Pre-senescence Harvest of Switchgrass Inhibits Xylose Utilization by Engineered Yeast

Proper timing of switchgrass harvest for bioenergy is important to maximize yield and optimize end use conversion. Proposed windows range from peak biomass to the following spring after overwintering in the field. There are various pros and cons associated with harvest timing: earlier harvests maximize yield but can remove nutrients from the field that may require replacement, while later harvests have reduced biomass yields due to weathering but maximize nutrient resorption in belowground tissues. Switchgrass composition changes during the harvest period, with losses of potential fermentation nutrients (amino acids and minerals), and sources of pretreatment-derived inhibitors (soluble sugars), which could affect downstream conversion by microorganisms. For this work we investigated whether switchgrass harvest could be timed to maximize beneficial impacts on fermentation. Switchgrass samples were harvested from five replicate field plots in Wisconsin, roughly every 2–3 weeks from peak biomass (Aug. 20) until after the killing frost (Nov. 7). Cell wall composition showed little consistent variation with harvest date while bulk biomass analysis showed a relative increase in cell wall content (lignin and structural sugars) and loss of extractives (minerals, protein, soluble sugars, and others). Following high or low severity AFEX pretreatment and high solids enzymatic hydrolysis (6% glucan loading), two field replicates were fermented using Saccharomyces cerevisiae 424A, a strain engineered to utilize xylose in addition to glucose. For both pretreatment severities, S. cerevisiae 424A grown in hydrolysates from the three earlier harvests utilized only a small fraction of available xylose, while almost complete utilization occurred within 96 hr for the last three harvest dates. Detailed analysis of the hydrolysate low molecular weight aromatics did not indicate any compounds potentially responsible for the inhibition, with most of the observed variation in their concentration due to pretreatment severity. Amino acid composition also did not appear to be limiting. Current indications point to a plant-generated compound that degrades during senescence, which future work will attempt to identify. Ultimately this work demonstrates that, although an attractive option to maximize yield, harvesting switchgrass before it begins senescing could have a negative effect on downstream conversion processes

Deconstructed Plastic Substrate Preferences of Microbial Populations from the Natural Environment

Over half of the world\u27s plastic waste is landfilled, where it is estimated to take hundreds of years to degrade. Given the continued use and disposal of plastic products, it is vital that we develop fast and effective ways to utilize plastic waste. Here, we explore the potential of tandem chemical and biological processing to process various plastics quickly and effectively. Four samples of compost or sediment were used to set up enrichment cultures grown on mixtures of compounds, including disodium terephthalate and terephthalic acid (monomers of polyethylene terephthalate), compounds derived from the chemical deconstruction of polycarbonate, and pyrolysis oil derived from high-density polyethylene plastics. Established enrichment communities were also grown on individual substrates to investigate the substrate preferences of different taxa. Biomass harvested from the cultures was characterized using 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing. These data reveal low-diversity microbial communities structured by differences in culture inoculum, culture substrate source plastic type, and time. Microbial populations from the classes , , , and were significantly enriched when grown on substrates derived from high-density polyethylene and polycarbonate. The metagenomic data contain abundant aromatic and aliphatic hydrocarbon degradation genes relevant to the biodegradation of deconstructed plastic substrates used here. We show that microbial populations from diverse environments are capable of growth on substrates derived from the chemical deconstruction or pyrolysis of multiple plastic types and that paired chemical and biological processing of plastics should be further developed for industrial applications to manage plastic waste. The durability and impermeable nature of plastics have made them a popular material for numerous applications, but these same qualities make plastics difficult to dispose of, resulting in massive amounts of accumulated plastic waste in landfills and the natural environment. Since plastic use and disposal are projected to increase in the future, novel methods to effectively break down and dispose of current and future plastic waste are desperately needed. We show that the products of chemical deconstruction or pyrolysis of plastic can successfully sustain the growth of low-diversity microbial communities. These communities were enriched from multiple environmental sources and are capable of degrading complex xenobiotic carbon compounds. This study demonstrates that tandem chemical and biological processing can be used to degrade multiple types of plastics over a relatively short period of time and may be a future avenue for the mitigation of rapidly accumulating plastic waste

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 werepretreated 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 cerevisiae Y128) or bacteria (Zymomonas mobilis 8b). 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 ferulicacids) 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. mobilis 8b. 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

Integration of Pretreatment With Simultaneous Counter-Current Extraction of Energy Sorghum for High-Titer Mixed Sugar Production

Sorghum (Sorghum bicolor L. Moench) offers substantial potential as a feedstock for the production of sugar-derived biofuels and biochemical products from cell wall polysaccharides (i. e., cellulose and hemicelluloses) and water-extractable sugars (i.e., glucose, fructose, sucrose, and starch). A number of preprocessing schemes can be envisioned that involve processes such as sugar extraction, pretreatment, and densification that could be employed in decentralized, regional-scale biomass processing depots. In this work, an energy sorghum exhibiting a combination of high biomass productivity and high sugar accumulation was evaluated for its potential for integration into several potential biomass preprocessing schemes. This included counter-current extraction of water-soluble sugars followed by mild NaOH or liquid hot water pretreatment of the extracted bagasse. A novel processing scheme was investigated that could integrate with current diffuser-type extraction systems for sugar extraction. In this approach, mild NaOH pretreatment (i.e., <90°C) was performed as a counter-current extraction to yield both an extracted, pretreated bagasse and a high-concentration mixed sugar stream. Following hydrolysis of the bagasse, the combined hydrolysates derived from cellulosic sugars and extractable sugars were demonstrated to be fermentable to high ethanol titers (>8%) at high metabolic yields without detoxification using a Saccharomyces cerevisiae strain metabolically engineered and evolved to ferment xylose

Water-soluble saponins accumulate in drought-stressed switchgrass and may inhibit yeast growth during bioethanol production

Background: Developing economically viable pathways to produce renewable energy has become an important research theme in recent years. Lignocellulosic biomass is a promising feedstock that can be converted into second-generation biofuels and bioproducts. Global warming has adversely affected climate change causing many environmental changes that have impacted earth surface temperature and rainfall patterns. Recent research has shown that environmental growth conditions altered the composition of drought-stressed switchgrass and directly influenced the extent of biomass conversion to fuels by completely inhibiting yeast growth during fermentation. Our goal in this project was to find a way to overcome the microbial inhibition and characterize specific compounds that led to this inhibition. Additionally, we also determined if these microbial inhibitors were plant-generated compounds, by-products of the pretreatment process, or a combination of both. Results: Switchgrass harvested in drought (2012) and non-drought (2010) years were pretreated using Ammonia Fiber Expansion (AFEX). Untreated and AFEX processed samples were then extracted using solvents (i.e., water, ethanol, and ethyl acetate) to selectively remove potential inhibitory compounds and determine whether pretreatment affects the inhibition. High solids loading enzymatic hydrolysis was performed on all samples, followed by fermentation using engineered Saccharomyces cerevisiae. Fermentation rate, cell growth, sugar consumption, and ethanol production were used to evaluate fermentation performance. We found that water extraction of drought-year switchgrass before AFEX pretreatment reduced the inhibition of yeast fermentation. The extracts were analyzed using liquid chromatography–mass spectrometry (LC–MS) to detect compounds enriched in the extracted fractions. Saponins, a class of plant-generated triterpene or steroidal glycosides, were found to be significantly more abundant in the water extracts from drought-year (inhibitory) switchgrass. The inhibitory nature of the saponins in switchgrass hydrolysate was validated by spiking commercially available saponin standard (protodioscin) in non-inhibitory switchgrass hydrolysate harvested in normal year. Conclusions: Adding a water extraction step prior to AFEX-pretreatment of drought-stressed switchgrass effectively overcame inhibition of yeast growth during bioethanol production. Saponins appear to be generated by the plant as a response to drought as they were significantly more abundant in the drought-stressed switchgrass water extracts and may contribute toward yeast inhibition in drought-stressed switchgrass hydrolysates

Inhibition of microbial biofuel production in drought-stressed switchgrass hydrolysate

Additional file 2. Maps of significant gene ontology terms for chemical genomics data. Untreated biomass composition. Detailed hydrolysate composition

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 latitudinal 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

Response to Comment on “Plant diversity increases with the strength of negative density dependence at the global scale”

Hülsmann and Hartig suggest that ecological mechanisms other than specialized natural enemies or intraspecific competition contribute to our estimates of conspecific negative density dependence (CNDD). To address their concern, we show that our results are not the result of a methodological artifact and present a null-model analysis that demonstrates that our original findings—(i) stronger CNDD at tropical relative to temperate latitudes and (ii) a latitudinal shift in the relationship between CNDD and species abundance—persist even after controlling for other processes that might influence spatial relationships between adults and recruits