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In vitro gas production as a surrogate measure of the fermentability of cellulosic biomass to ethanol.

Current methods for measuring ethanol yields from lignocellulosic biomass are relatively slow and are not well geared for analyzing large numbers of samples generated by feedstock management and breeding research. The objective of this study was to determine if an in vitro ruminal fermentation assay used in forage quality research was predictive of results obtained using a conventional biomass-to-ethanol conversion assay. In the conventional assay, herbaceous biomass samples were converted to ethanol by Saccharomyces cerevisiae cultures in the presence of cellulase enzymes. Cultures were grown in sealed serum bottles and gas production monitored by measuring increasing head space pressure. Gas accumulation as calculated from the pressure measurements was highly correlated (r(2)>0.9) with ethanol production measured by gas chromatography at 24 h or 7 days. The same feedstocks were also analyzed by in vitro ruminal digestion, as also measured by gas accumulation. Good correlations (r(2) approximately 0.63-0.82) were observed between ethanol production during simultaneous saccharification and fermentation and gas accumulation in parallel in vitro ruminal fermentations. Because the in vitro ruminal fermentation assay can be performed without sterilization of the medium and does not require aseptic conditions, this assay may be useful for biomass feedstock agronomic and breeding research.

Biotechnology↗

Sequencing and expression analysis of the sakacin P bacteriocin produced by a Lactobacillus sakei strain isolated from naturally fermented sausages.

A Lactobacillus sakei strain, designated as I151 and isolated from naturally fermented sausages, was found to produce the sakacin P bacteriocin which is active against Listeria monocytogenes. In this study, we performed the sequencing of the gene cluster involved in the production of the sakacin P, and we followed the expression of the sppA gene, encoding for the bacteriocin, in vitro, using Rogosa-Sharpe medium, and in situ, inoculating the strain in fermented sausages as starter culture. The results obtained underlined the high similarity (>99%) of the entire sakacin P gene cluster from the L. sakei studied here with others present in strains of L. sakei already described. Moreover, from the expression experiments, it was shown that the gene is expressed during the exponential phase and that production procedures typical of fermented sausages are not turning off the expression of the gene encoding the bacteriocin. The capability of the strain studied to produce sakacin P during production is considered an advantage for its use as starter culture to improve the safety aspect of traditional fermented sausages produced in Italy.

Animals↗

Characterization of very high gravity ethanol fermentation of corn mash. Effect of glucoamylase dosage, pre-saccharification and yeast strain.

Ethanol was produced from very high gravity mashes of dry milled corn (35% w/w total dry matter) under simultaneous saccharification and fermentation conditions. The effects of glucoamylase dosage, pre-saccharification and Saccharomyces cerevisiae strain on the growth characteristics such as the ethanol yield and volumetric and specific productivity were determined. It was shown that higher glucoamylase doses and/or pre-saccharification accelerated the simultaneous saccharification and fermentation process and increased the final ethanol concentration from 106 to 126 g/kg although the maximal specific growth rate was decreased. Ethanol production was not only growth related, as more than half of the total saccharides were consumed and more than half of the ethanol was produced during the stationary phase. Furthermore, a high stress tolerance of the applied yeast strain was found to be crucial for the outcome of the fermentation process, both with regard to residual saccharides and final ethanol concentration. The increased formation of cell mass when a well-suited strain was applied increased the final ethanol concentration, since a more complete fermentation was achieved.

Cell Culture Techniques↗

Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation.

In recombinant Pichia pastoris fermentation for hirudin production, copious cells were not viable and most of the secreted hirudin molecules were C-terminally truncated at the end of fermentation. In this work, the influences of reactive oxygen species (ROS) on cell viability and hirudin production were subsequently studied. In contrast to the untreated control condition, the addition of ascorbic acid at the methanol fed-batch phase could obviously relieve the damage of intracellular ROS to cell membranes. As a result, the cell viability could be increased to 91% from 74% in control at the end of fermentation and the extracellular proteolysis of hirudin reduced. Intact and total hirudin production, by supplying ascorbic acid, could reach 2.90 and 5.03 g/l, respectively, in contrast to 1.75 and 4.70 g/l at the control condition. Ascorbic acid, 4 mmol/l or more, in the fermentation broth increased markedly the production of the intact hirudin, despite a little effect on total hirudin production.

Ascorbic Acid↗

Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide-assimilating yeast strain.

The sulfuric acid hydrolysate of lignocellulosic biomass, such as wood chips, from the forest industry is an important material for fuel bioethanol production. In this study, we constructed a recombinant yeast strain that can ferment xylose and cellooligosaccharides by integrating genes for the intercellular expressions of xylose reductase and xylitol dehydrogenase from Pichia stipitis, and xylulokinase from Saccharomyces cerevisiae and a gene for displaying beta-glucosidase from Aspergillus acleatus on the cell surface. In the fermentation of the sulfuric acid hydrolysate of wood chips, xylose and cellooligosaccharides were completely fermented after 36 h by the recombinant strain, and then about 30 g/l ethanol was produced from 73 g/l total sugar added at the beginning. In this case, the ethanol yield of this recombinant yeast was much higher than that of the control yeast. These results demonstrate that the fermentation of the lignocellulose hydrolysate is performed efficiently by the recombinant Saccharomyces strain with abilities for xylose assimilation and cellooligosaccharide degradation.

Aldehyde Reductase↗

Simultaneous control of apparent extract and volatile compounds concentrations in low-malt beer fermentation.

Volatile compounds cause undesirable flavor when their concentrations exceed threshold values in beer fermentation. The objective of this study is to develop a system for controlling apparent extract concentration, which indicates the fermentation degree and which should be decreased below a targeted value at a fixed time under a constraint of tolerable amounts of volatile compounds. In beer fermentation, even though the production of volatile compounds is suppressed by maintaining a low fermentation temperature, a low temperature causes a delay in the control of apparent extract concentration. Volatile compound concentration was estimated on-line, and the simulation of apparent extract consumption and volatile compound production was performed. To formulate various beer tastes and conserve energy for attemperation, optimal temperature profiles were determined using a genetic algorithm (GA). The developed feedback control of the brewing temperature profile was successfully applied, and apparent extract and volatile compound concentrations at a fixed time reached their target concentrations. Additionally, the control technique developed in this study enables us to brew a wide variety of beers with different tastes.

Acids↗

Fermentation of 1,3-propanediol by a lactate deficient mutant of Klebsiella oxytoca under microaerobic conditions.

Klebsiella oxytoca M5al is an excellent 1,3-propanediol (1,3-PD) producer, but too much lactic acid yielded greatly lessened the fermentation efficiency for 1,3-PD. To counteract the disadvantage, four lactate deficient mutants were obtained by knocking out the ldhA gene of lactate dehydrogenase (LDH) of K. oxytoca M5al. The LDH activities of the four mutants were from 3.85 to 6.92% of the parental strain. The fed-batch fermentation of 1,3-PD by mutant LDH3, whose LDH activity is the lowest, was studied. The results showed that higher 1,3-PD concentration, productivity, and molar conversion rate from glycerol to 1,3-PD can be gained than those of the wild type strain and no lactic acid is produced under both anaerobic and microaerobic conditions. Sucrose fed during the fermentation increased the conversion and sucrose added at the beginning increased the productivity. In fed-batch fermentation with sucrose as cosubstrate under microaerobic conditions, the 1,3-PD concentration, conversion, and productivity were improved significantly to 83.56 g l-1, 0.62 mol mol-1, and 1.61 g l(-1) h-1, respectively. Furthermore, 60.11 g l(-1) 2,3-butanediol was also formed as major byproduct in the broth.

Aerobiosis↗

High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae.

Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high xylose reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the xylose consumption rate. The results indicate that ethanolic xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which xylose is introduced in the central metabolism.

Aldehyde Reductase↗

Online recovery of nisin during fermentation and its effect on nisin production in biofilm reactor.

An online removal of nisin by silicic acid coupled with a micro-filter module was proposed as an alternative to reduce detrimental effects caused by adsorption of nisin onto producer, enzymatic degradation by protease, and product inhibition during fermentation. In this study, silicic acid was successfully used to recover nisin from the fermentation broth of Lactococcus lactis subsp. lactis NIZO 22186. The effect of pH (at 6.8 and 3.0) during adsorption process and several eluents (deionized water, 20% ethanol, 1 M NaCl, and 1 M NaCl + 20% ethanol) for desorption were evaluated in a small batch scale. Higher nisin adsorption onto silicic acid was achieved when the adsorption was carried out at pH 6.8 (67% adsorption) than at pH 3.0 (54% adsorption). The maximum recovery was achieved (47% of nisin was harvested) when the adsorption was carried out at pH 6.8 and 1 M NaCl + 20% ethanol was used as an eluent for desorption. Most importantly, nisin production was significantly enhanced (7,445 IU/ml) when compared with the batch fermentation without the online recovery (1,897 IU/ml). This may possibly be attributed to preventing the loss of nisin due the detrimental effects and a higher biomass density achieved during online recovery process, which stimulated production of nisin during fermentation.

Adsorption↗

Contribution of the fermenting yeast strain to ethyl carbamate generation in stone fruit spirits.

Fermented fruit and beverages frequently contain ethyl carbamate (EC), a potentially carcinogenic compound that can be formed by the reaction of urea with ethanol. Both are produced by the yeast Saccharomyces cerevisiae with ethanol as the major end product of hexose fermentation and urea as a by-product in arginine catabolism. In spirit production, EC can also be derived from cyanide introduced by stone fruit. To determine the relative contribution of yeast metabolism to EC production, we genetically engineered a diploid laboratory strain to reduce the arginase activity, thus blocking the pathway to urea production. For this purpose, strains with either a heterozygous CAR1/car1 deletion or a homozygous defect (car1/car1) were constructed. These strains were compared to the parental wild type and to an industrial yeast strain in cherry mash fermentations and spirit production. The strain with the homozygous car1 deletion showed a significant reduction of EC in the final spirits in comparison to the non-engineered controls. Nevertheless, using this strain for fermentation of stoneless cherry mashes did not completely impede EC formation. This indicates another, as yet unidentified, source for this compound.

Arginase↗

The effect of heat-shocking on batch fermentation by Clostridium beijerinckii NRRL B592.

In spite of the large-scale industrial use of the acetone-butanol fermentation process earlier this century (until 1983 in South Africa), very little has been published on the inoculum preparation techniques required for successful fermentation using these bacteria. In particular, heat-shocking has often been referred to as "useful" but no quantitative data are available. Data presented in this paper demonstrate and quantify the effect of heat-shocking on batch fermentation yields using one organism capable of this fermentation.

Clostridium↗

Aqueous two-phase: the system of choice for extractive fermentation.

Extractive fermentation in aqueous two-phase systems is a meaningful approach to overcome low product yield in a conventional fermentation process, and by proper design of the two-phase system it is possible to obtain the product in a cell-free stream. The characteristics of an aqueous two-phase system, various polymers used for forming an aqueous two-phase system, the physicochemical parameters of the aqueous two-phase system, partitioning of biomolecules and cell mass and the effect of individual phase forming polymers on cell growth and product formation are reviewed in this article. The various extractive fermentation processes are also summarised here. At the end, the economic viability and scope of aqueous two-phase fermentation are briefly discussed in relation to the wider application of this topic.

Anti-Bacterial Agents↗

Fibrobacter succinogenes S85 ferments ball-milled cellulose as fast as cellobiose until cellulose surface area is limiting.

Fibrobacter succinogenes S85 grew rapidly on cellobiose (0.31 h(-1) and the absolute rate of increase in fermentation acids was 0.68 h(-1). Cultures that were provided with ball-milled cellulose initially produced fermentation acids and microbial protein as fast as those provided with cellobiose, but the absolute cellulose digestion rate eventually declined. If the inoculum size was increased, the kinetics decayed from first to zero order (with respect to cells) even sooner, but in each case the absolute rate declined after only 20 to 30% of the cellulose had been fermented. Congo red binding indicated that the cellulose surface area of individual cellulose particles was not decreasing, and the transition of ball-milled cellulose digestion corresponded with the appearance of unbound cells in the culture supernatant. When bound cells from partially digested cellulose were removed and the cellulose was re-incubated with a fresh inoculum, the initial absolute fermentation rate was as high as the one observed for undigested cellulose and cellobiose. Based on these results, cellulose digestion by F. succinogenes S85 appears to be constrained by cellulose surface area rather than cellulase activity per se.

Animals↗

Anaerobic growth and improved fermentation of Pichia stipitis bearing a URA1 gene from Saccharomyces cerevisiae.

Respiratory and fermentative pathways coexist to support growth and product formation in Pichia stipitis. This yeast grows rapidly without ethanol production under fully aerobic conditions, and it ferments glucose or xylose under oxygen-limited conditions, but it stops growing within one generation under anaerobic conditions. Expression of Saccharomyces cerevisiae URA1 (ScURA1) in P. stipitis enabled rapid anaerobic growth in minimal defined medium containing glucose when essential lipids were present. ScURA1 encodes a dihydroorotate dehydrogenase that uses fumarate as an alternative electron acceptor to confer anaerobic growth. Initial P. stipitis transformants grew and produced 32 g/l ethanol from 78 g/l glucose. Cells produced even more ethanol faster following two anaerobic serial subcultures. Control strains without ScURA1 were incapable of growing anaerobically and showed only limited fermentation. P. stipitis cells bearing ScURA1 were viable in anaerobic xylose medium for long periods, and supplemental glucose allowed cell growth, but xylose alone could not support anaerobic growth even after serial anaerobic subculture on glucose. These data imply that P. stipitis can grow anaerobically using metabolic energy generated through fermentation but that it exhibits fundamental differences in cofactor selection and electron transport with glucose and xylose metabolism. This is the first report of genetic engineering to enable anaerobic growth of a eukaryote.

Anaerobiosis↗

Overexpression of the OLE1 gene enhances ethanol fermentation by Saccharomyces cerevisiae.

The fermentation characteristics of Saccharomyces cerevisiae strains which overexpress a constitutive OLE1 gene were studied to clarify the relationship between the fatty acid composition of this yeast and its ethanol productivity. The growth yield and ethanol productivity of these strains in the medium containing 15% dextrose at 10 degrees C were greater than those of the control strains under both aerobic and anaerobic conditions but this difference was not observed under other culture conditions. During repeated-batch fermentation, moreover, the growth yield and ethanol productivity of the wild-type S. cerevisiae increased gradually and then were similar to those of the OLE1-overexpressing transformant in the last batch fermentation. However, the unsaturated fatty acid content (77.6%) of the wild-type cells was lower than that (86.2%) of the OLE1-recombinant cells. These results suggested that other phenomena caused by the overexpression of the OLE1 gene, rather than high unsaturated fatty acid content, are essential to ethanol fermentation by this yeast.

Culture Media↗

Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor.

Changes in product formation during carbohydrate fermentation by anaerobic microflora in a continuous flow stirred tank reactor were investigated with respect to the dilution rate in the reactor. In the fermentation by methanogenic microflora, stable methane fermentation, producing methane and carbon dioxide, was observed at relatively low dilution rates (less than 0.33 d(-1) on glucose and 0.20 d(-1) on cellulose). Decomposition of cellulose in the medium was a rate-limiting step in the reaction, because glucose was easily consumed at all applied dilution rates (0.07-4.81 d(-1)). Intermediate metabolites of methane fermentation, such as lactate, ethanol, acetate, butyrate, formate, hydrogen, and carbon dioxide, were accumulated as dilution rate increased. Maximum yield of hydrogen was obtained at 4.81 d(-1) of dilution rate (0.1 mol/mol glucose on glucose or 0.7 mol/mol hexose on cellulose). Lactate was the major product on glucose (1.2 mol/mol glucose), whereas ethanol was predominant on cellulose (0.7 mol/mol hexose). An analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S rDNA of the microflora indicated that changes in the microbial community took place at various dilution rates, and these changes appeared to correspond to the changes in product distributions. Sequence analyses of the DGGE fragments revealed the probable major population of the microflora. A band closely related to the microorganisms of thermophilic anaerobic bacteria was detected with strong intensity on both glucose and cellulose. Differences in the production yield of hydrogen could have been caused by different populations of microorganisms in each microflora. In the case of cellulose, increasing the dilution rate brought about an accumulation of microorganisms related to Clostridia species that have cellulolytic activity, this being in accordance with the notion of cellulose decomposition being the rate-limiting reaction.

Base Sequence↗

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures to ethanol and sorbitol.

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures were carried out at different operational conditions (aeration, feed rate and substrate concentration) to test their effects on the system productivity. In these fermentations, the main products were ethanol and sorbitol. Kinetic parameters were calculated using the experimental data. However, parameters in the sorbitol synthesis rate were estimated from data recorded in different experiments in order to avoid the effect of the simultaneous cell growth and ethanol synthesis. In this case, the crude cell extract was used as source of the enzyme responsible for the sorbitol synthesis. The highest degree of conversion of fructose into sorbitol obtained with the extract was equal to 71% in a sugar mixture with an initial concentration of 200 g/l. Results obtained in the fed-batch fermentations showed that aeration of the culture has a positive effect on the final biomass concentration. However, final ethanol concentration is lower under aerated conditions. The best sugar yields to biomass and ethanol were 0.032 and 0.411 g/g, respectively. On the other hand, the highest sorbitol yield in the fed-batch fermentations was 0.148 g/g.

Biomass↗

Glucose fermentation by Propionibacterium microaerophilum: effect of pH on metabolism and bioenergetic.

pH affected significantly the growth and the glucose fermentation pattern of Propionibacterium microaerophilum. In neutral conditions (pH 6.5-7.5), growth and glucose fermentation rate (qs) were optimum producing propionate, acetate, CO(2), and formate [which together represented 90% (wt/wt) of the end products], and lactate representing only 10% (wt/wt) of the end products. In acidic conditions, propionate, acetate, and CO(2) represented nearly 100% (wt/wt) of the fermentation end products, whereas in alkaline conditions, a shift of glucose catabolism toward formate and lactate was observed, lactate representing 50% (wt/wt) of the fermentation end products. The energy cellular yields ( Y(X/ATP)), calculated (i) by taking into account extra ATP synthesized through the reduction of fumarate into succinate, was 6.1-7.2 g mol(-1). When this extra ATP was omitted, it was 11.9-13.1 g mol(-1). The comparison of these values with those of Y(X/ATP) in P. acidipropionici and other anaerobic bacteria suggested that P. microaerophilum could not synthesize ATP through the reduction of fumarate into succinate and therefore differed metabolically from P. acidipropionici.

Carbon Dioxide↗