**4. Microbial consortia for biomass conversion and biofuel/chemical production**

Recent advances in synthetic biology, metabolic engineering, and systems biology have enabled rapid progress in developing microbial cell factories [6, 30, 31] and novel enzyme cascade systems [32–34] for the conversion of biomass feedstocks and synthesis of biofuels and other platform chemicals. Although there are some successful examples of developing 'superbugs' capable of multiple functions, engineering a single microbe to simultaneously perform multiple tasks is still quite challenging and bioenergetically costly under most situations. Because of the complexity and the multisubstrate nature of the biomass feedstocks, it is especially challenging to engineer a single microbe to efficiently convert the diverse substrates (carbohydrates, proteins, fatty acids, oils, etc.) of the biomass to produce value-added products. In contrast to the 'superbug' paradigm, in nature microbes rarely live in isolation, but rather exist in highly diverse and complex communities known as consortia. The microbes in these communities interact in numerous ways ranging from cooperation to competition and are often capable of performing tasks that are far too complex for any single organism to complete themselves [35]. Besides the ability to perform complex biosynthetic tasks, microbial consortia exhibit many other appealing properties such as stability, productivity and functional robustness. Inspired by the powerful features of the natural consortia, there are rapidly growing efforts been undertaken to understand natural consortia and to engineer synthetic consortia for biotechnology applications [36, 37]. Well-designed microbial consortia involving two or more microbes can take advantage of the functions of individual microbes and their interactions to realize synergistic division of labor and more efficient utilization of biochemical substrates than monocultures. Natural and synthetic microbial consortia developed for the conversion of different biomass feedstocks for biofuel and chemical production will be discussed below.

#### **4.1. Anaerobic digestion**

**3.3. Microalgae**

106 Biofuels - Challenges and opportunities

Data from [27].

While microalgae research has generally focused on high lipid strains and cultivation strategies for oil extraction and biodiesel production, recent studies have demonstrated the technoeconomic necessity for higher productivity algal systems which generally corresponds to lower quantities of lipids and a higher fraction of proteins and carbohydrates [29]. Because of the wide diversity of potential strains and cultivation conditions, microalgal biomass has a wide range of biochemical possibilities. This is further magnified by the distinct difference in biomass composition at different stages in microalgal cultures growth cycle. As a part of a study to understand the impact of this variation on a dilute acid hydrolysis pretreatment process, the composition of three microalgae strains at different points in their growth cycle

The challenges caused by feedstock variability provide a unique opportunity for consortium bioconversion strategies. For many biomass sources, techno-economic reports conclude that the cost of the biomass itself is the main economic sustainability driver [29], so a growing field is developing around the use of consortium conversion strategies that enable utilization of the vast majority of the biomass for production of a biofuel or petroleum displacing commodity chemical in an integrated biorefinery scheme. Clearly, if the biomass itself is the key cost driver at scale, then as much value as possible needs to be extracted from of the biomass. Consortium strategies allow for different organisms to specialize in efficient conversion of a particular substrate and collectively convert as much of the diverse and variable biomass-derived biochemical intermediates to a fuel or chemical product as possible. If done effectively, this strategy will

**Species Growth stage Lipids Ash Carbohydrate Protein** *N. granulata* Early 12.28 (0.16) 14.2 8.92 (0.13) 32.7

**Table 3.** Biochemical composition of DGS as a dry mass % (range of values) from four different studies as reported by [24].

*C. vulgaris* Early 12.07 (0.09) 6.7 11.12 (0.12) 43.2

**Table 4.** Biochemical composition in mass of dry cell weight (Std Dev) for two microalgae species.

Mid 25.6 (0.20) 13.6 11.12 (0.48) 23.1 Late 57.33 (0.09) 5.1 10.89 (0.11) 9.4

**Liu [26] Belyea et al. [25] Cromwell and Herkelman [27]**

Mid 15.02 (0.16) 4.4 35.69 (0.01) 24.0 Late 23.14 (0.19) 5.3 38.00 (0.36) 15.2

was investigated by Peinkos et al. and is summarized in **Table 4** [27].

**Spiehs and Whitney** 

Protein 30.2 (2.9) 27.4 (3.3) 31.4 (2.5) 29.7 (5.7) Oil 10.9 (1.2) 11.7 (2.2) 12.0 (1.7) 10.7 (5.0) starch — 4.9 (2.5) 5.3 (0.7) — Total carbohydrate 53.1 56.5 52.1 54.3 Crude fiber 8.8 (1.4) — 10.2 (1.0) —

**[28]**

When considering consortium-based bioconversion technologies, anaerobic digestion offers a model process due to its long history of industrial application of a complex, albeit unsupervised, microbial consortium. Anaerobic digestion is a biochemical process that converts organic material into a mixture of methane and CO2 (biogas) in an anaerobic environment. Its widespread use since the middle of the nineteenth century and the extensive research around the details of the process make it an important benchmark technology as well a valuable resource for future consortium conversion technologies [38]. The anaerobic digestion process is generally split into five stages, creating an interconnected web of processes each utilizing naturally adapted microorganisms that play a critical role in the overall conversion process. The stages can generally described as disintegration, hydrolysis, acidogenesis, acetogenesis, and methanogenesis which describes the sequential process of the organic polymers comprising biomass (such as carbohydrates and proteins) being disintegrated, those polymers being hydrolyzed to their corresponding monomers, the monomers being fermented to organic acids and hydrogen, the long carbon chain organic acids being further broken down into acetic acid and hydrogen, and finally the conversion of acetic acid to the fully reduced methane product.

The microbial consortia for CBP involve a cellulolytic strain that hydrolyzes hemicellulosic biomass to fermentable sugars and a fermentation strain that utilizes the cellulosic sugars for growth and conversion to biofuel products through the natural or engineered metabolic pathways. Native cellulosic microorganisms such as fungi and some anaerobic bacteria are usually selected as the cellulosic strains in the consortia. For example, the thermophilic anaerobe *Clostridium thermocellum* which is capable of hydrolyzing cellulose through the activity of the cellulosome multiprotein complex was cocultured with the engineered ethanol producing strain *Thermoanaerobacterium saccharolyticum* for the production of ethanol from Avicel [43]. By genetically engineering the two strains to be acetate and lactic acids-deficient, the yield was greatly improved, resulting in the coculture producing 38 g/L ethanol from 92 g/L Avicel. In another study, Minty et al. designed a synthetic fungal/bacterial consortium that involved a cellulolytic fungus *Trichoderma reesei* and an engineered *E. coli* strain for bioconversion of lignocellulosic feedstocks [44]. In the consortium, the cellulolytic fungus secreted cellulase enzymes to hydrolyze pretreated corn stover into fermentable sugars and the *E. coli* converted soluble saccharides into isobutanol with titers up to 1.88 g/L. The *T. reesei*/*E. coli* consortia developed in this study showed a cooperator-cheater dynamics which lead to stable equilib-

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The main challenges of using native cellulolytic microorganisms as the cellulolytic strains are the availability of tools for genetic manipulation and the application of these tools to engineer the strains with high yield, titer and robustness under industrial conditions. Industrial strains that are not naturally capable of hydrolyzing cellulose have also been genetically engineered as the cellulolytic strains. For example, *E. coli* was engineered to express cellulase, xylanase, β-glucosidase and xylobiosidase enzymes extracellularly [45]. The resulting two *E. coli* strains were able to grow on cellulose and xylan substrates, respectively. Subsequent incorporation of biofuel production pathways into each cellulolytic and hemicellulolytic strain enabled production of biofuel products such as fatty-acid ethyl ester, butanol and pinene from ionic liquid pretreated switchgrass by the *E. coli* coculture. In this *E. coli* based CBP, cellulose and hemicellulose in the pretreated biomass were hydrolyzed into soluble oligosaccharides by the secreted cellulases and hemicellulases from the two *E. coli* strains. The oligosaccharides were further converted to monomeric sugars by the expressed β-glucosidase enzymes and were eventually metabolized into biofuels via the heterologous pathways of the same *E. coli* strains. The *E. coli* strains developed in this study incorporated the biomass-degrading capability and biofuel-producing ability into one strain, unlike in most typical CBP systems where cellulolytic and fermentation strains are engineered separately. Instead, this *E. coli* coculture divided the labor of producing advanced biofuels from cellulose and hemicellulose fractions of the lignocellulosic biomass between the two engineered strains.

Besides biofuels, valuable chemicals are also produced from nonfood agricultural resources by the CBP approach. For example, Bayer et al. used a synthetic metagenomics approach to identify methyl halide transferase (MHT) genes from various organisms and screened these enzymes in *E. coli* for MHT activity [46]. MHT with the highest activity was cloned in *Saccharomyces cerevisiae* and the resulting strain was able to produce methyl halides from monomeric sugars such as glucose and xylose. The production of methyl halide from unprocessed switchgrass, corn stover, sugar cane bagasse, and poplar was then demonstrated by

rium population.

Anaerobic digestion offers a touchstone example of the technological potential of consortium bioconversion processes because many microorganisms involved are strongly dependent on other organisms in the consortium. This is exemplified by the interspecific transfer of hydrogen, i.e. hydrogenotrophs utilize the hydrogen being produced by syntrophic organisms oxidizing organic acids that require low pressures of H2 [39]. These syntrophic organisms are not able to grow in pure cultures because of this interdependency. While these symbiotic relationships are in many ways beneficial, they leave the entire process vulnerable to varying environmental conditions. If the temperature, pH, or another important environmental factor becomes unfavorable to an important organism in the anaerobic consortium the whole process is stalled and operates less efficiently. Furthermore, because of the wide variety of microorganisms involved in the anaerobic digestion process and the high level at which conditions can be controlled, it is difficult to engineer the system for any organism or class of organisms. The diversity of microbes involved in anaerobic digestion has minimized the potential roles for including metabolic engineering into the process, with most recent works simply looking to understand the population dynamics of the organisms involved [40]. The robustness of the technology makes it a base case bioconversion route for almost any biomass source, many of which have been investigated to varying levels of detail. Each biomass source presents unique challenges and difficulties to the overall anaerobic digestion process; microalgal biomass is a key example, since cells have been observed to remain intact after a 30 day retention time in a digester [41].

#### **4.2. Consolidated bioprocessing of lignocellulosic biomass**

Microbial conversion of lignocellulosic biomass requires multiple biological functionalities, including production of saccharifying enzymes, enzymatic hydrolysis of lignocellulose to soluble fermentable sugars, and metabolism of sugars to desired biofuel products. Cellulose and hemicellulose in lignocellulosic biomass can be hydrolyzed by cellulase and hemicellulase enzymes such as endoglucanase, exoglucanase, β-glucosidase, xylanase, xylosidase, and arabinofuranosidase. Microorganisms that are able to hydrolyze lignocellulosic biomass or secret cellulolytic hydrolysate enzymes are important for this step. Most biological process employed fungi such as *Trichoderma reesei*, *Penicillium echinulatum*, *Penicillium purpurogenum*, *Aspergillus niger*, and *Aspergillus fumigatus* [4] which secrete cellulolytic enzymes for saccharification. Some anaerobic bacteria such as *Clostridium thermocellum*, *Ruminococcus flavefaciens* and *Clostridium cellulovorans* developed large, extracellular multienzyme complexes called the cellulosomes, which are highly structured and consist of multiple cellulolytic enzyme units that interact with each other synergistically [42]. These anaerobic bacteria are also found to degrade cellulosic substrates efficiently. To reduce the number of unit operations and capital costs, a nascent approach called consolidated bioprocessing (CBP) has been developed for simultaneous enzyme production, hydrolysis, and fermentation by employing the microbial consortia biocatalyst strategy.

The microbial consortia for CBP involve a cellulolytic strain that hydrolyzes hemicellulosic biomass to fermentable sugars and a fermentation strain that utilizes the cellulosic sugars for growth and conversion to biofuel products through the natural or engineered metabolic pathways. Native cellulosic microorganisms such as fungi and some anaerobic bacteria are usually selected as the cellulosic strains in the consortia. For example, the thermophilic anaerobe *Clostridium thermocellum* which is capable of hydrolyzing cellulose through the activity of the cellulosome multiprotein complex was cocultured with the engineered ethanol producing strain *Thermoanaerobacterium saccharolyticum* for the production of ethanol from Avicel [43]. By genetically engineering the two strains to be acetate and lactic acids-deficient, the yield was greatly improved, resulting in the coculture producing 38 g/L ethanol from 92 g/L Avicel. In another study, Minty et al. designed a synthetic fungal/bacterial consortium that involved a cellulolytic fungus *Trichoderma reesei* and an engineered *E. coli* strain for bioconversion of lignocellulosic feedstocks [44]. In the consortium, the cellulolytic fungus secreted cellulase enzymes to hydrolyze pretreated corn stover into fermentable sugars and the *E. coli* converted soluble saccharides into isobutanol with titers up to 1.88 g/L. The *T. reesei*/*E. coli* consortia developed in this study showed a cooperator-cheater dynamics which lead to stable equilibrium population.

those polymers being hydrolyzed to their corresponding monomers, the monomers being fermented to organic acids and hydrogen, the long carbon chain organic acids being further broken down into acetic acid and hydrogen, and finally the conversion of acetic acid to the

Anaerobic digestion offers a touchstone example of the technological potential of consortium bioconversion processes because many microorganisms involved are strongly dependent on other organisms in the consortium. This is exemplified by the interspecific transfer of hydrogen, i.e. hydrogenotrophs utilize the hydrogen being produced by syntrophic organisms oxidizing

to grow in pure cultures because of this interdependency. While these symbiotic relationships are in many ways beneficial, they leave the entire process vulnerable to varying environmental conditions. If the temperature, pH, or another important environmental factor becomes unfavorable to an important organism in the anaerobic consortium the whole process is stalled and operates less efficiently. Furthermore, because of the wide variety of microorganisms involved in the anaerobic digestion process and the high level at which conditions can be controlled, it is difficult to engineer the system for any organism or class of organisms. The diversity of microbes involved in anaerobic digestion has minimized the potential roles for including metabolic engineering into the process, with most recent works simply looking to understand the population dynamics of the organisms involved [40]. The robustness of the technology makes it a base case bioconversion route for almost any biomass source, many of which have been investigated to varying levels of detail. Each biomass source presents unique challenges and difficulties to the overall anaerobic digestion process; microalgal biomass is a key example, since cells have been observed to remain intact after a 30 day retention time in a digester [41].

Microbial conversion of lignocellulosic biomass requires multiple biological functionalities, including production of saccharifying enzymes, enzymatic hydrolysis of lignocellulose to soluble fermentable sugars, and metabolism of sugars to desired biofuel products. Cellulose and hemicellulose in lignocellulosic biomass can be hydrolyzed by cellulase and hemicellulase enzymes such as endoglucanase, exoglucanase, β-glucosidase, xylanase, xylosidase, and arabinofuranosidase. Microorganisms that are able to hydrolyze lignocellulosic biomass or secret cellulolytic hydrolysate enzymes are important for this step. Most biological process employed fungi such as *Trichoderma reesei*, *Penicillium echinulatum*, *Penicillium purpurogenum*, *Aspergillus niger*, and *Aspergillus fumigatus* [4] which secrete cellulolytic enzymes for saccharification. Some anaerobic bacteria such as *Clostridium thermocellum*, *Ruminococcus flavefaciens* and *Clostridium cellulovorans* developed large, extracellular multienzyme complexes called the cellulosomes, which are highly structured and consist of multiple cellulolytic enzyme units that interact with each other synergistically [42]. These anaerobic bacteria are also found to degrade cellulosic substrates efficiently. To reduce the number of unit operations and capital costs, a nascent approach called consolidated bioprocessing (CBP) has been developed for simultaneous enzyme production, hydrolysis, and fermentation by employing the microbial

[39]. These syntrophic organisms are not able

fully reduced methane product.

108 Biofuels - Challenges and opportunities

consortia biocatalyst strategy.

organic acids that require low pressures of H2

**4.2. Consolidated bioprocessing of lignocellulosic biomass**

The main challenges of using native cellulolytic microorganisms as the cellulolytic strains are the availability of tools for genetic manipulation and the application of these tools to engineer the strains with high yield, titer and robustness under industrial conditions. Industrial strains that are not naturally capable of hydrolyzing cellulose have also been genetically engineered as the cellulolytic strains. For example, *E. coli* was engineered to express cellulase, xylanase, β-glucosidase and xylobiosidase enzymes extracellularly [45]. The resulting two *E. coli* strains were able to grow on cellulose and xylan substrates, respectively. Subsequent incorporation of biofuel production pathways into each cellulolytic and hemicellulolytic strain enabled production of biofuel products such as fatty-acid ethyl ester, butanol and pinene from ionic liquid pretreated switchgrass by the *E. coli* coculture. In this *E. coli* based CBP, cellulose and hemicellulose in the pretreated biomass were hydrolyzed into soluble oligosaccharides by the secreted cellulases and hemicellulases from the two *E. coli* strains. The oligosaccharides were further converted to monomeric sugars by the expressed β-glucosidase enzymes and were eventually metabolized into biofuels via the heterologous pathways of the same *E. coli* strains. The *E. coli* strains developed in this study incorporated the biomass-degrading capability and biofuel-producing ability into one strain, unlike in most typical CBP systems where cellulolytic and fermentation strains are engineered separately. Instead, this *E. coli* coculture divided the labor of producing advanced biofuels from cellulose and hemicellulose fractions of the lignocellulosic biomass between the two engineered strains.

Besides biofuels, valuable chemicals are also produced from nonfood agricultural resources by the CBP approach. For example, Bayer et al. used a synthetic metagenomics approach to identify methyl halide transferase (MHT) genes from various organisms and screened these enzymes in *E. coli* for MHT activity [46]. MHT with the highest activity was cloned in *Saccharomyces cerevisiae* and the resulting strain was able to produce methyl halides from monomeric sugars such as glucose and xylose. The production of methyl halide from unprocessed switchgrass, corn stover, sugar cane bagasse, and poplar was then demonstrated by the consortium of the engineered yeast and a cellulolytic bacterium *Actinotalea fermentans*. In the coculture, the cellulolytic bacterium produced monomeric sugars from hemicellulosic biomass which were further converted to methyl halide by the engineered yeast.

to optimize the product yield from feed streams with variable sugar compositions. Xia et al. extended the consortium strategy for mixed sugar utilization to a synthetic mixture composed of three sugars-glucose, xylose and arabinose as well as the growth inhibitor acetic acid [54]. In this case one strain engineered to utilize acetate but not sugar was used to selectively remove acetate in the first stage of the bioprocess. In the subsequent stage, three *E. coli* strains which were each engineered to utilize only one sugar were cocultured and consumed glucose, xylose and arabinose simultaneously. Although reduction of the net growth rate of the strains was observed as a result of the chromosomal gene deletions that were required to generate the strains, the consortium showed significant improvement in multiple sugar utilization than

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Substrate-selective consortia have also been engineered for the production of biofuels from hemicellulose-derived sugar mixtures. In a recent study, the synthetic pathway of n-butanol was engineered into a glucose-selective strain and a xylose-selective strain and the resulting *E. coli* coculture produced n-butanol from the sugar mixture [55]. The system was further improved by distributing the n-butanol production pathway into the two strains for improved redox balance and 5.2 g/L n-butanol was produced by the coculture from a glucose-xylose mixture. In another related study, *Saccharomyces cerevisiae* was genetically engineered to utilize L-arabinose. By coculturing the L-arabinose-utilizing yeast and the yeast that could ferment glucose and xylose, the hexose and pentose sugars present in corn stover hydrolysates

An *E. coli* coculture system with two substrate-selective strains has also been developed to produce commodity chemical such as *cis, cis*-muconic acid from a glucose/xylose sugar mixture [57]. To overcome the high-level intermediate accumulation during the synthesis, the entire muconic acid synthetic pathway was split into the two *E. coli* cells. Therefore, one strain was only able to utilize xylose and convert it to the intermediate 3-dehydroshikimic acid (DHS) which is the precursor for muconic acid. The second strain incorporated a DHS transporter and could utilize glucose and convert DHS to the final product muconic acid through the rest of the split pathway. The *E. coli*-*E. coli* coculture system has shown to use sugar mixtures efficiently even at increased scale, and produce significantly higher amount of

muconic acid than previous studies, with a yield of 51% of the theoretical maximum.

Besides sugar mixtures, many biomass resources are also rich in proteins. For example, distillers grains with solubles (DGS) produced from the first-generation bioethanol process are considered as a rich source of cellulosic polysaccharides (52–57%) and proteins (27–31%) [58]. Microalgae are another example of the biomass resource that is rich in polysaccharides and proteins. Therefore, developing biochemical techniques for simultaneous utilization of the sugar and protein fractions of the biomass is important for efficient bioconversion of these

Microbes normally cannot utilize amino acids as carbon source for growth. However, an *E. coli* strain that was able to use 13 individual amino acids as the sole carbon source for growth was developed after several rounds of chemical mutagenesis. A strategy was subsequently developed to deaminate amino acids and convert the remaining carbon backbones of amino acids to

any single-organism approach.

was efficiently fermented to ethanol [56].

**4.4. Microbial consortia for sugar and protein mixture**

biomass feedstocks.

Inspired by the natural cellulosome in some anaerobic bacteria for the hydrolysis of cellulose, Tsai et al. developed a synthetic yeast consortium displaying an artificial minicellulosome for the synergistic saccharification and fermentation of cellulose to ethanol [47]. In the consortium, one yeast strain displayed a miniscaffolding consisting of three different cohesin domains and three other yeast strains secreted dockerin-tagged endoglucanase, exoglucanase, and β-glucosidase, respectively. Because of the specific interaction between the orthogonal cohesin-dockerin pairs, dockerin-tagged cellulolytic enzymes bound onto the scaffolding and a minicellulosome structure formed on the yeast cell surface. Cellulose was hydrolyzed to glucose by the three cellulases in the yeast displayed minicellulosome and glucose was then utilized by the yeast and fermented to ethanol. In this work, it was found that the ethanol yield could be fine-tuned by adjusting the ratio of the different yeast strains in the consortium. The consortium with the optimized ratio of the different populations produced ethanol up to 93% of the theoretical value from cellulose.

#### **4.3. Microbial consortia for variable sugar mixture feeds**

Hydrolysis of lignocellulosic biomass by chemical or enzymatic methods usually results in a mixture of C6 sugars (glucose, mannose, and galactose) and C5 sugars (xylose and arabinose) [15, 48]. The relative proportion of these sugars varies among different hemicellulosic biomass sources. Although glucose is the most abundant hexose and xylose is typically the major pentose, the fraction of other sugars found in hydrolysates can also be significant depending on the biomass and the pretreatment process. Natural microbes commonly consume hexose sugars prior to initiating the consumption of a pentose sugar. This phenomenon is known as carbon catabolite repression, in which the presence of a preferred substrate represses the expression of genes in the microbes required for the metabolism of other substrates [49]. Although efforts have been made to develop a single organism that can consume glucose and xylose simultaneously [50–52], sugar mixtures remain inefficiently consumed in these singleorganism processes. Moreover, since the sugar composition and proportion vary in different biomass hydrolysates, a single organism has very limited ability to adjust to this variation. Instead, multiorganism coculture systems with each organism selectively consuming only one particular sugar substrate have been developed to eliminate the carbon catabolite repression and convert the mixed sugar feed streams more efficiently and completely. The multiorganism systems also have the ability to adjust the ratio of the different individual populations in the coculture in order to adapt to the fluctuation in the mixed sugar compositions of the feedstocks.

As a recent example, two substrate-selective strains of *E. coli* were metabolically engineered with one strain only able to consume glucose and the other one only able to utilize xylose [53]. To construct the xylose selective (glucose deficient) strain, three genes *ptsG*, *manZ* and *glk* involved in glucose uptake were knocked out from the chromosome and the glucose-selective (xylose deficient) strain has the *xylA* gene encoding for xylose isomerase deleted. This coculture was demonstrated for simultaneous conversion of glucose and xylose for acetate production. Moreover, the relative biocatalyst concentrations in the coculture could be adjusted in order to optimize the product yield from feed streams with variable sugar compositions. Xia et al. extended the consortium strategy for mixed sugar utilization to a synthetic mixture composed of three sugars-glucose, xylose and arabinose as well as the growth inhibitor acetic acid [54]. In this case one strain engineered to utilize acetate but not sugar was used to selectively remove acetate in the first stage of the bioprocess. In the subsequent stage, three *E. coli* strains which were each engineered to utilize only one sugar were cocultured and consumed glucose, xylose and arabinose simultaneously. Although reduction of the net growth rate of the strains was observed as a result of the chromosomal gene deletions that were required to generate the strains, the consortium showed significant improvement in multiple sugar utilization than any single-organism approach.

Substrate-selective consortia have also been engineered for the production of biofuels from hemicellulose-derived sugar mixtures. In a recent study, the synthetic pathway of n-butanol was engineered into a glucose-selective strain and a xylose-selective strain and the resulting *E. coli* coculture produced n-butanol from the sugar mixture [55]. The system was further improved by distributing the n-butanol production pathway into the two strains for improved redox balance and 5.2 g/L n-butanol was produced by the coculture from a glucose-xylose mixture. In another related study, *Saccharomyces cerevisiae* was genetically engineered to utilize L-arabinose. By coculturing the L-arabinose-utilizing yeast and the yeast that could ferment glucose and xylose, the hexose and pentose sugars present in corn stover hydrolysates was efficiently fermented to ethanol [56].

An *E. coli* coculture system with two substrate-selective strains has also been developed to produce commodity chemical such as *cis, cis*-muconic acid from a glucose/xylose sugar mixture [57]. To overcome the high-level intermediate accumulation during the synthesis, the entire muconic acid synthetic pathway was split into the two *E. coli* cells. Therefore, one strain was only able to utilize xylose and convert it to the intermediate 3-dehydroshikimic acid (DHS) which is the precursor for muconic acid. The second strain incorporated a DHS transporter and could utilize glucose and convert DHS to the final product muconic acid through the rest of the split pathway. The *E. coli*-*E. coli* coculture system has shown to use sugar mixtures efficiently even at increased scale, and produce significantly higher amount of muconic acid than previous studies, with a yield of 51% of the theoretical maximum.

#### **4.4. Microbial consortia for sugar and protein mixture**

the consortium of the engineered yeast and a cellulolytic bacterium *Actinotalea fermentans*. In the coculture, the cellulolytic bacterium produced monomeric sugars from hemicellulosic

Inspired by the natural cellulosome in some anaerobic bacteria for the hydrolysis of cellulose, Tsai et al. developed a synthetic yeast consortium displaying an artificial minicellulosome for the synergistic saccharification and fermentation of cellulose to ethanol [47]. In the consortium, one yeast strain displayed a miniscaffolding consisting of three different cohesin domains and three other yeast strains secreted dockerin-tagged endoglucanase, exoglucanase, and β-glucosidase, respectively. Because of the specific interaction between the orthogonal cohesin-dockerin pairs, dockerin-tagged cellulolytic enzymes bound onto the scaffolding and a minicellulosome structure formed on the yeast cell surface. Cellulose was hydrolyzed to glucose by the three cellulases in the yeast displayed minicellulosome and glucose was then utilized by the yeast and fermented to ethanol. In this work, it was found that the ethanol yield could be fine-tuned by adjusting the ratio of the different yeast strains in the consortium. The consortium with the optimized ratio of the different populations produced ethanol up to

Hydrolysis of lignocellulosic biomass by chemical or enzymatic methods usually results in a

[15, 48]. The relative proportion of these sugars varies among different hemicellulosic biomass sources. Although glucose is the most abundant hexose and xylose is typically the major pentose, the fraction of other sugars found in hydrolysates can also be significant depending on the biomass and the pretreatment process. Natural microbes commonly consume hexose sugars prior to initiating the consumption of a pentose sugar. This phenomenon is known as carbon catabolite repression, in which the presence of a preferred substrate represses the expression of genes in the microbes required for the metabolism of other substrates [49]. Although efforts have been made to develop a single organism that can consume glucose and xylose simultaneously [50–52], sugar mixtures remain inefficiently consumed in these singleorganism processes. Moreover, since the sugar composition and proportion vary in different biomass hydrolysates, a single organism has very limited ability to adjust to this variation. Instead, multiorganism coculture systems with each organism selectively consuming only one particular sugar substrate have been developed to eliminate the carbon catabolite repression and convert the mixed sugar feed streams more efficiently and completely. The multiorganism systems also have the ability to adjust the ratio of the different individual populations in the coculture in order to adapt to the fluctuation in the mixed sugar compositions of the

As a recent example, two substrate-selective strains of *E. coli* were metabolically engineered with one strain only able to consume glucose and the other one only able to utilize xylose [53]. To construct the xylose selective (glucose deficient) strain, three genes *ptsG*, *manZ* and *glk* involved in glucose uptake were knocked out from the chromosome and the glucose-selective (xylose deficient) strain has the *xylA* gene encoding for xylose isomerase deleted. This coculture was demonstrated for simultaneous conversion of glucose and xylose for acetate production. Moreover, the relative biocatalyst concentrations in the coculture could be adjusted in order

sugars (xylose and arabinose)

sugars (glucose, mannose, and galactose) and C5

biomass which were further converted to methyl halide by the engineered yeast.

93% of the theoretical value from cellulose.

110 Biofuels - Challenges and opportunities

mixture of C6

feedstocks.

**4.3. Microbial consortia for variable sugar mixture feeds**

Besides sugar mixtures, many biomass resources are also rich in proteins. For example, distillers grains with solubles (DGS) produced from the first-generation bioethanol process are considered as a rich source of cellulosic polysaccharides (52–57%) and proteins (27–31%) [58]. Microalgae are another example of the biomass resource that is rich in polysaccharides and proteins. Therefore, developing biochemical techniques for simultaneous utilization of the sugar and protein fractions of the biomass is important for efficient bioconversion of these biomass feedstocks.

Microbes normally cannot utilize amino acids as carbon source for growth. However, an *E. coli* strain that was able to use 13 individual amino acids as the sole carbon source for growth was developed after several rounds of chemical mutagenesis. A strategy was subsequently developed to deaminate amino acids and convert the remaining carbon backbones of amino acids to fusel alcohols through metabolic engineering [59]. In a follow-on study, the engineered *E. coli* was further improved by modifying the cofactor specificity of two enzymes involved in the metabolic pathway and the resulting strain can produce fusel alcohols with significantly improved yield [60].

and provide CO2

for photosynthesis by the microalgae. At the same time, microalgae act as

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.

an oxygen generator which benefits yeast growth. The mixed cultures usually showed higher yields of lipid production and higher growth rates and biomass concentration of the microalgae. Examples of oleaginous yeast and microalgae coculture include the culture of yeast *Rhodotorula glutinis* with microalgae *Chlorella vulgaris* [67] and yeast *Rhodotorula glutinis* with cyanobacterium *Spirulina platensis* [68]. In these studies, the lipid production and algae growth was reported to be higher than that in the pure cultures. Applying a similar strategy, cocultivation of filamentous fungus *A. fumigatus* and microalgae has also been demonstrated to increase biomass production and lipid yield [69]. Moreover, because filamentous fungi are bioflocculating agents, cocultivation with microalgae assisted the flocculation and therefore the harvesting of microalgae.

In the oleaginous yeast and microalgae coculture, organic carbon feedstocks are usually needed to feed the yeasts to produce biofuel products. A phototrophic sucrose-secreting cyanobacteria *Synechococcus elongatus* was engineered and cocultured with oleaginous yeast strain *Cryptococcus curvatus* or *Rhodotorula glutinis* which is capable of producing biofuel products

to yeasts while yeasts assist cyanobacteria growth and survival by eliminating oxidative stress. The oleaginous yeast strain was shown to have increased lipid production in the coculture and could be engineered to produce biofuel products. This synthetic coculture presents a potential

Algae-bacterium-archaeon consortia have also been developed for the production of oil-like mixtures under anaerobic, thermophilic, and atmospheric conditions. Thermostable bacterium *Thermosipho globiformans* and archaeon *Methanocaldococcus jannaschii* were cocultured at 68°C with different species of microalgae under anaerobic conditions, followed by pyrolysis at 300°C and the consortia produced n-alkane rich biofuels and isoprenoids [71]. The composition and quantities of n-alkanes produced by pyrolysis were found to be closely related to

Stability and tunable population compositions are highly desirable for microbial consortia developed for bioprocessing applications, because these properties could expand possible process configurations and improve efficiency. For examples, stability allows the use of continuous reactors and avoids eliminating one strain in the coculture during fermentation, whereas tunability would allow the optimization of the population composition for desired performance. Because of the multisubstrate nature of the biomass, microbial consortia with tunable population compositions are especially important to be adapted to the variable substrate compositions. Understanding the population dynamics and interactions between the members in the microbial consortia is important to develop a coculture with stable population

Real-time PCR assays have been used to study the population dynamics of the consortia developed for biofuel production. In the study of simultaneous conversion of sugar and protein fractions of the hydrolysates by an *E. coli* coculture, specific primers targeting the unique genes in the chromosome of the two different *E. coli* strains were designed and q-PCR based

sustainable production platform for biofuels production directly from sunlight and CO2

[70]. In this case, *S. elongatus* utilized sunlight and CO2

the lipid contents and composition of the microalgae.

**5. Tools for studying microbial consortia**

and to tune the composition of the consortia.

Liu et al. has demonstrated the feasibility of one-pot bioconversion of the protein and carbohydrate fractions of the DGS hydrolysates and algae hydrolysates into mixed fusel alcohols by an *E. coli* coculture [61]. In the consortia, one strain is dedicated for bioconversion of hexose and pentose sugars in the hydrolysates into isobutanol (C4) and isopentanol (C5) fusel alcohols, whereas the other strain was engineered to convert free amino acids into mixed fusel alcohols. At the optimized inoculation ratio of the two strains, the consortium produced the highest titer of total fusel alcohols, up to 10.3 g/L, including 6.5 g/L isobutanol which comprised 63.1% of the total alcohols. Correspondingly, the consortium with the optimized inoculation ratio consumed the highest amount of DGS carbohydrates and proteins in the hydrolysates, including near complete consumption of the glucose and arabinose and 85.1% of the xylose, as well as 31.3% of the total proteins in the hydrolysates. Evaluation of the biofuel properties of the fusel alcohols produced using this strategy indicates that the higher carbon chain length alcohol mixture (especially C3-C5) provides increased energy densities and a variety of improved physical properties, such as reduced water solubility and corrosivity, than ethanol [16]. Therefore, the mixed fusel alcohols produced in this coculture have promising potential applications as a fuel upgrading feedstock in gasoline, diesel, and jet fuel or as a neat fuel of itself.

Similar *E. coli* cocultures were also developed for the production of terpene mixtures from the carbohydrate and protein fractions of algae hydrolysates [62, 63]. In these works, the caryophyllene biosynthesis pathway was engineered into the carbohydrate conversion strain and the protein conversion strain respectively. The engineered coculture produced up to 507.4 mg/L of total terpene mixture including sesquiterpene, monoterpene, and caryophyllene from algae hydrolysates. Terpenes are considered potential "drop-in" candidates for aviation fuels based on their high energy density. Importantly, the terpene yield produced from the *E. coli* consortia is significantly higher than those from plant tissue.

#### **4.5. Microalgae cocultures**

In addition to lignocellulosic biomass, microalgae are an attractive biomass resource for biofuel production. Microalgae can provide several types of biofuels, including methane produced by anaerobic digestion of the algae biomass [64], biodiesel derived from microalgae oil [65], and biohydrogen produced photobiologically [66]. Many microalgae species can accumulate substantial quantities of lipids and contribute to a high oil yield. The average lipid content of microalgae varies between 1 and 70% and some can accumulate up to 90% of dry weight under certain conditions [65]. Furthermore, the lipids extracted from microalgae biomass often consist of triglycerides and can be converted to biodiesel by transesterification reactions in which three fatty acid molecules are esterified with a glycerol molecule. Synthetic consortia with microalgae cocultured with heterotrophs have been developed to take benefit from their mutualistic interactions.

Oleaginous yeast that can also accumulate high account of lipids has been cocultured with microalgae in several studies. In the mixed culture, yeasts use a vast variety of organic matter and provide CO2 for photosynthesis by the microalgae. At the same time, microalgae act as an oxygen generator which benefits yeast growth. The mixed cultures usually showed higher yields of lipid production and higher growth rates and biomass concentration of the microalgae. Examples of oleaginous yeast and microalgae coculture include the culture of yeast *Rhodotorula glutinis* with microalgae *Chlorella vulgaris* [67] and yeast *Rhodotorula glutinis* with cyanobacterium *Spirulina platensis* [68]. In these studies, the lipid production and algae growth was reported to be higher than that in the pure cultures. Applying a similar strategy, cocultivation of filamentous fungus *A. fumigatus* and microalgae has also been demonstrated to increase biomass production and lipid yield [69]. Moreover, because filamentous fungi are bioflocculating agents, cocultivation with microalgae assisted the flocculation and therefore the harvesting of microalgae.

In the oleaginous yeast and microalgae coculture, organic carbon feedstocks are usually needed to feed the yeasts to produce biofuel products. A phototrophic sucrose-secreting cyanobacteria *Synechococcus elongatus* was engineered and cocultured with oleaginous yeast strain *Cryptococcus curvatus* or *Rhodotorula glutinis* which is capable of producing biofuel products [70]. In this case, *S. elongatus* utilized sunlight and CO2 and produced sucrose as a carbon source to yeasts while yeasts assist cyanobacteria growth and survival by eliminating oxidative stress. The oleaginous yeast strain was shown to have increased lipid production in the coculture and could be engineered to produce biofuel products. This synthetic coculture presents a potential sustainable production platform for biofuels production directly from sunlight and CO2 .

Algae-bacterium-archaeon consortia have also been developed for the production of oil-like mixtures under anaerobic, thermophilic, and atmospheric conditions. Thermostable bacterium *Thermosipho globiformans* and archaeon *Methanocaldococcus jannaschii* were cocultured at 68°C with different species of microalgae under anaerobic conditions, followed by pyrolysis at 300°C and the consortia produced n-alkane rich biofuels and isoprenoids [71]. The composition and quantities of n-alkanes produced by pyrolysis were found to be closely related to the lipid contents and composition of the microalgae.
