Jean S. VanderGheynst

MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis

Data-independent acquisition (DIA) in liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) provides comprehensive untargeted acquisition of molecular data. We provide an open-source software pipeline, which we call MS-DIAL, for DIA-based identification and quantification of small molecules by mass spectral deconvolution. For a reversed-phase LC-MS/MS analysis of nine algal strains, MS-DIAL using an enriched LipidBlast library identified 1,023 lipid compounds, highlighting the chemotaxonomic relationships between the algal strains.

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ∼10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50°C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

Rapid Quantitative Analysis of Lipids Using a Colorimetric Method in a Microplate Format

A colorimetric sulfo-phospho-vanillin (SPV) method was developed for high throughput analysis of total lipids. The developed method uses a reaction mixture that is maintained in a 96-well microplate throughout the entire assay. The new assay provides the following advantages over other methods of lipid measurement: (1) background absorbance can be easily corrected for each well, (2) there is less risk of handling and transferring sulfuric acid contained in reaction mixtures, (3) color develops more consistently providing more accurate measurement of absorbance, and (4) the assay can be used for quantitative measurement of lipids extracted from a wide variety of sources. Unlike other spectrophotometric approaches that use fluorescent dyes, the optimal spectra and reaction conditions for the developed assay do not vary with the sample source. The developed method was used to measure lipids in extracts from four strains of microalgae. No significant difference was found in lipid determination when lipid content was measured using the new method and compared to results obtained using a macro-gravimetric method.

Glycoside Hydrolase Activities of Thermophilic Bacterial Consortia Adapted to Switchgrass

ABSTRACT Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

The effect of microbial inoculation and pH on microbial community structure changes during composting

Abstract Phospholipid fatty acid (PLFA) analysis was used to characterize microbial community structure during the composting of grape pomace and rice straw. Composting studies were completed in 30 litre static-bed reactors with continuous temperature and oxygen monitoring. The effects of inoculation and pH adjustment on microbial community structure and level of decomposition during composting were investigated. Principal components analysis (PCA) of the PLFA data showed that inoculation had little effect on the microbial community structure of the compost once temperature had peaked, while process temperature and the adjustment of initial pH had a significant effect. Adjustment of pH and inoculation did not significantly increase the level of decomposition as measured by oxygen consumption.

Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost.

Aims: Three previously published fungal specific PCR primer sets, referred to as the NS, EF and NL primer sets, were evaluated for use in compost microbial community analysis by PCR and denaturing gradient gel electrophoresis (DGGE).

Energy Transport in a High-Solids Aerobic Degradation Process: Mathematical Modeling and Analysis

An energy transport model has been developed to capture the temporal and spatial changes in temperature observed within a high‐solids degradation process employing forced aeration. The model components include terms for energy removed through the bulk flow of air, heat generated from microbial activity, and energy accumulated in the matrix. The heat generation component was expressed as an empirical equation developed from pilot‐scale experimental oxygen depletion rate and temperature data. The model was simplified to permit a semianalytical solution using the method of characteristics. The model was validated by comparing temperature profiles simulated by the model to pilot‐scale experimental profiles. The time‐dependent solution of the characteristic equation was found to follow the experimental profiles well. Using the time‐dependent solution, a regression analysis was performed on pilot‐scale data to determine a heat generation yield of 9500 kJ/kg of O2 consumed. The spatial‐dependent solution of the characteristic equation was found to follow the experimental profiles well during the early stages of the process. Deviations were observed as temperature and time increased. These deviations were concluded to be a result of the empirical heat generation component of the model.

Qualitative analysis of algal secretions with multiple mass spectrometric platforms

Lipid secretions from algae pose a great opportunity for engineering biofueler feedstocks. The lipid exudates could be interesting from a process engineering perspective because lipids could be collected directly from the medium without harvesting and disrupting cells. We here report on the extracellular secretions of algal metabolites from the strain UTEX 2341 (Chlorella minutissima) into the culture medium. No detailed analysis of these lipid secretions has been performed to date. Using multiple mass spectrometric platforms, we observed around 1000 compounds and were able to annotate 50 lipids by means of liquid chromatography coupled to accurate mass quadrupole time-of-flight mass spectrometry (LC-QTOF), direct infusion with positive and negative electrospray ion trap mass spectrometry and gas chromatography coupled to mass spectrometry (GC-MS). These compounds were annotated by tandem mass spectral (MS/MS) database matching and retention time range filtering. We observed a series of triacylglycerols (TG), sulfoquinovosyldiacylglycerols (SQDG), phosphatidylinositols and phosphatidylglycerols, as well as betaine lipids diacylglyceryl-N,N,N-trimethylhomoserines (DGTS).

THE ROLE OF PERIODIC AGITATION AND WATER ADDITION IN MANAGING MOISTURE LIMITATIONS DURING HIGH-SOLIDS AEROBIC DECOMPOSITION

Abstract The results of this study were obtained from a pilot-scale experimental system designed to mimic an agitated bed composting process. This choice of scale represents a compromise between a better controlled and reproducible bench-scale system-and a reality-based full-scale system. Two substrates were studied: a synthetic food waste (dry dog food) and anaerobically digested, polymer–dewatered biosolids. The goal was to evaluate the degree to which periodic agitation: (1) lessened spatial gradients in moisture and temperature; and (2) thereby improved sustained degradation rates and cumulative extent of biodegradation. Coefficient of variation for cumulative O 2 measurements in the three replications of the 55% dog food and wood chips experiment with an aeration rate of 100 l min −1 were of the order of 25–30% which was consistent with other bench and pilot-scale studies. Drying occurred in both static-bed and agitated-bed studies. However, moisture gradients in the agitated-bed were smaller than in the static-bed experiments. Also, drying of the solids matrix increased with increased aeration rates for all the experiments. For the 45% dog food and wood chips experiments, a moisture content of 30% was reached in 180–300 h for aeration rates of 50–100 l min −1 , respectively; while for the 55% dog food and wood chips experiments, 30% was reached in 320–480 h for aeration rates of 50–100 l min −1 . The results of the water addition study showed that adding water three times per week resulted in a cumulative O 2 uptake at 622 g O 2 kg −1 of TS at 496 h, a 48% increase over no water addition for an additional 96 h of decomposition, and a 26% increase over adding water once a week.