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  1. High temperatures and low soil moisture synergistically reduce switchgrass yields from marginal field sites and inhibit fermentation

    ‘Marginal lands’ are low productivity sites abandoned from agriculture for reasons such as low or high soil water content, challenging topography, or nutrient deficiency. To avoid competition with crop production, cellulosic bioenergy crops have been proposed for cultivation on marginal lands, however on these sites they may be more strongly affected by environmental stresses such as low soil water content. In this study we used rainout shelters to induce low soil moisture on marginal lands and determine the effect of soil water stress on switchgrass growth and the subsequent production of bioethanol. Five marginal land sites that span a latitudinalmore » gradient in Michigan and Wisconsin were planted to switchgrass in 2013 and during the 2018–2021 growing seasons were exposed to reduced precipitation under rainout shelters in comparison to ambient precipitation. The effect of reduced precipitation was related to the environmental conditions at each site and biofuel production metrics (switchgrass biomass yields and composition and ethanol production). During the first year (2018), the rainout shelters were designed with 60% rain exclusion, which did not affect biomass yields compared to ambient conditions at any of the field sites, but decreased switchgrass fermentability at the Wisconsin Central–Hancock site. In subsequent years, the shelters were redesigned to fully exclude rainfall, which led to reduced biomass yields and inhibited fermentation for three sites. When switchgrass was grown in soils with large reductions in moisture and increases in temperature, the potential for biofuel production was significantly reduced, exposing some of the challenges associated with producing biofuels from lignocellulosic biomass grown under drought conditions.« less
  2. Versatile microbial communities rapidly assimilate ammonium hydroxide-treated plastic waste

    Abstract Waste plastic presently accumulates in landfills or the environment. While natural microbial metabolisms can degrade plastic polymers, biodegradation of plastic is very slow. This study demonstrates that chemical deconstruction of polyethylene terephthalate (PET) with ammonium hydroxide can replace the rate limiting step (depolymerization) and by producing plastic-derived terephthalic acid and terephthalic acid monoamide. The deconstructed PET (DCPET) is neutralized with phosphoric acid prior to bioprocessing, resulting in a product containing biologically accessible nitrogen and phosphorus from the process reactants. Three microbial consortia obtained from compost and sediment degraded DCPET in ultrapure water and scavenged river water without addition ofmore » nutrients. No statistically significant difference was observed in growth rate compared to communities grown on DCPET in minimal culture medium. The consortia were dominated by Rhodococcus spp., Hydrogenophaga spp., and many lower abundance genera. All taxa were related to species known to degrade aromatic compounds. Microbial consortia are known to confer flexibility in processing diverse substrates. To highlight this, we also demonstrate that two microbial consortia can grow on similarly deconstructed polyesters, polyamides, and polyurethanes in water instead of medium. Our findings suggest that microbial communities may enable flexible bioprocessing of mixed plastic wastes when coupled with chemical deconstruction.« less
  3. A high solids field-to-fuel research pipeline to identify interactions between feedstocks and biofuel production

    Abstract Background Environmental factors, such as weather extremes, have the potential to cause adverse effects on plant biomass quality and quantity. Beyond adversely affecting feedstock yield and composition, which have been extensively studied, environmental factors can have detrimental effects on saccharification and fermentation processes in biofuel production. Only a few studies have evaluated the effect of these factors on biomass deconstruction into biofuel and resulting fuel yields. This field-to-fuel evaluation of various feedstocks requires rigorous coordination of pretreatment, enzymatic hydrolysis, and fermentation experiments. A large number of biomass samples, often in limited quantity, are needed to thoroughly understand the effectmore » of environmental conditions on biofuel production. This requires greater processing and analytical throughput of industrially relevant, high solids loading hydrolysates for fermentation, and led to the need for a laboratory-scale high solids experimentation platform. Results A field-to-fuel platform was developed to provide sufficient volumes of high solids loading enzymatic hydrolysate for fermentation. AFEX pretreatment was conducted in custom pretreatment reactors, followed by high solids enzymatic hydrolysis. To accommodate enzymatic hydrolysis of multiple samples, roller bottles were used to overcome the bottlenecks of mixing and reduced sugar yields at high solids loading, while allowing greater sample throughput than possible in bioreactors. The roller bottle method provided 42–47% greater liquefaction compared to the batch shake flask method for the same solids loading. In fermentation experiments, hydrolysates from roller bottles were fermented more rapidly, with greater xylose consumption, but lower final ethanol yields and CO 2 production than hydrolysates generated with shake flasks. The entire platform was tested and was able to replicate patterns of fermentation inhibition previously observed for experiments conducted in larger-scale reactors and bioreactors, showing divergent fermentation patterns for drought and normal year switchgrass hydrolysates. Conclusion A pipeline of small-scale AFEX pretreatment and roller bottle enzymatic hydrolysis was able to provide adequate quantities of hydrolysate for respirometer fermentation experiments and was able to overcome hydrolysis bottlenecks at high solids loading by obtaining greater liquefaction compared to batch shake flask hydrolysis. Thus, the roller bottle method can be effectively utilized to compare divergent feedstocks and diverse process conditions.« less
  4. Diverse lignocellulosic feedstocks can achieve high field‐scale ethanol yields while providing flexibility for the biorefinery and landscape‐level environmental benefits

    Increasing the diversity of lignocellulosic feedstocks accepted by a regional biorefinery has the potential to improve the environmental footprint of the facility; harvest, storage, and transportation logistics; and biorefinery economics. However, feedstocks can vary widely in terms of their biomass yields and quality characteristics (chemical composition, moisture content, etc.). To investigate how the diversity of potential biofuel cropping systems and feedstock supply might affect process and field-scale ethanol yields, we processed and experimentally quantified ethanol production from five different herbaceous feedstocks: two annuals (corn stover and energy sorghum) and three perennials (switchgrass, miscanthus, and mixed prairie). The feedstocks were pretreatedmore » using ammonia fiber expansion (AFEX), hydrolyzed at high solid loading (~17%–20% solids, depending on the feedstock), and fermented separately using microbes engineered to utilize xylose: yeast (Saccharomyces cerevisiaeY128) or bacteria (Zymomonas mobilis8b). The field-scale ethanol yield from each feedstock was dependent on biomass quality and cropping system productivity; however, biomass yield had a greater influence on the ethanol yield for low-productivity crops, while biomass quality was the main driver for ethanol yields from high-yielding crops. The process ethanol yield showed similar variability across years and feedstocks. A low process yield for corn stover was determined to result from inhibition of xylose utilization by unusually elevated levels of hydroxycinnamates (p-coumaric and ferulic acids) in the untreated biomass and their acid and amide derivatives in the resulting hydrolyzate. This finding highlights the need to better understand factors that influence process ethanol yield and biomass quality. Ultimately we provide evidence that most feedstocks fall within a similar range of process ethanol yield, particularly for the more resistant strain Z. mobilis8b. This supports the claim that the refinery can successfully diversify its feedstock supply, enabling many social and environmental benefits that can accrue due to landscape diversification.« less
  5. Strategies for the production of cell wall‐deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

    Summary Microbial cell wall‐deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall‐deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall‐deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzymemore » yields and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue‐specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. We also summarize methods that have been used to access and maintain activity of plant‐generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.« less

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