Christopher W. Simmons

A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression

Agrobacterium-mediated gene transfer, or agroinfiltration, can be a highly efficient method for transforming and inducing transient transgene expression in plant tissue. The technique uses the innate DNA secretion pathway of Agrobacterium tumefaciens to vector a particular plasmid-encoded segment of DNA from the bacteria to plant cells. Vacuum is often applied to plant tissue submerged in a suspension of A. tumefaciens to improve agroinfiltration. However, the effects of vacuum application on agroinfiltration and in planta transient transgene expression have not been well quantified. Here we show that vacuum application and release act to drive A. tumefaciens suspension into the interior of leaf tissue. Moreover, the amount of suspension that enters leaves can be predicted based on the vacuum intensity and duration. Furthermore, we show that transient expression levels of an agroinfiltrated reporter gene vary in response to the amount of A. tumefaciens vacuum infiltrated into leaf tissue, suggesting that vacuum infiltration conditions can be tailored to achieve optimal transient transgene expression levels after agroinfiltration.

Managing compost stability and amendment to soil to enhance soil heating during soil solarization

Soil solarization is a method of soil heating used to eradicate plant pathogens and weeds that involves passive solar heating of moist soil mulched (covered) with clear plastic tarp. Various types of organic matter may be incorporated into soil prior to solarization to increase biocidal activity of the treatment process. Microbial activity associated with the decomposition of soil organic matter may increase temperatures during solarization, potentially enhancing solarization efficacy. However, the level of organic matter decomposition (stability) necessary for increasing soil temperature is not well characterized, nor is it known if various amendments render the soil phytotoxic to crops following solarization. Laboratory studies and a field trial were performed to determine heat generation in soil amended with compost during solarization. Respiration was measured in amended soil samples prior to and following solarization as a function of soil depth. Additionally, phytotoxicity was estimated through measurement of germination and early growth of lettuce seedlings in greenhouse assays. Amendment of soil with 10%(g/g) compost containing 16.9 mg CO2/gdry weight organic carbon resulted in soil temperatures that were 2-4 °C higher than soil alone. Approximately 85% of total organic carbon within the amended soil was exhausted during 22 days of solarization. There was no significant difference in residual respiration with soil depth down to 17.4 cm. Although freshly amended soil proved highly inhibitory to lettuce seed germination and seedling growth, phytotoxicity was not detected in solarized amended soil after 22 days of field solarization.

Discovery of Microorganisms and Enzymes Involved in High-Solids Decomposition of Rice Straw Using Metagenomic Analyses

High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C) and thermophilic (55°C) conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2) were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production.

Thermophilic enrichment of microbial communities in the presence of the ionic liquid 1-ethyl-3-methylimidazolium acetate.

The aim of the study was to develop an approach to enrich ionic liquid tolerant micro‐organisms that efficiently decompose lignocellulose in a thermophilic and high‐solids environment.

Ensilage and Bioconversion of Grape Pomace into Fuel Ethanol

Two types of grape pomace were ensiled with eight strains of lactic acid bacteria (LAB). Both fresh grape pomace (FrGP) and fermented grape pomace (FeGP) were preserved through alcoholic fermentation but not malolactic conversion. Water leaching prior to storage was used to reduce water-soluble carbohydrates and ethanol from FrGP and FeGP, respectively, to increase malolactic conversion. Leached FeGP had spoilage after 28 days of ensilage, whereas FrGP was preserved. Dilute acid pretreatment was examined for increasing the conversion of pomace to ethanol via Escherichia coli KO11 fermentation. Dilute acid pretreatment doubled the ethanol yield from FeGP, but it did not improve the ethanol yield from FrGP. The ethanol yields from raw pomace were nearly double the yields from the ensiled pomace. For this reason, the recovery of ethanol produced during winemaking from FeGP and ethanol produced during storage of FrGP is critical for the economical conversion of grape pomace to biofuel.

Yeast tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate

Lignocellulosic plant biomass is the target feedstock for production of second-generation biofuels. Ionic liquid (IL) pretreatment can enhance deconstruction of lignocellulosic biomass into sugars that can be fermented to ethanol. Although biomass is typically washed following IL pretreatment, small quantities of residual IL can inhibit fermentative microorganisms downstream, such as the widely used ethanologenic yeast, Saccharomyces cerevisiae. The aim of this study was to identify yeasts tolerant to the IL 1-ethyl-3-methylimidazolium acetate, one of the top performing ILs known for biomass pretreatment. One hundred and sixty eight strains spanning the Ascomycota and Basidiomycota phyla were selected for screening, with emphasis on yeasts within or closely related to the Saccharomyces genus and those tolerant to saline environments. Based on growth in media containing 1-ethyl-3-methylimidazolium acetate, tolerance to IL levels ranging 1-5% was observed for 80 strains. The effect of 1-ethyl-3-methylimidazolium acetate concentration on maximum cell density and growth rate was quantified to rank tolerance. The most tolerant yeasts included strains from the genera Clavispora, Debaryomyces, Galactomyces, Hyphopichia, Kazachstania, Meyerozyma, Naumovozyma, Wickerhamomyces, Yarrowia, and Zygoascus. These yeasts included species known to degrade plant cell wall polysaccharides and those capable of ethanol fermentation. These yeasts warrant further investigation for use in saccharification and fermentation of IL-pretreated lignocellulosic biomass to ethanol or other products.

Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high-solids decomposition of rice straw

BackgroundNew lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched on rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes.ResultsDifferential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora.ConclusionsCoupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production.

Assessment of tomato and wine processing solid wastes as soil amendments for biosolarization.

Pomaces from tomato paste and wine production are the most abundant fruit processing residues in California. These residues were examined as soil amendments for solarization to promote conditions conducive to soil disinfestation (biosolarization). Simulated biosolarization studies were performed in both aerobic and anaerobic soil environments and soil temperature elevation, pH, and evolution of CO2, H2 and CH4 gases were measured as metrics of soil microbial activity. Tomato pomace amendment induced conditions associated with soil pest inactivation, including elevation of soil temperature by up to 2°C for a duration of 4days under aerobic conditions and a reduction of soil pH from 6.5 to 4.68 under anaerobic conditions. White wine grape pomace amendment showed similar trends but to a lesser extent. Red wine grape pomace was generally less suitable for biosolarization due to significantly lower soil temperature elevations, reduced acidification relative to the other pomaces and induction of methanogenesis in the soil.

Bacillus coagulans tolerance to 1-ethyl-3-methylimidazolium-based ionic liquids in aqueous and solid-state thermophilic culture.

The use of ionic liquids (ILs) to disrupt the recalcitrant structure of lignocellulose and make polysaccharides accessible to hydrolytic enzymes is an emerging technology for biomass pretreatment in lignocellulosic biofuel production. Despite efforts to reclaim and recycle IL from pretreated biomass, residual IL can be inhibitory to microorganisms used for downstream fermentation. As a result, pathways for IL tolerance are needed to improve the activity of fermentative organisms in the presence of IL. In this study, microbial communities from compost were cultured under high‐solids and thermophilic conditions in the presence of 1‐ethyl‐3‐methylimidazolium‐based ILs to enrich for IL‐tolerant microorganisms. A strain of Bacillus coagulans isolated from an IL‐tolerant community was grown in liquid and solid‐state culture in the presence of the ILs 1‐ethyl‐3‐methylimidazolium acetate ([C2mim][OAc]) or 1‐ethyl‐3‐methylimidazolium chloride ([C2mim][Cl]) to gauge IL tolerance. Viability and respiration varied with the concentration of IL applied and the type of IL used. B. coagulans maintained growth and respiration in the presence of 4 wt% IL, a concentration similar to that present on IL‐pretreated biomass. In the presence of both [C2mim][OAc] and [C2mim][Cl] in liquid culture, B. coagulans grew at a rate approximately half that observed in the absence of IL. However, in solid‐state culture, the bacteria were significantly more tolerant to [C2mim][Cl] compared with [C2mim][OAc]. B. coagulans tolerance to IL under industrially relevant conditions makes it a promising bacterium for understanding mechanisms of IL tolerance and discovering IL tolerance pathways for use in other microorganisms, particularly those used in bioconversion of IL‐pretreated plant biomass.

Effect of inoculum source on the enrichment of microbial communities on two lignocellulosic bioenergy crops under thermophilic and high-solids conditions.

Culturing compost‐derived microbial communities on biofuel feedstocks under industrial conditions is a technique to enrich for organisms and lignocellulolytic enzymes for bioenergy feedstock deconstruction. In this study, microbial communities from green waste compost (GWC) and grape pomace compost (GPC) were cultured on switchgrass and eucalyptus to observe the impact of inoculation on feedstock decomposition and microbial community structure.