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Review

Wastewater treatment using filamentous algae – A review

Jiajun Liu, Bill Pemberton, Justin Lewis, Peter J. Scales, Gregory J.O. Martin

PII: S0960-8524(19)31786-9
DOI: https://doi.org/10.1016/j.biortech.2019.122556
Reference: BITE 122556

To appear in: Bioresource Technology

Received Date: 30 September 2019

Revised Date: 2 December 2019
Accepted Date: 2 December 2019

Please cite this article as: Liu, J., Pemberton, B., Lewis, J., Scales, P.J., Martin, G.J.O., Wastewater treatment using
filamentous algae – A review, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122556

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 Wastewater treatment using filamentous algae – A review

Jiajun Liu1, Bill Pemberton2, Justin Lewis2, Peter J. Scales1, Gregory J.O. Martin1*

1Algal Processing Group, Department of Chemical Engineering, The University of Melbourne,

Parkville, Victoria 3010, Australia.

2Melbourne Water Corporation, 990 La Trobe Street, Docklands, 3008, Australia.

*Corresponding Author: Gregory J.O. Martin

Postal Address: Department of Chemical Engineering, University of Melbourne, Parkville, 3010,

Victoria, Australia.

Email: [email protected]; Telephone: +61 3 8344 6613

Abstract

Wastewater treatment using algae is a promising approach for efficient removal of contaminating

nutrients and their conversion into useful products. Monocultures of filamentous algae provide easier

harvesting compared to microalgae, and better control of biomass quality than polyculture systems such

as algal turf scrubbers. In this review, recent research into wastewater treatment using freshwater

filamentous algae is compiled and critically analysed. Focus is given to filamentous algae monocultures,

with key relevant findings from microalgae and polyculture systems discussed and compared. The

application of monocultures of filamentous algae is an emerging area of research. Gaps are identified in

our understanding of key aspects important to large-scale system design, including criteria for species

selection, influence of nutrient type and loading, inorganic carbon supply, algae-bacteria interactions,

and parameters such as pond depth, mixing and harvesting regimes. This technology has much promise,

however future research is needed to maximise productivity and wastewater treatment efficiency.

Key words: Filamentous algae; wastewater treatment; nitrogen removal; macroalgae; algal turf scrubber

1
1. Introduction

Wastewater treatment involves the removal of organic carbon, nitrogen, phosphorus, heavy metals and

other contaminants using physical, chemical and biological processes. Conventional wastewater

treatment processes require intensive aeration to drive bacterial oxidation of organic carbon and the

nitrification stage of biological nitrogen removal. Aeration of large volumes of wastewater is energy

intensive and can account for over 50% of the total energy costs of a wastewater treatment plant (Groves

et al., 1992; Martin et al., 2011). The oxidation of organic carbon directly releases CO2 into the

atmosphere, while the energy used for aeration results in indirect CO2 emissions. In addition, significant

amounts of the potent greenhouse gas N2O, are released during the biological nitrogen removal process

(Campos et al., 2016; Kampschreur et al., 2009). Another limitation of conventional wastewater

treatment is that although the nitrogen and phosphorous may be removed, they are not typically

recovered in a useful form.

In contrast, wastewater treatment with algae has been proposed as a more environmentally friendly

approach (Hoffmann, 1998), particularly for the removal and recovery of nitrogen and phosphorus. As

photosynthetic organisms, algae generate oxygen via photosynthesis and assimilate CO2. This can

support a beneficial cycle between bacteria and algae, in which the oxygen generated by algae can be

used by bacteria to oxidise organic carbon, and the CO2 generated by bacterial respiration can be

assimilated by the algae. In this way the aeration requirements and associated CO2 emissions can be

reduced by incorporating algae into wastewater treatment processes. During growth, algae assimilate

nitrogen and phosphorus along with photosynthetically fixed carbon. This can reduce the requirements

for bacterial nitrogen and phosphorus removal and the associated aeration demands and N2O emissions.

Further, the harvested algal biomass can be processed into useful products such as animal feed (Vucko et

al., 2017), slow-release fertiliser (Mulbry et al., 2005) or biofuel (Neveux et al., 2016; Park et al., 2011),

thus turning waste into resources.

2
Pressure to reduce greenhouse gas emissions and discharge of nutrients into waterways is increasing,

with algal wastewater treatment reemerging as an active area of research and application, having been

reported over 60 years ago (Oswald et al., 1957). The term algae spans an array of aquatic

photosynthetic organisms. Strictly, algae refers to eukaryotic species, however it is often also used to

refer to prokaryotic cyanobacteria, or so-called blue-green algae. Beyond phylogenetic classifications,

algae can be categorised based on their size, as either microalgae or macroalgae. Microalgae are

microscopic and typically unicellular, whereas macroalgae are multicellular, with individual organisms

visible without microscopic aids. For wastewater treatment applications, microalgae have historically

been considered over macroalgae. Microalgae can be cultivated outdoors as suspended cells in systems

such as high-rate algal ponds (HRAP) or raceway ponds. In addition to wastewater treatment,

microalgae cultivation is used for commercial purposes including biodiesel and bioethanol production

(Abinandan and Shanthakumar, 2015). While there is potential to produce microalgae for production of

commodity fuel and feed products, the high cost of production currently limits commercial viability to

high-value pigments and nutraceutical products. HRAP in wastewater treatment have both economic and

environmental advantages compared to commercial algal ponds. While supplies of nitrogen and

phosphorus are a cost to commercial facilities, the removal of these nutrients from wastewater represents

the primary aim of HRAPs (Park et al., 2011). In addition, the requirement to provide an external CO2

source is off-set by the presence of organic carbon and bacterial CO2 production in the wastewater. In

terms of infrastructure costs and land use, wastewater treatment using algal ponds is an intermediate

between more costly but intensive activated sludge systems (with effective nitrogen removal), and

cheaper but less land efficient and less effective nitrogen removal in facultative lagoon-based

wastewater treatment (Craggs et al., 2011; Park et al., 2011).

While microalgal HRAPs have been shown to be technically feasible for wastewater treatment

applications (Park et al., 2011), harvesting of the small microalgal cells remains a major issue.

Microalgae are naturally difficult to harvest due to their small cell size, close to neutral buoyancy and

3
negative surface charge (Moraine et al., 1979; Vandamme et al., 2013). As a result, harvesting

microalgae can represent over 90% of the equipment costs (Amer et al., 2011) and 20-30% of the overall

production costs in an operational facility (Rawat et al., 2011). Microalgae in HRAPs are also subject to

zooplankton grazing, which reduces biomass productivity and the reliability of nutrient removal

efficiency (Park et al., 2013). The use of different cultivation systems and photobioreactors (PBR) has

also been investigated for wastewater treatment applications to improve system performance. Various

configurations including submerged membrane PBR (Luo et al., 2017), tubular PBR (González et al.,

2008; Shangguan et al., 2015) and rotating algal biofilm reactors (Christenson and Sims, 2012) have

been investigated for wastewater treatment purposes. However, there are also limitations to these

systems, such as complex construction, intensive energy consumption and fouling (Liu et al., 2017).

Although less researched, macroalgae, in particular filamentous freshwater algae, have significant

advantages over microalgae for wastewater treatment including their amenability to harvesting and their

greater resistance to predation (Liu et al., 2016; Yun et al., 2015). The facilitation of biomass harvesting

due to their macroscopic size reduces the cost and complexity of the implementing an algae-based

wastewater treatment process. In addition, the ability to physically retain filamentous algae while

washing out bacteria and microalgae, allows strong culture enrichment and the opportunity to maintain a

monoculture of a desired strain. The ability to increase the solids retention time (SRT) of the filamentous

algae above that hydraulic residence time (HRT) of the wastewater ensures robust operations despite

flow fluctuations, including during high rain events that can be particularly problematic for microalgae.

There are two main approaches for utilising freshwater filamentous algae for wastewater treatment -

Algal Turf Scrubbers (ATS) and raceway pond monocultures. The ATS process was developed in the

1980s as a technology for wastewater treatment based on attached, complex communities of

microorganisms, called a periphyton (Adey, 1983; Adey et al., 2011). The periphyton consist of self-

seeding microbial communities that include filamentous algae that attach to a screen. The screen is

immersed in shallow flowing wastewater so that nutrients can be taken up by the organisms in the
4
periphyton (Adey et al., 2011). The biomass can then be physically removed using a scraper or vacuum,

by and potentially used for biofuel, applications. ATS can be used to treat a range of contaminated water

including agricultural runoff (Kangas and Mulbry, 2014), aquaculture wastewater (Ray et al., 2015),

dairy and swine manure effluent (Kebede-Westhead et al., 2004, 2006; Mulbry et al., 2008a), primary

domestic wastewater (Marella et al., 2019), and secondary effluent (Craggs et al., 1996a; Sandefur et al.,

2011), with high biomass productivity and efficient nutrient removal being reported in these studies.

The second approach to using filamentous algae for wastewater treatment is via cultivation of

monocultures in algal ponds. This is an emerging area of research. One main advantage of this system

compared to ATS is that it allows harvesting of the chosen species of cultivated macroalgae, which gives

control of the biomass quality for potential downstream applications (Neveux et al., 2016). Species from

the genera Oedogonium, Cladophora, Spirogyra, Klebsormidium and Stigeoclonium have been

established as potential species for wastewater treatment applications (Lawton et al., 2013; Liu et al.,

2016). As an emerging technology, knowledge of the effects of key design parameters on monoculture

performance remains incomplete and is yet to be reviewed.

The aim of this paper is to help advance wastewater treatment using freshwater filamentous algae by

providing a systematic and critical review of recent research into monoculture cultivation. Key research

findings and trends are summarised and research gaps and opportunities identified. Relevant research on

microalgae systems and ATS is also compared and discussed. To help guide process design, focus is

given to key parameters including species selection, nutrient loading, inorganic carbon supply, and

algae-bacteria interaction in relation to algal productivity and wastewater treatment (nominally nutrient

removal) efficiency.

2. Species Selection

2.1 Candidate Algae Species

5
The term filamentous algae refers to a group of algae from the division Chlorophyta (Green Algae)

whose cells are connected longitudinally to form long filaments. The filaments are usually one cell thick

with a diameter of order micrometers and can be seen with the naked eye. They are variously referred to

both as microalgae (Wang et al., 2013) and macroalgae (Lawton et al., 2013) in existing literature. For

studies involving wastewater treatment, the term ‘macroalgae’ is used to distinguish their feature of

easier harvesting compared to unicellular algae that have been more extensively studied. There are

numerous types of freshwater filamentous algae that exist in nature, however only some of them have so

far been identified as having advantages for wastewater treatment applications. Filamentous algae from

the genera Oedogonium, Cladophora, Spirogyra, Rhizoclonium, Microspora, Klebsormidium and

Stigeoclonium have been identified as dominative species in algae wastewater treatment systems or have

been specifically studied for wastewater treatment purposes. Some descriptions of these filamentous

algae are provided in Table 1. As described, different filamentous algae have significantly different

microscopic and macroscopic features. Some of the physical characteristics, such as the length and

strength of the filaments, the adhesion properties, and the growth behaviour at high cell density, can

affect large-scale cultivation of these algae and their application to wastewater treatment. For example,

self-adhesion of filamentous algae to form clumps or mats allows protection from shear and predation as

well as easier retention and harvesting. However, filamentous algae can also adhere to piping outlets,

paddle wheels or pond walls, which may require periodic removal. If extensive mats are allowed to

develop on the water surface, self-shading can result in a reduction in light utilisation in the deeper

sections of the ponds.

Table 2 summarises research of ATS for which the composition of the algal community was reported.

Only filamentous algae species are listed despite the complex biological composition in ATS. A variety

of filamentous algae have been found in ATS systems, with various species reported as being dominant.

While ATS systems include varied and often unidentified mixtures of algae species (Table 2), suspended

cultures of filamentous algae offer the possibility of cultivating chosen strains. A number of genera of

6
filamentous algae have been reported for application to wastewater as monocultures, including

Oedogonium, Cladophora, Spirogyra, Stigeoclonium and Klebsormidium. Oedogonium, Cladophora,

Spirogyra have also been reported in ATS systems (Table 2). Selection of a suitable algae species is one

of the most important parameters as it forms the basis of all subsequent research and applications.

Lawton et al. (2013) listed four key criteria for algae selection for wastewater treatment purposes: high

areal productivity, adaptability to varying conditions, competitive dominance over undesired species,

and consistent biochemical composition. The potential of various candidate freshwater filamentous algae

species is reviewed here based on these four criteria and compared against the mixed communities found

in ATS. In addition to the four listed criteria, other species-dependent factors need to be considered in

relation wastewater treatment. These include the ability of the algae to grow in wastewater containing

much higher concentrations of organic carbon, nutrients and heavy metals than are typically present in

the natural algae habitats; the ability to grow in the presence of bacteria are present in wastewater; and

the algae having filament structures that facilitate retention and harvesting, while minimising clogging

and predation. These additional factors will be discussed in more depth in later sections of the review.

2.2 Algal Productivity

Algal productivity is the most important parameter as it reflects the amount of nutrient removed from

wastewater and the amount of product that can be recovered downstream. Areal productivity, expressed

in biomass dry weight produced per unit area per unit time, is often reported for algae in literature.

Reported areal productivity values for ATS systems vary significantly between different studies,

spanning from 0.8 gdry algae/(m2.d) up to around 50 gdry algae/(m2.d) (Table 3). The variation is associated

with parameters such as wastewater type, nutrient loading, climate, system configurations, amongst a

long list. Nonetheless, the reported productivity range for these systems can be used as a benchmark

when selecting monoculture species.

Candidate monoculture species must be able to achieve comparable productivities to polyculture

systems in order to be advantageous. They must also achieve higher productivity compared to undesired
7
species at given conditions in order to maintain competitive dominance. Table 4 summarises areal

productivity values that have been reported for monoculture freshwater filamentous algae genera for

wastewater treatment purposes. Other units such as specific growth rate and volumetric productivity are

also commonly reported for lab-scale experiments. Although not directly translatable to areal

productivity, they are included in Table 4 for comparison purposes.

Based on Table 3 and 4, filamentous algae from the genera Oedogonium show comparable levels of

areal productivity to polyculture systems. As with ATS, the reported areal productivity values vary

significantly depending on differences in the growth conditions, hampering a comparative analysis

between species. Only one study appears to have directly compared species under the same conditions,

finding Oedogonium, Cladophora, and Spirogyra to have similar productivities under the tested

laboratory conditions. There are limited reports of areal productivity for filamentous algae other than

Oedogonium, such as Cladophora and Spirogyra. One reason is that research is still in an experimental

phase, for which specific growth rate and volumetric productivity are the main parameters reported (for

example in Liu et al, 2016; Ross et al, 2018).

2.3 Adaptability

Adaptability refers to the ability of candidate algal species to survive and maintain good performance

under varying climatic conditions and wastewater composition encountered in outdoor environments.

Research aimed at understanding the performance of algal species in relation to different growth

conditions can enable the most suitable species to be selected for given situations. This section will

focus on the effect of climate on algae growth, while the influence of wastewater composition will be

discussed in later sections. It is worth noting that an assimilation period is typically provided in most

experiments involving filamentous algae to treat wastewater. This allows the algae to adapt to the

wastewater and the different conditions imposed on the algae compared to this of the original stock

culture or natural habitat. This avoids making conclusions based on observations of the lag phase so that

the long-term adaptability of the selected strain can be observed.

8
It is well known that algae growth is affected by temperature and light intensity. Each algae species has

distinct optimal growth conditions that make it more suitable for growth in certain locations and seasons.

For outdoor growth of algae, solar irradiance is typically coupled with temperature based on alternation

of seasons, with summers combining longer day time hours, higher solar irradiance and higher

temperatures than winter. Light and temperature cannot practically be controlled in outdoor cultures and

will vary geographically, seasonally and diurnally. Nonetheless, algal ponds are usually designed to

optimise the amount of light received by the cultures (Cole et al., 2018). For example, the depth of algal

ponds is carefully selected to provide an optimal light intensity for algae growth. Mixing allows

suspended algal cells to frequently traverse the pond depth, which reduces the length of time an

individual cell must spend either experiencing light inhibition on the pond surface or light deficiencies at

the bottom of the pond. This provides a more suitable light profile for all cells within the pond. For

cultivation of filamentous algae, the light intensity profile can be different to that experienced by

microalgae, as they are multicellular and can form floating or adhered mats rather than being evenly

suspended. Depending on their physical behaviour, the distribution of cells and self-shading regime can

be very different to those in microalgae systems. The response of filamentous algae to these parameters

is therefore critical for outdoor cultivation and wastewater treatment.

It is not possible to correlate the composition of filamentous algae species in ATS with temperature

based on existing studies (Table 1). Therefore, additional research focusing on monoculture systems are

required to understand their adaptability to temperature change. Only limited studies have directly

investigated the relationship between temperature and filamentous algae productivity, especially in

relation to large-scale cultivation and wastewater treatment. In a temperature- and light-controlled lab-

scale study conducted by Cole et al. (2018) to examine on the suitability of Oedogonium intermedium to

large-scale cultivation in Australia, this species showed consistent growth between 15 ºC – 40 ºC, with

an optimum temperature range of 20 ºC – 35 ºC. It was therefore concluded that only tropical Australia

(latitudes ≤25º) is suitable for year-long cultivation of Oedogonium intermedium (Cole et al., 2018).

9
Other studies conducted at larger scale in an outdoor study in tropical Australia showed a positive

correlation between temperature and Oedogonium productivity between 17 and 32 ºC (Cole et al., 2016;

Neveux et al., 2016), also suggesting this genus is better suited to warmer climates. Ecological studies

show that Spirogyra tend to bloom during spring (Berry and Lembi, 2000; Graham et al., 1995).

Controlled laboratory studies showing that Spirogyra can grow between 5 ºC to 35 ºC, with an optimum

temperature for growth around 25 ºC (Graham et al., 1995; O’Neal and Lembi, 1995). In comparison,

Cladophora glomerate was found to have a maximal net photosynthesis rate at lower temperatures of 13

ºC – 17 ºC (Graham et al., 1982). These studies indicate that Spirogyra and Cladophora could perform

better than other algae such as Oedogonium in cooler climates. In addition, there is the possibility that

different species could be favoured (deliberately or naturally) in winter and summer, to maximise

performance across a year. Although temperature is a critical parameter, there is still only limited

information available on filamentous algae. Further studies are required to understand the adaptability of

filamentous algal species to different temperatures and climates.

2.4 Competitive Dominance

ATS systems have uncontrolled mixed algal populations, with the dominant species varying in response

to growth conditions and not typically being actively controlled. In suspended filamentous algae cultures

there is the possibility of maintaining a ‘monoculture’ in which one chosen species is dominant. This

offers advantages including the ability to produce a more consistent biomass (Neveux et al., 2016) with

a desirable biochemical composition (see Section 2.5) and the ability to predict and control process

performance. In relation to competitive dominance, the desired species should be dominant over other

filamentous algae as well as microalgae and cyanobacteria.

Competitive dominance of filamentous algae is largely dependent on their response to growth conditions

and adaptability to surrounding environments. Lawton et al. (2013) have observed competitive

dominance of Oedogonium over Cladophora and Spirogyra under relatively high temperature of 30 ºC

and high light intensity of 35.5 mol/(m2.d). This further demonstrates the adaptability and suitability of
10
Oedogonium in tropical regions compared to the other two genera. Another study by Liu et al. (2016)

showed competitive dominance of Stigeoclonium over Klebsormidium during horticultural wastewater

treatment at moderate temperature of 23 ºC and light intensity of ~5 mol/(m2.d). However, as there is no

other research that has compared the adaptability of Stigeoclonium and Klebsormidium to other

temperatures or climates, it is difficult to generalise these findings. Since competitive dominance of

filamentous algae is related to their adaptability, future studies should focus on revealing the relationship

between temperature/climate conditions and algae growth, preferably with direct comparisons between

promising species. Experiments on competitive dominance within mixed cultures can be performed

subsequently to validate the findings on adaptability.

Maintaining competitive dominance of filamentous algae over other algae types is naturally difficult in

open systems. Nearly all studies on ATS have shown a mixture of microalgae, macroalgae, diatoms and

cyanobacteria growing as part of the wastewater treatment community. The use of a screen in ATS

allows self-seeding and attachment of various contaminants from the surrounding environment. In

contrast, contamination from undesired species has not been reported in the key studies that utilise

monoculture filamentous algae for wastewater treatment in outdoor culture (Cole et al., 2015, 2016; Kim

et al., 2018; Neveux et al., 2016). The apparently low level of contamination is possibly related to the

algal pond configuration in monoculture systems. Filamentous algae naturally form attached or floating

mats which can be easily retained in the system (Lawton et al., 2013). Mesh screens can also be installed

at the outlet to prevent excessive loss of filamentous algae (Cole et al., 2015; Neveux et al., 2016). As a

result, the desired filamentous algae are given a competitive advantage by being kept in the system while

smaller sized contaminants such as microalgae and diatoms are washed out. The selective retention of a

desired species of filamentous algae represents a major potential advantage of monoculture filamentous

algal ponds over ATS.

2.5 Biochemical Composition

11
The algal biomass generated during wastewater treatment represents a potentially valuable resource and

a recovery of nitrogen and phosphorus. The filaments comprise of protein, carbohydrates and lipids that

have nutritive and calorific value. For example, biofuel in the form of biogas can be generated via

anaerobic digestion, or biocrude from hydrothermal liquefaction. The protein nitrogen and biologically-

fixed phosphorus could potentially be used as a feed supplement for aquaculture or animal feed, or as a

fertiliser for crops.

The biochemical composition of the algal biomass generated during wastewater treatment influences the

type and quality of the downstream products that can be produced. Of particular importance is the ash

content of harvested biomass, which should be minimised to avoid issues for downstream applications

including use as a feed, fertilizer or feedstock for biofuel production. Ash can be a major limitation for

ATS systems, with ash content values over 70% having been reported (Aston et al., 2018; Kangas and

Mulbry, 2014). In ATS there are two sources of ash: biogenic ash that is mainly silica contained in the

walls of diatoms present in the system, and exogenous ash in the form of dust and sand particles

entrapped amongst the filamentous algae (Aston et al., 2018; Yun et al., 2015). The self-seeding and

periphytic nature of ATS increases the chance of both biogenic and exogenous sources of ash

contamination. Both these sources can be practically eliminated in monoculture pond systems in which

to diatoms and inorganic contaminants are washed out, as mentioned previously.

In terms of species selection for monoculture systems, the elemental composition of proposed candidate

species can be used to calculate the higher heating value (HHV) that indicates the amount of energy

stored in the biomass and the potential for biofuel production (Channiwala and Parikh, 2002). In a

comparison between Oedogonium, Cladophora and Spirogyra, Oedogonium was found to have a

slightly higher carbon content, lower ash content and higher HHV (Lawton et al., 2013). Elemental

analysis and subsequent HHV calculations for Spirogyra grown in municipal wastewater were also

performed by Ge et al. (2018), showing similar results to the above study. The theoretical hydrothermal

12
liquefaction biocrude yield from Oedogonium grown for municipal wastewater treatment was found to

be 26-27 wt% or 35% on ash-free basis based on its carbon content (Neveux et al., 2016).

In terms of feed applications, the amino acid profile of Oedogonium grown in aquaculture effluent was

shown to include the full spectrum and a balanced ratio of essential and non-essential amino acids, with

an overall protein content of about 18% (Cole et al., 2015). In another more detailed analysis,

Oedogonium biomass was shown to have a comparable nutritional composition to a commercial feed for

herbivorous fishes, and demonstrated in a long-term study to be equivalent to commercial pellet as feed

for the herbivorous fish, Ancistrus cirrhosis (Vocko et al., 2017). These studies indicate that

Oedogonium biomass has a promising composition for both feed and biofuel applications.

It is also important to consider the potential toxicity of algal biomass grown on wastewater, an issue

which has not yet been properly studied. As will be discussed in section 3.4, adsorption or biological

uptake of heavy metals or other emerging contaminants can occur during cultivation of filamentous

algae in wastewater, potentially resulting in toxicity for feed applications. In addition, various pathogens

present in the wastewater may also compromise the quality and safety of the biomass as a feed, which

may require downstream disinfection or pasteurisation to be performed. The issues of toxicity and

pathogens require future investigation and possible regulation to ensure safe application of wastewater-

grown filamentous algae as an animal or aquaculture feed.

To date most studies of the biochemical composition of filamentous algae have been limited to

elemental analysis and determination of protein content and amino acid profiles. Future studies should

include more detailed biochemical analysis of the lipid content, polysaccharides and pigments, and the

conditions in which the productivity of these substances can be maximised.

3. Nutrient Type and Loading

Nitrogen, phosphorus and trace amounts of metals are the essential nutrients for algae growth. They are

also key elements that the algae are desired to remove during wastewater treatment. This section will

13
discuss how nitrogen, phosphorus and metals can be removed by algae in wastewater treatment, and how

system performance can be affected by the type and loading of these nutrients.

3.1 Options for Algal Nitrogen Removal

Regulatory discharge of nitrogen often targets both inorganic nitrogen (including ammonium/ammonia,

nitrite and nitrate) and organic nitrogen (such as urea). These can all be utilised for filamentous algae

growth (Ross et al., 2018). Figure 1 illustrates a typical wastewater treatment process, including the

major process stages and the key components present in the wastewater as it goes through processing.

Raw wastewater typically contains mostly organic nitrogen, which is rapidly broken down to

ammonium. While it is theoretically possible to grow suspended cultures of filamentous algae in raw

wastewater, the presence of suspended matter makes the water highly turbid, blocking light and

impeding algae photosynthesis. The suspended solids in raw wastewater are usually removed via

primary sedimentation or dissolved air flotation, leaving a relative clear primary effluent containing

soluble organic carbon and ammonium. As demonstrated in previous studies (Table 5), it is possible to

used filamentous algae to remove ammonium from the resulting primary effluent. The presence of high

concentrations of soluble organic carbon promotes proliferation of bacteria that can reduce light

penetration into the algae cultures, and which needs to be considered in relation to potential utilisation of

the algal biomass. In conventional wastewater treatment, the soluble organic carbon is removed by

aerobic treatment using bacteria as well as during the denitrification stage of nitrogen removal.

Maintaining aerobic conditions requires active aeration, which consumes significant amounts of energy,

resulting in indirect CO2 emissions. Applying algae to remove nitrogen from primary effluent could

potentially reduce aeration demands by converting organic carbon to biomass via mixotrophic

metabolism (utilisation of organic carbon and photosynthesis) or through the generation of dissolved

oxygen via photosynthesis. The synergistic exchange of oxygen and carbon dioxide between algae and

bacteria is well known in wastewater treatment and can also feature in high-rate algal ponds (Mñoz and

Guieysse, 2006). This will be discussed further in section 5.

14
Conventionally, nitrogen is removed from primary effluent via a two-step bacterial process involving

nitrification and denitrification (Figure 1). During nitrification the ammonium is converted through to

nitrate via aerobic bacteria. As the nitrifying bacteria are relatively slow-growing, nitrification is usually

promoted by operating at a high solids residence time and providing aeration to wastewater that is low in

soluble organic carbon. As with aerobic removal of organic carbon, nitrification requires significant

energy for aeration. Applying filamentous algae to remove ammonium from wastewater following

aerobic removal of organic carbon could replace bacterial nitrification and avoid the need for aeration as

well as subsequent denitrification. In this case the filamentous algae would again have to use ammonium

for growth, but with minimal amounts of bacteria present, given the greatly reduced concentrations of

soluble organic carbon.

Nitrification results in the conversion of ammonium to nitrate, which is usually subsequently converted

to nitrogen gas during denitrification. While denitrification is anoxic and therefore doesn’t require

energy-intensive aeration, it instead results in significant emissions of the potent greenhouse gas, nitrous

oxide. Another possibility is to use filamentous algae to replace denitrification to avoid nitrous oxide

emissions. In this case, they would grow on nitrate instead of ammonium.

3.2 Growth of Filamentous Algae on Different Forms of Nitrogen and Phosphorus

Many algae can utilise various types of organic nitrogen, including urea, amino acid, purines and

pyrimidines, for their growth (Berman and Chava, 1999; Neilson and Larsson, 1980). However, in

wastewater treatment, the organic nitrogen is readily broken down to ammonium. Therefore, the focus of

research for nitrogen uptake of algae should be on inorganic nitrogen, particularly ammonium and

nitrate/nitrite. While algae can assimilate ammonium and nitrate/nitrite, anabolic metabolism requires

reduction of nitrite and nitrate, which is energy-intensive for the cell. Therefore, most algae prefer

ammonium as a nitrogen source when both ammonium and nitrate/nitrite are present because of the

lower metabolic energy requirements (Monfet and Unc, 2017; Ross et al., 2018). As discussed above,

for wastewater sources containing a large proportion of nitrogen as ammonium, filamentous algae
15
systems can replace conventional nitrification processes to reduce aeration costs. However, although

ammonium is a preferred nitrogen sources for many algae, it is in equilibrium with free ammonia, which

is known to be toxic to algae by triggering photodamage of photosystem II (Drath et al., 2008; Gutierrez

et al., 2016). The equilibrium between the ammonium ions and free ammonia is dependent on pH and

temperature. For algae cultivation, pH is a very important parameter since algal activity can rapidly

increase the pH of the solution via the consumption of carbon dioxide (Cole et al., 2014b). Typically,

only millimolar levels of ammonia in wastewater are needed to inhibit algae growth, with the effect

becoming more pronounced at higher pH and during summer (Azov and Goldman, 1982). For

wastewater treatment applications of filamentous algae, it is crucial to understand the tolerance of

various species to different ammonia concentrations. The growth rate of Cladophora coelothrix and

Cladophora parriaudii was found to decrease with increasing total ammonium concentrations ranging

from 160 to 2560 μM at an initial pH of 7.95±0.08 (translating to initial ammonia concentrations of 8 to

129 μM at pH 8 and 24 ºC), while this decrease in growth rate was not observed when other nitrogen

sources (nitrate, nitrite, urea) were used for algae growth (Ross et al., 2018). In a study of Oedogonium

species the maximum growth rate was found at an ammonium-N concentration of 10 mg/L within the

range of 1-100 mg/L (Francke and Den Oude, 1983). For the same range of ammonium-N, optimal

growth of Stigeoclonium occurred at 5, 10 or 50 mg/L ammonium-N depending on the actual species

(Francke and Den Oude, 1983). Other studies have also shown growth and ammonium-N removal from

wastewater for Oedogoniuim and Spirogyra, as summarised in Table 5. However, most of these studies

involve relatively low initial ammonium-N concentrations and/or constant pH below 8 through buffering

or CO2 bubbling. For studies involving higher initial ammonium-N concentrations, there were no

negative controls in place to quantify the amount of biological nitrogen uptake compared to free

ammonia stripping or nitrification by bacteria. There does not yet appear to have been any studies that

have investigated the performance of freshwater filamentous algae under measured and controlled levels

of free ammonia. Due to relatively long residence times involved in wastewater treatment processes and

16
the typical pH elevation in algal ponds, it is very likely that algae will be subject to prolonged exposure

to free ammonia. Therefore, it is crucial for future studies to focus on quantifying ammonia tolerance of

different filamentous algae species, which is necessary information for both species selection and system

design. Currently, ammonia tolerance has only been examined for a few well-studied algae such as

Oedogonium and Spirogyra. Information on a greater range of species with potential application to

wastewater treatment is required to aid species selection for different wastewater conditions.

Phosphorus exists in both organic and inorganic forms in wastewater. This includes orthophosphate,

polyphosphate, pyrophosphate, metaphosphate and corresponding organic forms, with orthophosphate

being the form most readily utilisable by algae (Markou et al., 2014). As both nitrogen and phosphorus

are essential nutrients for algae growth and are assimilated by algae simultaneously, the study of

nitrogen and phosphorus removal by algae is usually coupled. The nitrogen to phosphorus ratio (N:P

ratio) is commonly reported in literature to understand whether nitrogen or phosphorus is the limiting

nutrient for algae growth in certain wastewater. This is a critical parameter for process design and

performance and will be discussed in more depth in the following sections.

3.3 Nutrient Loading and N:P Ratio

Nitrogen and phosphorus loading rate refers to the application rate of nitrogen or phosphorus into the

treatment system per unit area and time (e.g. kg-N/(m2.d). As such, this parameter is intuitively parallel

to the areal biomass productivity (e.g. kg-biomass/(m2.d). It is necessary to understand how the algae

respond to varying nitrogen and phosphorus loading rates in order to design perform large scale

filamentous algae systems. Loading rate is defined as the product of nutrient concentration and flow rate

per unit area of ATS, and studies have shown that this parameter is closely related to the growth rate of

filamentous algae. Yun et al. (2015) summarised and performed regression on data from various ATS

studies and found that both algal productivity and nutrient removal rate were parabolically related to the

nitrogen/phosphorus loading rate. The maximum productivity and total nitrogen (TN)/total phosphorus

(TP) removal rates were found to occur at total nitrogen and phosphorus loadings of 3 g-TN/(m2.d) and
17
0.4–0.5 g-TP/(m2.d) respectively (Yun et al., 2015). Other researchers have also claimed Michaelis-

Menten type relationships, where algae productivity reaches a plateau at high nitrogen concentration

(Ross et al., 2018). However, a decrease in algal productivity is expected to occur at excessively high

nutrient loadings, either due to high concentration of inhibitory substances (such as ammonia), or

excessive shear at high flow rates. A generalised relationship between algal productivity, nutrient uptake

rate and nutrient loading is shown in Figure 2. Wastewater treatment systems should operate in the

region between nutrient-limited and nutrient-saturated loading rates to allow sufficient biological uptake

of nutrients while maximising the percentage removal of the nutrients from the wastewater.

The relationship between nutrient loading and algal productivity was also studied for monocultures of

filamentous algae. In a study of Oedogonium used for aquaculture wastewater treatment, it was shown

that increasing the water exchange rate (equivalent to the dilution rate or the reciprocal of hydraulic

residence time) between 0.1 to 5 day-1 (representing an increase in the nutrient loading rate), slightly

decreased the algal productivity (Cole et al., 2014a). This indicated nitrogen saturation occurred even at

low water exchange rates (Cole et al., 2014a). The same study also showed low productivity at low

water exchange rates when ammonium-enriched wastewater was used. However, this can be attributed

to the experimental design, which resulted in high TAN levels that were inhibitory to the algae at low

water exchange rates (Cole et al., 2014a). Another study with the same species and wastewater type used

a cascade system comprising of three cultivation tanks in series (upstream, midstream and downstream),

allowing comparison of the performance as a function of different nitrogen concentrations while

maintaining a constant water exchange rate of 1 day-1 (Cole et al., 2015). The results showed that both

ash-free dry weight productivity and nitrogen uptake decreased as nitrogen concentration decreased,

with the majority of the nitrogen uptake occurring within the upstream tank (Cole et al., 2015). Both

studies are consistent with the relationship shown in Figure 2, however the relationship this will vary

quantitatively for different species of filamentous algae and for different wastewater sources.

18
As mentioned above, the N:P ratio of the wastewater is an important parameter. An imbalanced N:P

ratio will result in premature depletion of one of the essential nutrients, thus reducing algae growth and

wastewater treatment efficiency in relation to removal of the other nutrient. A summary of various

microalgae studies showed a large variation in the reported optimal N:P ratio, ranging from 7.1 up to

133.3 on a molar basis (Monfet and Unc, 2017). However, very few studies have investigated the

optimal N:P ratio for filamentous algae species. One such study involved growing different filamentous

algae at varying N:P ratios to obtain a range within which over 99% of the nitrogen and phosphorus

could be removed simultaneously (Liu and Vyverman, 2015). The optimal range of N:P ratio for

Cladophora, Klebsormidium and Pseudanabaena was found to be 5-15, 7-10 and 7-20 respectively, on a

weight basis (Liu and Vyverman, 2015). In addition to variations in the optimal N:P ratio between

different algal species, even for the same species of microalgae the optimal N:P ratio has been shown to

depend on external factors such as temperature or light conditions (Rhee and Gotham, 1980). Such

variations may also exist for freshwater filamentous algae species. Further research into the effect of N:P

ratio for different filamentous algae is required to aid species selection and process design for

wastewater treatment.

Although the input level of nitrogen and phosphorus for a particular wastewater treatment plant is

usually within certain range, the exact value cannot be predicted or controlled directly. Nevertheless, it

may be feasible to keep within a desired range of nitrogen and phosphorus concentration by

manipulating the streams that are diverted into the algal ponds. For example, high-strength centrate

water or primary effluent can be mixed with low-strength secondary effluent to achieve nutrient levels

suitable for algae growth. Developing dynamic growth models would therefore be useful in helping to

predict system performance and control flow regimes based on influent wastewater nutrient profiles.

3.4 Heavy Metal Removal by Filamentous Algae

The ability of filamentous algae to adsorb heavy metals from wastewater is important in relation to both

cleaning of the wastewater and contamination of the algal biomass. There are two main directions of
19
research that involve both “heavy metal removal” and “filamentous algae” – biosorption using dried

algal biomass, and bioaccumulation using living algae cultures. The majority of studies have focused on

biosorption, in which dried filamentous algae biomass is added to the wastewater as a biological

adsorbent material. Removal of Pb, Cd, Cr, Co, Ni and Hg through biosorption have been reported using

dried Oedogonium, Spirogyra or Cladophora biomass (Gupta and Rastogi, 2008a, 2008b; Gupta et al.,

2001, 2006, 2010; Lee and Chang, 2011; Rezaee et al., 2006). Numerous brown, green and red algae

species have also been found to be effective biosorbents for heavy metals, as summarised by He and

Chen (2014). In this regard, it is conceivable that the filamentous algae biomass generated for the

purpose nitrogen removal from wastewater could be subsequently used as a biosorbent for heavy metal

removal from other contaminated water, thereby serving a dual function.

Bioaccumulation or bioconcentration is more directly relevant to this review. It has been claimed that

bioconcentration is advantageous over simple biosorption as it involves both storage of metal elements

in various cellular compartments, and simultaneous production of new algal biomass to provide extra

metal uptake capacity (Saunders et al., 2012). A few studies have shown various filamentous algae

species to have high capacity to uptake heavy metals. A study of Oedogonium, Rhizoclonium and

Hydrodictyon species cultivated in heavy metal contaminated ash dam water demonstrated accumulation

of most heavy metal types, and total heavy metal uptake of over 60 mg/g for all three species, which is

comparable to typical metal binding capacity in biosorption studies (Saunders et al., 2012). In batch and

semi-batch experiments, Cladophora grown on synthetic wastewater removed over 80% of the

cadmium, resulting in a biomass with a cadmium composition of 1.7±0.3% on a dry weight basis

(Sternberg and Dorn, 2002). In addition, ecological studies on heavy metal polluted rivers have also

shown the ability of various filamentous algae species to bioconcentrate heavy metals (Foster, 1982;

Tang et al., 2014). The above-mentioned studies demonstrate the promise of heavy metal removal using

filamentous algae. However, a major limitation in the above studies is the lack of analysis in the actual

mechanism of heavy metal removal. Although elemental analysis showed high concentrations of heavy

20
metals in the harvested biomass, it was not determined whether the heavy metals were taken into algae

cells or simply adsorbed on the cell surface. In addition, some key aspects of heavy metal

bioconcentration essential for system design have not yet been fully described. This includes the

tolerance level of different species, the competitive or synergistic uptake of different heavy metal types,

the maximum uptake capacity, and the effect of external environment conditions.

While removal of heavy metals from wastewater is beneficial, the ability of filamentous algae to

bioconcentrate heavy metals during N/P removal could also compromise the quality of the biomass as a

fertiliser or animal feedstock. For example, during pilot scale treatment of secondary municipal

wastewater using Oedogonium the algae were shown to uptake various metals, resulting in 67%, 42%

and 38% removal of Mn, Zn and Cd respectively (Cole et al., 2016). However, only a low concentration

of heavy metals was identified in the algal biomass, due to the low heavy metal concentration in the

treated secondary wastewater and the frequent harvesting of biomass (twice per week) (Cole et al.,

2016). Since heavy metal uptake by filamentous algae depends on biomass density and contact time

(Sternberg and Dorn, 2002), problematic concentrations of heavy metals may eventuate in the algal

biomass depending on the wastewater type and system design. Therefore, attention should be given to

heavy metal levels in the biomass during both research and implementation of algal wastewater

treatment systems.

4. Inorganic Carbon Supply

As photosynthetic organisms, the growth of filamentous algae is dependent on the supply of inorganic

carbon. Inorganic carbon can be the primary growth-limiting factor in large-scale algae cultivation due

to the very limited supply of CO2 provided by diffusion from the atmosphere relative to the rapid rate of

consumption by dense algae cultures (Cole et al., 2014b). While some of the organic carbon in

wastewater can potentially be used by algae, or indirectly following conversion into CO2 by bacteria, the

C:N ratio in wastewater is significantly lower than the typical biological C:N ratio in algal biomass,

meaning an external carbon source must be supplied to achieve complete removal of nitrogen
21
(Benemann, 2003; Craggs et al., 2011). Various studies on wastewater treatment using high-rate

microalgal ponds have shown increasing productivity when CO2 is supplied, as summarised by Park &

Craggs (2010). Similarly, some studies on filamentous algae have shown improved productivity when

CO2 was provided. For example, the productivity of Oedogonium in ash dam water was shown to

increase from <5 gdry biomass/(m2.d) to 17.0 and 22.5 g/(m2.d) when 3 L/min and 6 L/min of 99.9% CO2

were provided to 60L tanks, respectively (Roberts et al., 2013). Similarly, the productivity of

Oedogonium was increased from 3.4 to 8.3 gdry biomass/(m2.d) when 99.9% CO2 was bubbled along 15 m2

tanks to maintain a pH of 7.5 during daytime hours (Cole et al., 2014b). It is worth noting that the

positive correlation between CO2 supply and algae growth rate is not necessarily solely due to the

increased amount of dissolved inorganic carbon (DIC). Aeration also creates turbulence that allows a

higher exchange rate of nutrients and reduces accumulation of metabolic waste products around the algal

biomass. Addition of CO2 also lowers the pH of the solution, which is beneficial to algae growth in

several ways. At lower pH, the equilibrium of total DIC is shifted towards higher proportions of free

CO2 and HCO3-. Above a certain pH, there is no net uptake of DIC, since the majority of DIC exists in

the form of CO32- which is not utilizable. The pH at which DIC uptake was in equilibrium with CO2

release through respiration was found to be 10.7 and 10.5 for Oedogonium and Cladophora respectively

(Choo et al., 2002; Cole et al., 2014b). There has been no observation of CO32- utilisation by any algae,

and CO2 is more readily utilisable than HCO3- by most algae including Oedogonium and Cladophora

(Choo et al., 2002; Cole et al., 2014b). Therefore, lowering pH and liberating more readily utilisable

forms of DIC can promote filamentous algae growth. Lowering the pH also reduces the proportion of

toxic free ammonia relative to the ammonium ions. Research on microalgae has shown that supplying

CO2 can suppress ammonia volatilisation and improve algae tolerance to NH4-N (Yao et al., 2015). High

pH values above 9 are often reported in algae wastewater treatment systems such as ATS when no pH

control is applied (Craggs, 2001; Craggs et al., 1996b; Liu et al., 2016). In pond systems such as

22
monocultures of filamentous algae, sparging of CO2-enriched air can be a viable option to both reduce

pH stress and avoid carbon limitation, thereby enhancing wastewater treatment efficiency.

A large number of studies have focused on combining CO2 capture and wastewater treatment (Razzak et

al., 2013). In many wastewater treatment systems, biogas is obtained from anaerobic digestion to

recover energy and reduce greenhouse gas emissions (Whiting and Azapagic, 2014). The flue gas

generated from biogas combustion provides a cheap and easily accessible source of CO2 for wastewater

treatment systems with algae. However, studies with microalgae have shown that supply of flue gas does

not necessarily increase algae growth rate and wastewater treatment efficiency (de Godos et al., 2010).

Both H2S and the high concentrations CO2 in flue gas can be inhibitory to algae growth, with inhibitory

effects observed even at a typical CO2 concentration of 15% v/v for certain microalgae species (Kumar

et al., 2014; Yun et al., 1997). The high temperature of flue gas can also have a negative effect,

depending on the temperature tolerance of the algae species (Razzak et al., 2013). However, the heat

may also be beneficial if the flue gas is provided in winter to maintain a more optimum growth

temperature within the algal ponds. In terms of filamentous algae, no studies to date have investigated

the effect of flue gas on algae growth and wastewater treatment efficiency. Therefore, future research

can target this aspect, although the results can be site-specific, as they depend on the composition on flue

gas and wastewater.

5. Algae-Bacteria Interaction

Bacteria are invariably present during algal wastewater treatment, given the systems are open and that

the wastewater contains organic carbon that can support heterotrophic growth. The relative abundance of

bacteria to algae will depend to a large extent on the relative concentrations of organic and inorganic

carbon and the availability of light to drive photosynthesis. In addition, the ability to selectively retain

algae in preference to bacteria can increase the relative purity of the algae cultures, which is an

advantage that filamentous algae have over microalgae. The presence of bacteria is important in terms of

system performance and the consistency and composition of the harvested algal biomass in relation to
23
potential utilisation. As highlighted above, algae and bacteria do not exist in isolation, but interact

beneficially via nutrient and gas exchange, and potentially negatively through blocking of light and

competition for nitrogen and organic carbon (Figure 3).

Algae-bacteria interactions and broader aspects of algae-bacteria consortia, have been extensively

studied in various wastewater treatment contexts, including waste stabilisation ponds and high-rate algal

ponds. Complex biotic and abiotic as well as symbiotic and competitive interactions exist within algae-

bacteria consortia, as recently summarised by Liu et al. (2017). As referred to above, a major symbiotic

interaction between algae and bacteria is the CO2-O2 exchange process. Algae provides O2 for aerobic

bacterial metabolism, which in turn decomposes organic matters and releases CO2 for algae growth. This

is considered the major BOD removal process in algae-bacteria consortium (Muñoz and Guieysse,

2006). Although various microalgae are also able to utilise organic carbon sources and grow

mixotrophically/heterotrophically (Perez-Garcia et al., 2011), bacteria can easily outcompete algae under

heterotrophic conditions during nighttime hours (Lee, 2001). Most studies on wastewater treatment

using filamentous algae have observed decreases in the COD/BOD levels to varying extents (Adey et al.,

1996; Bohutskyi et al., 2016; Neveux et al., 2016; Wang et al., 2013). However, there have been no

fundamental studies that have directly investigated organic carbon uptake by filamentous algae, so it is

currently unknown how much organic carbon removal is due to bacteria compared to algae.

Interestingly, two studies involving Spirogyra observed constant or even increasing levels of COD

following lab-scale treatment of secondary effluent (Ge et al., 2018) and outdoor pilot-scale treatment of

a nutrient-enriched rural stream (Kim et al., 2018). Possible explanations provided by the authors

include the already low COD level in influent and the relatively sterile laboratory conditions (Ge et al.,

2018), as well as inhibition of microbial enzymes at high pH and release of organic matter from dead

algae cells during outdoor cultivation (Kim et al., 2018).

System performance of filamentous algae-bacteria consortia is usually reported as a whole, with little

focus on the individual contributions of the algae and bacteria. Nevertheless, some observations of
24
filamentous algae wastewater systems, such as diurnal fluctuation in pH (Cole et al., 2014b), do indicate

dominance of algal photosynthesis during the daytime and bacteria respiration during the night. In

addition to organic carbon removal, algae-bacteria consortia can also affect nitrogen removal, which can

occur via multiple pathways. Nitrification/denitrification by bacteria can occur simultaneously with the

biosynthetic uptake of nitrogen by the algae (Liu et al., 2017). For wastewater that is rich in ammonium,

it has been reported that nitrification may be suppressed by microalgae and cyanobacteria (Choi et al.,

2010); however, no research has yet been performed to investigate whether this will also occur when

filamentous algae are used. There exists the possibility of implementing a heterotrophic nitrifying

bacteria-augmented system to assist algal treatment of ammonium-rich wastewater. This can help reduce

the level of total ammonium nitrogen to relieve ammonia inhibition, while utilising the CO2-O2

exchange process of an algae-bacteria consortium. Overall, mixotrophic/heterotrophic growth of

filamentous algae, filamentous algae-heterotrophic bacteria interactions and filamentous algae-nitrifying

bacteria interactions remain uninvestigated areas of research. Future studies can aim to understand and

test the effects of increasing the concentration of certain bacteria on the performance of filamentous

algae wastewater treatment systems. Developing an understanding of the mixotrophic growth behaviour

of filamentous algae and the complex interactions with bacteria will be important to maximising the

productivity and yield of algal biomass and will help guide species selection.

6. Engineering Parameters and System Design

6.1 Turbulence and Shear

Turbulence and shear are inevitably generated in algal ponds by paddle wheel mixing, gas sparging,

natural convection due to temperature differences, and the inflow and outflow of the wastewater. In ATS

systems, an appropriate level of turbulence can provide higher nutrient flux and reduce self-shading

effects, while excess shear can damage the algae and result in loss of biomass (Blersch et al., 2013).

Algae produce a complex extracellular matrix used for protection, adhesion, and sexual reproduction of

25
the cells (Domozych et al., 2012). Excessive shear may damage the extracellular matrix, adversely

affecting algal productivity. Turbulence may also affect evaporation of water from outdoor ponds.

It is very difficult to quantify the amount of shear exerted to algae cells, which can vary significantly

depending on algal species, reactor setup and external environmental factors. There are in fact only a

limited number of studies that have even qualitatively mentioned the effect of turbulence on filamentous

algae. In relation to ATS systems, productivity has been found to be strongly influenced by wave surge

frequency, with maximal productivity achieved at a medium wave surge frequency of 17 min-1 (Blersch

et al., 2013). The wave amplitude, determined by the volume of flow in each wave, can positively affect

productivity, but only under nitrogen-saturated conditions (Blersch et al., 2013). This provides some

insight on the effect of turbulence on attached systems, however the concept of wave surge may not

apply to pond systems where the effect of incoming wave is diminished and the shear from mechanical

mixing is more pronounced. A lab-scale study on Spirogyra found that applying a high aeration rate of

206 ± 14 cm3/min to 2L column type photobioreactor significantly reduced productivity, which was

attributed to cell fracture under the high shear resulting from the aeration (Ge et al., 2018). In contrast,

no adverse effect of aeration has been reported in studies using Oedogonium for wastewater treatment

(Cole et al., 2014a, 2014b, 2015, 2016). However, the exact air flow rate has not been mentioned in most

of these studies, and the reactors were much larger (from about 850 L up to 80 m3) than in the study by

Ge et al., 2018. Future studies should quantitatively analyse the shear in algal ponds and investigate the

fundamental mechanisms of the effects of shear and turbulence on filamentous algae.

6.2 Pond Depth

Pond depth is a key design parameter for algal ponds that can affect algae productivity. It influences the

light and nutrient profile, fluid dynamics and mixing, and gas exchange. For example, in a study of

HRAPs using microalgae for wastewater treatment, an increase in pond depth from 200mm to 400mm

resulted in 134-200% higher areal productivity and about twice the nutrient removal rate (Sutherland et

al., 2014). In this context, the key advantages of the increased pond depth were better light utilisation,
26
larger CO2 storage capacity, less chance of algae wash-out during heavy rains, as well as reduced land

requirements and capital costs (Sutherland et al., 2014). More generally, it has shown by mathematical

modelling that the optimum depth of an HRAP should be ~1 cm deeper than the layer at which oxygen

production and respiration are in equilibrium (Kroon et al., 1989), or at which 1% of the incident light is

perceived (Sutherland et al., 2014). However, previous findings relating to the depth of microalgal ponds

may not be applicable for large-scale cultivation of filamentous algae due to differences in their

morphology and physical behaviour when in suspension. Unlike microalgae which can be evenly

suspended in water, filamentous algae tend to form floating mats or clumps, or attach to surfaces

(Lawton et al., 2013). Complete and even suspension of algae filaments may not be feasible depending

on their susceptibility to turbulence, as well as the energy input required for thorough mixing at large-

scale. These factors will greatly influence the optimum pond depth. Pond depths ranging from 30 cm to

89 cm have been reported in studies using filamentous algae for wastewater treatment under outdoor

conditions (Cole et al., 2014a, 2016; Kim et al., 2018; Neveux et al., 2016), yet no studies have yet

compared system performance as a function of pond depth, leaving this an open area for future research.

This should include fundamental studies of filament aggregation and movement in relation to fluid

dynamics, coupled with an understanding of the effects of shear on filament biology and related to

targeted experimental studies investigating performance in relation to pond depth and mixing.

6.3 Residence Time and Harvesting Frequency

In addition to pond depth and turbulence, hydraulic residence time (HRT) and biomass concentration

can also affect algal productivity and nutrient removal efficiency (Sutherland et al., 2014). However,

unlike microalgae systems where harvesting of algae usually occurs downstream via flocculation,

centrifugation or filtration (Abinandan and Shanthakumar, 2015), harvesting of filamentous algae is

relatively easy and low-cost. In addition, filamentous algae can be readily retained in the ponds by

installing mesh screens at the outlet (Cole et al., 2015; Neveux et al., 2016). Therefore, the HRT and

solids residence time (SRT) can be uncoupled for filamentous algae systems, allowing the process to be
27
intensified and the filamentous algae to be selectively retained over potential competitors. Some

previous research has been focussed on microalgal MBR systems that also uncouple HRT and SRT for

wastewater treatment, showing higher algal productivity and wastewater treatment efficiency compared

to CSTR configurations (Tang and Hu, 2016; Xu et al., 2015). For algal MBR systems, both HRT and

SRT were found to be key parameters that can affect system performance (Xu et al., 2015). The

retention of filamentous algae requires a much simpler and cheaper setup than a microalgal MBR

system. For both systems the HRT and SRT are both key parameters in system design. Under this

configuration, the SRT is controlled by occasional removal of filamentous algae biomass from the

ponds, therefore the extent and frequency of harvesting are the key parameters controlling the SRT. For

a given SRT, altering the balance between the extent and frequency of harvesting will affect the

difference in the peak and trough biomass concentration in the ponds, the operational requirements for

harvesting, and the regularity of biomass supply for downstream processing. Many long-term

demonstrations of wastewater treatment systems with filamentous algae have typically employed a

harvesting frequency of once per week, but it can range from monthly for slow-growing algae in winter

up to twice a week for fast-growing biomass (Cole et al., 2016; Craggs, 2001; Kangas et al., 2017;

Neveux et al., 2016; Ray et al., 2015). The benefits of more frequent harvesting include physical

removal of pollutants, control of self-shading effects, control of grazer population, and rejuvenation of

the filament population to help maintain exponential growth by preferential removal or large, mature,

slow-growing filaments (Craggs, 2001; Ray et al., 2015). Harvesting frequency is also likely to affect

accumulation of heavy metals in the algal biomass. When designing wastewater treatment systems with

filamentous algae, the HRT, SRT, and the proportion of algal biomass being harvested need to be

optimised in connection with each other. Fundamental studies can focus on understanding how these

parameters are affected by different species and by external factors such as wastewater composition and

climate variations.

6.4 Assessment of Cost and Land Requirements

28
The purchase of land on which to site the cultivation ponds is likely the major capital cost in many

situations. The land area requirements can be readily estimated, however the cost of land purchase is

highly site specific. All the above-mentioned engineering design parameters affect the areal productivity

and nutrient removal rate from wastewater. It is these two parameters that determine how much

cultivation area would be required to remove a desired amount of nitrogen from a given flowrate of

wastewater as follows:

𝐶𝑢𝑙𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 𝑎𝑟𝑒𝑎 (ℎ𝑒𝑐𝑡𝑎𝑟𝑒𝑠) 𝑅𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑖𝑛 𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝑔 ― 𝑁/𝐿) 1

= ×
𝑊𝑎𝑠𝑡𝑒𝑤𝑎𝑡𝑒𝑟 𝑡ℎ𝑟𝑜𝑢𝑔ℎ𝑝𝑢𝑡 (𝑀𝐿/𝑑) 𝐴𝑟𝑒𝑎𝑙 𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑟𝑒𝑚𝑜𝑣𝑎𝑙 𝑟𝑎𝑡𝑒 (𝑔 ― 𝑁/(𝑚2.𝑑)) 10

The areal nitrogen removal rates that have reported for filamentous algae range from about 0.4 to 1.3 g-

N/(m2.d) (Lawton et al., 2017). Taking a nitrogen removal rate of 1g-N/(m2.d) as an example, 3.6

hectares of filamentous algae cultivation area would be required to remove 90% of the nitrogen from 1

ML/d of a typical municipal wastewater containing about 40 mg/L total nitrogen (Neveux et al., 2016).

This estimation is in line with a field study using Oedogonium to treat primary municipal wastewater,

which reported an estimated land requirement of 94 hectares to treat 29 ML/d of wastewater (Neveux et

al., 2016). Based on these areal nitrogen removal rates, the amount of required land is likely to be a

limiting factor for the implementation of filamentous algal ponds, especially in densely populated areas

where the land costs are high. It is therefore important to investigate ways to improve the areal

productivity and nitrogen uptake rates of filamentous algae to reduce the land requirements. This

requires developing an understanding of which factors limit the growth of a given species under given

cultivation conditions, which would enable an appropriate improvement to be implemented. For

instance, temperature limitations could be mitigated by seasonal rotation of high-temperature and low-

temperature tolerant strains. If there is incomplete photosynthetic utilisation of the available sunlight,

this could perhaps be improved by better depth selection or mixing of the ponds, or by ensuring

adequate supplies of inorganic carbon. Ideally filamentous algae productivity can even exceed that

possible via photosynthesis alone, via mixotrophic utilisation of the organic carbon present in the

29
wastewater. As discussed, the algae will be in competition with bacteria for this carbon, however,

manipulation of SRT through retention of the filamentous algae could help ensure dominant algae

populations that can outcompete bacteria for this resource.

Beyond the cost of land purchase, implementing a filamentous algae treatment system requires various

capital works and ongoing operating expenses. Again, these costs will be case specific, varying greatly

depending on the scale and location of implementation. Many life cycle analyses (LCA) and techno-

economic assessments (TEA) have been performed in relation to microalgae production and wastewater

treatment (Lundquist et al., 2010; Quinn & Davis, 2015; Xin et al., 2011) that estimate the

environmental impact and economic feasibility of these systems. There are only a few studies that have

performed LCA and cost analysis on ATS systems applied to dairy wastewater treatment (Higgins &

Kendall, 2012; Pizarro et al., 2006), and there appears to be only one study that has qualitatively

described the industrial ecology of wastewater treatment using filamentous algal ponds (Lawton et al.,

2017). Detailed TEA and LCA studies remain to be performed for filamentous algae pond systems,

preferably in direct comparison to ATS and microalgae systems. Major similarities across these systems,

which are also advantages of algae wastewater treatment compared to traditional systems, are the cost

reduction from claiming GHG emission offsets and the revenue from downstream products recovered

from algal biomass. However, there are significant differences between filamentous algae and

microalgae systems including the reactor setup, harvesting methods, and the products that can be

recovered from the biomass. In particular, mechanical harvesting is applied for filamentous algae, which

is easier to operate and potentially has lower costs compared to flocculation and centrifugation, which

are typically required for microalgae.

6.5 Summary

Various engineering system design parameters mentioned in this section can affect the areal productivity

of filamentous algae. Based on currently reported values of areal nitrogen uptake rate the estimated land

area is high and can potential limit the implementation of such systems. Since the land requirement is
30
directly related to the areal productivity and nutrient removal rate, it is crucial for future studies to aim at

improving the areal productivity by informing improvements in system design and species selection.

The comparative costs of implementing a filamentous algae wastewater treatment process compared to a

microalgae system have yet to be determined. However, the simpler harvesting and retention of

filamentous algae are major advantages that could help lower the costs. As with microalgae and ATS

wastewater treatment, the potential benefits of avoiding nitrous oxide emissions, reducing energy

requirements and the conversion of waste nutrients into useful biomass appear likely to outweigh the

costs in some situations.

7. Conclusions

Filamentous algae have advantages over microalgae due to their size, which allows easier harvesting and

retention. This facilitates maintenance of monocultures, separation of HRT and SRT, and robust

operations despite flow fluctuations. Various filamentous algae species are candidates for monoculture

wastewater treatment systems, including algae from the genera Oedogonium, Cladophora and

Spirogyra. However, there are considerable research gaps in areas including species selection, the effect

of nutrient and carbon loading, algae-bacteria interactions, and engineering design parameters. Despite

the great potential of filamentous algae for wastewater treatment applications, more fundamental

research is required to prove the value of implementing such systems.

Acknowledgements

The authors are grateful to the Melbourne Water for providing financial support for this work and to The

University of Melbourne School of engineering for a scholarship to support J. Liu.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Declaration of authors’ contribution

31
J. Liu drafted the initial manuscript with contributions from G. Martin. All authors helped to develop the

conceptual outline of the work and revised and approved the final manuscript. G. Martin takes

responsibility for the integrity of the entire work and can be contacted as [email protected].

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Algal Cultivation Using Wastewater Nutrients. Journal of Chemical Technology & Biotechnology, 69(4),

451-455.

41
Table 1. Characteristics of some freshwater filamentous algae (adapted from Entwisle et al., 1997).

Algae Genera Biological Microscopic Features Natural Habitat

Classification
Oedogonium Division Unbranched filament, 2-90 Free floating or attached to
Chlorophyta, µm in diameter. Easily various substrates.
Family identified by the globular-
Oedogoniaceae shaped female gametangia
and the ‘ring’ structure at
the end of old cells after cell
division.
Spirogyra Division Unbranched filament, 20- Free floating or attached, slimy
Chlorophyta, 160 µm in diameter. to touch, and sometimes forms
Family Readily identified by the mats and blocks channels.
Zygnemataceae, spiral-shaped chloroplasts
within the cells.
Subfamily
Spirogyroideae
Cladophora Division Branched filament with 80- Coarse to touch, and form turfs
Chlorophyta, 90 µm long cells, 25-80 µm or large mats in warm and
Family in diameter and thick cell temperate climates, causing
Cladophoraceae walls. clogging issues.
Rhizoclonium Division Unbranched filament, 10-50 Mixed with other filamentous
Chlorophyta, µm in diameter with thick algae or form extensive mats
Family cell walls. on its own. Often found in
Cladophoraceae alkaline or saline water
conditions.
Microspora Division Unbranched filament, 10-20 Usually mixed with other
Chlorophyta, µm in diameter with thick filamentous algae and
Family H-shaped cell walls. unattached. Commonly found
Microsporaceae in cool and unpolluted
conditions.
Klebsormidium Division Unbranched filament, 4-15 Loosely attached or floating
Chlorophyta, µm in diameter. Identified at/above water surface in
Family by one single nucleus and cooler climates.
Klebsormidiaceae chloroplast covering less
than half of the width but
extending along the length
of a cell.
Stigeoclonium Division Irregularly branched Closely attached to substrates.
Chlorophyta, filament, 3-10 µm in Usually found in fast-moving
Family diameter. Can be identified streams in winter or spring.
Chaetophoraceae by the sharp, erected Commonly growing in urban
branches. creeks but does not cause
clogging.

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Table 2. Reported filamentous algae genera and corresponding temperature range in ATS.
Temperature Range (oC) Filamentous Algae Present Reference
5-30 Spirogyra Kangas et al. (2017)
19-24 Microspora willeana (major) Kebede-Westhead et
Rhizoclonium hieroglyphicum al. (2003);
Oedogonium sp. Mulbry and Wilkie
(2001)
5-30* Spirogyra (major) Mulbry et al. (2010)
Ulothrix
Microspora
Claophora
20-26** Spirogyra Marella et al. (2019)
15-20 (influent) Rhizoclonium hieroglyphicum (major) Mulbry et al. (2008b)
<32 (effluent) Microspora willeana
Ulothrix ozonata
Oedogonium sp.
25 ± 3 Ulva (major) Ray et al. (2015)
Cladophora
Rhizoclonium
22 Microspora willeana (major) Wilkie and Mulbry
Ulothrix zonata (2002)
Ulothrix aequalis
Rhizoclonium hieroglyphicum
Oedogonium sp.
* Annual range. Filamentous algae dominated during summer/autumn
** Temperature in winter/rainy season when filamentous algae were observed

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Table 3. Areal biomass productivity of ATS in different studies.

Areal Productivity Wastewater Type Scale Reference

(gdry algae/(m2.d))
23.8±16.4 Secondary effluent Large pilot Craggs et al. (1996b)
scale
26 ± 16 Secondary effluent Pilot scale Sandefur et al. (2011)
29.7-48.9 Raw domestic Lab scale Marella et al. (2019)
4.9 ± 1.6 Agricultural drainage water Pilot scale Kangas and Mulbry
(2014)
7.1-9.6 Swine manure Lab scale Kebede-Westhead et al.
(2006)
2.5-24 Dairy manure Pilot scale Mulbry et al. (2008b)
5-23 Anaerobically digested dairy Lab scale Kebede-Westhead et al.
manure (2003)
6.8-21.3 (lab scale) Dairy and swine manure Lab and pilot Mulbry et al. (2008a)
4.9-14.6 (pilot scale) scale
5 Dairy manure (anaerobically Lab scale Wilkie and Mulbry
digested and undigested) (2002)
88.8 Oyster aquaculture wastewater Lab scale Ray et al. (2015)
0.8-2.0 Horticultural wastewater Lab scale Liu et al. (2016)
* lab scale refers to growth area of 1m2 or less

44
1 Table 4. Areal biomass productivity of monocultures of filamentous algae for wastewater treatment.
Algae Areal Productivity Wastewater Type Light Water pH Bomass Water Exchange Reference
(gdry biomass/(m2.d)) and Scale Intensity Temperature Concentration Rate
(mol/(m2.d)) and Harvesting
Oedogoni ~5 (no aeration) MAF growth 30.9 ±3.0 27.7±1.6 oC 8.2± 2.0 (with CO2) Weekly harvest to Equivalent to 10% Lawton et
um ~8 (low aeration) medium; outdoor 10.5±1.5 (without CO2) 0.5 g/L per day al. (2013)
~10 (high aeration)* small-scale study
3.8-23.8 Aquaculture 23.2 ±1.5 daily 18.9 - 24.3 oC 7.7 ±0.1 (without CO2) Initially 0.35 g/L, 10% to 500% per Cole et al.
effluent; outdoor average; 6.8 ±0.2 (with CO2) harvested after 4 day (2014a)
small scale 90-165 peak Peak 9.3-9.4 days
17.1-23.6* Aquaculture 49.3 ±1.3 daily 20.9-32.1 oC Influent: 8.0 ±0.2 Weekly harvest to 33% for the whole Cole et al.
effluent; outdoor average; Maximum culture pH: 0.23 g/L system (2015)
small scale 170-218 peak 9.4-10.3
12.7-13.8 Primary effluent; 36-60 weekly 25–27 oC 8.5 (sunrise) Biweekly harvest 5%, 10% and 20% Neveux et
outdoor small scale average 10.8 (sunset) to 0.25 g/L per day al. (2016)
0-6 Secondary effluent; 36-60 weekly 25–27 oC 8.5 (sunrise) Biweekly harvest 5%, 10% and 20% Neveux et
outdoor average 10.8 (sunset) to 0.25 g/L per day al. (2016)
experimental scale
6.8-9.9 Primary effluent; 18-33 weekly 17.4–24.5 oC 9.3 (sunrise) Weekly harvest to 5% per day Neveux et
outdoor pilot scale average 9.9 (sunset) 0.25 g/L al. (2016)
8.9-15.8 Secondary effluent; 32.3 daily 27.6 ±0.8 °C Influent: 6.7-7.1 Biweekly harvest 100% per day Cole et al.
outdoor pilot scale average (min. 14.2°C, Effluent: 9.0-9.9 to 0.18-0.37 g/L (2016)
max. 36.5 °C)
Cladopho ~5 (no aeration) MAF growth 30.9 ±3.0 27.7±1.6 oC 8.2± 2.0 (with CO2) Weekly harvest to Equivalent to 10% Lawton et
ra ~8 (low aeration) medium; outdoor 10.5±1.5 (without CO2) 0.5 g/L (f.w.) per day al. (2013)
~8 (high aeration)* small-scale study
Spirogyra ~3 (no aeration) MAF growth 30.9 ±3.0 27.7±1.6 oC 8.2± 2.0 (with CO2) Weekly harvest to Equivalent to 10% Lawton et
~5.5 (low aeration) medium; 10.5±1.5 (without CO2) 0.5 g/L (f.w.) per day al. (2013)
~8 (high aeration)* outdoor small-scale
study
6.0 ± 0.9 Secondary effluent 10.4 ± 1.3 23 oC - Initially 0.625 g/L Batch culture Ge et al.
(low mixing), continuous (2018)
~0.6 (high mixing)* light
9.1 ± 2.2 (secondary Various; lab study 6.5-10.4 - - Initially 0.625 g/L Batch culture Ge et al.
eff.), continuous (2018)
7.1 ± 2.3 (diluted light
primary eff.),
Negative (others)*
2.9-4.0 (secondary eff.), Secondary effluent, 4.3-7.3 20-25 oC - Initially 2.5 g/L Repeat batch; Ge et al.
0.1-0.4 (diluted 2% diluted centrate continuous medium changed (2018)
centrate)* water; lab study light fortnightly or
nutrients <1 mg/L
2 *ash-free basis
45
3 Table 5. Growth of Oedogonium and Spirogyra under different ammonium-N levels.

Algae Wastewater Type Initial Ammonium- Ammonium- Initial pH Final pH Algae Growth Reference
Genera N concentration N removal
(mg/L) (%)
Oedogonium Digested piggery 59.4 95.9 ± 1.3 Not Not Not mentioned Wang et
wastewater mentioned mentioned al. (2013)
Spirogyra Diluted primary About 8 >99 About 7 About 7 2.17 ± 0.76 g/(m2.d) Ge et al.
effluent (20%) (2018)
16.4 ± 2.0% /d
Diluted Centrate About 9 38.0% About 7 About 6 Negative growth Ge et al.
Water (1%) (2018)
Diluted Centrate About 15 40.2% About 7 About 6 Negative growth Ge et al.
Water (2%) (2018)
Artificially 13.0 - 15.0 73.2% 7.9 8.5-10.6 ~17 g/(m2.d) Kim et al.
Enriched rural (2018)
stream
4

46
6
7 Figure 1. Diagram showing stages in a typical wastewater treatment process (shaded boxes) and

8 the different wastewater streams (white boxes) in which filamentous algae could be grown. Key

9 components in the wastewater streams are listed (white boxes) and major sources of greenhouse

10 gas emissions in conventional processes that could be mitigated by algae are identified in red

11 text.

47
12

13 Figure 2. Generalised relationship between algal productivity, nutrient uptake rate and nutrient

14 loading based on studies of ATS systems.

15

48
16

17 Figure 3. Schematic diagram summarising some key aspects of suspended monocultures of

18 filamentous algae for wastewater treatment.

19

20 Highlights:

21  Filamentous algae have promise for wastewater treatment

22  The literature is reviewed in relation to species selection and engineering design

23  The size of filamentous algae provides advantages over microalgae

24  Filamentous algae can be readily harvested and retained to control solids residence time

25  Future research is needed to understand algae-bacteria interactions and techno-economics

26

27

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28 Declaration of interests

29

30 ☒ The authors declare that they have no known competing financial interests or personal relationships
31 that could have appeared to influence the work reported in this paper.

32

33 ☐The authors declare the following financial interests/personal relationships which may be considered
34 as potential competing interests:
35

36
37
38

39

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