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We report here the improved draft genome sequence of Pseudomonas poae strain A2-S9, a bacterium that was originally isolated from switchgrass plants and exhibited the capacity for plant growth promotion. Its genome has a size of 6.68 Mbp and a GC content of 61.3%.
Screening the genetic diversity of 45 Yarrowia lipolytica strains identified five candidates with unique metabolic capability and robustness in undetoxified switchgrass hydrolysates, including superior lipid production and efficient pentose sugar utilization. Here, we report the genome sequences of these strains to study their robustness and potential to produce fuels and chemicals.
Zymomonas mobilis is a promising biocatalyst for the sustainable conversion of lignocellulosic sugars into biofuels and bioproducts, yet its response to lignocellulosic hydrolysates remains poorly understood. Here, we investigate the physiological response of Z. mobilis to ammonia fiber expansion (AFEX)-pretreated switchgrass hydrolysate using a systems-level approach integrating LC–MS/MS-based lipidomics and shotgun proteomics. Growth on hydrolysate induced substantial shifts in fatty acid and membrane phospholipid composition, alongside broad proteomic remodeling. Notably, Z. mobilis exhibited a stress response characterized by the upregulation of heat shock proteins and efflux transporters and the downregulation of cell motility proteins. Unexpectedly, hydrolysate exposure also led to a robust upregulation of the Entner–Doudoroff pathway, the ethanol fermentation pathway, and other central carbon metabolism enzymes, indicating a substantial cellular investment potentially driven by additional nutrient availability in hydrolysate. These findings provide new insights into the metabolic adaptations of Z. mobilis to lignocellulosic hydrolysates, informing strategies to enhance its biofuel production capabilities.
In this work we identify changes in high-resolution zones across the globe linked by environmental similarity that have implications for agriculture, bioenergy, and zoonosis. We refine exhaustive vector comparison methods with improved similarity metrics as well as provide multiple methods of amalgamation across 744 months of climatic data. The results of the vector comparison are captured as networks which are analyzed using static and longitudinal comparison methods to reveal locations around the globe experiencing dramatic changes in abiotic stress. Specifically we (i) incorporate updated similarity scores and provide a comparison between similarity metrics, (ii) implement a new feature for resource optimization, (iii) compare an agglomerative view to a longitudinal view, (iv) compare across 2-way and 3-way vector comparisons, (v) implement a new form of analysis, and (vi) demonstrate biological applications and discuss implications across a diverse set of species distributions by detecting changes that affect their habitats. Species of interest are related to agriculture (e.g., coffee, wine, chocolate), bioenergy (e.g., poplar, switchgrass, pennycress), as well as those living in zones of concern for zoonotic spillover that may lead to pandemics (e.g., eucalyptus, flying foxes).
In short, the GENESPACE pipeline conducts analysis of orthology networks, constrained within syntenic regions. Since analyses are limited to local tests conducted within syntenic blocks, GENESPACE is agnostic to ploidy, duplicated regions, inversions or other whole-genome chromosomal complexities that are common across many evolutionary lineages. This advantage allows for evolutionary tests in polyploids (e.g. switchgrass, manuscript in review), species with ancient, but retained whole-genome duplications (e.g. pecan, manuscript in prep), high levels of tandem array proliferation (e.g. eukalypts, manuscript in review) and many other factors that can confound comparative genomic analyses. The major advances of GENESPACE are three-fold: First, this is the first R package to integrate visualization and analysis of large-scale comparative genomics. R, which offers a high-level environment for graphical and statistical exploration of data, is often speed- and memory-limited and not used for computationally intensive tasks such as comparative genomics. The highly efficient C++ scripts used in GENESPACE (via data.table) permit a much faster and computationally lightweight implementation of comparative genomics than is currently available. Second, the pipeline itself is novel. To the best of our knowledge, no other program accomplishes synteny-constrained and ploidy-agnostic comparative genomics. Since nearly all plants and many animals have a history of whole-genome duplications, this is a major and necessary advance to the field. Third, GENESPACE offers high-level and intuitive multi-genome graphical outputs. The dotplots and 'riparian' plots produced herein, which are produced entirely through original R code, are publication-ready and easily customizable.
Biofuels policies induce land use changes (LUC), including cropland expansion and crop switching, and this in turn alters water and soil management practices. Policies differ in the extent and type of land use changes they induce and therefore in their impact on water resources. We quantify and compare the spatially varying water impacts of biofuel crops stemming from LUC induced by two different biofuels policies by coupling a biophysical model with an economic model to simulate the economically viable mix of crops, land uses, and crop management choices under alternative policy scenarios. We assess the outputs of an economic model with a high-resolution crop-water model for major agricultural crops and potential cellulosic feedstocks in the US to analyze the impacts of three alternative policy scenarios on water balances: a counterfactual ‘no-biofuels policy’ (BAU) scenario, a volumetric mandate (Mandate) scenario, and a clean fuel-intensity standard (CFS) scenario incentivizing fuels based on their carbon intensities. While both biofuel policies incentivize more biofuels than in the counterfactual, they differ in the mix of corn ethanol and advanced biofuels from miscanthus and switchgrass (more corn ethanol in Mandate and more cellulosic biofuels in CFS). The two policies differ in their impact on irrigated acreage, irrigation demand, groundwater use and runoff. Net irrigation requirements increase 0.7% in Mandate and decrease 3.8% in CFS, but in both scenarios increases are concentrated in regions of Kansas and Nebraska that rely upon the Ogallala aquifer for irrigation water. Our study illustrates the importance of accounting for the overall LUC and shifts in agricultural production and management practices in response to policies when assessing the water impacts of biofuels.
Second patch release of v2.0.0 Changes since v2.0.1 [Important change] Add and update SSP370 and SSP585 cases, add tests, fix use-case files, Change ocean and sea-ice IC for ARRM60to10. [EAM] allow thetaxx as a CAM_TARGET, enable northamericax4v1pg2_WC14to60E2r3 for AMIP, fix ndrop initialization, allow up to 15 history files. [EAMxx] allow use of readnl [EAM-MMF] allow transient SST case for C++ MMF, remove specific task/thread count for ESMT test, modify the variance transport diagnostic, reorg tests, [HOMME] Allow optional sponge layer. [ELM] add 2 land-atm compsets, update FATES to API 17.0.0, allow FATES sp mode, allow ELM harvest to drive FATES harvest, fix memleaks, reduce test build times, modified parameters for miscanthus and switchgrass based on calibration [MPAS-Ocean] turn on ocean BGC in BGC cases, fix interface locations for 60L PHC grid, add mode spec to conservation check streams, allow oRRS18to6v3 grid to run with JRA, update ocean and sea ice ICs for ARRM60to10 (needed for v2), GPU port of thickness tendency, add CMPASO-JRA1p4, [MPAS framework] Add new reproducible global sum module for MPAS components, Add mostRecentAccessTime attribute to streams [MPAS-landice] Update MALI version and Greenland mesh [MPAS-seaice] add single-cell test case, fix BGC restart, Adds omp critical directives for ice warnings seen on cori. [CIME] Fix component namelist creation bugs when NINST>1, add OpenACC, OpenMP and CXX GPU tests, fix nonBFB tests, stop using config_compilers, reduce ELM test build times with shared executable, update OpenMPI on Chrysalis. fix baseline handling, provenance handling with update to cime6.0.33, also use component-specific config_pes files and add the ones from master [Machine updates] cori modules after maint, Chrysalis to OpenMPI-4.1.3 [run_e3sm] replace default case name and group. [Externals] update SCORPIO to 1.3.2
Background: Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. Results: Twenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo , purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C. Conclusions: Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.
Background: 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. We present an approach to produce these chemicals based on the selective breakdown of lignin during ionic liquid pretreatment. Results: The lignin breakdown products generated are found to be dependent on the starting biomass, and significant levels were generated on dissolution at 160°C for 6 hrs. Guaiacol was produced on dissolution of biomass and technical lignins. Vanillin was produced on dissolution of kraft lignin and eucalytpus. Syringol and allyl guaiacol were the major products observed on dissolution of switchgrass and pine, respectively, whereas syringol and allyl syringol were obtained by dissolution of eucalyptus. Furthermore, it was observed that different lignin-derived products could be generated by tuning the process conditions. Conclusions: We have developed an ionic liquid based process that depolymerizes lignin and converts the low molecular weight lignin fractions into a variety of renewable chemicals from biomass. The generated chemicals (phenols, guaiacols, syringols, eugenol, catechols), their oxidized products (vanillin, vanillic acid, syringaldehyde) and their easily derivatized hydrocarbons (benzene, toluene, xylene, styrene, biphenyls and cyclohexane) already have relatively high market value as commodity and specialty chemicals, green building materials, nylons, and resins.
Obtaining accurate chemical composition and reactivity (measures of carbohydrate release and yield) information for biomass feedstocks in a timely manner is necessary for the commercialization of biofuels. Our objective was to use near-infrared (NIR) spectroscopy and partial least squares (PLS) multivariate analysis to develop calibration models to predict the feedstock composition and the release and yield of soluble carbohydrates generated by a bench-scale dilute acid pretreatment and enzymatic hydrolysis assay. Major feedstocks included in the calibration models are corn stover, sorghum, switchgrass, perennial cool season grasses, rice straw, and miscanthus. Here are the results: We present individual model statistics to demonstrate model performance and validation samples to more accurately measure predictive quality of the models. The PLS-2 model for composition predicts glucan, xylan, lignin, and ash (wt%) with uncertainties similar to primary measurement methods. A PLS-2 model was developed to predict glucose and xylose release following pretreatment and enzymatic hydrolysis. An additional PLS-2 model was developed to predict glucan and xylan yield. PLS-1 models were developed to predict the sum of glucose/glucan and xylose/xylan for release and yield (grams per gram). The release and yield models have higher uncertainties than the primary methods used to develop the models. In conclusion, it is possible to build effective multispecies feedstock models for composition, as well as carbohydrate release and yield. The model for composition is useful for predicting glucan, xylan, lignin, and ash with good uncertainties. The release and yield models have higher uncertainties; however, these models are useful for rapidly screening sample populations to identify unusual samples.
Lignocellulosic biomass is an abundant, renewable feedstock useful for the production of fuel-grade ethanol via the processing steps of pretreatment, enzyme hydrolysis, and microbial fermentation. Traditional industrial yeasts do not ferment xylose and are not able to grow, survive, or ferment in concentrated hydrolyzates that contain enough sugar to support economical ethanol recovery since they are laden with toxic byproducts generated during pretreatment. Repetitive culturing in two types of concentrated hydrolyzates was applied along with ethanol challenged xylose-fed continuous culture to force targeted evolution of the native pentose fermenting yeast Scheffersomyces (Pichia) stipitis strain NRRL Y-7124 maintained in the ARS Culture Collection, Peoria, IL. Isolates collected from various enriched populations were screened and ranked based on relative xylose uptake rate and ethanol yield. Ranking on hydrolyzates with and without nutritional supplementation was used to identify those isolates with best performance across diverse conditions. Robust S. stipitis strains adapted to perform very well in enzyme hydrolyzates of high solids loading ammonia fiber expansion-pretreated corn stover (18% weight per volume solids) and dilute sulfuric acid-pretreated switchgrass (20% w/v solids) were obtained. Improved features include reduced initial lag phase preceding growth, significantly enhanced fermentation rates, improved ethanol tolerance and yield, reduced diauxic lag during glucose-xylose transition, and ability to accumulate >40 g/L ethanol in <167 h when fermenting hydrolyzate at low initial cell density of 0.5 absorbance units and pH 5 to 6.
Microbial electrolysis is a promising technology for converting aqueous wastes into hydrogen. However, substrate adaptability is an important feature, seldom documented in microbial electrolysis cells (MECs). In addition, the correlation between substrate composition and community structure has not been well established. This study used an MEC capable of producing over 10 L/L-day of hydrogen from a switchgrass-derived bio-oil aqueous phase and investigated four additional substrates, tested in sequence on a mature biofilm. The additional substrates included a red oak-derived bio-oil aqueous phase, a corn stover fermentation product, a mixture of phenol and acetate, and acetate alone. The MECs fed with the corn stover fermentation product resulted in the highest performance among the complex feedstocks, producing an average current density of 7.3 ± 0.51 A/m 2 , although the acetate fed MECs outperformed complex substrates, producing 12.3 ± 0.01 A/m 2 . 16S rRNA gene sequencing showed that community structure and community diversity were not predictive of performance, and replicate community structures diverged despite identical inoculum and enrichment procedure. The trends in each replicate, however, were indicative of the influence of the substrates. Geobacter was the most dominant genus across most of the samples tested, but its abundance did not correlate strongly to current density. High-performance liquid chromatography (HPLC) showed that acetic acid accumulated during open circuit conditions when MECs were fed with complex feedstocks and was quickly degraded once closed circuit conditions were applied. The largest net acetic acid removal rate occurred when MECs were fed with red oak bio-oil aqueous phase, consuming 2.93 ± 0.00 g/L-day. Principal component analysis found that MEC performance metrics such as current density, hydrogen productivity, and chemical oxygen demand removal were closely correlated. Net acetic acid removal was also found to correlate with performance. However, no bacterial genus appeared to correlated to these performance metrics strongly, and the analysis suggested that less than 70% of the variance was accounted for by the two components. This study demonstrates the robustness of microbial communities to adapt to a range of feedstocks and conditions without relying on specific species, delivering high hydrogen productivities despite differences in community structure. The results indicate that functional adaptation may play a larger role in performance than community composition. Further investigation of the roles each microbe plays in these communities will help MECs to become integral in the 21st-century bioeconomy to produce zero-emission fuels.
Abstract Background Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called “esters” pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the “reverse” direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis. Loss of function of HCT gives severe growth phenotypes in several dicot plants, but less so in some monocots, questioning whether this enzyme, and therefore the shikimate shunt, plays the same role in both monocots and dicots. The model grass Brachypodium distachyon has two HCT genes, but lacks a classical CSE gene. This study was therefore conducted to evaluate the utility of HCT as a target for lignin modification in a species with an “incomplete” shikimate shunt. Results The kinetic properties of recombinant B. distachyon HCTs were compared with those from Arabidopsis thaliana , Medicago truncatula , and Panicum virgatum (switchgrass) for both the forward and reverse reactions. Along with two M. truncatula HCTs, B. distachyon HCT2 had the least kinetically unfavorable reverse HCT reaction, and this enzyme is induced when HCT1 is down-regulated. Down regulation of B. distachyon HCT1, or co-down-regulation of HCT1 and HCT2, by RNA interference led to reduced lignin levels, with only modest changes in lignin composition and molecular weight. Conclusions Down-regulation of HCT1, or co-down-regulation of both HCT genes, in B. distachyon results in less extensive changes in lignin content/composition and cell wall structure than observed following HCT down-regulation in dicots, with little negative impact on biomass yield. Nevertheless, HCT down-regulation leads to significant improvements in biomass saccharification efficiency, making this gene a preferred target for biotechnological improvement of grasses for bioprocessing.
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 effect 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.
Abstract Background The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. Results We obtained eight independent sorghum ( Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p -coumarate ( p CA), a higher amount of p -coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of p CA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. Conclusions Expression of OsAT10 in sorghum leads to an increase of xylan-bound p CA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall p CA remains unchanged indicating that most p CA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound p CA and lignification in cell walls.
Abstract Background The aim of this study was to increase the accessibility and accelerate the breakdown of lignocellulosic biomass to methane in an anaerobic fermentation system by mechanicalcotreatment: milling during fermentation, as an alternative to conventionalpretreatment prior to biological deconstruction. Effluent from a mesophilic anaerobic digester running with unpretreated senescent switchgrass as the predominant carbon source was collected and subjected to ball milling for 0.5, 2, 5 and 10 min. Following this, a batch fermentation test was conducted with this material in triplicate for an additional 18 days with unmilled effluent as the ‘status quo’ control. Results The results indicate 0.5 – 10 min of cotreatment increased sugar solubilization by 5– 13% when compared to the unmilled control, with greater solubilization correlated with increased milling duration. Biogas concentrations ranged from 44% to 55.5% methane with the balance carbon dioxide. The total biogas production was statistically higher than the unmilled control for all treatments with 2 or more minutes of milling (α = 0.1). Cotreatment also decreased mean particle size. Energy consumption measurements of a lab-scale mill indicate that longer durations of milling offer diminishing benefits with respect to additional methane production. Conclusions Cotreatment in anaerobic digestion systems, as demonstrated in this study, provides an alternative approach to conventional pretreatments to increase biogas production from lignocellulosic grassy material.
The compound middle lamella (CML) of woody angiosperm species, such as poplar (Populus spp.), birch (Betula spp.), and alder (Alnus spp.), and gymnosperm species, such as pine (Pinus spp.), Douglas fir (Pseudotsuga menziesii), and spruce (Picea spp.), is an amalgam of lignin, hemicellulosic xylans, and pectic polysaccharides that is the major site of cell-cell adhesion of fibre cells and tracheary elements. In grass species, such as sorghum (Sorghum bicolor), switchgrass (Panicum virgatum), and sugarcane (Saccharum officinarum), a phenylpropanoid network deposited in xylan-rich primary walls extends into the lignified CML. This review traces the early history of discovery of the molecular components of the CML and presents the current state of knowledge on the development of the CML in bioenergy-relevant woody species and grasses. This chapter emphasises the role of the CML in the recalcitrance of biomass to deconstruction by mechanical and enzymatic means for conversion to biofuels and valuable bioproducts. The use of genetic engineering to alter CML composition and structure to facilitate biomass deconstruction and to redesign wood to generate materials with novel properties is discussed.
This zip file contains a UNIX-format DayCent model executable, input files, automation code, and associated directory structure necessary to re-produce the DayCent analysis underlying the manuscript. The main script “autodaycent.py” (written for Python 2.7) opens an interactive command line routine that facilitates: Calibrating the DayCent pine growth model; Initializing DayCent for a set of case studies sites; Executing an ensemble of model runs representing case study site reforestation, grassland restoration, or conversion to switchgrass cultivation; and Results analysis & generation of manuscript Fig. 3. Note that the interactive analysis code requires that all input files to be contained in the directory structure as uploaded, without modification. Executable versions of the DayCent model compatible with other operating systems are available upon request.