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

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

Mutagenesis and Functional Characterization of the Four Domains of GlnD, a Bifunctional Nitrogen Sensor Protein▿

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of PII protein; PII in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants

MOESM4 of Chemical genomic guided engineering of gamma-valerolactone tolerant yeast

Additional file 4. Proteomic profile of Y133 pad1∆ fdc1∆ vs Y133 grown in YPD medium plus 1% GVL

MOESM1 of Chemical genomic guided engineering of gamma-valerolactone tolerant yeast

Additional file 1. GVL hydrolysate inhibitors

Controlling microbial contamination during hydrolysis of AFEX-pretreated corn stover and switchgrass: effects on hydrolysate composition, microbial response and fermentation

Abstract Background Microbial conversion of lignocellulosic feedstocks into biofuels remains an attractive means to produce sustainable energy. It is essential to produce lignocellulosic hydrolysates in a consistent manner in order to study microbial performance in different feedstock hydrolysates. Because of the potential to introduce microbial contamination from the untreated biomass or at various points during the process, it can be difficult to control sterility during hydrolysate production. In this study, we compared hydrolysates produced from AFEX-pretreated corn stover and switchgrass using two different methods to control contamination: either by autoclaving the pretreated feedstocks prior to enzymatic hydrolysis, or by introducing antibiotics during the hydrolysis of non-autoclaved feedstocks. We then performed extensive chemical analysis, chemical genomics, and comparative fermentations to evaluate any differences between these two different methods used for producing corn stover and switchgrass hydrolysates. Results Autoclaving the pretreated feedstocks could eliminate the contamination for a variety of feedstocks, whereas the antibiotic gentamicin was unable to control contamination consistently during hydrolysis. Compared to the addition of gentamicin, autoclaving of biomass before hydrolysis had a minimal effect on mineral concentrations, and showed no significant effect on the two major sugars (glucose and xylose) found in these hydrolysates. However, autoclaving elevated the concentration of some furanic and phenolic compounds. Chemical genomics analyses using Saccharomyces cerevisiae strains indicated a high correlation between the AFEX-pretreated hydrolysates produced using these two methods within the same feedstock, indicating minimal differences between the autoclaving and antibiotic methods. Comparative fermentations with S. cerevisiae and Zymomonas mobilis also showed that autoclaving the AFEX-pretreated feedstocks had no significant effects on microbial performance in these hydrolysates. Conclusions Our results showed that autoclaving the pretreated feedstocks offered advantages over the addition of antibiotics for hydrolysate production. The autoclaving method produced a more consistent quality of hydrolysate, and also showed negligible effects on microbial performance. Although the levels of some of the lignocellulose degradation inhibitors were elevated by autoclaving the feedstocks prior to enzymatic hydrolysis, no significant effects on cell growth, sugar utilization, or ethanol production were seen during bacterial or yeast fermentations in hydrolysates produced using the two different methods