The plant polymer lignin is the most abundant renewable source of aromatics on the planet and conversion of it to valuable fuels and chemicals is critical to the economic viability of a lignocellulosic biofuels industry and to meeting the DOE’s 2022 goal of $\$2.50$/gallon mean biofuel selling price. Presently, there is no efficient way of converting lignin into valuable commodities. Current biological approaches require mixtures of expensive ligninolytic enzymes and engineered microbes. This project was aimed at circumventing these problems by discovering commensal relationships among fungi and bacteria involved in biological lignin utilization and using this knowledge to engineer microbial communities capable of converting lignin into renewable fuels and chemicals. Essentially, we aimed to learn from, mimic and improve on nature. We discovered fungi that synergistically work together to degrade lignin, engineered fungal systems to increase expression of the required enzymes and engineered organisms to produce products such as biodegradable plastics precursors.
Background: Due to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks. In contrast, the oleaginous red yeast Rhodosporidium toruloides is capable of efficiently metabolizing both xylose and glucose, suggesting that it may be a suitable host for the production of lignocellulosic bioproducts. In addition, R. toruloides naturally produces several carotenoids (C40 terpenoids), indicating that it may have a naturally high carbon flux through its mevalonate (MVA) pathway, providing pools of intermediates for the production of a wide range of heterologous terpene-based biofuels and bioproducts from lignocellulose. Results: Sixteen terpene synthases (TS) originating from plants, bacteria and fungi were evaluated for their ability to produce a total of nine different monoterpenes in R. toruloides. Eight of these TS were functional and produced several different monoterpenes, either as individual compounds or as mixtures, with 1,8-cineole, sabinene, ocimene, pinene, limonene, and carene being produced at the highest levels. The 1,8-cineole synthase HYP3 from Hypoxylon sp. E74060B produced the highest titer of 14.94 ± 1.84 mg/L 1,8-cineole in YPD medium and was selected for further optimization and fuel properties study. Production of 1,8-cineole from lignocellulose was also demonstrated in a 2L batch fermentation, and cineole production titers reached 34.6 mg/L in DMR-EH (Deacetylated, Mechanically Refined, Enzymatically Hydorlized) hydrolysate. Finally, the fuel properties of 1,8-cineole were examined, and indicate that it may be a suitable petroleum blend stock or drop-in replacement fuel for spark ignition engines. Conclusion: Our results demonstrate that Rhodosporidium toruloides is a suitable microbial platform for the production of non-native monoterpenes with biofuel applications from lignocellulosic biomass.
Background: First generation bioethanol production utilizes the starch fraction of maize, which accounts for approximately 60% of the ash-free dry weight of the grain. Scale-up of this technology for fuels applications has resulted in a massive supply of distillers' grains with solubles (DGS) coproduct, which is rich in cellulosic polysaccharides and protein. It was surmised that DGS would be rapidly adopted for animal feed applications, however, this has not been observed based on inconsistency of the product stream and other logistics-related risks, especially toxigenic contaminants. Therefore, efficient valorization of DGS for production of petroleum displacing products will significantly improve the techno-economic feasibility and net energy return of the established starch bioethanol process. In this study, we demonstrate 'one-pot' bioconversion of the protein and carbohydrate fractions of a DGS hydrolysate into C4 and C5 fusel alcohols through development of a microbial consortium incorporating two engineered Escherichia coli biocatalyst strains. Results: The carbohydrate conversion strain E. coli BLF2 was constructed from the wild type E. coli strain B and showed improved capability to produce fusel alcohols from hexose and pentose sugars. Up to 12 g/L fusel alcohols was produced from glucose or xylose synthetic medium by E. coli BLF2. The second strain, E. coli AY3, was dedicated for utilization of proteins in the hydrolysates to produce mixed C4 and C5 alcohols. To maximize conversion yield by the co-culture, the inoculation ratio between the two strains was optimized. The co-culture with an inoculation ratio of 1:1.5 of E. coli BLF2 and AY3 achieved the highest total fusel alcohol titer of up to 10.3 g/L from DGS hydrolysates. The engineered E. coli co-culture system was shown to be similarly applicable for biofuel production from other biomass sources, including algae hydrolysates. Furthermore, the co-culture population dynamics revealed by quantitative PCR analysis indicated that despite the growth rate difference between the two strains, co-culturing didn't compromise the growth of each strain. The q-PCR analysis also demonstrated that fermentation with an appropriate initial inoculation ratio of the two strains was important to achieve a balanced co-culture population which resulted in higher total fuel titer. Conclusions: The efficient conversion of DGS hydrolysates into fusel alcohols will significantly improve the feasibility of the first generation bioethanol process. The integrated carbohydrate and protein conversion platform developed here is applicable for the bioconversion of a variety of biomass feedstocks rich in sugars and proteins.
The feasibility of converting algal protein to mixed alcohols has recently been demonstrated with an engineered E. coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening, more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the four highest activity YqhD mutants were selected for combination with two IlvC mutants, both accepting NADH as a redox cofactor, for modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E. coli strain, subtype AY3, with increased fusel alcohol yield of ~ 60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.
Large-scale open microalgae cultivation has tremendous potential to make a significant contribution to replacing petroleum-based fuels with biofuels. Open algal cultures are unavoidably inhabited with a diversity of microbes that live on, influence, and shape the fate of these ecosystems. However, there is little understanding of the resilience and stability of the microbial communities in engineered semicontinuous algal systems. To evaluate the dynamics and resilience of the microbial communities in microalgae biofuel cultures, we conducted a longitudinal study on open systems to compare the temporal profiles of the microbiota from two multigenerational algal cohorts, which include one seeded with the microbiota from an in-house culture and the other exogenously seeded with a natural-occurring consortia of bacterial species harvested from the Pacific Ocean. From these month-long, semicontinuous open microalga Nannochloropsis salina cultures, we sequenced a time-series of 46 samples, yielding 8804 operational taxonomic units derived from 9,160,076 high-quality partial 16S rRNA sequences. We provide quantitative evidence that clearly illustrates the development of microbial community is associated with microbiota ancestry. In addition, N. salina growth phases were linked with distinct changes in microbial phylotypes. Alteromonadeles dominated the community in the N. salina exponential phase whereas Alphaproteobacteria and Flavobacteriia were more prevalent in the stationary phase. We also demonstrate that the N. salina-associated microbial community in open cultures is diverse, resilient, and dynamic in response to environmental perturbations. This knowledge has general implications for developing and testing design principles of cultivated algal systems.
Lignin is often overlooked in the valorization of lignocellulosic biomass, but lignin-based materials and chemicals represent potential value-added products for biorefineries that could significantly improve the economics of a biorefinery. Fluctuating crude oil prices and changing fuel specifications are some of the driving factors to develop new technologies that could be used to convert polymeric lignin into low molecular weight lignin and or monomeric aromatic feedstocks to assist in the displacement of the current products associated with the conversion of a whole barrel of oil. Our project of understanding microbial lignolysis for renewable platform chemicals aimed to understand microbial and enzymatic lignolysis processes to break down lignin for conversion into commercially viable drop-in fuels. We developed novel lignin analytics to interrogate enzymatic and microbial lignolysis of native polymeric lignin and established a detailed understanding of lignolysis as a function of fungal enzyme, microbes and endophytes. Bioinformatics pipeline was developed for metatranscryptomic analysis of aridland ecosystem for investigating the potential discovery of new lignolysis gene and gene products.