Patricia J. Slininger

Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1 , GND1 , RPE1 , and TKL1 in Saccharomyces cerevisiae

Engineering yeast to be more tolerant to fermentation inhibitors, furfural and 5-hydroxymethylfurfural (HMF), will lead to more efficient lignocellulose to ethanol bioconversion. To identify target genes involved in furfural tolerance, a Saccharomyces cerevisiae gene disruption library was screened for mutants with growth deficiencies in the presence of furfural. It was hypothesized that overexpression of these genes would provide a growth benefit in the presence of furfural. Sixty two mutants were identified whose corresponding genes function in a wide spectrum of physiological pathways, suggesting that furfural tolerance is a complex process. We focused on four mutants, zwf1, gnd1, rpe1, and tkl1, which represent genes encoding pentose phosphate pathway (PPP) enzymes. At various concentrations of furfural and HMF, a clear association with higher sensitivity to these inhibitors was demonstrated in these mutants. PPP mutants were inefficient at reducing furfural to the less toxic furfuryl alcohol, which we propose is a result of an overall decreased abundance of reducing equivalents or to NADPHs role in stress tolerance. Overexpression of ZWF1 in S. cerevisiae allowed growth at furfural concentrations that are normally toxic. These results demonstrate a strong relationship between PPP genes and furfural tolerance and provide additional putative target genes involved in furfural tolerance.

Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae

BackgroundBiofuels offer a viable alternative to petroleum-based fuel. However, current methods are not sufficient and the technology required in order to use lignocellulosic biomass as a fermentation substrate faces several challenges. One challenge is the need for a robust fermentative microorganism that can tolerate the inhibitors present during lignocellulosic fermentation. These inhibitors include the furan aldehyde, furfural, which is released as a byproduct of pentose dehydration during the weak acid pretreatment of lignocellulose. In order to survive in the presence of furfural, yeast cells need not only to reduce furfural to the less toxic furan methanol, but also to protect themselves and repair any damage caused by the furfural. Since furfural tolerance in yeast requires a functional pentose phosphate pathway (PPP), and the PPP is associated with reactive oxygen species (ROS) tolerance, we decided to investigate whether or not furfural induces ROS and its related cellular damage in yeast.ResultsWe demonstrated that furfural induces the accumulation of ROS in Saccharomyces cerevisiae. In addition, furfural was shown to cause cellular damage that is consistent with ROS accumulation in cells which includes damage to mitochondria and vacuole membranes, the actin cytoskeleton and nuclear chromatin. The furfural-induced damage is less severe when yeast are grown in a furfural concentration (25 mM) that allows for eventual growth after an extended lag compared to a concentration of furfural (50 mM) that prevents growth.ConclusionThese data suggest that when yeast cells encounter the inhibitor furfural, they not only need to reduce furfural into furan methanol but also to protect themselves from the cellular effects of furfural and repair any damage caused. The reduced cellular damage seen at 25 mM furfural compared to 50 mM furfural may be linked to the observation that at 25 mM furfural yeast were able to exit the furfural-induced lag phase and resume growth. Understanding the cellular effects of furfural will help direct future strain development to engineer strains capable of tolerating or remediating ROS and the effects of ROS.

Formulation of Bacillus spp. for Biological Control of Plant Diseases

ABSTRACT Maximizing the potential for successfully developing and deploying a biocontrol product begins with a carefully crafted microbial screening procedure, proceeds with developing mass production protocols that optimize product quantity and quality, and ends with devising a product formulation that preserves shelf-life, aids product delivery, and enhances bioactivity. Microbial selection procedures that require prospective bio-control agents to possess both efficacy and amenability to production in liquid culture increase the likelihood of selecting agents with enhanced commercial development potential. Scale-up of biomass production procedures must optimize product quantity without compromise of product efficacy or amenability to stabilization and formulation. Formulation of Bacillus spp. for use against plant pathogens is an enormous topic in general terms but limited in published specifics regarding formulations used in commercially available products. Types of formulations include dry products such as wettable powders, dusts, and granules, and liquid products including cell suspensions in water, oils, and emulsions. Cells can also be microencapsulated. Considerations critical to designing successful formulations of microbial biomass are many fold and include preserving biomass viability during stabilization, drying, and rehydration; aiding biomass delivery, target coverage, and target adhesion; and enhancing biomass survival and efficacy after delivery to the target. Solutions to these formulation considerations will not necessarily be compatible. Data from several biocontrol systems including the use of B. subtilis OH 131.1 (NRRL B-30212) to reduce Fusarium head blight of wheat are used to illustrate many of these issues. Using our recently described assay for efficiently evaluating biomass production and formulation protocols, we demonstrate the effectiveness, in vitro, of UV protectant compounds lignin (PC 1307) and Blankophor BBH in reducing OH 131.1 morbidity when cells were exposed to UV light from artificial sunlight.

Multiple gene-mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae

Furfural and 5-hydroxymethylfurfural (HMF) are representative inhibitors generated from biomass pretreatment using dilute acid hydrolysis that interfere with yeast growth and subsequent fermentation. Few yeast strains tolerant to inhibitors are available. In this study, we report a tolerant strain, Saccharomyces cerevisiae NRRL Y-50049, which has enhanced biotransformation ability to convert furfural to furan methanol (FM), HMF to furan di-methanol (FDM), and produce a normal yield of ethanol. Our recent identification of HMF and development of protocol to synthesize the HMF metabolic conversion product FDM allowed studies on fermentation metabolic kinetics in the presence of HMF and furfural. Individual gene-encoding enzymes possessing aldehyde reduction activities demonstrated cofactor preference for NADH or NADPH. However, protein extract from whole yeast cells showed equally strong aldehyde reduction activities coupled with either cofactor. Deletion of a single candidate gene did not affect yeast growth in the presence of the inhibitors. Our results suggest that detoxification of furfural and HMF by the ethanologenic yeast S. cerevisiae strain Y-50049 likely involves multiple gene mediated NAD(P)H-dependent aldehyde reduction. Conversion pathways of furfural and HMF relevant to glycolysis and ethanol production were refined based on our findings in this study.

Enhanced Biotransformation of Furfural and Hydroxymethylfurfural by Newly Developed Ethanologenic Yeast Strains

Furfural and hydroxymethylfurfural (HMF) are representative inhibitors among many inhibitive compounds derived from biomass degradation and saccharification for bioethanol fermentation. Most yeasts, including industrial strains, are susceptible to these inhibitory compounds, especially when multiple inhibitors are present. Additional detoxification steps add cost and complexity to the process and generate additional waste products. To promote efficient bioethanol production, we studied the mechanisms of stress tolerance, particularly to fermentation inhibitors such as furfural and HMF. We recently reported a metabolite of 2,5-bis-hydroxymethylfuran as a conversion product of HMF and characterized a dose-dependent response of ethanologenic yeasts to inhibitors. In this study, we present newly adapted strains that demonstrated higher levels of tolerance to furfural and HMF. Saccharomyces cerevisiae 307-12H60 and 307-12H120 and Pichia stipitis 307 10H60 showed enhanced biotransformation ability to reduce HMF to 2,5-bis-hydroxymethylfuran at 30 and 60 mM, and S. cerevisiae 307-12-F40 converted furfural into furfuryl alcohol at significantly higher rates compared to the parental strains. Strains of S. cerevisiae converted 100% of HMF at 60 mM and S. cerevisiae 307-12-F40 converted 100% of furfural into furfuryl alcohol at 30 mM. The results of this study suggest a possible in situ detoxification of the inhibitors by using more inhibitor-tolerant yeast strains for bioethanol fermentation. The development of such tolerant strains provided a basis and useful materials for further studies on the mechanisms of stress tolerance.

Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations

SummaryThree strains ofPichiastipitis and three ofCandidashehatae were compared withPachysolentannophilus in their abilities to ferment xylose at concentrations as high as 200 g/L when subjected to both aerobic and microaerophilic conditions. Evaluations based on accumulated ethanol concentrations, ethanol productivities, xylose consumption, and ethanol and xylitol yields were determined from batch culture time courses. Of the strains considered,P.stipitis NRRL Y-7124 seemed most promising since it was able to utilize all but 7 g/L of 150 g/L xylose supplied aerobically to produce 52 g/L ethanol at a yield of 0.39 g per gram xylose (76% of theoretical yield) and at a rate comparable to the fastest shown byC.shehatae NRRL Y-12878. For all strains tested, fermentation results from aerobic cultures were more favorable than those from microaerophilic cultures.

Effects of Antagonist Cell Concentration and Two-Strain Mixtures on Biological Control of Fusarium Dry Rot of Potatoes

ABSTRACT Eighteen bacterial strains were individually assayed against Gibberella pulicaris (5 x 10(5) conidia per ml) by coinoculating antagonist and pathogen in wounds in cv. Russet Burbank potatoes. All antagonist concentrations (10(6), 10(7), and 10(8) CFU/ml) decreased disease (38 to 76% versus control, P < 0.05). When four strains were assayed at 11 concentrations (range 10(5) to 10(8) CFU/ml) against G. pulicaris, linear regression of the log-dose, log-response data was significant for all four strains (P < 0.001 to 0.01, R(2) = 0.50 to 0.74). Challenging G. pulicaris with all possible antagonist pairings within 2 sets of 10 antagonist strains (5 x 10(5) CFU of each strain per ml) resulted in 16 of 90 pairs controlling disease better than predicted based on averaging the performance of the individual strains making up the pair (P < 0.10). Successful pairs reduced disease by ~70% versus controls, a level of control comparable to that obtained with 100 times the inoculum dose of a single antagonist strain. Neither strain genus nor soil of origin were useful in predicting successful antagonist pairs. Factors potentially influencing dose-response relationships and the effectiveness of antagonist pairs in controlling disease are discussed.

Microbial lipid-based lignocellulosic biorefinery: feasibility and challenges

Although single-cell oil (SCO) has been studied for decades, lipid production from lignocellulosic biomass has received substantial attention only in recent years as biofuel research moves toward producing drop-in fuels. This review gives an overview of the feasibility and challenges that exist in realizing microbial lipid production from lignocellulosic biomass in a biorefinery. The aspects covered here include biorefinery technologies, the microbial oil market, oleaginous microbes, lipid accumulation metabolism, strain development, process configurations, lignocellulosic lipid production, technical hurdles, lipid recovery, and technoeconomics. The lignocellulosic SCO-based biorefinery will be feasible only if a combination of low- and high-value lipids are coproduced, while lignin and protein are upgraded to high-value products.

Greenhouse and Field Evaluation of Biological Control of Fusarium Head Blight on Durum Wheat

Fusarium head blight (FHB) is a devastating disease that causes extensive yield and quality losses to wheat and barley. In durum wheat, the pathogen-produced toxin deoxynivalenol (DON) is retained in semolina at ˜50%, and the causal agent of FHB, Gibberella zeae, has a strong adverse effect on pasta color. Two bacteria and two yeast strains with known efficacy against G. zeae on hexaploid wheats were produced in liquid culture and assayed on two cultivars of durum wheat in greenhouse bioassays. All antagonists reduced FHB severity on cultivar Renville, and three of the four reduced severity on cultivar Ben, with Bacillus subtilis strain AS 43.3 decreasing FHB severity by as much as 90%. In separate greenhouse bioassays, the car-bon:nitrogen ratio of the medium used to produce antagonists did not consistently influence antagonist efficacy. All antagonist/production medium combinations but one were effective in reducing disease on both durum cultivars. Of six antagonists tested at field sites, Cryptococcus sp. OH 71.4 and C. nodaensis OH 182.9 reduced disease severity by as much as 57% in Peoria, IL, while Cryptococcus sp. OH 181.1 reduced disease severity by as much as 59% in a trial at Langdon, ND. Antagonists did not influence the DON content of grain in the Peoria trial. Relative performance indices for four antagonists calculated from greenhouse and field results on the two durum cultivars demonstrated that the bioassay location, but not the cultivar of durum, influenced the relative performance of antagonists. Yeast antagonists OH 71.4, OH 181.1, and OH 182.9 appear to have the highest potential for contributing to the reduction of FHB on du-rum wheat in the field.

Selection and Evaluation of Microorganisms for Biocontrol of Fusarium Head Blight of Wheat Incited by Gibberella zeae

Gibberella zeae incites Fusarium head blight (FHB), a devastating disease that causes extensive yield and quality losses to wheat and barley. Of over 700 microbial strains obtained from wheat anthers, 54 were able to utilize tartaric acid as a carbon source when the compound was supplied as choline bitartrate in liquid culture. Four tartaric acid-utilizing and three nonutilizing strains reduced FHB in initial tests and were selected for further assays. Antagonists were effective against three different isolates of G. zeae when single wheat florets were inoculated with pathogen and antagonist inoculum. All seven antagonists increased 100-kernel weight when applied simultaneously with G. zeae isolate Z3639 (P ≤ 0.05). Bacillus strains AS 43.3 and AS 43.4 and Cryptococcus strain OH 182.9 reduced disease severity by >77, 93, and 56%, respectively. Five antagonists increased 100-kernel weight of plants inoculated with G. zeae isolate DAOM 180378. All antagonists except one increased 100-kernel weight, and four of seven antagonists reduced disease severity (P ≤ 0.05) when tested against G. zeae isolate Fg-9-96. In spray-inoculation experiments, Bacillus strains AS 43.3 and AS 43.4 and Cryptococcus strains OH 71.4 and OH 182.9 reduced disease severity, regardless of the sequence, timing, and concentration of inoculum application (P ≤ 0.05), though 100-kernel weight did not always increase when antagonists were applied 4 h after inoculum of G. zeae. Overall, 4 of 54 isolates that utilized tartaric acid in vitro were effective against G. zeae versus only 3 of 170 isolates tested that did not utilize tartaric acid (P ≤ 0.05, χ-square test of goodness of fit), demonstrating the potential benefit of prescreening candidate antagonists of FHB for their ability to utilize tartaric acid. Biological control shows promise as part of an integrated pest management program for managing FHB.