BioEnergy Research (2022) 15:1–26

https://doi.org/10.1007/s12155-021-10254-8

Microalgal Co-cultivation for Biofuel Production and Bioremediation:

Current Status and Benefits
Prabir Kumar Das 1 & Jyoti Rani 1 & Shweta Rawat 1,2 & Sanjay Kumar 1

Received: 16 September 2020 / Accepted: 26 January 2021 / Published online: 1 March 2021
# The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021

Abstract
Microalgae have been reported to exhibit mutualistic interactions with other microorganisms like bacteria, filamentous fungi, and
yeast and help each other co-exist. The potential of microalgae to perform photosynthesis and accumulate lipids make them
suitable candidates for lipid production. Biofuel production from various single oleaginous microorganisms is already in practice.
However, the high cost of biomass harvesting, extraction of lipids, and contamination issues are significant challenges of biofuel
bioprocess commercialization. Recent microalgal co-culture studies showed considerable potential for easy biomass harvesting
and reduction in overall energy consumption cost. Therefore, microalgal co-culture could be an alternative to overcome these
constraints and enhance biomass and lipid production. Additionally, the integration of the nutrient sequestration process from
potential agro-industrial wastewater using microalgal co-culture can reduce the cost of the substrate requirement for cultivation as
well as ecological load. The co-culture in wastewater has shown excellent total phosphate removal efficiencies by microalgae
Chlorella sorokiniana and yeast Rhodotorula glutinis, nitrogen removal by microalgae C. sorokiniana with activated sludge, and
ammonium-nitrogen removal by C. vulgaris and fungi Aspergillus sp. co-culture. This review summarized the current advances
towards biofuel and its value-added production from various microalgae co-culture and compared it with monoculture fermen-
tation. It also includes some critical challenges of co-culturing for the economically viable bioprocess development for biofuel
production. Furthermore, techno-economic analysis and life-cycle assessment of co-culture technology were also discussed for
biofuel production feasibility from microalgal co-culture.

Keywords Co-culture . Mutualism . Agro-industrial . Lipid productivity . Waste removal . Techno-economic . Feasibility

Introduction energy-related CO2 emission has increased by 1.7% to 33

Gigatonnes in 2018 [3]. Besides fossil fuels, other renewable
The increasing global warming and climate changes have cre- energy resources like solar, wind, hydropower, geothermal,
ated a global energy crisis and have raised concern on the etc. are also a major contributor to this power generation ex-
sustainability issues of fossil fuel utilization [1]. pansion, accounting for nearly half of the electricity demand
Undoubtedly, increasing environmental pollution with rapidly growth. In some recent studies, a green approach to prepare
growing energy demand is a future challenge to be answered nanoparticles is explored as energy storage strategies to miti-
in the near future [2]. Energy demand has grown up by 2.3% gate the energy crisis issue. Recently, TiO2 nanotubes were
worldwide as per the 2018 statistics. The demand for fuels of prepared from unannealed TiO2 nanoparticles which exhibit
all types is increasing gradually and fossil fuels meet nearly strong UV absorption with 7.2% power conversion efficiency
70% of this ever-increasing demand. As a result, global for the prepared solar cell [4]. Despite major growth in renew-
able energy sources, global emissions are still rising. To re-
duce global warming and to develop the renewable, clean, and
* Sanjay Kumar green energy supply, some strict actions need to be taken on
[email protected] all fronts—developing clean energy solutions including cap-
1
turing carbons from the combustion of renewable energies,
School of Biochemical Engineering, Indian Institute of Technology curbing emissions, improving the efficiency of energy-
(BHU) Varanasi, U.P, Varanasi 221005, India
2
intensive processes, and spurring investments and innovations
Biochemical Engineering Department, Bipin Tripathi Kumaon for better production, utilization, and storage process of
Institute of Technology, Dwarahat, Uttarakhand 263653, India
2 Bioenerg. Res. (2022) 15:1–26

energy should be well established [3]. The development of still under development and not fully commercialized due to
microbial technologies are the promising approaches for the major obstacles faced by microalgae-based industries. At
conversion of biomass into clean and renewable energy is present time, microalgae-based biofuel industries are facing
sustainable development to the security of energy and the key challenges listed as; a) very less oil content and substan-
environment too [2]. dard composition, b) High economic costs for the harvesting/
Among various clean energy solutions, renewable biofuels dewatering of microalgal cells from large volumes of culture
have received the greatest attention. Since biofuels from food c) high cost of extraction of lipids from biomass, and d) poor
crops and lignocellulosic feedstocks could not meet the supply of nutrients [14]. Researchers are focusing on devel-
existing demand and economic parameters, microalgae appear oping various microalgal co-cultivation strategies that will be
to be one of the most promising feedstock for energy produc- advantageous to increase the total biomass and lipid yield
tion [5, 6]. Oleaginous microorganisms are popular for their which can minimize the overall production cost. Co-culture
excess lipid productivity and oil content that exceeds up to is a cell cultivation setup where two or more different micro-
20% of the total biomass on a dry weight basis. These oils organisms are grown together with some degree of contact
produced by oleaginous microorganisms are called single cell between them [15]. The motivation for the co-cultivation tech-
oil (SCO) [7]. Production of biofuel from algae was initially nique came from the studies: the study of natural interaction
proposed for producing methane gas in the early 1950s, but between different populations, improving the success of sur-
curiosity in the application of algae for biofuel production was vival for certain cultures, or establishing the synthetic interac-
revitalized during the oil embargo and the oil price surge of tion between populations [15]. This co-culture system has
the 1970s [8]. The Department of Energy (DOE), USA, been recognized as an efficient technique for different cultures
established various programs for renewable energy produc- growing together in a natural environment [16]. Naturally,
tion from solar to plant life as the source of transportation clusters of microbial cells can grow and survive in close asso-
fuels. DOE supported the Aquatic Species Program (ASP), a ciation in the same biocoenosis either by mutualism or antag-
research endeavor desired to look into the aquatic life as a onist relationship. Interactions observed in many microbial
source of energy supply and a specific goal was the production communities depend upon molecular diffusions such as
of oil from microalgae by relatively small efforts (about $25 cross-fed metabolites, quorum-sensing molecules, and antimi-
million over almost 20 years) from 1980 to 1996 [9]. crobial compounds [16]. Most of the microorganisms not only
During the last decade, algal biofuel production has drawn interact with each other but also interfere with higher organ-
keen attention as a third-generation biofuel due to certain lim- isms. A well-known example is the symbiotic interaction of
itations of first and second-generation biofuels. Food security nitrogen-fixing bacteria present in the root nodules of plants
issues, rising stress on free available land, and social conflicts [17]. The co-culture technique has been used for decades,
are the main causes to question the sustainability of first and primarily in wastewater treatment, producing biomass and
second-generation biofuels for the future. Further, it provokes bioactive compounds, and to degrade hydrocarbons and car-
the urgent requirement for third-generation biofuel. In this bohydrates into biofuels [14]. In microalgal co-culture, an
regard, algae may be considered an excellent candidate due effective increase in lipid production along with the changes
to very simple and feasible algae culture conditions, i.e., suf- in the composition of lipid has also been observed in co-
ficient light, CO2, and naturally available dissolved nutrients culture [18]. One of the aims of the co-cultivation technique
[10]. Algae have tremendous applications other than biofuels, is to mix two or more particular species in such a way that one
industrially important co-products and several value-added strain has an enzymatic activity that is absent in the other
products are extracted, sequestration of CO2, and treatment strain so that both will be benefited from each other. An ex-
of wastewater [11]. periment was carried out where an amylolytic enzyme-
Most of the studies for algal biofuel production have been producing strain was mixed with a non-amylolytic producer
done using single-cell culture (monoculture), but recently the strain to hydrolyze the starch used as a carbon source and the
focus is shifting to co-culture techniques for biodiesel produc- results were highly representative [16]. Some marine
tion. In this direction, co-cultivation of yeast with microalgae microalgae and heterotrophic bacteria, when co-cultured in a
in simplest and low-cost cultivation conditions, even grown in single culture, secreted some extracellular enzymes and
wastewater may be considered a potential feedstock for bio- growth factors that helped in the growth of each strain. The
diesel production [12]. Similarly, algae-bacteria co-culture is co-culture study of Bacillus halmapalus and Alexandrium
an advantageous process for higher biomass and lipid produc- tamarense also show some effect of mutualism among the
tion along with the potential for waste removal [13]. Such co- two strains [19].One recent study analyzed seaweeds from
cultivation systems are also utilized for wastewater treatment Abu Qir Bay for biofuel production. In this study, twenty-
along with biofuel production to reform the economic and two macroalgal species were collected and screened based
environmentally feasible system [13].Although algae-based on lipid and carbohydrate profile with biomass productivity
biofuel production started in the early 1950s, the process is [20]. This sequential biodiesel- bioethanol production study
Bioenerg. Res. (2022) 15:1–26 3

concluded Ulva intestinalis as a potential feedstock for en- production. Later on, oleaginous microbes were explored for
hanced energy conversion [20]. Another novel strategy to im- the production of lipids which were trans-esterified to fatty
prove biomass and lipid productivity is the co-culturing of an acid methyl esters (FAMEs) [27].
oleaginous yeast with algae. Microalgae accumulate lipids mainly in the plastid and
Some oleaginous yeast like Scenedesmus obtusiusculus, endoplasmic reticulum (ER). The biosynthesis of lipid starts
Cutaneotrichosporon oleaginosus, Cryptococcus curvatus, through photosynthesis where CO2 is converted into a three-
Rhodosporidium toruloides, Lipomyces starkeyi, Yarrowia carbon compound glyceraldehyde-3-phosphate (G3P) and
lipolytica, and Rhodotorula glutinis are gaining attention for then into pyruvate [28]. The pyruvate is further converted into
the production of biofuels as these can convert lignocellulosic acetyl-CoA by the pyruvate dehydrogenase complex, which is
sugars into lipids (35–61% on a dry weight basis) [21, 22]. the initiator of lipid biosynthesis in the plastid. Other bio-
Recently, a co-culture study between microalgae Chlorella chemical pathways that allow conversion of stored polysac-
vulgaris and yeast Saccharomyces cerevisiae in non-aerated charides can also generate acetyl-CoA which leads to the bio-
photobioreactor has shown in situ CO2 mitigation and these synthesis of lipid in many heterotrophic oleaginous microor-
types of the fermentation process are widely used in food, ganisms [28]. In environmental stress conditions like phos-
high-value-added compounds, and bioenergy industries [23]. phate and nitrogen limitations, the microalgal tricarboxylic
A co-culture study of filamentous fungi Aspergillus niger and acid (TCA) pathway gets imbalanced which leads to the ac-
microalgae Chlorella vulgaris has shown a potential tech- cumulation of citrate in mitochondria and finally discharged
nique for microalgae harvesting with more than 90% harvest- into the cytosol. Citrate lyase, an ATP-dependent cytosolic
ing efficiency [24]. So, co-culture between different microbial enzyme helps in the conversion of citrate into acetyl-CoA
communities either algae, bacteria, yeast, or filamentous fungi and oxaloacetate that is later converted into malonyl-CoA by
can offer enhanced lipid production. acetyl-CoA carboxylase (ACC); therefore, the elongation of
Thus, in this review, different types of algal co-culture with fatty acids occur in ER [29]. Lipid content in some common
bacteria, fungi, yeast, and algae are discussed and compared microalgal species belonging to genera such as Chlorella and
with monoculture of algae on the basis of lipid productivity as Isochrysis varies within 20–50% Nanochloropsis 59% [30],
well as wastewater treatment effectiveness. Furthermore, the Dunaliella, and Schizochytrium 72% on a dry weight basis
other related benefits associated with the co-cultivation sys- [31]. Oleaginous microorganisms can accumulate lipids up
tem and the key challenges that need to overcome for the to 70% by controlling the physical parameters such as tem-
scaling up of current microalgal biofuel production strategies perature, pH, and nutrient availability during the culturing
are also summarized. period [7]. Recently, researchers have been focused on the
genetic engineering of oleaginous microorganisms to enhance
lipid accumulation. Among various genetic engineering strat-
Naturally Available Microorganisms egies, the main concern was the overexpression of certain
for Biofuel Production enzymes that play a key role in the synthesis of fatty acids
and TAGs. Overexpression of ATP-dependent citrate lyase,
Biofuels are clean sustainable liquid fuels produced from ole- acetyl-CoA carboxylase, acetyl-CoA oxidase, malic enzymes,
aginous biological feedstocks that can be helpful in replacing glycerol-3-phosphate dehydrogenase, etc., and partial or full
the conventional fossil fuels used for transportation and other blocking of few competitive lipid biosynthesis pathways can
energy-intensive activities. Biofuel research is receiving more increase the lipid accumulating properties in oleaginous mi-
attention globally due to various concerns that can be ad- croorganisms [32]. Therefore, the management of environ-
dressed like energy security, environmental deterioration, lim- mental conditions and the selection of proper strains can im-
itation on imports, agricultural economy, and rural applica- prove the lipid accumulation in microorganisms. Light is one
tions [25]. Initially, attempts for biodiesel production were of the most important factors for microalgal growth where the
made by using edible plant oils such as rapeseed, peanut, different light spectrum on microalgae affects their size,
safflower, corn, cottonseed, palm, coconut, sunflower, etc. growth, biomass yield, and lipid content. However, in all liv-
But, continuous use of edible plants for biofuel production ing creatures, different color light qualities, as well as dark-
led to competition with the food supply. So, the utilization ness, significantly affect biomass yield and extracellular as
of non-edible plants as a biofuel feedstock assists the sustain- well as intracellular pigment production. [33]. A study reveals
ability of fuel production and, minimizes the use of edible the effect of different light spectrum on Chlorella vulgaris
plant sources [26]. The cost of biodiesel extraction from edible where it showed the maximum growth rate can be obtained
plant seeds accounts for up to 70–85% of the overall cost [16]. in white (0.70 divisions per day) and blue (0.67 divisions per
To address the problems related to biofuel production from day) light along with the highest lipid yield of 18.74% (w/w)
plant sources, researchers proposed microbial sources as a on the dry weight basis in the blue light spectrum [34]. The
sustainable and economically viable source for biofuel intensity of the light spectrum is also a key factor to achieve
4 Bioenerg. Res. (2022) 15:1–26

maximum algal biomass yield. The highest microalgal bio- industries [44]. The growth of microalgal cells and accumula-
mass productivity (367.82 ± 21.63 mg L−1 day−1) can be ob- tion of lipids under a phototrophic environment is influenced
tained when light is provided at 200 μmol photons m−2 s−1 by the intensity of light sources, dissolved oxygen concentra-
and the highest lipid productivity (126.72 ± 3.27 mg L−1 tion, pH of the medium, carbon dioxide present in sparing gas,
day−1) can be obtained in 400 μmol photons m−2 s−1 [35]. the concentration of nutrient supplements such as nitrogen,
The intensity of light is also responsible for the variation in phosphorus, iron, silicon and presence of organic carbon
fatty acid profile, as the ratio of saturated fatty acids increased sources [45]. One of the main advantages of microalgae cul-
with the enhanced light intensity [35]. A recent study sug- tivation is that they can easily grow in wastewater and help to
gested that replacing the red light instead of the white light remove waste components by utilizing them for their growth.
in the autotrophic mode of marine diatom (Phaeodactylum Wastewater sources like sewage, distillery, dairy, etc. have
tricornutum) cultivation enhances the total biomass (2-fold) high biological oxygen demand (BOD), chemical oxygen de-
and lipid content (2.3-fold) and showed the fatty acid compo- mand (COD), dissolved oxygen (DO), and high nitrogen and
sition is altered without changing the morphology of lipid phosphorus concentration which favors the growth of
droplets [36]. microalgae [46]. The hydrolysate of sewage sludge which
consists of volatile fatty acids (VFAs) is a convenient carbon
Oleaginous Microalgae source for the maturation of microalgal cells; a biomass con-
centration of 0.5 g L−1 and a lipid yield of 21.5% were attained
There are different groups of prokaryotic and eukaryotic mi- by the cultivation of microalgae Chlorella protothecoides
croorganisms but microalgae are becoming a promising [46]. Microalgae can grow by resisting different emerging
source for biofuel production and have the ability to meet pollutants (EPs) present in wastewater, so they have the po-
the international demand for fuel for transportation along with tential for wastewater treatment. In an experiment, three
the possibility of complete replacement of the use of fossil microalgae species Spirulina platensis, Scenedesmus
fuels [37]. The microalgae require minimal inputs for carrying obliquus, and Chlorella homosphaera were grown in a medi-
out metabolic processes such as sunlight, water, and CO2 with um containing different EPs like paracetamol, caffeine, fluox-
few required mineral nutrients. The efficiency of capturing etine, diazepam, and acetylsalicylic acid with a range of 1–
solar energy and converting it into biomass and finally into 100 mg L−1 where microalgae can support their growth up to a
lipids exceeds the potential of terrestrial plants. The average certain concentration of EPs like each microalga can grow in a
lipid content of algal cells varies between 1 and 70% but it diazepam concentration of up to 30 mg L−1 and above is toxic
may reach up to 90% of dry weight under certain conditions for all, paracetamol in a range of 50–100 mg L−1 causes the
(nutrient limitation, high salinity, high temperature, etc.) [38]. death of S. platensis and C. homosphaera, and fluoxetine is
Lipid content of 30–50% is quite familiar in microalgae, such most toxic as those can survive up to 1 mg L−1 only [47].
as Chlorophyta and Bacillariophyceae, which have higher oil Cultivation of green microalgae Coelastrum sp. in synthetic
content and easier to cultivate in autotrophic mode, especially and industrial wastewaters as a culture medium enhances the
Chlorella, which can be used for commercial production. biomass and lipid yield 2.29 and 0.71 g L−1 respectively [48].
There are several other aspects of using microalgae derived A study was carried out by using shrimp farm wastewater
products in the food, chemical, and pharmaceutical industries. (SFW) for the cultivation of few microalgal species including
Microalgae is an important source of proteins, vitamins, vita- Dunaliella sp., Nanochloropsis sp., Chlorella sp., Tetraselmis
min precursors like tocopherol, riboflavin, ascorbic acid, etc., sp, and two different strains of Navicula sp. and observed a
and other value-added products besides lipid production [39]. high accumulation of polyunsaturated fatty acids including
Butanol can also be produced by the acetone-butanol-ethanol eicosapentaenoic acid and most of the fatty acids are the
(ABE) fermentation of microalgae Chlorella vulgaris and the C16 and C18 groups [49]. Fish farm wastewater also can be
study showed the highest butanol was extracted 8.05 g L−1 used as a substrate for microalgae cultivation. Fish farm efflu-
[40]. A distinct bioactive compound lectin can be produced ent has been used for the culturing of certain Nanochloropsis
from Nanochloropsis oculata [41] and taurine, an important strains in stirred tank reactor (STR) and open pond–
cardiovascular compound can be produced from stimulating reactor (OPSR) where a certain strain
Ostreococcus tauri [42]. Various polysaccharides like man- N. gaditana was proliferated very well, obtaining dry cell
nose, β-1,3 glucan, N-acetylgalactosamine, rhamnose, etc. are mass of 847 mg L−1 and increase of lipid yield 7.8% (w/w)
also synthesized from Chlorella sp. which have antioxidant to 24.7% (w/w) [50].
properties and other immunological applications [43]. Although microalgae have turned up as a potential biofuel
Microalgal species are a rich source of pigments. Halophiles feedstock, the commercialization of microalgal biofuel is still
like the Dunaliella sp. are cultured for the production of ca- a constraint due to the deficiency of lipogenic strains and the
rotenoids like zeaxanthin, cryptoxanthin, α and β-carotene, lack of cost-effective oil extraction methodologies. These
violaxanthin, etc. that are used in the food and cosmetic drawbacks have to be overcome to achieve an economically
Bioenerg. Res. (2022) 15:1–26 5

feasible microalgal oil production process. The microalgal carbon sources like glucose, glycerol, and few micronutrients
strains have been engineered by targeting various key meta- that help in lipid accumulation of 15–25% w/w and synthesis
bolic enzymes like glycerol-3-phosphate acyltransferase, 1- of other value-added products like ethanol (31.3 and 21.9 g/L)
acyl-sn-glycerol-3-phosphate acyltransferase, and diacylglyc- from S. cerevisiae and C. tropicalis respectively, mannitol
erol acyltransferase for the overproduction of lipids [51]. (13.4 g/L), and citric acid (30.3 g/L) from Y. lipolytica [95].
There are so many different microalgal strains of wild type Other than recent strategies like overexpression of key en-
and engineered type that were cultured at various conditions zymes, modification of substrates, suppression of other com-
that support optimum lipid yields (Table 1). petitive pathways of lipid biosynthesis, etc. for the enhance-
ment of lipid synthesis, the evolutionary strategy is one of the
Oleaginous Yeast important techniques to select the improved strain by evolu-
tion. A study shows that the evolution of Y. lipolytica after 77
Among other microorganisms, yeast is one of the most prom- generations was able to produce 44% lipid (w/w) which is
ising in biotechnological applications as these lack endotoxins 30% higher than the lipid accumulation in the same strain
which are generally common in most bacterial species. In the without evolution [96]. Various fatty acids’ profiles are ob-
existence of excess carbon sources, few well-known bacteria tained from different species, however, the main fatty acids
can accumulate poly-β-hydroxybutyrate and poly-β- commonly produced by yeasts are myristic acid (C14:0),
hydroxyalkanoates as carbon reserve materials in intracellular palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid
granules while yeast accumulates extra carbon as lipid or gly- (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic
cogen in the form of triacylglycerides (TAGs) [88]. acid (C18:3), similar to fatty acids derived from algae and
Lignocellulosic biomass is an important substrate for the plants [97].
growth of yeast, moreover, pretreatment of lignocellulosic
biomass with different enzymes or acids allows better growth Oleaginous Bacteria
and lipid accumulation [89]. Thermal pretreatment with enzy-
matic hydrolysis of wheat bran containing sugars (glucose, Like yeast and fungi, some strains of bacteria can also accu-
xylose, and arabinose) has been used for the cultivation of mulate lipids in certain environmental conditions. The com-
Lipomyces starkeyi where lipid yield and oleic acids yield position of lipids synthesized from bacteria is a little different
were 37.3% and 70% respectively [90]. Recent studies show from other microbial oils. The most plentiful neutral lipids
oleaginous yeast use volatile fatty acids (VFAs) which are synthesized by bacteria are polyhydroxyalkanoic acids
produced in anaerobic fermentation of wastes as a substrate (PHAs) serving as intracellular carbon and energy repository,
and accumulate the optimum amount of lipids. Llamas et al. but few species can accumulate TAGs which mostly belong to
(2020) has shown in a study where few oleaginous yeasts the actinomycetes genera Streptomyces, Nocardia,
Yarrowia lipolytica, Lipomyces lipofer, and Mycobacterium, Rhodococcus, etc [98]. It was found that
Cutaneotrichosporon curvatum were allowed to grow in Rhodococcus opacus and Gordonia sp. can accumulate lipids
VFAs in the concentration range of 5 to 15 g L−1, biomass up to 80% under special conditions but biomass obtained is
yield of 4.6, 4.9, and 5.2 g L−1 respectively was observed and only 1.88 g/L [45]. Various agro-industrial wastewaters are
the highest lipid content was observed to be 30–37% of dry used for the growth of oleaginous bacteria and the production
biomass [91]. Other than glucose, glycerol can also be used as of lipids has been carried out in recent years. A culture of
a substrate for the growth of a few oleaginous yeasts such as Cunninghamella echinulata in tomato waste hydrolysate hav-
Mucor, Mortierella, Candida sp. etc. A study showing the ing glucose as a carbon source can produce a lipid of 8.7 g/L
comparison on lipid yield by Mortierella isabellina and [99]. E. coli is also an important model organism to produce
Mucor sp. was 8.5 and 5.4 g L−1 when grown in glucose biofuel as it has the potential to carry out genetic modifications
and glycerol respectively [92]. An oleaginous yeast as well as gene expression. Currently, ethanol is dominating
Yarrowia lipolytica was grown under double nitrogen and the biofuel industry and is commercially being produced by
magnesium limitation and was able to produce 12.2 g L−1 E. coli through the endogenous process called anaerobic fer-
biomass containing 47.5% lipid (w/w) that leads to lipid pro- mentation [100]. Ethanol is synthesized through two sequen-
duction of 5.8 g L−1 [93]. Different agro-industrial wastewater tial NADH-dependent reductions that are catalyzed by alcohol
resources are used these days for the culturing of yeast. Arous dehydrogenase (adhE gene product), and the genetic modifi-
et al. (2016) used Debaryomyces etchellsii strain BM1 for the cation for the upregulation of the adhE gene in E.coli enhances
treatment of different wastewater sources from cheese, olive the ethanol biosynthesis [101]. A cellulolytic Streptomyces sp.
oil, soft drinks, confectionery, etc., and surprisingly the lipid has the potential to hydrolyze agricultural wastes and the cel-
content estimated from this oleaginous yeast was around 26% lulolytic activities were obtained 73% and 65% when incubat-
of dry biomass [94]. Olive mill wastewater is considered a ed at 60 °C for 4 and 5 h respectively, and 98% activity when
fermentation media for various yeasts as it has a variety of incubated at 50 °C for 8 h [102].
6 Bioenerg. Res. (2022) 15:1–26

Table 1 Oleaginous microorganisms and their lipid profile in monocultures

Microorganism Genus Species Lipid yield (w/w, %) Cultivation References

Microalgae Botryococcus brauni 20.8 A [52]

Chaetoceros muelleri, calcitrans 39.8, 33.6 A [53]
Chlorella emersonii, protothecoides, sorokiniana, 25–63, 43.6–46.8, A, B, A, A, B, A [53–57]
vulgaris, pyrenoidosa, minutissima 19.0–22.0, 33–38,
38–52, 31–57
Neochloris oleoabundans 7.0–40.3 A [58]
Schizochytrium mangrovei 16.0 A [59]
Nannochloropsis sp. 11.0 A [60]
Dunaliella salina, tertiolecta 16–45, 60–67 A [61, 62]
Euglena gracilis 14–20 A [63]
Scenedesmus quadricauda, bijuga 27.3, 30.7 A [64, 65]
Isochrysis sp. 27.4 A [63]
Bacteria Rhodococcus opacus 54.3 B [66]
Bacillus subtilis 39.8 B [67]
Gordonia sp. 70.0 B [68]
Arthrobacter sp. 40.0 B [7]
Filamentous fungi Aspergillus niger, terreus 9.6, 37.4 B [69]
Cunninghamella elegans 33.6 B
Mucor circinelloides, plumbeus 23.8, 20.6 B
Neosartorya fischeri 8.8 B
Rhizopus oryzae 34.8 B
Thermomyces lanuginosus 21.0 B
Mortierella isabellina, vinacea 67.0, 51.9 B
Chaetomium globosum 5.10 B
Fusarium oxysporum 43.0 B [70]
Colletotrichum sp. 73.0 B [71]
Cunninghamella echinulata 28.0–35.0 B [72]
Yeast Myxozyma melibiosi, udenii 23.0, 20.3 B [73]
Lipomyces lipofer, tetrasporus, starkeyi, doorenjongii, 43.0, 66.5, 62.0, 72.3, 77.8 B [74–76]
kockii
Kodamaea ohmeri 53.3 B [77]
Metschnikowia pulcherrima 30.0 B [78]
Candida freyschussii 32.0 B [79]
Kurtzmaniella cleridarum 33.0 B [73]
Candida diddensiae, tropicalis 37.0, 23.0 B [80]
Galactomyces pseudocandidus 28.04 B [81]
Torulaspora delbrueckii 40.00 B [81]
Trigonopsis variabilis 20.00–43.70 B [82]
Yarrowia lipolytica 37.60-54.80 B [83]
Rhodotorula minuta, mucilaginosa, colostri, graminis, 24.6, 28.0, 26.9, 41.1, 35.4, B [84]
glutinis, babjevae,diobovatum, toruloides, 65.0,20–41, 19–51,25, [85]
fluvial, glacialis, terpenoidalis 68, 27 [73]
[78]
Leucosporidiella creatinivora 61–62 B [86]
Rhodosporidium paludigenum, sphaerocarpum 31.00, 43.00 B [81]
Cryptococcus terreus, terricola, aerius, adeliensis, albidus 51, 69.3, 63.3, 32.1, 40.1 B [73]
B [76]
Trichosporon coremiiforme, domesticum, 37.8, 35.16, 25.6, 40.1 B [73]
montevideense, dermatis B [87]

A phototrophic, B hetrotrophic
Bioenerg. Res. (2022) 15:1–26 7

Oleaginous Filamentous Fungi natural phenomena that are observed in the microbial popula-
tion of the biological ecosystem. In the field of drug research,
Few research reports support the idea of biofuel production a co-culture study is highly relevant as it allows monitoring
from fungi because of its rapid growth in a short span, easy the effect of the drug in cell-cell interaction in human in-vivo
scalability, and most importantly light independence [103]. tissue-like structure. [112]. Mostly monocultures are used in
Oleaginous fungi can grow and accumulate lipids by utilizing bio-manufacturing for consistent product yield but the risk of
different renewable substrates like glycerol, whey, molasses, contamination is quite high which increase operating and cap-
sewage water, etc. [104]. Sweet sorghum extract and rice hull ital cost [113]. So, the controlled, symbiotic co-culture process
hydrolysate are also used for the growth of oleaginous fila- can provide an extra advantage over the traditional monocul-
mentous fungal species. Lipid accumulation by oleaginous ture technique as the mixed consortium is less prone to con-
Mortierella isabellina was observed 43–51% and 36–64% tamination. Though the co-culture technique may not be glob-
on the dry weight basis when cultured in sweet sorghum and ally accepted for all cellular systems, yet it has shown en-
rice hull hydrolysate respectively [105]. Moustogianni et al. hancement in the yield of total biomass, lipid productivity,
(2014) has shown that glycerol can also be used for the growth production of enzymes, and other value-added products like
and lipid production by Zygomycetes where he used an initial secondary metabolites, polymer, organic acids, etc. [114].
glycerol concentration of 50 g L−1 and allowed growth in both Microbial associations are mainly of three types: mutualism,
aseptic and non-aseptic conditions [106]. The accumulation of commensalism, and parasitism or predator-prey [115].
lipids for M. isabellina, M. ramanniana, and Mucor sp. were Bacterial co-culture between Bacillus sp. and Clostridium
16% and 30%, 33% and 45%, and 22% and 23% in aseptic sp. showed a mutualistic interaction for the production of
and non-aseptic conditions respectively [106]. Culturing of bioenergy from lignocellulosic biomass where multienzyme
filamentous fungus Thamnidium elegans in low-cost carbon producing Bacillus compensated the confined ability of
sources (crude glycerol) derived from sugar refinery plants Clostridium for the enzymatic hydrolysis of lignocellulosic
obtained a maximum lipid yield 71% (w/w) on a dry weight biomass and turn into the fermentable precursor [17]. One
basis [107]. Few fungal species have been used recently by the commensalism-type interaction is observed in a consortium
researchers for biodiesel production and most of them focus of naphthalene degrading bacteria Pseudomonas sp. and a soil
mainly on synthesizing polyunsaturated fatty acids (PUFA) bacteria Acinetobacter oleivorans which cannot survive alone
like arachidonic acid and γ-linolenic acid. One of the most in naphthalene containing media but when co-cultured with
relevant examples is the exploitation of filamentous fungi Pseudomonas sp. it can survive by utilizing salicylic acid, a
Mortierella alpine to produce n-1, n-3, n-4, n-6, n-7, and n-9 metabolic intermediate produced by Pseudomonas sp.
PUFAs [108]. Mucor indicus, a zygomycete fungus is one of through naphthalene degradation [17]. Communication be-
the major lipid (rich in gamma-linolenic acid) producers, tween microorganisms in a single population through metab-
which also has commercial, pharmaceutical, and nutraceutical olites plays an important role in interpreting the relationships
importance [109]. Lipid accumulation in Mucor between interacting species, protection, evolution, selection of
cercinelloides is also studied where biodiesel can be produced partners, and division of labor [116]. A mutual relationship of
by direct transesterification of lipids and its TAGs have been protection was observed in a co-culture between obligate an-
proposed as a potential feedstock [110]. Aspergillus is also a aerobic bacteria and yeasts which ferments cello-dextrin,
well-known fungal strain for the production of biofuels. where yeasts protect bacteria from oxygen inhibition by main-
Different types of fatty acids accumulated in Aspergillus taining an anoxic environment in the culture, and in return,
oryzae include palmitic (C16:0), palmitoleic (C 16:1), stearic bacteria supplied fermentable carbohydrates by hydrolyzing
(C18:0), oleic (C 18:1), linoleic (C 18:2), linolenic (C 18:2), cellulosic compounds [17]. The selection of partners is an
and Arachidic acid (C 20:0) in proportions of 11.6%, 15.6%, important criterion for various types of mixed cultures and
19.3%, 30.3%, 6.5%, 5.5%, and 2% respectively [111]. one of the plant and microbe interaction studies showed that
Different fungal species were cultured at various conditions rhizobia have limited scopes for the host root infection so a
that support optimum lipid yields (Table 1). particular partner microbe can give a positive signal to the
infected, otherwise a mismatch will cause incompatibility be-
tween host and symbiont [117]. One of the examples of
Microbial Co-culture and Communication protecting a species from another in a microbial population
Between the Cells is shown in a study that co-culturing of fungus Aspergillus
nidulans, microalgae Chlamydomonas reinherdtii, and an al-
The co-culture system has been studied for decades to observe gicidal bacteria Streptomyces iranensis where it was observed
the interaction between different microbial communities and that A. nidulans attracts C. reinherdtii which are embedded
the underlying importance of cell-cell interaction in the field and surrounded by fungal mycelium and shielded from algi-
of synthetic biology. The co-culture study is based on some cidal bacterium [118]. The mechanism behind the various
8 Bioenerg. Res. (2022) 15:1–26

modes of microbial communication is shown in Fig. 1. microbial consortium has been manifested to be flexible in
Communication within microbial populations through metab- adverse conditions and withstand incursion from other species
olite exchange facilitates various modes of transport in which [120]. A co-culture can conquer challenges via communica-
microorganisms show intra- or inter-species intercommunica- tion and division of labor, evolving into a stable assemblage
tions [119]. Communication is used for several functions: (a) like biofilms [119].
quorum sensing that regulates the population density in a con-
sortium by the expression of genes and elucidates the afflu-
ence of each species where primary metabolites shape growth, Advantages of Microalgal Co-cultivation
development, and reproduction; (b) relationship that show the
type of relationships (mutualism, commensalism, and/or par- Microbial activities play important role in the versatile field,
asitism) and the role played by the partner; (c) protection (one i.e., food security, biofuel production, climate change issues,
species may protect other species from being affected by third human health, and bioremediation, etc. to achieve sustainable
species); (d) nutrient acquisition and division of labor where developments [121]. Different strategies were applied to in-
the whole workload is distributed among organisms. Further, crease the biomass and lipid productivity which are important
when the community evolves, some changes may occur which for surpassing the economic viability of microalgal biofuel
recruit new partners, (e) selection of a suitable partner species production. There are various conventional approaches to im-
leads to a mutualistic growth (compatibility issues may occur prove biomass and lipid yield like manipulation of nutrients
between different species when co-existing), (f) evolving into (e.g., carbon, nitrogen, and phosphorus), environmental pa-
a stable microbial consortium by acquiring new characteristics rameters (e.g., temperature, light intensity, salinity, etc.), the
[119]. When compared with a single taxonomic group, the addition of plant hormones, genetic modifications, and

Fig. 1 Mechanism of communication within different microbial effect of another group of species and (D) division of labor: showing that
communities. Exchange of metabolites (arrows) simplifies various the work load is divided among species, like photosynthetic organisms
modes of communication where microorganisms (geometrical shapes) can take energy from sunlight and release metabolites which may be
indicate intra- or inter-species interactions. Communication is used for consumed by other group of species or they can take energy from other
(A) quorum sensing: explaining the regulation of each other’s growth sources and support each other’s growth (E) selection of partner: selection
depending on the species available in a population (B) relationship: of compatible partner species will help to grow in same bio-coenosis (F)
type of symbiosis and roles played by companions, like in (C) evolution: as the community evolves, species will acquire new characters
protection: where one species can protect second species from harmful that may affect the communication.
Bioenerg. Res. (2022) 15:1–26 9

metabolic engineering, etc. To accept these conventional ap- investigated, it was found that algae coexist in mixed culture
proaches, a more recent strategy for co-cultivation of in the natural ecosystem [125].
microalgae with yeast, bacteria, and filamentous fungi have
received more attention to obtaining more biomass and lipid Microalgae-Microalgae Co-culture
yield [122]. In incorporation, the use of inexpensive substrates
like agricultural or industrial wastewater for the co-cultivation Currently, researchers are using microalgae co-culture in the
will be a contemporary approach to reduce the biofuel produc- laboratory to study their growth, biomass production, and also
tion cost. Such systems not only enhance the biomass and the effect of various parameters on lipid synthesis. To mini-
lipid yield but are also helpful in the bioremediation of waste- mize the price of raw materials and overall production cost,
waters [123]. Collaboration between microbial communities the co-culture system is an efficient alternative for microalgal
acts as an umbrella under which sustainable development oil production [16]. Sometimes in a co-cultivation system, the
goals may be achieved by targeting all current issues like biomass and accumulated lipid content increase significantly
energy crisis, greenhouse gas (GHG) emissions, wastewater along with the change in lipid composition as the mutualistic
treatment, and soil fertility for agriculture [121]. Concerning relationship enhances each other’s growth and, as a result,
the current energy system, microbial interactions can be uti- improves the metabolic activities related to lipid biosynthesis
lized for two parallel actions: first is the conversion of biomass [114]. Co-cultivation of halophilic microalgae in saline water
resources to green energy molecules and, second, reduction of to recycle nutrients can be the best alternative to reduce the
environmental pollution [2]. A proper balance between these unreasonable use of natural resources like fresh water and
two aspects may be achieved by utilizing microbial technolo- manures, and the produced biofuel by this process may be
gies to reform the long-term sustainability of energy and the potential to compete in the fuel market [126]. Microalgae with
environment [2]. The properties of the lipids extracted from tolerance towards a varying range of salinity can adapt to both
the co-culture systems are different from the lipids that are low and high salt concentrations, e.g., Tetraselmis indica can
extracted from the conventional monocultures. It has been tolerate salinity level from 35000 ppm of NaCl to a salt satu-
reported that the higher percentage of short-chain unsaturated ration state are identified as a potential feedstock biofuel pro-
fatty acids as compared with saturated FAMEs, enhances the duction [127]. In the co-cultivation system, if culture mode is
fuel properties like cetane number, heating value, octane num- semi-continuous throughout the culturing period, maximum
ber, iodine number, density, kinematic viscosity, etc. [123]. productivity can be attained. The semi-continuous model for
From various microalgal co-cultures, it has been observed that the microalgal co-culture should maintain an accurate inocu-
the percentage of unsaturated fatty acids is higher in FAMEs lum volume and also secure a stable production rate [128].
as compared with saturated fatty acids. So, microalgal co- A co-culture of Chlorella sp. and Monoraphidium sp. was
culture enhances not only the biomass and lipid productivity tested under a photoautotrophic environment and it was found
but also the fuel properties of the biofuel. Microalgal co- that it can be used as a potential source of biodiesel production
culture is also an efficient technique for wastewater treatment. [129]. This combination of co-culture is advantageous and
Waste removal efficiencies also increased in the case of increases the lipid productivity of Monoraphidium sp. by 20
microalgal co-culture as compared with the monoculture of folds [129]. The co-culture technique has been proposed to
microalgae in this review. Besides nutrient removal, co- curtail the biofuel production cost. Naturally, microalgae can
cultures are also used to remove toxic heavy metals like Cd, produce extracellular polymeric substances (EPSs), and in the
Pb, Hg, Mn, Ni, Zn, etc., where yeasts can degrade heavy case of a co-culture, an improved amount of EPSs was pro-
metals and the process is called bioaugmentation [124]. A duced due to more interactions between two different
schematic diagram of different microalgal co-cultures and microalgal cells and this will support the microalgae metabol-
their benefits is shown in Fig. 2. ically to grow and accumulate lipids in the nutrient starvation
conditions [130]. Recent studies are focusing on investigating
different co-culture models for higher lipid production that
would have a low operating cost. Under photoautotrophic,
Co-culture of Microalgae with Different heterotrophic, and mixotrophic conditions, the co-cultivation
Microorganisms of Chlorella sp. and Monoraphidium sp. has driven to the lipid
productivity of 29.52, 93.4–223.42, and 21.23 g m−3 day−1
In comparison with first and second-generation biofuels, respectively [131]. In a dairy wastewater treatment by
microalgae as a source of third-generation biofuel have much Scenedesmus dimorphus and Chlorella vulgaris co-culture
higher biomass and oil productivity. Researchers have also experiment, lipid yield observed was 22.2% and 9.9%, respec-
found that algae can use wastewater from different sources tively, on a dry weight basis when those were cultured indi-
like dairy, agriculture, municipal, and industrial waste for their vidually, but a 25.5% (DW) yield was observed in the mixed
growth. While the monoculture of algae has been extensively culture at 25% manure concentration [132]. The nutrient
10 Bioenerg. Res. (2022) 15:1–26

Fig. 2 Microalgal co-cultures and

their benefits

sequestration profile along with biomass and lipid yield of the major objectives of wastewater treatment is the removal of
both the microalgal species were analyzed and further con- total suspended solids (TSS) and volatile suspended solids
cluded that S. dimorphus has more potential for waste degra- (VSS) which were achieved at a maximum level of 66.7%
dation over C. vulgaris which causes the differences in nutri- and 67.6% respectively, at 10% manure concentration [136].
ent utilization and biomass yield [132]. A recent study of two Microalgal consortia (Chlorella sp., Nannochloropsis sp.,
different microalgal consortia (MAC1 and MAC2) were cul- Chlamydomonas reinhardtii, Scenedesmus bijugatus, and
tured in sewage wastewater and achieved a good lipid content Scenedesmus dimorphus) in sewage wastewater has shown
(31.33% dry weight basis) where the abundant microalgae removal of % total organic carbon (TOC), % of COD, 94%
present in MAC1 consortia are Chlorella sp., of both NO3− and PO4− along with the efficient removal of
Nannochloropsis sp., Chlamydomonas reinhardtii, heavy metals such as 85.06% Cu, 98.2% Pb, 75.2% Cr, and
Scenedesmus bijugatus, and Oscillatoria and in MAC2 99.6% Cd [133]. The waste removal efficiency can be in-
Na nnochlorop sis s p . , Chlorella sp., Kirc hnella, creased by maintaining several factors like (1) microalgal cul-
Scenedesmus dimorphus, and Microcoleus [133]. ture, (2) growth conditions, (3) initial nutrient concentration,
Microalgae-based treatment is one of the best biological (4) nitrogen to phosphorus ratio (N/P), (5) nutrient source, and
solutions that can solve environmental problems such as glob- (6) wastewater characteristics [137]. There are fewer studies
al warming, ozone layer depletion, and climate change due to on the interaction of all variables and the synergized knock on
its ability to sequester a high quantity of CO2 in the process of both algal biomass and lipid production, so the optimization of
photosynthesis [134]. However, co-culture of microalgae has algal co-cultivation in wastewater is quite problematic. To
been carried out for a long time for several purposes. One of overcome such problems, the use of response surface meth-
the important purposes of co-culture is bioremediation and odology (RSM) is very much helpful. RSM is a polynomial
treatment of municipal, agricultural, animal, farm, and dairy equation with a combination of statistical and mathematical
wastewater [135]. Microalgae can remove 99.7–99.9% phos- data that are constructed to fit in the experimental data and
phorus and 88.6–96.4% nitrogen from the medium when cul- helps in investigating the composition of exogenous and en-
tivated in wastewater [136]. In a dairy wastewater treatment dogenous materials of algae for wastewater-based algal biofu-
by S. dimorphus and Chlorella vulgaris individually and a co- el production where mainly three parameters like light inten-
culture experiment, good waste removal efficiencies were ob- sity, CO2 concentration, and harvesting time were considered
served: Nitrogen removal as nitrate equivalents (NO3−-N) [138]. A recent study on the coupling of microalgal culture
from the dairy wastewater when treated with 10% manure with the flue-gas emissions from coal-based industries has
concentration was achieved 83.2% (by S. dimorphus), 4.1% shown an economically feasible technique of reusing CO2
(by C. vulgaris), 65.7% (by S. dimorphus + C. vulgaris) and gas to enhance algal growth and biomass. In this study, a
at 25% manure concentration removals were 67.7%, 55.3%, novel photobioreactor was designed for algae cultivation and
70.7%, respectively, after 7 days of growth. Similarly, phos- models have developed that estimate the potential of carbon
phate removal as total orthophosphate equivalents (PO4−-P) captured in this process which is equivalent to 1.16 × 104
were 82.9%, 20.8%, and 84.6% when treated with 10% ma- metric ton of CO2 per year [139]. Researchers are focusing
nure concentration and 22.0%, 12.0%, and 56.8% when treat- on the scaling up of biofuel production from microalgae and
ed with 25% manure concentration respectively [132]. One of trying to make it a feasible process by developing efficient
Bioenerg. Res. (2022) 15:1–26 11

strategies for the microalgae-microalgae co-cultivation which metabolites especially indole-3-acetic acid which promotes
is easier to handle, less contamination risk, and overall im- lipid production [144].
prove biomass and lipid productivity. The co-cultivation Many microalgal species are auxotrophic for certain vita-
should be carried out in photoautotrophic conditions so that mins, a study shows that algal species require an external
freely available sunlight and CO2 can be utilized as much as supply of vitamin B12 that helps in the proper functioning of
possible for enhanced biomass growth. If the culturing medi- an iso-form of methionine synthase enzyme in microalgae
um is heterotrophic then to enhance the accumulation of lipid, [145]. A co-culture study on microalgae Lobamanas rostrata
a nutrient starvation condition specifically nitrogen limitation and the bacterium Mesorhizobium loti were cultured in a me-
should be maintained. It is observed that the consumption of dium containing neither vitamin B12 nor any organic carbon
nitrogen in microalgal co-culture is quite stronger than in sources, but the mutualism of bacteria and microalgae allowed
monoculture, so nitrogen depletion may occur early or easily. the supply of vitamin B12 from the bacterium and finally, both
Too much early depletion of nitrogen may affect the synthesis reached to a stable equilibrium in terms of population size
of other important metabolites such as amino acids, carbohy- where the initial ratio of L. rostrata to M. loti was 1:30
drates, peptides, etc., so the regulation of nutrient supply [143]. The interaction of bacteria and microalgae also occurs
should be maintained. Microalgae co-culture in the aspect of in the marine environment. Bacteria can metabolize almost
wastewater treatment is significantly efficient but the selection half of the carbon which is fixed by phytoplankton in the
of suitable partner species for the co-cultivation is one of the sea. To study the bacterial and microalgal interaction, a co-
important criteria as different wastewater resources containing culture was designed between diatom Thalassiosira
various types of waste compounds which might not be neces- pseudonana CCMP-1335 and the roseobacterium
sarily degraded by those microalgal species who have poten- Ruegerium pomeroyi DSS-3 where R. pomeroyi provides vi-
tial in biofuel production. tamin B12 to T. pseudonana which discharges 2, 3-
dihydroxypropane-1-sulphonate into the broth and bacteria
used it for metabolism [146]. It was also observed that the
Microalgae-Bacteria Co-culture growth of the diatom T. pseudonana was slower when grow-
ing in the absence of R. pomeroyi [146].
Both microalgae and bacteria influence the ecosystem togeth- According to the literature, biomass growth inhibition by
er by different modes of interaction like mutualism, commen- oxygen is a serious problem in high-rate algal ponds (HRAPs)
salism, and/or parasitism. Bacteria are often considered mere and in summers, during day time, the dissolved oxygen con-
contaminants of algal culture, but the establishment of algae- centration increases very fast that exceeds 200% saturation,
bacteria co-culture is beneficial by reducing the risk of con- causing a reduction in biomass by 25% [147]. Bilanovi et al.
tamination, the total cost of operation, and simultaneously the [148] showed that the co-culture of microalgae and nitrifying
recovery of various by-products from the co-culture [140]. bacteria is a cautious, non-invasive method for the elimination
Algae are an admirable feedstock for biofuel production and of oxygen inhibition which helps in higher biomass produc-
naturally, coexist with the bacterial population accompanying tion. It was also proven in this article that the co-culture of
algal growth and lipid accumulation. The algae-bacterial sym- C. vulgaris and nitrifying bacteria enhance biomass produc-
biotic relationship becomes beneficial as algae stimulate bac- tion by 23% as compared with the control monoculture of
terial growth by supplying dissolved organic carbon (DOC) axenic C. vulgaris [148].
[141]. The microscale mucus region surrounding the algae is With an increasing crisis in current global energy demand
rich in organic matter, known as phycosphere and it is the base and environmental pollution, the importance of renewable en-
for bacterial decomposition [142]. Conversely, bacteria also ergy has been highlighted, especially for hydrogen energy.
stimulate the growth of algae in a mutualistic way. The pres- Chlamydomonas reinhardtii, single-cell green algae is effi-
ence of bacteria in algal culture maintains the DOC level of the cient in hydrogen production due to its higher hydrogenase
broth at a steady-state concentration and in an axenic culture activity [149]. Nowadays, hydrogen production has been im-
of microalgae, the DOC gradually increases [141]. According proved by co-culturing algae and bacteria. Miura et al. report-
to Gause’s principle about community ecology, two species ed that the co-cultivation of C. reinhardtii with photosynthetic
cannot coexist if they are competing for the same limited bacteria achieved a 10-fold increase in hydrogen production
resources. So, any particular microbial species can be found [150]. Kawaguchi et al. [151] used a mixed culture of
to prevent the same species from invading as the ecological C. reinhardtii, Lactobacillus amylophilus, and Duchenella
habitat has already been occupied [143]. This strategy is salinifera and achieved an increase in hydrogen production
known as “synthetic ecology” which helps the algae-bacteria by 15.5%. Nowadays, algae-bacteria consortia are also an
co-culture from being contaminated with other bacteria when advantageous way for biofuel production as it enhances the
they will be grown in open photobioreactors. Hence, bacteria microalgal biomass and lipid production. The green algae
support microalgal growth as well as the synthesis of C. reinhardtii is one of the most famous model microalgae
12 Bioenerg. Res. (2022) 15:1–26

for lipid production, and it was added to bacterium hydrolysate, grass hydrolysate, and second cheese whey
Azotobactor chroococcum in nitrogen-deficient conditions; where the co-culture showed a significant waste removal from
the maximum percentage of lipid content and production were poplar sawdust hydrolysate and grass hydrolysate. The re-
65% and 387 mg/L, respectively, which is 2.3 and 6 times of moval rates of COD, TN, and PO4− were 88.7% and 34%,
the C. reinhardtii monoculture [19]. A similar type of study 72–91% and 25%, and 97.5% and 26.4% from grass hydro-
was also carried out by Contreras-Angulo et al. [152] with lysate and polar sawdust hydrolysate respectively [153]. A
symbiotic co-culture of Azospirillum brasilense study of co-culturing microalgae C. vulgaris and endophytic
(ATCC29145) and green algae Scenedesmus sp. (UTEX bacteria mainly Bacillus sp. is capable of purifying biogas and
1589) in nitrogen-deficient media and fatty acid content ob- biogas slurry where the removal efficiencies achieved 88.29 ±
tained was 57 ± 3% of the dry weight. Tsolcha et al. (2018) 5.03% of COD, 88.31 ± 4.29% of TN, 88.21 ± 4.51% of TP,
used a consortium of cyanobacteria Leptolyngbya sp. and and 68.13 ± 1.69% of CO2 [163].
microalgae Ochromonas sp. for the treatment of different
agro-industrial wastewaters and biodiesel production [153]. Microalgae-Fungi Co-culture
The study used a rectangular glass photobioreactor under aer-
obic and non-aseptic batch cultivation conditions where the Researchers have indicated that a positive correlation is ob-
lipid productivity obtained from each different substrate such served when filamentous fungi are used alongside algae cul-
as synthetic medium, polar sawdust hydrolysate, and second tivation. The naturally occurring symbiotic relationship be-
cheese whey was 38.4, 38, and 34.9 mg L−1 day−1 respectively tween microalgae and fungi or cyanobacteria and fungi also
[153]. Co-culturing of bacterium Rhizobium sp. and known as lichens has existed for more than 600 million years
microalgae Ankistrodesmus sp. has shown a 30% increment [164]. Among various microorganisms used in co-cultivation
of lipid accumulation and it was observed that the effects of with algae, filamentous fungi are advantageous in wastewater
vitamin B12 and/or indole 3-acetic acid secreted from the treatment as they can reduce the biodiesel production cost by
bacterium, stimulates the accumulation of lipid [144]. their easy, feasible, and efficient harvesting method [165].
Microalgae Chlorella sp. and bacterium Azospirillum sp., Many studies are focusing on the harvesting of microalgal
when co-cultured, the lipid content increased significantly cells by the fungal pellets. Fungal pellets are formed due to
from 6.76% (monoculture) to 32.94% (co-culture) [144]. the aggregation of various types of filamentous fungi at their
Different types of microalgae-bacteria co-culture and their lip- early stage of spores and the types of pellets are fluffy pellets,
id productivities are summarized in Table 2. smooth-compact pellets, and smooth-yellow pellets [166].
Microalgae-bacteria consortia not only enhance the bio- Harvesting of microalgal cells from the large culture is not
mass and lipid production but also help in wastewater treat- economically viable as it accounts for 50% of the total biodie-
ment. Co-culture under light conditions is more effective as sel production cost in just only one step which is the harvest-
algae remove pollutants by degrading organic matter such as ing of tiny microalgal cells from a liquid medium [155].
NH4+-N, total phosphate (TP), and total nitrogen (TN) and Harvesting of algal cells by conventional methods like centri-
generate oxygen via photosynthesis which is consumed by fugation, filtration, flocculation, flotation, etc. requires high
bacteria to break down the pollutants and thereby decrease energy input or the addition of chemicals is quite a cost input
the chemical oxygen demand [160]. The effect of light inten- stage. To minimize the biofuel production cost, specifically to
sity on microalgae-bacterial interaction was studied and it was reduce the cost investment for the harvesting purposes, the co-
observed that the nitrogen removal rate reached the highest culture of algae and fungi is introduced as the filamentous
i.e., 8.5 mg L−1 day−1, under the light intensity of 244 μmol fungi act as an attractive bioflocculant because of their self-
m−2 s−1 [161]. A co-culture study of filamentous algae pelletization nature by which it entraps the microalgal cells
Chlorella sp. and Selenastrum bibraianum with activated [154]. Self-pelletization of fungi has been observed in various
sludge from printing and dyeing industry containing bacteria fungal strains and the mechanism behind it is of two types i)
to algae ratio of 1:3 (w/w) in the sludge was carried out in a coagulation (spores are aggregated) ii) non-coagulation
multifunctional integrated aerobic bioreactor for 95 days (spores germinate into hyphae and then intertwine into pellets)
[162]. The results showed that the algal-bacterial symbiotic [167]. It is reported that microalgae carry a net negative charge
system demonstrated an average COD, NH4+-N, and TP re- (-23.7 mV) on their surface as phosphoric, phosphodiester,
moval rate of 83%, 93%, and 37% respectively and the color amine, hydroxyl, and proton-active carboxylic functional
of the wastewater was reduced by 60–80 times [162]. A con- groups are present. Hyphae and mycelia carry a net positive
sortium of cyanobacteria Leptolyngbya sp. and microalgae charge (+ 46.1 mV) as contain polysaccharides on their sur-
Ochromonas sp. showed efficient waste removal efficiencies faces [167]. Therefore, filamentous fungi can potentially neu-
when the co-culture was used to treat agro-industrial waste- tralize the negative surface charge of algae enabling attach-
waters. Different sets of substrates were used for the co- ment to the fungal cell wall when are co-cultured and finally
culture and these are synthetic media, poplar sawdust precipitated by co-pelletization [155]. The use of fungi also
Bioenerg. Res. (2022) 15:1–26 13

Table 2 Comparison of lipid productivity between various microalgal monocultures and co-cultures

Culture type Monoculture or co-culture Species name Lipid productivity (mg L−1 day−1) References

Algae-algae Algae Scenedesmus dimorphus 17.1 ± 4.7 [132]

Algae Chorella vulgaris 5.2 ± 1.6
Algae-algae S. dimorphus + C. vulgaris 22.3 ± 9.3
Algae-fungi Algae Dunaleilla salina 7.6 ± 0.9 [154]
Algae Dunaleilla tertrioleta 10.1± 1.2
Algae Taustochytrium sp. 50.9 ± 12.8
Algae Chlorella vulgaris 22.9 ± 2.75
Algae Chlorella reinherdtii 7.7 ± 0.9
Algae Nanochloropsis oculata 25.4 ± 3.1
Algae Chlorella protothecoides 349.5 ± 60.2 [155]
Algae Tetraselmis suecica 107.7 ± 25.3
Fungi Aspergillus fumigatus/PDB 120.5 ± 20.1
Fungi Aspergillus fumigatus/GLU 12.7 ± 2.1 [154]
Algae-fungi C. protothecoides + A. fumigatus/PDB 1020.4 ± 220.1 [155]
T. suecica + A. fumigatus/PDB 289.1 ± 110.4
D. salina + A. fumigatus 31.55 ± 3.8 [154]
D. tertrioleta + A. fumigatus/GLU 34.75 ± 4.1
Thaustochytrium sp +A. fumigatus/GLU 122.98 ± 18.1
C. vulgaris + A. fumigatus/GLU 73 ± 8.8
C. reinherdtii + A. fumigatus/GLU 36.67 ± 4.4
N. oculata + A. fumigatus/GLU 54.6 ± 6.5
Algae-yeast Algae Scenedemus obliquus 68.3 ± 1.5 [156]
Algae Chlorella pyrenoidosa 120 ± 3.3 [157]
Yeast Saccharomyces cerevisiae – [156]
Yeast Candida tropicalis –
Yeast Rhodotorula glutinis 108.33 ± 1
Algae-yeast S. obliquus + S. cerevisiae 79.36 ± 4.3
S. obliquus + C. tropicalis 97.8 ± 1.3
C. pyrenoidosa + R. glutinis 206.67 ± 7.5 [157]
Algae-bacteria Algae Chlamydomonas reinhardtii 65.99 [19]
Algae Chlorella variabilis 6.28 [158]
Algae Characium sp. 8.96 [159]
Bacteria Azotobacter chroococcum 7.33 [19]
Bacteria Pseudomonas composti – [159]
Bacteria Idimarina loihiensis – [158]
Algae-bacteria C. reinhardtii + A. chroococcum 141.86 [19]
C. variablis + I. loihiensis 10.9 [158]
Characium sp.+ P. composti 8.24 [159]

PDB potato dextrose broth; GLU glucose; “–” data not available; data ± experimental standard error

has another advantage of enhanced lipid production in co- changes in a co-culture may bring changes in the interaction
culture. Oleaginous fungi can store lipids more than 20% between filamentous fungi and algae that may modulate the
(DW) which contains mostly saturated and monounsaturated fatty acid composition of synthesized lipids [169].
fatty acids like palmitic acid (C16:1), stearic acid (C18:0), and Muradov et al. [155] in a study on fungal-assisted algal
oleic acid (C18:1), which are generally used for the production flocculation screened 33 fungal strains and checked the floc-
of biodiesel [168]. Co-culture of fungi and algae require culation efficiency against two commercially important
mixotrophic conditions as the fungi are obligate heterotrophs microalgal strains Chlorella protothecoides and Tetraselmis
and green algae are photoautotrophs so, the relative selectivity suecica. Among screened strains, Mucor circinelloides and
of nutrients and mode of tropism for both may vary. These Aspergillus fumigatus showed the maximum lipid yield of
14 Bioenerg. Res. (2022) 15:1–26

14.7% and 9.9% respectively and the flocculation efficiency accumulation of ~ 10% (dry weight) compared with ~ 6%
of A. fumigatus was the best. A. fumigatus showed the floccu- (DW) of the wild type under nutrient replete conditions
lation efficiency of 90% after 24 h of co-cultivation and there [172]. Different types of microalgae-fungi co-cultures and
was no significant difference observed in flocculation efficien- their lipid productivities are summarized in Table 2.
cy of seawater and freshwater microalgal species and it was It has been reported in articles that among various co-
reported that the highest lipid yield of 2041 ± 440 mg/L was culture between microalgae and other microorganisms,
observed in C.protothecoides/A.fumigatus after 48 h of co- algae-fungi co-culture is the most effective for wastewater
cultivation [155]. Co-culturing of algae and fungi has shown treatment. A co-culture study of unicellular protist
fungal-assisted pelletization of algae C. sorokiniana by the Thaustochytrium sp. and filamentous fungi Aspergillus
filamentous fungi A. flavus and a harvesting efficiency was fumigatus was carried out using swine wastewater and the
achieved 96% within 48 h [170]. A co-culture study also removal rates of PO43−-P and NH4+-N achieved were 84%
showed similar kind of results when 11 microalgal strains and 96% respectively [154]. A similar kind of study was car-
were co-cultivated against A. fumigatus where C. vulgaris ried out with C. protothecoides and A. fumigatus using swine
and Thaustochytrium sp. showed promising results [154]. In wastewater for 48 h and the removal of NH4+-N and PO43—P
this study, A. fumigatus was treated in two different condi- achieved were 93% and 87% respectively [155]. Another co-
tions: A. fumigatus grown in the presence of excess glucose culture study of Chlorella sp. and Aspergillus sp. was carried
(20 g/L) and A. fumigatus grown in the presence of 1% acid- out using wastewater from the sugar industry which has a dark
treated wheat straw, to enhance the flocculation efficiency up brown color and there was a decrease in the color intensity by
to 90% and lipid production. The results showed that the co- 69.98% after 5 days of co-cultivation. In the case of decolor-
culture of algae with A. fumigatus/TWS (acid-treated wheat ization and suspended solid removal, the co-culture of algae-
straw) showed higher flocculation efficiency and algae with fungi is a promising way when compared with other microbial
A.fumigatus/GLU (glucose added) showed higher lipid pro- co-cultures [173]. This study also showed the removal of nu-
ductivity [154]. A co-culture study between filamentous fungi trients by algal-fungal pellets indicating removal of TN, and
A. fumigatus and green microalgae C. vulgaris reported a har- NH3-N by 67.09%, and 94.7% respectively and thereby de-
vesting efficiency of > 90% when those were co-cultured in creasing the COD by 70.68% [173]. Algal-fungal co-culture is
excess glucose (2 g/L) concentration. This result was observed very much effective for waste removal because enzymes re-
when the co-cultivation was carried out in microalgae to fila- leased by fungi can degrade solid organics into low molecular
mentous fungi ratio of 300:1 and the numbers of pellets pres- weight consumable nutrients for microalgae and especially the
ent were 5 ± 1.5 pellets/ml where the average diameter of nitrogen uptake is enhanced so that microalgae are more prone
pellets was 3.3 ± 0.3 mm [167]. Bhatnagar et al. [171] de- to nitrogen assimilation thus the removal efficiency of total
signed a study to elucidate the effect of an endophytic fungus nitrogen is higher [173]. Various types of microalgae-fungi
Piriformospora indica on the marine algal biomass and lipid co-cultures and their nutrient removal efficiencies are summa-
profile of Parachlorella kessleri-I under a co-culture environ- rized in Table 3. Microalgae and filamentous fungi consor-
ment. The average biomass (DW) of the co-culture obtained tium is very advantageous in terms of harvesting, lipid yield,
was 704.4 ± 6.02 mg/L after 12 days of cultivation, which is and waste removal efficiency but the major drawback behind
almost 1.5-folds compared with control. The result of the this co-culture is choosing a compatible partner and fixing the
FAMEs profile reported that the two important components ratio of the inoculation. Fungal spore-forming pellets require
of biodiesel, i.e., oleic acid and elaidic acid, increased by 1.8 optimum agitation because higher rpm interrupt pellets com-
and 1.4 fold respectively [171]. In Du et al. [172], co-culture pactness and also cause cell damage, so for the large scale
was done by combining marine algae Nanochloropsis culture in bioreactors, various models should be tested for
oceanica and oleaginous filamentous fungi Mortierella the assessment of morphological characters of fungal pellets
elongate to enhance biofuel production and harvesting effi- as well as microalgae.
ciency. In this study, the algae and fungi were grown sepa-
rately and then combined for the bio-flocculation as the Microalgae-Yeast Co-culture
M. elongata mycelium could expeditiously capture
N. oceanica by an abstruse interaction between each other. The most promising third-generation biofuel production tech-
The co-culture showed that the TAGs and total fatty acid nologies involve the use of microorganisms where the lipid is
accumulation were 15 and 22% (DW) respectively, and the extracted from the biomass. It has been already proven that
accumulation of a high concentration of PUFAs was also ob- multi-species culturing systems with their complementary
served. In this study, the researchers genetically modified the metabolic capabilities enhance overall productivity [182].
strain N. oceanica CCMP1779 to overexpress DGTT5, a gene Microbial consortia of microalgae and other oleaginous mi-
that encodes type II acyl-CoA: diacylglycerol acyltransferase croorganisms like bacteria, fungi, and yeast for the production
5, to increase the production of TAGs and reported an of biofuel is already reported. Oleaginous yeasts are now
Bioenerg. Res. (2022) 15:1–26 15

Table 3 Comparison of wastes removal efficiencies between various microalgal monocultures and co-cultures

Culture type Monoculture or Species Removal efficiency (%) References

co-culture
TP TN COD NH4- TSS VSS
N

Algae-algae Algae Scenedesmus dimorphus 22.0 67.7 – – 33.3 54.1 [132]

Algae Chorella vulgaris 12.0 55.3 – – 36.8 34.2
Algae-algae S. dimorphus + C. vulgaris 56.8 70.7 – – 52.3 52.0
Algae-fungi Algae Chlorella vulgaris – 44.4 26.0 – – – [173]
Algae Chlorella protothecoides 35.4 – – 36.2 – – [174]
Algae Tetraselmis suecica 31.1 – – 26.5 – –
Fungi Aspergillus sp. 18.2 59.0 – – [173]
Fungi Aspergillus fumigatus 50.9 – – 41.2 – – [155]
Algae-fungi C. vulgaris + Asprgillus sp. 88.4 67.1 70.7 94.7 – – [173]
C. protothecoides + A. fumigatus 55.6 – – 73.7 – – [155]
T. suecica + A. fumigatus 57.8 – – 62.1 – –
Algae-yeast Algae Parachlorella beijerinckii – – 59.0 – – – [175]
Algae Chlorella sorokiniana – – – – – – [176]
Yeast Cryptococcus curvatus 96.2 40.7 59.5 – – –
Yeast Rhodotorula glutinis 89.4 46.8 63.1 – – –
Yeast Saccharomyces cerevisiae – – 91.0 – – – [175]
Algae-yeast P. beijerinckii + S. cerevisiae – – 88.0 – – –
C. sorokiniana + C. curvatus 99.5 96.4 95.2 – – – [176]
C. sorokiniana + R. glutinis 99.8 96.2 94.4 – – –
Algae-bacteria Algae Chlorella vulgaris < 20.0 59.0 – – – – [177]
Algae Chlorella vulgaris 70.0 75.0 – – – – [178]
Bacteria Azospirillum brasilense – – – – – – [177]
Bacteria Pseudomonas putida – – – – – – [178]
Algae-bacteria C. vulgaris + A. brasilense 83.0 91.0 – – – – [177]
C. vulgaris +B. licheniformis 93.0 86.0 – – – – [[179]
C. vulgaris + P. putida 60.0 80.0 80.0 – – – [178]
C. microsporum + wastewater bacteria 89.0 88.0 – – – –
C. pyrenoidosa + wastewater bacteria 95.0 96.0 – – – –
C. sorokiniana + activated sludge 86.0 99.0 – – – –
C. sorokiniana + activated sludge 86.0 – 75.0 99.0 – – [180]
S. obliquus + activated sludge 65.0 36.0 – – – – [181]
Scenedesmus sp. + anaerobic sludge 80.0 89.0 – – – –

“–” Data not available

becoming one of the best-supporting microorganisms with Scenedesmus sp. and yeast Candida pimensis co-culture study
microalgae for not just biofuel production but also as aquacul- reveals that there are certain inorganic micronutrients like so-
ture feed, production of biochemicals, and wastewater treat- dium nitrate (NaNO3), ferrous sulfate (FeSO4), and potassium
ment [183]. Recent studies show that the mutualistic relation phosphate (K2HPO4) that influence the growth of biomass,
in a co-culture between oleaginous yeast and microalgae en- lipid, protein, and carbohydrate yield when these are present
hances biomass and lipid accumulation. The mutualism relies at certain concentrations in the culture medium and it showed
on interchanging carbon and nitrogen where fermentation of a significant amount of biomass production of 1.68 g L−1 and
mannose by yeast produces carbon dioxide, which is taken up the maximum lipid content of 27.77% was obtained from this
by microalgae and the microalgae provide nitrogen to the co-culture [184].
yeast by metabolizing nitrite to ammonium [174]. RSM study The symbiotic relationship of a mixed culture of two algae
can identify the best-suited compositions of inorganic nutri- (C. vulgaris and C. pyrenoidosa) and two yeast (Rhodotorula
ents should require to achieve a promising amount of biomass glutinis and Candida tropicalis) species showed enhanced
and lipid yield from microbial culture. A microalgae biomass and oil production of 3626 and 1219 g L −1
16 Bioenerg. Res. (2022) 15:1–26

respectively [185]. Similar work has also been reported in R. glutinis with piggery wastewater in a 1000-L pilot-scale
another article, where dual-species cultivation of C. vulgaris bioreactor is capable of removing NH3-N, TN, TP, and
and C. pyrenoidosa with Rhodotorula glutinis in a ratio of 3:1 COD of 93.48%, 82.65%, 53.51%, and 85.44% respectively
led to maximum biomass and lipid yield [157]. It is also re- [194]. Different types of microalgae-yeast co-cultures and
ported that at higher carbon to nitrogen ratio (C/N), the max- their nutrient removal efficiencies are summarized in
imum lipid productivity of 206.7 ± 7.5 mg L−1 day−1 can be Table 3. Yeast cells are also used nowadays for the removal
achieved [157]. A co-culture study of Spirulina platensis and of heavy metals from any waste sources by bioaccumulation
R. glutinis reported a significant increase in biomass and total or bioaugmentation process where selected microbial strain or
lipid production of 1600 and 467 mg L−1 respectively along mixed cultures were used to remove the specific pollutants.
with the COD and TN removal efficiency of 73% and 35% The yeast-based bioaugmentation approach is advantageous
respectively [186]. A co-culture study between algae for soil and water cleanup, where the contamination is due to
S. obliquus and yeast R. glutinis was carried out in a 5-L heavy metals and/or organic pollutants [195]. The soil bioaug-
photobioreactor where the total lipid was increased 60–70% mentation by Cyberlindnera fabianii removed 60% of chro-
than the single culture of algae [18]. A photoautotrophic co- mium from soil (40 mg/kg). Saccharomyces cerevisiae has the
culture of C. vulgaris and yeast R. glutinis in industrial waste- removal efficiency of 100%, 95%, and 67–82% for Zn, Cd,
water has shown fourfold higher lipid production (117.73 mg and Pb respectively [124]. As yeast can grow easily on various
L−1) than its photoautotrophic monoculture [187]. Another low-cost substrates and rotten and waste materials, the cost
report of a co-culture between Chlorella sp. and oleaginous minimization for nutrients is easier. Immobilization of
yeast Torulaspora maleeae or Torulaspora globosa has microalgae can be a cost-effective technique as cells can be
shown an enhanced lipid yield of 96% as compared with the reused and harvesting is easier than normal culturing. So, the
single cultures [188]. In a study of co-culture between differ- microalgae encapsulation in alginate beads and other matrices
ent Chlorella sp. and Trichosporonoides sapthulata JU4-57, it can help for the co-culturing in a large-scale reactor.
was reported that the highest biomass yield of 12 g/L with
lipid content of 47% (DW) was achieved from one of the
Chlorella sp. TISTR 8261 [189]. A symbiotic growth of Key Challenges for Scaling Up of Microalgal
microalgae C. pyrenoidosa and yeast Cryptococcus sp. at an Biofuel Production
initial ratio of 20:1 (algae: yeast) has shown the final biomass
with the algae content of 83.5% and 93.2% in heterotrophic Microalgae are one of the best sustainable and renewable
and mixotrophic culture respectively. The lipid content of this feedstock for biofuel production, so researchers are focusing
symbiotic growth was achieved by 27.1% (w/w) and 28.2% on developing various strategies to make the process techno-
(w/w) in heterotrophic and mixotrophic cultures respectively economically affordable. Although biofuel production is ac-
[190]. A microalgae-yeast consortium where the abundance of companied by the production of other valuable products like
microalgae was Scenedesmus obliquus (68%), Scenedesmus healthcare and food supplements and environmental applica-
sp. (29%), and one yeast species mainly available Candida tions such as bioremediation, bio-fixation, biofertilizer pro-
pimensis (43%) in presence of inorganic nutrients achieved duction, heat or electricity generation, etc., industries are still
maximum biomass was obtained 1.43–1.68 g L−1 and the facing obstacles in scaling up the process [196]. One of the
maximum lipid content was 27.77% (w/w) [184]. major challenges is the selection of high-lipid-producing
Microalgae are preferred for biofuel production over any microalgal strain and the most effective solution is the modi-
other oleaginous microorganisms and are very efficient in car- fication of strains through genetic and metabolic engineering
bon dioxide uptake and removal of various waste components, for the fast growth and overproduction of lipids [196]. The
but not very effective in the removal of organic wastes. In next major obstacle is the large-scale cultivation of microalgae
comparison with algae, yeasts are very effective in organic as it requires a huge quantity of water and nutrient supply,
matter removal from wastewater in a short period [191]. A sophisticated pieces of equipment for controlling all the pa-
mixed culture of Lipomyces starkeyi with Chlorella sp. and rameters, energy requirements for the dewatering, and lipid
Scenedesmus sp. was grown in an open raceway pond for extraction processes. It is said that a large-scale algae culture
wastewater treatment and biodiesel production, and obtaining requires the same amount of nutrients as required for large-
15% increment in lipid and biomass after the end of the culti- scale agriculture [197]. However, the problems related to the
vation period [192]. Galactomyces geotrichum MTCC 1360, supply of water and nutrients are somehow minimized by
a yeast species, showed removal of 88% AMDI (American using agro-industrial wastewater for microalgal growth.
Dye Manufacturing Institute) mixture of structurally different Although, there are a few drawbacks such as microalgal path-
dyes (Methyl red, Brilliant blue, Remazol red, Golden yellow, ogens or other predators, the presence of toxic heavy metals,
etc.) and also decreased COD by 69% and TOC (total organic and other contaminants that affect the overall growth and pro-
carbon) by 43% [193]. A co-culture of C. pyrenoidosa and ductivity [197]. The next challenge is the harvesting or
Bioenerg. Res. (2022) 15:1–26 17

dewatering of tiny microalgal cells from the liquid broth Other Benefits of Microalgal Co-culture
which is economically expensive as the traditional harvesting
techniques including centrifugation (costs 0.43 € kg−1), mem- Besides, biofuel production, microalgae cultivation, and co-
brane filtration (1.47 € kg−1), spiral plate technology (10.95 € cultivation both offer value-added products in the form of
kg−1), flotation, sedimentation, flocculation (0.5-1.2 € kg−1) food, feed, and other significant biological products [203].
require high energy or chemical flocculants [198]. Biomass Recently nanoparticle formation is another exciting explora-
pretreatment and lipid extraction from the lysed biomass is tion in the microalgae research field. The presence of gold and
also a challenge to overcome. Pretreatment of algal biomass silver nanoparticles in microalgae offers enormous application
includes disruption and degradation of biomass to convert the in the biomedical area [204, 205]. A similar kind of study is
accumulated carbohydrates and lipids into desired products. also reported in the formation of titanium dioxide nanoparti-
Pretreatment of biomass can be done through chemical, me- cles doped with silver nanoparticles by utilizing Aloe vera gel
chanical, and enzymatic methods depending upon the prod- as a capping and reducing agent. These formulated nanopar-
ucts of interest [14]. Recently, researchers are applying elec- ticles exhibit significant cancer cell growth suppression [206].
tric pulse for the pretreatment and used microalgae Microalgal co-culture setup for the biotechnological appli-
C. protothecoides where the pulse applied was 60 kV/cm for cations requires the management of certain specific character-
60- and 300-ns duration and the observed lipid recovery was istics and growth conditions. To achieve the possible out-
increased by 16.1% and 13.3% respectively [199]. comes from the co-culture, the correct ratio of two microor-
Pretreatment is still a bottleneck and potentially the costliest ganisms (microalgae with microalgae/ bacteria/ fungi/ yeast)
step in biofuel production. So, researchers should develop having a maximum mutualistic effect, has to be fixed. A very
economically feasible strategies for the pretreatment which basic advantage was cited that the co-culture system maintains
can be applied irrespective of the products of interest. Few the pH throughout the culture, the exchange of many gaseous
conventional methods including Bligh & Dyer and Folch sol- and metabolic substances, etc. [207]. Besides higher biomass
vent extraction are being used for a long time for the lipid yield, the co-culture study also reported the release of many
extraction from biomass [200]. Various improved methods extracellular FFAs into the culture broth [208], which are con-
combined with conventional methods are being researched sidered potential precursors for lipid biosynthesis [209].
throughout the globe and it seems lipid extraction involving Although several microalgal co-culture studies have shown
organic solvents has its disadvantages when applied at a large the lipids isolated from co-culture are mainly subjected to
scale [200]. Other than solvent extraction, important thermo- the production of biodiesel, but many other different types
chemical methods including pyrolysis, gasification, combus- of biofuels could also be generated. The residuals (carbohy-
tion, and liquefaction are used for the rapid decomposition of drates and fraction of proteins) after the extraction of fatty
organic compounds at a higher temperature. Fundamental dif- acids from microalgal co-culture can be further consumed by
ferences in biomass feedstock containing a high amount of yeast for the production of biobutanol and bioethanol by the
oxygen as compared with petroleum feedstock results in low process called fermentation. The biomass obtained from
thermal stability and difficulty in conserving the functionality microalgae cultures could also be utilized for bacterial fermen-
[201]. Recent studies have shown a well-developed tation to produce biohydrogen [210]. Many articles have re-
ultrasonication with integrated response surface methodology ported various high-value-added biomolecules such as phyco-
for the recovery of lipid from dry as well as wet biomass. cyanin, β-carotene, astaxanthin, omega-3 fatty acid,
Research on the extraction of lipids from the dry and wet exopolysaccharides (EPS), organic acids, and some allelo-
biomass of Chlorella sp. culture by ultrasonication and sol- pathic chemicals from the co-culturing of these microorgan-
vent extraction (double extraction and single solvent recovery) isms which have a commercial interest in pharmaceutical and
process has shown a promising lipid yield with the lowest nutraceutical industries [119]. The tight-knit relationship be-
energy consumption [202]. However, many challenges have tween microalgae and bacteria relies on some essential vita-
to be overcome such as cost reduction, stability, and function- mins like cobalamin (Vit B12), thiamin (Vit B1), and/or biotin
ality of bio-oil, environmental health and protection affairs, (Vit B7) which are produced in the algae-bacteria co-culture
transportation, and usage, etc. for the scaling up of microalgal [211]. Microalgal co-culture techniques are recently used for
biofuel production. There are so many challenges may arise the production of secondary metabolites including antibiotics,
for the co-cultivation of microalgae with other species like terpenoids, flavonoids, alkaloids, and polyketides, etc. at the
bacteria, fungi, and yeast; such as the selection of specific late stationary phase of their growth. Co-culture of microalgae
microorganism for the co-culturing with algae, optimization and filamentous fungi give rise to the production of anticancer
of growth media for co-culturing, pretreatment of biomass and antibiotic substances [212]. A study has been reported that
cannot be effectively done as there is a different population co-culturing of Tsukumurella pulmonis or Corynebacteria
in mixed culture, extraction of individual lipid and fatty acid glutamicum with can activate a novel metabolic pathway in
composition is also rigorous for the mixed culture. Streptomyces endus which allows the synthesis of
18 Bioenerg. Res. (2022) 15:1–26

alchivemycin A, a chromophore-containing antibiotic. Apart also, the productivity will be increased by newly developed
from the production of secondary metabolites, the microbial engineering techniques in co-culture strategies. Along with
co-cultures provide biomimetic scaffolds in the studies of re- TEA, life-cyle assessment (LCA) is also an important tool
generative medicine and tissue engineering [212]. for assessing the environmental performances of the whole
chain of production or the strategy throughout the life cycle
from the uprooting of raw materials, requirement of energy for
Techno-economic Analysis of Algae-Based production, transportation of goods to the point of distribu-
Biofuel Production tion, use of products, and finally the disposal into the environ-
ment [217]. LCA is based on all the steps of input and output
Despite all the benefits of algal biofuel, there is still a lack of to scrutinize and identify the environmental burdens so that
comprehensive public consent on the long term economic more improved opportunities will be encountered [217].
feasibility of advanced biofuels along with the performance
of advanced engineering procedures due to the variability of
techniques related to scaling up of newly developed strategies Conclusion and Future Perspectives
for easy and efficient biofuel production. All the developed
techniques are followed by techno-economic analysis (TEA) Microbial symbiosis has great importance in nature over the
during the development of procedures where the direct invest- individual existence of microorganisms to maintain a balance
ments in each step are analyzed so that gratuitous expenditures in our ecosystem. The exploitation of such types of microbial
can be minimized. Different computer-based simulators such communications into microalgal co-cultivation has tremen-
as ASPEN PLUS, UniSim Design, and Chemcad are used in dous potential as positive interactions were observed in many
these analysis methods which help in chemical process simu- research articles. As discussed in this review, various advan-
lation and optimization of various uncertain inputs parameters tages of co-cultivation include higher biomass and lipid yield,
and assumptions for sensitive analysis of change in each mod- minimization of contamination, enhanced waste removal effi-
el and how their input affects the specific processes [213]. ciency, co-production of value-added biosimilar products.
TEA study of three algal strains Chlorella vulgaris, However, to set up a co-culture at a laboratory scale, we
Tetraselmis suecica, and Nannochloropsis sp. showed a sig- should keep in mind the basic mechanism of microbial com-
nificant difference in overall lipid production cost ($6.4, $7, munication which has already been discussed so that the se-
and $8.3 /kg lipid respectively) varies for each strain due to lection of partner species will be easier otherwise, slight dis-
different cell characteristics and their treatment processes crepancies in the culturing system could change the nature of
[214]. A study showed using municipal wastewater for the the co-culture and destabilize the synergetic balance, will lead
treatment and biofuel production by cultivating algae to a loss of final products. The limitations of existing technol-
(Chlorella sp. and Scenedesmus sp.) in open raceways under ogies and methodologies for the easy cultivation, recovery,
the optimum light wavelength the remarkable decrease in bio- and purification of biofuels and metabolites obtained from
mass production cost (73%) from 2.71 to 0.73 $/kg biomass as the co-culture system need to be overcome to make it com-
compared with normal photobioreactors [215]. The utilization mercially viable. Optimization of the up and downstream pro-
of various types of equipment, chemicals, labor, etc. counting cess is also an important criterion to obtain the uniform com-
from the cultivation to final lipid extraction and purification position of the biofuels and is still a challenge to overcome.
causes excessive cost investment for biofuel production. To Techno-economic analysis will help to establish the newly
balance or minimize the overall production cost, the focus developed procedures for enhanced biomass and lipid produc-
should be on the extraction of multiple products rather than tion. In the future, suitable bioreactors with appropriate cul-
the one single product from the algal mono or co-cultures turing conditions will be developed for microalgal co-
[216]. A recent study on algal biorefinery at an industrial scale cultivation to acquire a series of products with significant im-
with four microalgae Isochrysis galbana, Phaeodactylum portance for social welfare.
tricornutum, S. obliquus, and N. graditana showed that the
profit range increases in the case of a multi-product value
chain (more than one valuable marketable products) rather Abbreviations AMDI, American Dye Manufacturing Institute; ASP,
Aquatic Species Program; BOD, Biological oxygen demand; COD,
than a single product value chain (one main valuable market-
Chemical oxygen demand; DO, Dissolved oxygen; DOC, Dissolved or-
able product and the rest biomass is utilized for lower value ganic carbon; DOE, Department of Energy; DW, Dry weight; EPSs,
application) and the average potential profit expressed per unit Extracellular polymeric substances; FAMEs, Fatty acid methyl esters;
of initial biomass for single and multiple value chains were FFA, Free fatty acids; HRAPs, High rate algal ponds; NH 4+ -N,
Ammonium-nitrogen; NO3−-N, Nitrate-nitrogen; PHAs,
1.26–3.93 € kg−1 and 1.61–7.76 € kg−1 respectively [216]. By
Polyhydroxyalkanoic acids; PUFA, Polyunsaturated fatty acids; RSM,
the better management practices in every stage of algal biofuel Response surface methodology; SCO, Single cell oil; TAGs,
production, unnecessary cost investment can be reduced, and Triacylglycerides; TN, Total nitrogen; TOC, Total organic carbon; TP,
Bioenerg. Res. (2022) 15:1–26 19

Total phosphate; TSS, Total suspended solids; VFAs, Volatile fatty acids; 15. Pacheco AR, Segrè D (2019) A multidimensional perspective on
VSS, Volatile suspended solids microbial interactions. FEMS Microbiol Lett 366(11):fnz125.
https://doi.org/10.1093/femsle/fnz125
Funding PKD and JR are financially supported by the Ministry of 16. Magdouli S, Brar SK, Blais JF (2016) Co-culture for lipid produc-
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