Cellulosic Biofuel Production Research Articles

Engineering Compositionally Uniform Yeast Whole-Cell Biocatalysts with Maximized Surface Enzyme Density for Cellulosic Biofuel Production.

In recent decades, whole-cell biocatalysis has played an increasingly important role in the food, pharmaceutical, and energy sector. One promising application is the use of ethanologenic yeast displaying minicellulosomes on the cell surface to combine cellulose hydrolysis and fermentation into a single step for consolidated bioprocessing. However, cellulosic ethanol production using existing yeast whole-cell biocatalysts (yWCBs) has not reached industrial feasibility due to their inefficient cellulose hydrolysis. As prior studies have demonstrated enzyme density on the yWCB surface to be one of the most important parameters for enhancing cellulose hydrolysis, we sought to maximize this parameter at both the population and single-cell levels in yWCBs displaying tetrafunctional minicellulosomes. At the population level, enzyme density is limited by the presence of a nondisplay population constituting 25-50% of all cells. In this study, we identified the cause to be plasmid loss and successfully eliminated the nondisplay population to generate compositionally uniform yWCBs. At the single-cell level, we demonstrate that enzyme density is limited by molecular crowding, which hinders minicellulosome assembly. By adjusting the integrated gene copy number, we obtained yWCBs of tunable enzyme display levels. This tunability allowed us to avoid the crowding-limited regime and achieve a maximum enzyme density per cell. As a result, the best strain showed a cellulose-to-ethanol yield of 4.92 g/g, corresponding to 96% of the theoretical maximum and near-complete conversion (∼96%) of the starting cellulose (1% PASC). Our holistic engineering strategy that combines a population and single-cell level approach is broadly applicable to enhance the WCB performance in other biocatalytic cascade schemes.

Ethanol tolerance in engineered strains of Clostridium thermocellum

Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75–80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production.

The role of social support on midwestern farmers’ willingness to grow perennial bioenergy crops

The lack of farmers' willingness to grow perennial bioenergy crops (PBCs) presents a critical barrier to the emergence of cellulosic biofuel production. The willingness relies on a complex network of economic, environmental, and social drivers, among which the influence of social factors (e.g., the influence of neighborhood, community, and communication) is less understood. This study addresses this knowledge gap via a survey analysis of midwestern farmers. The survey data are analyzed through ordinary least square regression and structural equation model, which together investigate the individual and interactive impacts of multiple factors on farmers' decisions to adopt PBCs. Based on a farm-scale analysis, six statistically significant predictors of farmer willingness to grow PBCs are identified: perception of PBCs' environment benefits, education level, willingness to take risks, familiarity with PBCs, portion of peers already growing PBCs, and support of biorefineries locating in the local community. Among these, the latter three predictors are social support variables. It is found that familiarity with the crops is the most significant predictor of willingness; familiarity is also an important intermediate variable that mediates the influence of many other predictors. In addition, peer adoption can both directly and indirectly affect willingness via its influence on familiarity. These findings suggest that it is a pressing need to improve farmers’ knowledge of PBCs to promote the adoption of such crops.

Harvested winter rye energy cover crop: multiple benefits for North Central US

Cover crops (CCs) can reduce nitrogen (N) loss to subsurface drainage and can be reimagined as bioenergy crops for renewable natural gas production and carbon (C) benefits (fossil fuel substitution and C storage). Little information is available on the large-scale adoption of winter rye for these purposes. To investigate the impacts in the North Central US, we used the Root Zone Water Quality Model to simulate corn-soybean rotations with and without winter rye across 40 sites. The simulations were interpolated across a five-state area (IA, IL, IN, MN, and OH) with counties in the Mississippi River basin, which consists of ∼8 million ha with potential for rye CCs on artificially drained corn-soybean fields (more than 63 million ha total). Harvesting fertilized rye CCs before soybean planting in this area can reduce N loads to the Gulf of Mexico by 27% relative to no CCs, and provide 18 million Mg yr−1 of biomass-equivalent to 0.21 EJ yr−1 of biogas energy content or 3.5 times the 2022 US cellulosic biofuel production. Capturing the CO2 in biogas from digesting rye in the region and sequestering it in underground geologic reservoirs could mitigate 7.5 million Mg CO2 yr−1. Nine clusters of counties (hotspots) were identified as an example of implementing rye as an energy CC on an industrial scale where 400 Gg yr−1 of rye could be sourced within a 121 km radius. Hotspots consisted of roughly 20% of the region’s area and could provide ∼50% of both the N loss reduction and rye biomass. These results suggest that large-scale energy CC adoption would substantially contribute to the goals of reducing N loads to the Gulf of Mexico, increasing bioenergy production, and providing C benefits.

Thirteen‐year stover harvest and tillage effects on soil compaction in Iowa

AbstractCorn (Zea mays L.) stover is an abundant biomass source with multiple end‐uses including cellulosic biofuel production. However, stover removal may increase soil compaction by reducing organic matter inputs and increasing vehicle loads during harvest. While numerous studies have reported stover removal impacts on soil physical quality, few have assessed the role played by traffic compaction. Our objective was to quantify subsurface soil compaction after 13 years of chisel plow versus no‐till management and no, moderate (3.5 ± 1.1 Mg ha−1 year−1), or high (5.0 ± 1.7 Mg ha−1 year−1) stover harvest rates. Penetration resistance was measured in most‐ and least‐trafficked interrow spaces. Chisel plowed plots with moderate and high levels of stover removal had higher penetration resistance in trafficked areas relative to least‐trafficked areas, whereas there was no evidence of traffic compaction when stover was retained. Traffic compaction did not negatively impact yields, which were greater with high levels of stover removal compared to no removal. The no‐till practice led to very small increases in penetration resistance with wheel traffic and had no evidence of increased compaction with residue removal. This lack of traffic compaction indicated soils under no‐till practice have a higher load‐bearing capacity than soils under chisel plow practice. Overall, there were no yield‐limiting effects of tillage practice or stover removal, and no evidence of soil compaction below the plow layer, suggesting stover removal with both tillage practices can be effectively employed without detrimental effects on plant or soil health.

Modification of cellulase with smart-green polymers to promote low-cost and cleaner production of cellulosic ethanol

Cellulosic ethanol produced using the enzymatic method is promising; however, developing an efficient and green cellulase hydrolysis system to cut the cost of cellulosic ethanol and promote cleaner biofuel production remains challenging. Herein, based on the fact that multiple hydrogen bond can be generated between poly (methyl acrylic acid-co-acrylic acid) and gelatin, cellulase was first covalently bonded to the copolymer and then mixed with gelatin, to endow the cellulase with ideal upper critical solution temperature (UCST). As a result, the affinity between cellulase and the substrate did not decrease significantly after the modification (the Km value of modified cellulase (4.62 g/L) was not significantly different from that of free cellulase (4.55 g/L)). The enzymes can be switched on-demand between free state (above 36 °C), enabling high hydrolysis reactivity for insoluble substrates such as paper or corn stalks, and fixed state (at 4 °C), facilitating enzymes separation and reuse (the modified cellulase still has 58.2% of its initial activity for 20 batches). Thus, the cost of cellulosic ethanol production could be significantly reduced in the precondition of high catalytic efficiency. Furthermore, the system presents nontoxic and biodegradable properties. The system can be completely degraded in soil after 7 days and the inhibition rate of the degraded products on luminescent bacteria was only 2%, which can promote cleaner cellulosic biofuel production.

Application of Rhodococcus jostii RHA1 glycolate oxidase as an efficient accessory enzyme for lignin conversion by bacterial Dyp peroxidase enzymes.

Lignin oxidation by bacterial dye-decolorizing peroxidase enzymes requires hydrogen peroxide as a co-substrate, an unstable and corrosive oxidant. We have identified a glycolate oxidase enzyme from Rhodococcus jostii RHA1 that can couple effectively at pH 6.5 with DyP peroxidase enzymes from Agrobacterium sp. or Comamonas testosteroni to oxidise lignin substrates without addition of hydrogen peroxide. Rhodococcus jostii RHA1 glycolate oxidase (RjGlOx) has activity for oxidation of a range of α-ketoaldehyde and α-hydroxyacid substrates, and is also active for oxidation of hydroxymethylfurfural (HMF) to furandicarboxylic acid. The combination of RjGlOx with Agrobacterium sp. DyP or C. testosteroni DyP generated new and enhanced amounts of low molecular weight aromatic products from organosolv lignin substrates, and was able to generate high-value products from treatment of lignin residue from cellulosic biofuel production, and from a polymeric humin substrate.

Evaluation of cellulolytic fungal-bacterial biofilms for the enhancement of bioethanol production

Biodegradation of lignocellulosic materials by cellulolytic microbial consortia would be beneficial in cellulosic biofuel production. This study aimed at investigating the cellulose degradation potential of fungal-bacterial biofilms (FBBs) followed by bioethanol production. Fungi and bacteria were isolated from soil samples and their cellulolytic activity was analyzed in carboxy methylcellulose medium. FBBs were developed from the selected fungi and bacteria and their cellulolytic activity was evaluated using reducing sugar production through 3,5-Dinitro Salicylic acid method. Cellulolytic digestion followed by fermentation was performed with the selected biofilms by co-culturing Saccharomyces cerevisiae suspension. Ethanol content was quantified by dichromate method and confirmed through FTIR analysis. The highest significant (P < 0.05) cellulolytic activity was observed in Aspergillus niger (F6) and Pseudomonas aeruginosa (B7). Significantly (P < 0.05) the highest cellulolytic activity was recorded by A. niger-P. aeruginosa biofilm (FB2). Fermentation study confirmed higher production of ethanol by the biofilms co-cultured with S. cerevisiae over their single cultures and the highest was recorded by A. niger - Bacillus subtilis biofilm (FB1) co-cultured with S. cerevisiae. In this context, FBB can be considered as an important agent for bioethanol production from cellulosic materials through cellulolytic digestion followed by fermentation.

Agricultural and Forestry Biomass for Meeting the Renewable Fuel Standard: Implications for Land Use and GHG Emissions

Agricultural land and forestland are considered as two largest potential biomass sources for meeting the Renewable Fuel Standard (RFS) mandate for cellulosic biofuels. However, the land use change and greenhouse gas (GHG) savings with both agricultural and forest biomass production are yet to be examined systematically. This paper examines the effects of implementing a 16-billion gallon (60 billion liters) cellulosic biofuel mandate by 2035 on the mix of agricultural and forest biomass, land use change and GHG emissions by using a dynamic partial equilibrium model of the agricultural, forestry and transportation sectors in the US. Our results show that crop residues play a significant role in supplying cellulosic ethanol before 2030, while energy crops are the major feedstocks used for meeting the RFS cellulosic mandate after 2030. Milling and logging residues are economically viable supplements to agricultural biomass for cellulosic ethanol production, though their role in total biomass is small. Across different scenarios of cellulosic ethanol mandate that can be met with either agricultural biomass only or with both agricultural and forest biomass, we find GHG savings from displacing the gasoline range from 0.61 to 0.82 B MgCO2e over the 2015–2035 period. Induced land use change effects associated with expanded feedstock production are modest between and within the agricultural and forestry sectors. We conclude that a mixed feedstock base maximizes the economic and environmental benefits of cellulosic biofuel production. The mitigation potential of cellulosic biofuels is severalfold larger than natural-based solutions such as grassland restoration.

Metaproteomics reveals enzymatic strategies deployed by anaerobic microbiomes to maintain lignocellulose deconstruction at high solids

Economically viable production of cellulosic biofuels requires operation at high solids loadings—on the order of 15 wt%. To this end we characterize Nature’s ability to deconstruct and utilize mid-season switchgrass at increasing solid loadings using an anaerobic methanogenic microbiome. This community exhibits undiminished fractional carbohydrate solubilization at loadings ranging from 30 g/L to 150 g/L. Metaproteomic interrogation reveals marked increases in the abundance of specific carbohydrate-active enzyme classes. Significant enrichment of auxiliary activity family 6 enzymes at higher solids suggests a role for Fenton chemistry. Stress-response proteins accompanying these reactions are similarly upregulated at higher solids, as are β-glucosidases, xylosidases, carbohydrate-debranching, and pectin-acting enzymes—all of which indicate that removal of deconstruction inhibitors is important for observed undiminished solubilization. Our work provides insights into the mechanisms by which natural microbiomes effectively deconstruct and utilize lignocellulose at high solids loadings, informing the future development of defined cultures for efficient bioconversion.

Evaluating the potential of marginal lands available for sustainable cellulosic biofuel production in Italy

The European Union aims to provide as much as one quarter of its transportation fuels via biofuels derived from renewable sources by 2030. To put this into perspective, the Italian government has recently established an ambitious goal to support the wider uptake of advanced second-generation biofuels, including cellulosic biofuels for the transportation sector. A sustainable way forward is to grow perennial biomass crops on marginal lands, however the nationwide availability of those lands for lignocellulosic feedstock production remains uncertain. We identify and evaluate the potential of marginal lands in Italy to produce sizeable amounts of biomass for sustainable cellulosic biofuel production while limiting land use conflicts and negative ecological impacts. We applied spatial multi-criteria decision analysis techniques in geographic information systems to ultimately generate spatially-explicit national land suitability and availability maps at a fine resolution (250-m). We selected a broad range of leading cellulosic biomass crops that includes poplar (Populus × canadensis Moench), willow (Salix alba Linnaeus), black locust (Robinia pseudoacacia Linnaeus), giant reed (Arundo donax Linnaeus), and vetiver grass (Chrysopogon zizanioides Linnaeus). Based on marginality criteria, our results suggest that such biomass plantations of perennial grasses and short rotation trees may produce 3.1–27.4 billion liters of cellulosic ethanol per year from 462,265 to 2,811,064 million hectares of available marginal lands. This estimated production may fulfill 7.8–69.1% of Italy's current liquid transportation fuel consumption, constrained by the requirement that each modelled location be within 70 km of a potential cellulosic biorefinery. Collectively, this study provides the cornerstone of efforts to rationally meet Italy's need for renewable fuels in a sustainable low-carbon economy future.

Additional N application and ecotype affect yield and quality of ratoon harvested sorghum x sudangrass hybrid for temperate regions

Sorghum x sudangrass hybrid (Sorghum bicolor (L.) Moench) has excellent biomass yield and requires low maintenance. It can be harvested multiple times during the growing season. It has great potential to be used for cellulosic biofuel production in the temperate regions, including the Korean peninsula. However, N requirements during the regrowth have not been evaluated yet. Three years of the study with two ecotypes (early-flowering vs. late-flowering) were compared. After summer harvest, additional N (0, 50, 100, 150 kg N ha−1) were applied. Early-middle-late ratoon harvest treatments were executed to evaluate the ideal period for biomass yield and Theoretical Ethanol Yield (TEY). The late-flowering ecotype had greater biomass yield, TEY, and N-Agronomic Efficiency (NAE) due to longer vegetative growth. Maximum biomass yield was 7.44 Mg D.W. ha−1 (equivalent to TEY of 2657 L ha−1) at the additional 100 N kg ha−1 of middle ratoon harvest in 2018 when weather condition was unfavorable. However, with the favorable weather condition in 2019, additional 150 N kg ha−1 of late ratoon harvest outyielded (10.66 Mg ha−1; TEY of 4081 L ha−1). NAE was highest at 50 N kg ha−1 and then decreased with higher N rates. It indicates that additional N application is essential to achieve biomass yield and TEY under multiple harvest conditions. The appropriate harvest window for the ratooning crop was up to 14 weeks. Thus, additional N application and decision-making for harvest window are crucial factors affecting biomass yield and quality for biofuel with a choice of late-flowering ecotype.

Cassava (Manihot esculenta) dual use for food and bioenergy: A review

AbstractCassava (Manihot esculenta. Crantz) is a starch‐rich, woody tuberous, root crop important for food, with little being done to investigate its potential as a bioenergy crop despite its enormous potential. The major bottleneck in the crop being able to serve this dual role is the competition of its storage roots for both purposes. The major cassava production regions primarily use the tuberous roots for food, and this has resulted in its neglect as a bioenergy crop. The use of non‐food cassava parts as a feedstock in cellulosic biofuel production is a promising strategy that can overcome this challenge. However, in non‐tuber parts, most of the sugars are highly sequestered in lignin complexes making them inaccessible to bacterial bioconversion. Additionally, cassava production in these major growing areas is not optimal owing to several production constraints. The challenges affecting cassava production as a food and bioenergy crop are interconnected and therefore need to be addressed together. Cassava improvement against biotic and abiotic stresses can enhance productivity and cater for the high demand of the roots for food and bioenergy production. Furthermore, increased production will enhance the usability of non‐food parts for bioenergy as the bigger goal. This review addresses efforts in cassava improvement against stresses that reduce its productivity as well as strategies that enhance biomass production, both important for food and bioenergy. Additionally, prospective strategies that could ease bioconversion of cassava for enhanced bioenergy production are explored.

Long-Term Cellulose Enrichment Selects for Highly Cellulolytic Consortia and Competition for Public Goods.

ABSTRACTThe complexity of microbial communities hinders our understanding of how microbial diversity and microbe-microbe interactions impact community functions. Here, using six independent communities originating from the refuse dumps of leaf-cutter ants and enriched using the plant polymer cellulose as the sole source of carbon, we examine how changes in bacterial diversity and interactions impact plant biomass decomposition. Over up to 60 serial transfers (∼8 months) using Whatman cellulose filter paper, cellulolytic ability increased and then stabilized in four enrichment lines and was variable in two lines. Bacterial community characterization using 16S rRNA gene amplicon sequencing showed community succession differed between the highly cellulolytic enrichment lines and those that had slower and more variable cellulose degradation rates. Metagenomic and metatranscriptomic analyses revealed that Cellvibrio and/or Cellulomonas dominated each enrichment line and produced the majority of cellulase enzymes, while diverse taxa were retained within these communities over the duration of transfers. Interestingly, the less cellulolytic communities had a higher diversity of organisms competing for the cellulose breakdown product cellobiose, suggesting that cheating slowed cellulose degradation. In addition, we found competitive exclusion as an important factor shaping all of the communities, with a negative correlation of Cellvibrio and Cellulomonas abundance within individual enrichment lines and the expression of genes associated with the production of secondary metabolites, toxins, and other antagonistic compounds. Our results provide insights into how microbial diversity and competition affect the stability and function of cellulose-degrading communities.IMPORTANCE Microbial communities are a key driver of the carbon cycle through the breakdown of complex polysaccharides in diverse environments including soil, marine systems, and the mammalian gut. However, due to the complexity of these communities, the species-species interactions that impact community structure and ultimately shape the rate of decomposition are difficult to define. Here, we performed serial enrichment on cellulose using communities inoculated from leaf-cutter ant refuse dumps, a cellulose-rich environment. By concurrently tracking cellulolytic ability and community composition and through metagenomic and metatranscriptomic sequencing, we analyzed the ecological dynamics of the enrichment lines. Our data suggest that antagonism is prevalent in these communities and that competition for soluble sugars may slow degradation and lead to community instability. Together, these results help reveal the relationships between competition and polysaccharide decomposition, with implications in diverse areas ranging from microbial community ecology to cellulosic biofuels production.

Water Availability for Biorefineries in the Contiguous United States and the Implications for Bioenergy Production Distribution.

Renewable biofuel production depends on many factors, including feedstock availability, refinery and shipment infrastructure, and in particular, water availability. This study assesses water requirement and availability for mainstream biorefinery technologies in the contiguous United States (CONUS). The assessment is conducted in newly defined spatial units, namely, biorefinery planning boundaries, considering feedstock availability, transportation cost, and refinery capacity requirement for cost-effectiveness. The results suggest that the total biorefinery water use in the CONUS by 2030 will be low compared to the total water availability. However, biorefinery water requirements can aggravate the water stress situation in many regions, including the Great Plains, California Central Valley, and the upper Columbia-Snake River basin in Washington. Bioenergy productions in these regions can be largely constrained by water. It is projected that biofuel production will concentrate in Northern Plains, Lake States, and Corn Belt regions, which contribute 94.4% of the conventional, 86.1% of biodiesel, and 54.8% of cellulosic biofuel production mandated by the renewable fuel standard. If biorefineries are constrained to use less than 10% of the locally available water, up to 7% of planned cellulosic biofuel production will be affected. Findings from this study can aid the sustainable planning of national bioenergy production.

Lignocellulose Degradation and Enzymatic Hydrolysis of Soybean Incorporated Sorghum Silage Inoculated with Feruloyl-Esterase Producing Lactobacillus plantarum

Availability of feedstock is one of the uncertainties impeding cellulosic biofuel production, and conservation of whole crop biomass as silage is a promising method to ensure year-round feedstock availability for biofuel production. This study investigated lignocellulose degradation and subsequent enzymatic hydrolysis of a 90-d sorghum silage incorporated with soybean and inoculated with Lactobacillus plantarum A1 in a three (0, 25, and 50%; soybean inclusions) by two (uninoculated and inoculated) factorial experiment. The results revealed that L. plantarum A1 inoculated silages had improved fermentation characteristics. The silages’ total N and non-fiber carbohydrate increased with L. plantarum A1 and soybean inclusion (p < 0.05). Inoculation also increased the residual water-soluble carbohydrate by 33.9% (p < 0.05). Inoculation and soybean inclusion significantly hydrolyzed the biomass’ lignocellulose, altered its morphology and microstructural matrix, increased production of ferulic acid and reduced the biomass crystallinity by 15.60% (p < 0.05). L. plantarum A1 inoculation × soybean inclusion improved glucose yield and cellulose conversion during enzymatic saccharification compared to uninoculated treatments. Therefore, incorporating soybean into sorghum silage with L. plantarum A1 inoculation enhanced fermentation quality, lignocellulose degradation and enzymatic saccharification which could serve as a sure way for sustainable year-round feedstock supply for enhanced biofuel production.

A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype.

Clostridium thermocellum is a thermophilic, anaerobic bacterium that natively ferments cellulose to ethanol and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here, we reintroduced this mutation using recently developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation is hypothesized to function by interfering with metal ion coordination in the PHP (polymerase and histidinol phosphatase) domain, which is responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments. IMPORTANCE Cellulosic biofuels are a promising approach to decarbonize the heavy-duty-transportation sector. A longstanding barrier to cost-effective cellulosic biofuel production is the recalcitrance of cellulose to solubilization. Native cellulose-consuming organisms, such as Clostridium thermocellum, are promising candidates for cellulosic biofuel production; however, they often need to be genetically modified to improve product formation. One approach is adaptive laboratory evolution. Our findings demonstrate a way to increase the mutation rate in this industrially relevant organism, which can reduce the time needed for adaptive evolution experiments.

Gene Editing Technologies for Sugarcane Improvement: Opportunities and Limitations.

Plant-based biofuels present a promising alternative to depleting non-renewable fuel resources. One of the benefits of biofuel is reduced environmental impact, including reduction in greenhouse gas emission which causes climate change. Sugarcane is one of the most important bioenergy crops. Sugarcane juice is used to produce table sugar and first-generation biofuel (e.g., bioethanol). Sugarcane bagasse is also a potential material for second-generation cellulosic biofuel production. Researchers worldwide are striving to improve sugarcane biomass yield and quality by a variety of means including biotechnological tools. This paper reviews the use of sugarcane as a feedstock for biofuel production, and gene manipulation tools and approaches, including RNAi and genome-editing tools, such as TALENs and CRISPR-Cas9, for improving its quality. The specific focus here is on CRISPR system because it is low cost, simple in design and versatile compared to other genome-editing tools. The advance of CRISPR-Cas9 technology has transformed plant research with its ability to precisely delete, insert or replace genes in recent years. Lignin is the primary material responsible for biomass recalcitrance in biofuel production. The use of genome editing technology to modify lignin composition and distribution in sugarcane cell wall has been realized. The current and potential applications of genome editing technology for sugarcane improvement are discussed. The advantages and limitations of utilizing RNAi and TALEN techniques in sugarcane improvement are discussed as well.

Genomic Variation Shaped by Environmental and Geographical Factors in Prairie Cordgrass Natural Populations Collected across Its Native Range in the USA.

Prairie cordgrass (Spartina pectinata Link) is a native perennial warm-season (C4) grass common in North American prairies. With its high biomass yield and abiotic stress tolerance, there is a high potential of developing prairie cordgrass for conservation practices and as a dedicated bioenergy crop for sustainable cellulosic biofuel production. However, as with many other undomesticated grass species, little information is known about the genetic diversity or population structure of prairie cordgrass natural populations as compared to their ecotypic and geographic adaptation in North America. In this study, we sampled and characterized a total of 96 prairie cordgrass natural populations with 9315 high quality SNPs from a genotyping-by-sequencing (GBS) approach. The natural populations were collected from putative remnant prairie sites throughout the Midwest and Eastern USA, which are the major habitats for prairie cordgrass. Partitioning of genetic variance using SNP marker data revealed significant variance among and within populations. Two potential gene pools were identified as being associated with ploidy levels, geographical separation, and climatic separation. Geographical factors such as longitude and altitude, and environmental factors such as annual temperature, annual precipitation, temperature of the warmest month, precipitation of the wettest month, precipitation of Spring, and precipitation of the wettest month are important in affecting the intraspecific distribution of prairie cordgrass. The divergence of prairie cordgrass natural populations also provides opportunities to increase breeding value of prairie cordgrass as a bioenergy and conservation crop.

Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts

BackgroundFuture expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production between S. cerevisiae, O. polymorpha, and K. marxianus in glucose and cellulosic media at different temperatures.ResultsIn this work, we show that cellulosic emulsions hydrolyze rapidly at temperatures tolerable to yeast, reaching up to 40-fold higher conversion in the first hour compared to microcrystalline cellulose (MCC). To evaluate suitable conditions for the eSSF process, we explored the upper temperature limits for the thermotolerant yeasts Kluyveromyces marxianus and Ogataea polymorpha, as well as Saccharomyces cerevisiae, and observed robust fermentation at up to 46, 50, and 42 °C for each yeast, respectively. We show that the eSSF process reaches high ethanol titers in short processing times, and produces close to theoretical yields at temperatures as low as 30 °C. Finally, we demonstrate the transferability of the eSSF technology to other products by producing the advanced biofuel isobutanol in a light-controlled eSSF using optogenetic regulators, resulting in up to fourfold higher titers relative to MCC SSF.ConclusionsThe eSSF process addresses the main challenges of cellulosic biofuel production by increasing saccharification rate at temperatures tolerable to yeast. The rapid hydrolysis of these emulsions at low temperatures permits fermentation using non-thermotolerant yeasts, short processing times, low enzyme loads, and makes it possible to extend the process to chemicals other than ethanol, such as isobutanol. This transferability establishes the eSSF process as a platform for the sustainable production of biofuels and chemicals as a whole.