BackgroundLignocellulosic 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.ResultsRepetitive 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.ConclusionsRobust 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.
The antifungal compound 2,4-diacetylphloroglucinol (DAPG) is produced in the rhizosphere of wheat by pseudomonad populations responsible for the natural biological control phenomenon known as "take-all decline." Studies were conducted to elucidate the impact of DAPG and its co-product 2,4,6-trihydroxyacetophenone (THA) on the production of Pseudomonas fluorescens for biological control. Increasing DAPG from 0.1 g/l to 0.5 g/l and THA from 0.05 g/l to 0.5 g/l significantly inhibited the growth and lowered the yield of viable bacteria in liquid cultures. On further examination of these metabolites applied in seed coatings, levels of DAPG and THA exceeding 0.05 mg/g seed significantly reduced wheat germination percentages. The three-way interaction of DAPG, THA, and culture medium ingredients was significant, and greatest seed germination loss (40-50%) was observed when 0.5 mg DAPG and 0.25 mg THA were combined in a coating of 0.5 ml culture medium per gram of seed. Based on the results of Biolog GN microplate, flask, and fermentor screens of C sources, proline was found to optimize the viable cell yields of the P. fluorescens strains tested. The combination of proline with glucose and urea as C and N sources in growth media could be optimized to minimize DAPG production and maximize the vitality of P. fluorescens Q8R1-96 and Q69c-80:miniTn5:phl20 (DAPG over-producer). In production cultures, the proline supply rate offers a potentially useful means to optimize the biological control agent yield and quality.
Pseudomonas fluorescens strains S11:P:12, P22:Y:05, and S22:T:04 and Enterobacter cloacae strain S11:T:07 have been documented to suppress four important storage potato maladies Á dry rot, late blight, pink rot, and sprouting. This research investigates the efficacy and consistency of strain mixtures produced by co-culturing strains together in one vessel or by blending them together after separate cultivations in pure cultures. Pure and co-cultures were produced in flask or fermentor cultures, viable cell concentrations were assessed using a nutrientbased selective plating method to identify and enumerate strains, and the efficacy of treatments was assessed with respect to dry rot, pink rot, late blight or sprout suppression. Experiments were designed to analyze dry rot suppression versus all strain combinations and the combination method (co-culture or blend). Results of a two-way analysis of variance of disease with strain composition and combination method showed that significantly better dry rot suppression was obtained by co-cultures (30.392.4% relative disease) than by similar strain blends of pure cultures (41.392.4%) (P B0.001). During a 3-year study, both biocontrol efficacy and consistency were assessed in 16 laboratory and small pilot trials simulating commercial storages. Three-strain co-culture had a lower mean disease rating than the blend in 9 of 16 experiments examining control of the three diseases and sprouting. The co-culture led other treatments in incidences of significant malady reduction relative to the control: 14 of 16 attempts for coculture, 11 of 16 attempts for blend, 10 of 13 attempts for pure S11:P:12, 8 of 13 attempts for S22:T:04, and 9 of 13 attempts for P22:Y:05. Using relative performance indices to rank treatment performance across all experiments, the co-culture treatment ranked significantly higher than the blend. A synergy analysis suggested that co-culturing strains stimulated inter-strain activities to boost biocontrol efficacy and consistency, a feature not developed in strains grown separately and mixed just prior to addition to potatoes.
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