**5. Tools for studying microbial consortia**

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

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

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* consor-

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

improved yield [60].

112 Biofuels - Challenges and opportunities

or as a neat fuel of itself.

**4.5. Microalgae cocultures**

tia is significantly higher than those from plant tissue.

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 and to tune the composition of the consortia.

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 quantification method was developed to monitor the temporal profile of cell growth of the two strains in the coculture during fermentation [61]. The results indicated that an optimized coculture population which was tunable by changing the inoculation ratio of the two strains is essential for the consortium to achieve higher biofuel yield. q-PCR was also used to probe the dynamics of the yeast consortium which assembled the mini-cellulosome for ethanol production [72]. The primers were designed to specifically target a unique gene encoding for the endoglucanase, exoglucanase, β-glucosidase and the scaffolding expressed by each of the yeast strain. It was found that the final population ratio of the four yeast species did not change significantly compared with the initial inoculation ratio. By comparing the population of each strain in the assembled mini-cellulosome structure with that in the free enzyme system, the synergistic effect among the cellulases in the mini-cellulosome on cellulose hydrolysis was suggested. In another study, a real-time PCR assay based on the 16S rRNA gene sequence was performed to study the population abundance of each strain in a coculture consist of a celluloytic bacterium and a noncelluloytic, solventogenic bacterium during the production of butanol [73]. The competition and cooperation relationships between the two strains at different stages of the fermentation was revealed by the population dynamics study; it was found that the population of each strain was readily modulated by culture conditions such as pH and nutrient availability.

the general biorefinery concept include technoeconomic hurdles stemming from recalcitrance of the biomass to hydrolysis, feedstock variability, achieving high rate, yield, and titers of bioconversion of the bulk of the biomass to fuels, up- and down-stream separations, and minimizing resource inputs, including water, chemical additives, electricity, and infrastructure. Progress in development of consortium-based bioconversion technologies provides solutions to many of these challenges by consolidating pretreatment and biocatalysis, allowing flexibility for utilization of multiple substrates at variable concentrations, and supporting tunability for targeted end-products. New advances in synthetic biology and metabolic engineering in the context of microbial communities will be required to accelerate adoption and scale-up of

Engineering Microbial Consortia for Bioconversion of Multisubstrate Biomass Streams to Biofuels

http://dx.doi.org/10.5772/intechopen.80534

115

Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia LLC., a wholly owned subsidiary of Honeywell International Inc. for the US Department of Energy's National Nuclear Security

these strategies for an economically viable bio-based economy.

Administration under contract DE-NA0003525.

Fang Liu\*, Eric Monroe and Ryan W. Davis

States; 2016. DOI:10.2172/1245338

The authors declare that they have no competing interests.

\*Address all correspondence to: fliu006@gmail.com and fanliu@sandia.gov

Department of Biomass Science & Conversion Technologies, Sandia National Laboratories,

[1] Langholtz MH, Stokes BJ, Eaton LM. 2016 Billion-Ton Report: Advancing domestic resources for a thriving bioeconomy, Volume 1: Economic availability of feedstocks. Oak

[2] Schwab A. Bioenergy Technologies Office Multi-Year Program Plan. March 2016. United

[3] McCann MC, Carpita NC. Biomass recalcitrance: A multi-scale, multi-factor, and conversion-specific propertyo. Journal of Experimental Botany. 2015;**66**:4109-4118

Ridge National Laboratory, Oak Ridge, TN; 2016, 448p. DOI: 10.2172/1271651

**Acknowledgements**

**Conflict of interest**

**Author details**

Livermore, CA, USA

**References**

Beyond the experimental demonstrations of bioconversion consortia, a modeling framework based on comprehensive ordinary differential equation has been developed to gain insights into the behavior and dynamics of a fungal-bacterial consortium for isobutanol production [44]. The rate expressions for each of the reaction steps were derived and the parameter values were obtained from the literature or by experiment. The concentrations of cellulose, microbial biomass, and isobutanol during the coculture fermentation predicted by the model were validated by experimental data. The model suggested that the competition between the fungal and bacterial strain for soluble saccharides is the key interaction that drives the behavior of two strains in the coculture and the relationship between the two strains was recognized as the cooperator-cheater. The model could also predict the outcomes and stability of the interactions in the microbial consortia, which provided important information for tuning the coculture population and stabilizing the consortia.

Furthermore, genome-based metabolic networks using multispecies dynamic flux balance analysis were developed to build a process model for an open pond system involving the oleaginous yeast and microalgae consortia for biodiesel production [74]. The algal monoculture and yeast monoculture were modeled separately and compared to the algae and yeast coculture with cellulosic glucose and xylose feeds. The model predicted the biomass and lipid productivities of the coculture with results comparable to those reported in literature. The economic analysis of this system was also performed and indicated that the algae and yeast coculture can produce biodiesel at competitive prices.
