.3390/ijms12010633.
Latifi N, Marto A, Eisazadeh A. Structural characteristics of laterite soil treated by SH-85 Paige-Green P, Dealing with road subgrade problems in Southern Africa, In: the 12th
and TX-85 (non-traditional) stabilizers. EJGE 2013;18:1707–18. international conference of international association for computer methods and
Latifi N, Meehan CL, Majid MZA, Horpibulsuk S. Strengthening montmorillonitic and advances in geomechanics (IACMAG), India, 2008; 4345-4353.
kaolinitic clays using a calcium-based non-traditional additive: A micro-level study. Panchal S, Khan MM, Sharma A. Stabilization of soil using bio-enzyme. Int J Civ Eng
Appl Clay Sci 2016;132:182–93. https://doi.org/10.1016/j.clay.2016.06.004. 2017;8:234–7.
Le TM, Dang LC, Khabbaz H. Combined effects of bottom ash and lime on behaviour of Pei R, Liu J, Wang S. Use of bacterial cell walls as a viscosity-modifying admixture of
expansive soil. In: International Congress and Exhibition, Sustainable Civil concrete. Cem Concr Compos 2015;55:186–95. https://doi.org/10.1016/j.
Infrastructures: Innovative Infrastructure Geotechnology. Springer; 2018. p. 28–44. cemconcomp.2014.08.007.
10.1007/978-3-030-01914-3_3. Peng HT, Su HT, Zhang XP, Wang J. An Experimental Comparison of Compressive
Lim SM, Wijeyesekera D, Lim A, Bakar I. Critical review of innovative soil road Strengths of Soils Stabilized with Enzyme and Ground Quicklime. Adv Mater Res
stabilization techniques. IJEAT 2014;3:204–11. 2011;280:9–12. https://doi.org/10.4028/www.scientific.net/AMR.280.9.
Limoli DH, Jones CJ, Wozniak DJ. Bacterial extracellular polysaccharides in biofilm Plank J. Applications of biopolymers and other biotechnological products in building
formation and function. Microbiol Spectr 2015;3. https://doi.org/10.1128/ materials. Appl Micro Biotech 2004;66:1–9. https://doi.org/10.1007/s00253-004-
microbiolspec.MB-0011-2014. 1714-3.
Portilho M, Matioli G, Zanin GM, Moraes FF and Scamparini ARP. Production of
insoluble exopolysaccharide of Agrobacterium sp. (ATCC 31749 and IFO 13140). In:
17
V.M. Ramdas et al. Transportation Geotechnics 27 (2021) 100458
McMillan JD, Adney WS, Mielenz JR, Klasson KT, editors, Twenty-Seventh Specialised Protection Products, Gauteng, South Africa. Available: http://www.
Symposium on Biotechnology for Fuels and Chemicals. ABAB Symposium. Humana specializedproducts.co.za/. (Accessed 05.01.2020).
Press, 2006; 864-869. Doi: 10.1007/978-1-59745-268-7_70. Stabnikov V, Naeimi M, Ivanov V, Chu J. Formation of water-impermeable crust on sand
Rajoria V, Kaur S. A review on stabilization of soil using bio-enzyme. IJRET 2014;3:78. surface using biocement. Cem Concr Res 2011;41:1143–9. https://doi.org/10.1016/
Ramage MH, Burridge H, Busse-Wicher M, Fereday G, Reynolds T, Shah DU, et al. The j.cemconres.2011.06.017.
wood from the trees: The use of timber in construction. Renew Sustain Energy Rev Stocks-Fischer S, Galinat JK, Bang SS. Microbiological precipitation of CaCO3. Soil Biol
2017;68:333–59. Biochem 1999;31:1563–71. https://doi.org/10.1016/S0038-0717(99)00082-6.
Ramdas VM, Mandree P, Mgangira M, Mukaratirwa S, Lalloo R, Ramchuran S. Sukmak P, Horpibulsuk S, Shen SL. Strength development in clay–fly ash geopolymer.
Establishing miniaturised structural testing techniques to enable high-throughput Constr Build Mater 2013;40:566–74. https://doi.org/10.1016/j.
screening of microorganisms and microbial components for unpaved road conbuildmat.2012.11.015.
stabilisation application. J Adv Res 2020;21:151–9. https://doi.org/10.1016/j. Ta’negonbadi B, Noorzad R. Stabilization of clayey soil using lignosulfonate. Transp
jare.2019.11.002. Geotech 2017;12:45–55. https://doi.org/10.1016/j.trgeo.2017.08.004.
Rao MVS, Reddy VS, Sasikala C. Performance of microbial concrete developed using Taha MR, Khan TA, Jawad IT, Firoozi AA, Firoozi AA. Recent experimental studies in soil
Bacillus subtilus JC3. J Insti Eng (India): Series A. 2017;98(501–510). https://doi. stabilization with bio-enzymes-a review. J Geotech Eng 2013;18:3881–94.
org/10.1007/s40030-017-0227-x. Tastan EO, Edil TB, Benson CH, Aydilek AH. Stabilization of organic soils using fly ash.
Rauch A, Harmon J, Katz L, Liljestrand H. Measured effects of liquid soil stabilizers on J Geotech Geoenviron 2011;137:819–33. https://doi.org/10.1061/(ASCE)GT.1943-
engineering properties of clay. Transp Res Rec 2002:33–41. https://doi.org/ 5606.0000502.
10.3141/1787-04. Thomas AG, Rangaswamy BK. Strength behavior of enzymatic cement treated clay. I J
Raut SH, Sarode DD, Lele SS. Biocalcification using B. pasteurii for strengthening brick Geotech Eng 2019:1–14. https://doi.org/10.1080/19386362.2019.1622854.
masonry civil engineering structures. World J Microbiol Biotechnol 2014;30 Urrutia MM, Beveridge TJ. Formation of fine grained metal and silicate precipitates on a
(191–200). https://doi.org/10.1007/s11274-013-1439-5. bacterial surface (Bacillus subtilis). Chem Geol 1994;116:261–80. https://doi.org/
Rehm BH. Bacterial polymers: Biosynthesis, modifications and applications. Nat Rev 10.1016/0009-2541(94)90018-3.
Microbiol 2010;8:578–92. https://doi.org/10.1038/nrmicro2354. Vahabi A, Ramezanianpour AA, Sharafi H, Zahiri HS, Vali H, Noghabi KA. Calcium
Roa R, Kumar U, Vokunnaya S, Paul P, Orestis I. Effect of Bacillus flexus in healing carbonate precipitation by strain Bacillus licheniformis AK01, newly isolated from
concrete structures. IJIRSET 2015;4:7273–80. https://doi.org/10.15680/ loamy soil: A promising alternative for sealing cement-based materials. J Basic
IJIRSET.2015.0408106. Microbiol 2015;55:105–11. https://doi.org/10.1002/jobm.201300560.
Saleh S, Yunus NZM, Ahmad K, Ali N. Improving the strength of weak soil using Van Paassen LA, Ghose R, van der Linden TJ, van der Star WR, van Loosdrecht MC.
polyurethane grouts: A review. Constr Build Mater 2019;202:738–52. https://doi. Quantifying biomediated ground improvement by ureolysis: large-scale biogrout
org/10.1016/j.conbuildmat.2019.01.048. experiment. J Geotech Geoenviron 2010;136:1721–8.
Samang L, Harianto T, Djide MN. Bearing capacity improvement of soft soil subgrade Van Tittelboom K, De Belie N, De Muynck W, Verstraete W. Use of bacteria to repair
layer with bio stabilised Bacillus subtilis. MATEC Web Conferences EDP Sci 2018. cracks in concrete. Cem Concr Res 2010;40:157–66. https://doi.org/10.1016/j.
https://doi.org/10.1051/matecconf/201818101001. cemconres.2009.08.025.
Sarda D, Choonia HS, Sarode D, Lele S. Biocalcification by bacillus pasteurii urease: A Van Veelen M, Visser AT. The performance of unpaved road material using soil
novel application. J Ind Microbiol Biotechnol 2009;36:1111–5. https://doi.org/ stabilisers. J S Afr Inst Civ Eng 2007;49:2–9.
10.1007/s10295-009-0581-4. Vandevivere P, Baveye P. Effect of bacterial extracellular polymers on the saturated
Sardari RR, Kulcinskaja E, Ron EY, Björnsdóttir S, Friðjónsson ÓH, Hreggviðsson GÓ, hydraulic conductivity of sand columns. Appl Environ Microbiol 1992;58:1690–8.
et al. Evaluation of the production of exopolysaccharides by two strains of the https://doi.org/10.1128/AEM.58.5.1690-1698.1992.
thermophilic bacterium rhodothermus marinus. Carbohydr Polym 2017;156:1–8. Velasquez R, Marasteanu MO, Hozalski R. Investigation of the effectiveness and
https://doi.org/10.1016/j.carbpol.2016.08.062. mechanisms of enzyme products for subgrade stabilisation. Int J Pavement Eng
Schallmey M, Singh A, Ward OP. Developments in the use of bacillus species for 2006;7:213–20.
industrial production. Can J Microbiol 2004;50:1–17. https://doi.org/10.1139/w03- Velde KVD, Kiekens P. Biopolymers: Overview of several properties and consequences on
076. Doi: 10.1139/w03-076. their applications. Polym Test 2002;21:433–42. https://doi.org/10.1016/S0142-
Seco A, Ramírez F, Miqueleiz L, García B. Stabilization of expansive soils for use in 9418(01)00107-6.
construction. Appl Clay Sci 2011;51:348–52. https://doi.org/10.1016/j. Visser AT and Hudson WR. Design and maintenance criteria for unpaved roads. National
clay.2010.12.027. Institute for Transport and Road Research, Report (RR337), CSIR, Pretoria, South
Seto KC, Güneralp B, Hutyra LR. Global forecasts of urban expansion to 2030 and direct Africa. 1983.
impacts on biodiversity and carbon pools. Proc Natl Acad Sci 2012;109 Wang J, Soens H, Verstraete W, De Belie N. Self-healing concrete by use of
(16083–16088). https://doi.org/10.1073/pnas.1211658109. microencapsulated bacterial spores. Cem Concr Res 2014;56:139–52. https://doi.
Shehata H, Poulos H. Latest thoughts on ground improvement techniques. In: Conference org/10.1016/j.cemconres.2013.11.009.
proceedings GeoMEast, International congress and exhibition on sustainable civil Wang J, Van Tittelboom K, De Belie N, Verstraete W. Use of silica gel or polyurethane
infrastructures. 2018. immobilized bacteria for self-healing concrete. Constr Build Mater 2012;26:532–40.
Siyepu C. Identifying factors that have negative impact on labour efficiency within road https://doi.org/10.1016/j.conbuildmat.2011.06.054.
maintenance. Faculty of Engineering: Cape Peninsula University of Technology, Wijeyesekera DC. Overview of innovations in geotechnical engineering. IJIE 2014;6.
Cape Town; 2016. https://doi.org/10.1016/j.cemconcomp.2014.08.007.
Sorrell S, Speirs J, Bentley R, Miller R, Thompson E. Shaping the global oil peak: A Yu X, Qian C, Xue B, Wang X. The influence of standing time and content of the slurry on
review of the evidence on field sizes, reserves growth, decline rates and depletion bio-sandstone cemented by biological phosphates. Constr Build Mater 2015;82:
rates. Energy 2012;37:709–24. https://doi.org/10.1016/j.energy.2011.10.010. 167–72. https://doi.org/10.1016/j.conbuildmat.2015.02.038.
Spanò A, Gugliandolo C, Lentini V, Maugeri TL, Anzelmo G, Poli A, et al. A novel eps- Zhang T, Cai G, Liu S. Application of lignin-based by-product stabilised silty soil in
producing strain of bacillus licheniformis isolated from a shallow vent off panarea highway subgrade: A field investigation. J Clean Prod 2017;142:4243–57. https://
island (italy). Curr Microbiol 2013;67:21–9. https://doi.org/10.1007/s00284-013- doi.org/10.1016/j.jclepro.2016.12.002.
0327-4.
18