Michael A. Cotta

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular methods was performed on three strains of a Gram-stain positive, non-sporeforming, motile aerobic rod-shaped bacterium resistant to tylosin and tetracycline isolated from a swine-manure storage pit. On the basis of 16S rRNA gene sequence analyses, it was confirmed that these isolates are highly related to each other and form a hitherto unknown lineage within the Planococcaceae. In particular, pairwise analysis of the 16S rRNA gene sequence demonstrated that the novel organism is closely related to members of the genus Sporosarcina (92.8–94.5 %), Pyschrobacillus (93.5–93.9 %) and Paenisporosarcina (93.3–94.5 %). The predominant fatty acids were found to consist of iso-C_15:0 and iso-C_17:1 ω10c and the G+C mol% was determined to be 41.8. Based on biochemical, chemotaxonomic, and phylogenetic evidence, it is proposed that these novel strains be classified as a novel genus and species, Savagea faecisuis gen nov., sp. nov. The type strain is Con12^T (=CCUG 63563^T = NRRL B-59945^T = NBRC 109956^T).

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.

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular genetic methods was performed on six strains of an unknown Gram-positive, nonspore-forming, facultative anaerobic coccus-shaped bacterium isolated from a swine-manure storage pit. On the basis of 16S rRNA, RNA polymerase-subunit (rpoA), and the 60-kilodalton chaperonin (cpn60) gene sequence analyses, it was shown that all the isolates were enterococci but formed two separate lines of descent. Pairwise 16S rRNA sequence comparisons demonstrated that the two novel organisms were most closely related to each other (97.9 %) and to Enterococcus aquimarinus (97.8 %). Both organisms contained major amounts of C_16:0, C_16:1 ω7c, and C_18:1 ω7c/12t/9t as the major cellular fatty acids. Based on biochemical, chemotaxonomic, and phylogenetic evidence, the names Enterococcus lemanii sp. nov. (type strain PC32^T = CCUG 61260^T = NRRL B-59661^T) and Enterococcus eurekensis sp. nov. (type strain PC4B^T = CCUG 61259^T = NRRL B-59662^T) are proposed for the hitherto undescribed species.

Inclusion of low levels of antibiotics in the feed of domestic food animals promotes improved growth, animal performance and overall health benefits. However, this practice has come under scrutiny due to concerns over such feeding on bacterial antibiotic resistance (AR) and potential impact on human health. There is a paucity of data on the types and levels of AR genes that may be present in agricultural practices. Using PCR detection of AR genes, this study demonstrates that both stored swine manure and swine faeces harbour a variety of AR genes, and bacterial members of these communities contain genes that may move between micro-organisms. Thus, both ecosystems may serve as reservoirs of AR genes. Significance and Impact of the Study Subtherapeutic feeding of antibiotics to production animals in the United States has prompted concerns of the effects of such practices on development of AR in bacterial populations that could affect human health. However, little is known about antibiotic resistant bacteria, and genes present in the swine gastrointestinal tract and stored manure. The current research demonstrates through the PCR amplification of AR genes in swine manure and faeces, and pure cultures of bacteria isolated from these ecosystems, that a number of such genes are present, and that the commensal bacterial microbiota might serve as reservoirs of AR.

Saccharomyces cerevisiae strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-xylose fermentation.

Switchgrass (Panicum virgatum L., SG) is a perennial grass presently used for forage and being developed as a bioenergy crop for conversion of cell wall carbohydrates to biofuels. Up to 50% of the cell wall associated carbohydrates are xylan. SG was analyzed for xylan structural features at variable harvest maturities. Xylan from each of three maturities was isolated using classical alkaline extraction to yield fractions (Xyl A and B) with varying compositional ratios. The Xyl B fraction was observed to decrease with plant age. Xylan samples were subsequently prepared for structure analysis by digesting with pure endo-xylanase, which preserved side-groups, or a commercial carbohydrase preparation favored for biomass conversion work. Enzymatic digestion products were successfully permethylated and analyzed by reverse-phase liquid chromatography with mass spectrometric detection (RP-HPLC-MSⁿ). This method is advantageous compared to prior work on plant biomass because it avoids isolation of individual arabinoxylan oligomers. The use of RP-HPLC- MSⁿ differentiated 14 structural oligosaccharides (d.p. 3–9) from the monocomponent enzyme digestion and nine oligosaccharide structures (d.p. 3–9) from hydrolysis with a cellulase enzyme cocktail. The distribution of arabinoxylan oligomers varied depending upon the enzyme(s) applied but did not vary with harvest maturity.