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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 NADPH's 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.

Carbohydrate Epimerases↗

Genomic adaptation of ethanologenic yeast to biomass conversion inhibitors.

One major barrier to the economic conversion of biomass to ethanol is inhibitory compounds generated during biomass pretreatment using dilute acid hydrolysis. Major inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) inhibit yeast growth and subsequent fermentation. The ethanologenic yeast Saccharomyces cerevisiae demonstrated a dose-dependant inhibition by the inhibitors and has the potential to transform furfural and HMF into less toxic compounds of furfuryl alcohol and 2,5-bis-hydroxymethylfuran (also termed as furan-2,5-dimethanol (FDM)), respectively. For a sustainable and cost-competitive biomass-to-ethanol industry, it is important to develop more tolerant yeast strains that can, in situ, detoxify the inhibitors and produce ethanol. This study summarizes current knowledge and our understanding of the inhibitors furfural and HMF and discusses metabolic conversion pathways of the inhibitors and the yeast genomic expression response to inhibitor stress. Unlike laboratory strains, gene expression response of the ethanologenic yeast to furfural and HMF was not transient, but a continued dynamic process involving multiple genes at the genome level. This suggests that during the lag phase, ethanologenic yeasts undergo a genomic adaptation process in response to the inhibitors. The findings to date provide a strong foundation for future studies on genomic adaptation and manipulation of yeast to aid more robust strain design and development.

Antifungal Agents↗

Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae.

The physiological effects of 5-hydroxymethylfurfural (HMF) on Saccharomyces cerevisiae CBS 8066 in the presence and absence of furfural were studied. Experiments were carried out by pulse addition of HMF (2-4 g/l) as well as HMF (2 g/l) together with furfural (2 g/l) to batch cultivations of S. cerevisiae. Synthetic medium with glucose (50 g/l) as carbon and energy source was used. Addition of 4 g/l of HMF caused a decrease (approx. 32%) in the carbon dioxide evolution rate. Furthermore, the HMF was found to be taken up and converted by the yeast with a specific uptake rate of 0.14 (+/-0.03) g/g x h during both aerobic and anaerobic conditions, and the main conversion product was found to be 5-hydroxymethylfurfuryl alcohol. A previously unreported compound was found and characterized by mass spectrometry. It is suggested that the compound is formed from pyruvate and HMF in a reaction possibly catalysed by pyruvate decarboxylase. When HMF was added together with furfural, very little conversion of HMF took place until all of the furfural had been converted. Furthermore, the conversion rates of both furfural and HMF were lower than when added separately and growth was completely inhibited as long as both furfural and HMF were present in the medium.

Anaerobiosis↗

Methanogenesis from furfural by defined mixed cultures.

Methanogenesis from furfural by defined mixed cultures was studied. Under sulfate-reducing conditions, a Desulfovibrio strain was used as the furfural-degrading species producing acetic acid. This sulfate-reducing bacterium (SRB) Desulfovibrio strain B is an incomplete oxidizer, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. Introduction of acetate-utilizing methanogenic archaeon Methanosarcina barkeri 227 converted acetic acid to methane. This well-defined mixed consortium used furfural as its sole source of carbon and converted it to methane and CO(2). In the mixed culture, when a methanogen inhibitor was used in the culture medium, furfural was converted to acetic acid by the Desulfovibrio strain B, but acetic acid did not undergo further metabolism. On the other hand, when the growth of Desulfovibrio in the consortium was suppressed with a specific SRB inhibitor, namely molybdate, furfural was not degraded. Thus, the metabolic activities of both Desulfovibrio strain B and M. barkeri 227 were essential for the complete degradation of furfural.

Acetic Acid↗

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran.

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C6H8O3 and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.

Adaptation, Physiological↗

Mediated electrochemical measurement of the inhibitory effects of furfural and acetic acid on Saccharomyces cerevisiae and Candida shehatae.

The toxic effects of furfural and acetic acid on two yeasts, Saccharomyces cerevisiae and Candida shehatae, were evaluated using an electrochemical method. Intracellular redox activities were lowered by 40% and 78% for S. cerevisiae and C. shehatae, respectively, by 8 g furfural l(-1), and by 46% and 67%, respectively, by 8 g acetic acid l(-1). The proposed method can accurately measure the effects of inhibitors on cell cultures.

Acetic Acid↗

Fed-batch cultivation of Mucor indicus in dilute-acid lignocellulosic hydrolyzate for ethanol production.

Mucor indicus fermented dilute-acid lignocellulosic hydrolyzates to ethanol in fed-batch cultivation with complete hexose utilization and partial uptake of xylose. The fungus was tolerant to the inhibitors present in the hydrolyzates. It grew in media containing furfural (1 g/l), hydroxymethylfurfural (1 g/l), vanillin (1 g/l), or acetic acid (7 g/l), but did not germinate directly in the hydrolyzate. However, with fed-batch methodology, after initial growth of M. indicus in 500 ml enzymatic wheat hydrolyzate, lignocellulosic hydrolyzate was fermented with feeding rates 55 and 100 ml/h. The fungus consumed more than 46% of the initial xylose, while less than half of this xylose was excreted in the form of xylitol. The ethanol yield was 0.43 g/g total consumed sugar, and reached the maximum concentration of 19.6 g ethanol/l at the end of feeding phase. Filamentous growth, which is regarded as the main obstacle to large-scale cultivation of M. indicus, was avoided in the fed-batch experiments.

Acetic Acid↗

Quality evaluation of honey harvested from selected areas in Tanzania with special emphasis on hydroxymethyl furfural (HMF) levels.

The physicochemical properties of honey harvested from popular honey-producing areas in Tanzania were investigated. Honey from Shibe-Dodoma had the highest values of specific gravity, total acidity, free fatty acid content, diastatic number, overall acceptability, and lowest hydroxymethyl-furfural (HMF) level as compared to honey samples from other areas. There was no significant difference (p > 0.05) in terms of HMF in the other honey samples from Tanga, Morogoro, Same, Arusha, and Tabora. HMF levels in all honey samples were far below the maximum acceptable level of 40 mg/kg as recommended by the Codex Alimentarius Commission Standards before storage for 6 months. No traces of streptomycin and phenol were detected in all honey samples. It was concluded that according to the values of the studied quality parameters, the types of Tanzanian honey obtained from the popular honey producing areas may be judged to be of high quality.

Fatty Acids, Volatile↗

Automated fluorimetric determination of furfurals.

Hydroxymethylfurfural and furfural can be estimated automatically at 60 samples/h in a continuous-flow system. The hydrazone of the furfural with 5-hydroxybenzoylhydrazine is formed in aqueous solution and dialyzed into methanol or other lower alcohols on the recipient side of the dialyzer. The fluorescence of the zinc chelate of the hydrazone is measured. The procedure has applications for estimating the extent of hemoglobin glycosylation and for measuring the levels of hydroxymethylfurfural in some beverages. For more exacting work it can be used in conjunction with corrections from a blank channel or after preliminary specimen purification. Its specificity, though limited, compares favorably with other methods, while having advantages in sensitivity and capacity for high-throughput batch processing.

Acids↗

Automated colorimetric estimation of glycosylated haemoglobins.

The measurement of glycosylated haemoglobin is now widely used as a guide to glycaemic control in patients with diabetes mellitus. Present methods for measurement of glycosylated haemoglobin are either laborious, expensive or both. We describe a precise and inexpensive method for the automated analysis of glycosylated haemoglobin based on a colorimetric technique. The within-batch coefficient of variation ranged from 2.0 to 4.8% and between-batch was less than 11.0%. At least 200 samples can be analysed per day.

Colorimetry↗

Improvement of the thiobarbituric acid assay for serum glycosylprotein determination.

This study shows that the thiobarbituric acid (TBA) reaction as applied to the measurement of non-enzymatically glycosylated serum proteins(s) [1], yields erroneous results unless strictly standardized conditions are followed. Critical points are in particular (a) the concentrations of NaBH4 required for the preparation of the blanks by reduction of the ketoamine linkages; (b) the amounts of protein which should be identical in all serum samples to be compared; (c) dialysis for removal of glucose and NaBH4 prior to hydrolysis; (d) increase in the yield of 5-hydroxymethylfurfural (HMF) by appropriate conditions of hydrolysis. From our data obtained with an improved TBA-assay it appears that about 90% of total glycosylated serum protein is accounted for by glycosylated albumin. Thus changes in the latter used for diagnostic purposes in diabetes, may well be reflected by total serum glycosylprotein measurement.

Blood Glucose↗

Measurement of nonenzymatically glucosylated serum protein by an improved thiobarbituric acid assay.

We describe here some useful modifications of the thiobarbituric acid (TBA) assay for measurement of nonenzymatic glucosylation of serum protein. The modified assay minimizes interference by glucose without a lengthy dialysis step, and does not require an independent blank determination. These modifications should make the TBA assay more convenient for evaluating glycemic control in diabetes. Serum protein is first precipitated with cold ethanol to remove endogenous glucose. The protein is then hydrolyzed in an oxalic acid solution to release glucose as hydroxymethylfurfural (HMF). The HMF is reacted with TBA to form a chromophore which is extracted into isobutanol for spectrophotometric analysis (lambda max = 435 nm). The absorbance at 435 nm is corrected by subtracting a blank reading at 500 nm, and the nmol HMF released is determined using a standard curve prepared with pure HMF. Normal values of this assay for both adults and children are 0.38 +/- 0.10 nmol HMF/mg serum protein (means +/- 2 SD). When the assay was applied to serum samples from a group of 39 Type I diabetic children more than 90% of the children exceeded the normal range of the assay.

Adolescent↗