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Strategies for reducing solvent toxicity in extractive ethanol fermentation.

Extractive fermentation is a widely preferred technique in which the products of fermentation are removed from the fermentation medium by a proper solvent, in order to avoid the inhibitory effects of the products. In this work, decanol, which has a high distribution coefficient with respect to the biocompatible solvents, was used in extractive ethanol fermentation. In order to reduce decanol toxicity, Saccharomyces cerevisiae cells were immobilized in calcium alginate gel. Further, sunflower oil and Al2O3 were added to the immobilization media. Experiments were performed in 250-mL Erlenmeyer flasks that were placed in the constant-temperature bath of a constant stirring-rate shaker. Ethanol concentrations were measured to observe the effect of various parameters on ethanol production. Immobilization media included 10, 20, and 30% sunflower oil, or 5, 10, and 20% Al2O3, or Al2O3 and sunflower oil together. The ratio of the volume of aqueous phase to that of decanol phase ranged from 2:1 to 6:1. It was observed that protection depends on the oil, Al2O3, and decanol amounts. Utilization of sunflower oil (30%) and Al2O3 (5%) together yielded best results.

Aluminum Oxide↗

Penicillin fermentation in a 200-liter tower fermentor using cells confined to microbeads.

The scale-up of the penicillin fermentation through cell confinement in a 200-L tower fermentor is described. P. chrysogenum spores were adsorbed into Celite microbeads having diameters greater than 180 microns. Fed-batch fermentations were performed using both free and confined cells. Cell growth and penicillin concentrations were measured during the fermentation. In addition, the oxygen transfer rate, the aeration rate, and the level of dissolved oxygen were also measured. Significant improvement in the mass transfer coefficient was found when the cells were anchored onto the microbeads. This improved oxygen transfer rate was accompanied by higher production of penicillin at a lower aeration rate. Besides the improved oxygen transfer rate into the mycelial broth, a reduction of the energy input for the oxygen transfer was observed. The confinement of the cells to this microcarrier furthermore allowed the intermittent harvesting of fermentation broth without reducing the cell mass in the fermentor.

Fermentation↗

Multiobjective optimization and multivariable control of the beer fermentation process with the use of evolutionary algorithms.

This paper describes empirical research on the model, optimization and supervisory control of beer fermentation. Conditions in the laboratory were made as similar as possible to brewery industry conditions. Since mathematical models that consider realistic industrial conditions were not available, a new mathematical model design involving industrial conditions was first developed. Batch fermentations are multiobjective dynamic processes that must be guided along optimal paths to obtain good results. The paper describes a direct way to apply a Pareto set approach with multiobjective evolutionary algorithms (MOEAs). Successful finding of optimal ways to drive these processes were reported. Once obtained, the mathematical fermentation model was used to optimize the fermentation process by using an intelligent control based on certain rules.

Algorithms↗

Pollution potential reduction of cheese whey through yeast fermentation.

Batch and continuous pilot-scale aerobic fermenters of 4.8 L operating volume were designed and constructed from plexiglass materials. The fermenters were used to study the kinetics of cheese whey fermentation using the yeast K. fragilis for pollution potential reduction and single cell protein production. Four retention times (6, 12, 18, and 24 h) were used in this study. The fermentation process was successful in reducing the total chemical oxygen demand (COD) by 42%, the soluble COD by 65%, the total solids by 53%, and the ammonium nitrogen by 90%. There were also gains in the suspended solids and the organic nitrogen of 60 and 17%, respectively. The reductions in the COD, total solids, and ammonium nitrogen, and the gains in the suspended solids and organic nitrogen were affected by the hydraulic retention time. More soluble material was converted to insoluble microbial cells at the 12-h hydraulic retention time, whereas greater pollution potential reduction was achieved at the 24-h hydraulic retention time for both batch and continuous operations.

Cheese↗

The promotion of molasse alcoholic fermentation using Saccharomyces cerevisiae in the presence of gamma-alumina.

Using gamma-alumina pellets, more than threefold increase of the ethanol productivity in the fermentation molasse has been obtained in the present work. Also, molasse fermentation in the presence of gamma-alumina gave 78.4 g/L ethanol, ethanol yield factor 0.44 g/g, and conversion 89.4% at initial sugar concentration (ISC) 179.5 g/L, compared to 53.9 g/L, 0.30 g/g and 62.7% in its absence, respectively. Furthermore, it was found that gamma-alumina reduces the activation energy Ea of fermentation. This inorganic material does not promote the fermentation of raisin extract.

Aluminum Oxide↗

CO2-dependent fermentation of phenol to acetate, butyrate and benzoate by an anaerobic, pasteurised culture.

Fermentative degradation of phenol was studied using a non-methanogenic, pasteurised enrichment culture containing two morphologically different bacteria. Phenol was fermented to benzoate, acetate and butyrate and their relative occurrence depended on the concentration of hydrogen. Proportionately more benzoate was formed with high initial levels of H2. The influence of PH2 on the fermentation pattern was studied both in dense cell suspensions and in growing cultures by addition of hydrogen. An increase in growth yield (OD578) was observed, compared to controls, as a consequence of phenol degradation; however, the increase was less in H2-amended treatments, in which most of the phenol ended up as benzoate. The degradation of phenol in the dense cell suspension experiments was dependent on CO2. Benzoate was not degraded when added as a substrate to the growing culture. This is, to our knowledge, the first report concerning the fermentative degradation of phenol to nonaromatic products.

Acetates↗

Fermentation of trans-aconitate via citrate, oxaloacetate, and pyruvate by Acidaminococcus fermentans.

Growing cells of Acidaminococcus fermentans (DSM 20731 and ATCC 25085) fermented trans-aconitate via citrate, oxaloacetate, and pyruvate to approximately 2 CO2, 1.8 acetate, 0.1 butyrate and 0.9 H2. The carbon and electron recoveries were close to 100%. On citrate no growth was observed and washed cells were unable to ferment this tricarboxylate. In cell-free extracts, however, citrate as well as trans-aconitate were readily fermented to CO2 and acetate. Under these conditions, also cis-aconitate, oxaloacetate, and pyruvate were formed, whereas butyrate and intermediates of glutamate fermentation, 2-oxoglutatrate and glutaconate, could not be detected. Citrate Si-lyase, a Mg2+-dependent oxaloacetate decarboxylase, and pyruvate synthase were present in quantities that corresponded to the growth rate of the organism.

Aconitic Acid↗

Regulatory O2 tensions for the synthesis of fermentation products in Escherichia coli and relation to aerobic respiration.

In an oxystat, the synthesis of the fermentation products formate, acetate, ethanol, lactate, and succinate of Escherichia coli was studied as a function of the O2 tension (pO2) in the medium. The pO2 values that gave rise to half-maximal synthesis of the products (pO0. 5) were 0.2-0.4 mbar for ethanol, acetate, and succinate, and 1 mbar for formate. The pO0.5 for the expression of the adhE gene encoding alcohol dehydrogenase was approximately 0.8 mbar. Thus, the pO2 for the onset of fermentation was distinctly lower than that for anaerobic respiration (pO0.5 </= 5 mbar), which was determined earlier. An essential role for quinol oxidase bd in microaerobic growth was demonstrated. A mutant deficient for quinol oxidase bd produced lactate as a fermentation product during growth at microoxic conditions (approximately 10 mbar O2), in contrast to the wild-type or a quinol-oxidase-bo-deficient strain. In the presence of nitrate, the amount of lactate was largely decreased. Therefore, under microoxic conditions, the pO2 appears to be too high for (mixed acid) fermentation to function and too low for aerobic respiration by quinol oxidase bo.

Acetates↗

Thiosulfate as a metabolic product: the bacterial fermentation of taurine.

Thiosulfate (S2O32-) is a natural product that is widely utilized in natural ecosystems as an electron sink or as an electron donor. However, the major biological source(s) of this thiosulfate is unknown. We present the first report that taurine (2-aminoethanesulfonate), the major mammalian solute, is subject to fermentation. This bacterial fermentation was found to be catalyzed by a new isolate, strain GKNTAU, a strictly anaerobic, gram-positive, motile rod that formed subterminal spores. Thiosulfate was a quantitative fermentation product. The other fermentation products were ammonia and acetate, and all could be formed by cell-free extracts.

Acetates↗

Determination of yeast assimilable nitrogen content in wine fermentations by sequential injection analysis with spectrophotometric detetection.

An automated method for measuring the primary amino acid concentration in wine fermentations by sequential injection analysis with spectrophotometric detection was developed. Isoindole-derivatives from the primary amino acid were formed by reaction with o-phthaldialdehyde and N-acetyl- L-cysteine and measured at 334 nm with respect to a baseline point at 700 nm to compensate the observed Schlieren effect. As the reaction kinetic was strongly matrix dependent the analytical readout at the final reaction equilibrium has been evaluated. Therefore four parallel reaction coils were included in the flow system to be capable of processing four samples simultaneously. Using isoleucine as the representative primary amino acid in wine fermentations a linear calibration curve from 2 to 10 mM isoleucine, corresponding to 28 to 140 mg nitrogen/L (N/L) was obtained. The coefficient of variation of the method was 1.5% at a throughput of 12 samples per hour. The developed method was successfully used to monitor two wine fermentations during alcoholic fermentation. The results were in agreement with an external reference method based on high performance liquid chromatography. A mean-t-test showed no significant differences between the two methods at a confidence level of 95%.

Amino Acids↗

Effect of redox potential on stationary-phase xylitol fermentations using Candida tropicalis.

Redox potential was used to develop a stationary-phase fermentation of Candida tropicalis that resulted in non-growth conditions with a limited decline in cell viability, a xylitol yield of 0.87 g g(-1) (95% of the theoretical value), and a high maximum specific production rate (0.67 g g(-1) h(-1)). A redox potential of -100 mV was found to be optimum for xylitol production over the range 0-150 mV [correction]. A shift from ethanol to xylitol production occurred when the redox potential was reduced from 50 mV to 100 mV as cumulative ethanol (Y(ethanol)) decreased from 0.34 g g(-1) to 0.025 g g(-1) and Y(xylitol) increased from 0.15 g g(-1) to 0.87 g g(-1) (alpha=0.05). Reducing the redox potential to 150 mV did not improve the fermentation. Instead, the xylitol yield and productivity decreased to 0.63 g g(-1) and 0.58 g g(-1) h(-1) respectively and cell viability declined. The viable, stationary-phase fermentation could be used to develop a continuous fermentation process, significantly increasing volumetric productivity and reducing downstream separation costs, potentially by the use of a membrane cell-recycle reactor.

Bioreactors↗

Solid residues from Ruminococcus cellulose fermentations as components of wood adhesive formulations.

Residues from the fermentation of cellulose by the anaerobic bacteria Ruminococcus albus (strain 7) or Ruminococcus flavefaciens (strains FD-1 or B34b) containing residual cellulose, bacterial cells and their associated adhesins, were examined for their ability to serve as components of adhesives for plywood fabrication. The residues contained differing amounts of protein (0.4-4.2% of dry weight), but the ratios of monosaccharides recovered following two-stage treatment of the residue with detergent (pH 7) and TFA were similar for all three strains (0.71 glucose:0.18 xylose:0.08 mannose:0.02 galactose), suggesting similarities in exopolysaccharide composition. Three-ply aspen panels prepared with fermentation residues (FR) displayed better shear strength and wood failure under dry conditions than following a vacuum/pressure/soak/dry treatment, but adhesive properties were inferior to those prepared with conventional phenol-formaldehyde (PF) adhesives. However, panels prepared by incorporating the R. albus 7 FR into PF formulation, at 73% by weight of the total adhesive, exhibited shear strength and wood failure similar to that obtained with PF adhesive alone. Use of residues from fermentations by these bacteria as components of adhesives may add value to biomass fermentations aimed primarily at producing ethanol and other chemical products.

Adhesives↗

Metabolic engineering for improved fermentation of pentoses by yeasts.

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g(-1) sugar consumed, so commercialization seems feasible for some applications.

Aerobiosis↗

Biotechnological advantages of laboratory-scale solid-state fermentation with fungi.

Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented. This is due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O2, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology. This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF. In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.

Aspergillus oryzae↗

Effects of CO2 on the formation of flavour volatiles during fermentation with immobilised brewer's yeast.

Immobilised-cell fermentors offer great benefits compared to traditional free-cell systems. However, a major problem is unbalanced flavour production when these fermentors are used for the production of alcoholic beverages. One of the keys to obtaining better control over flavour formation may be the concentration of dissolved CO2, which has inhibitory effects on yeast growth and metabolism. This article demonstrates that the presence of immobilisation matrices facilitates the removal of CO2 from the liquid medium, which results in a low level of dissolved CO2 during fermentation. Moreover, the formation of volatile higher alcohols and esters was greatly enhanced in the immobilised-cell system when compared to the free cell system. By sparging a CO2 flow (45 ml/min) into the immobilised-cell system, cell growth was reduced by 10-30% during the active fermentation stage, while the fermentation rate was unaffected. The uptake of branched-chain amino acids was reduced by 8-22%, and the formation of higher alcohols and esters was reduced on average by 15% and 18%, respectively. The results of this study suggest that mismatched flavour profiles with immobilised-cell systems can be adjusted by controlling the level of dissolved CO2 during fermentation with immobilised yeast.

Alcoholic Beverages↗

Biotechnological lycopene production by mated fermentation of Blakeslea trispora.

A semi-industrial process (800-l fermentor) for lycopene production by mated fermentation of Blakeslea trispora plus (+) and minus (-) strains has been developed. The culture medium was designed at the flask scale, using a program based on a genetic algorithm; and a fermentation process by means of this medium was developed. Fermentation involves separate vegetative phases for (+) and (-) strains and inoculation of the production medium with a mix of both together. Feeding with imidazole or pyridine, molecules known to inhibit lycopene cyclase enzymatic activity, enhanced lycopene accumulation. Different raw materials and physical parameters, including dissolved oxygen, stirring speed, air flow rate, temperature, and pH, were checked in the fermentor to get maximum lycopene production. Typical data for the fermentation process are presented and discussed. This technology can be easily scaled-up to an industrial application for the production of this carotenoid nowadays widely in demand.

Bioreactors↗

Aerobic induction of respiro-fermentative growth by decreasing oxygen tensions in the respiratory yeast Pichia stipitis.

The fermentative and respiratory metabolism of Pichia stipitis wild-type strain CBS 5774 and the derived auxotrophic transformation recipient PJH53 trp5-10 his3-1 were examined in differentially oxygenated glucose cultures in the hermetically sealed Sensomat system. There was a good agreement of the kinetics of gas metabolism, growth, ethanol formation and glucose utilisation, proving the suitability of the Sensomat system for rapid and inexpensive investigation of strains and mutants for their respiratory and fermentative metabolism. Our study revealed respiro-fermentative growth by the wild-type strain, although the cultures were not oxygen-limited. The induction of respiro-fermentative behaviour was obviously due to the decrease in oxygen tension but not falling below a threshold of oxygen tension. The responses differed depending on the velocity of the decrease in oxygen tension. At high oxygenation (slow decrease in oxygen tension), ethanol production was induced but glucose uptake was not influenced. At low oxygenation, glucose uptake and ethanol formation increased during the first hours of cultivation. The transformation recipient PJH53 most probably carries a mutation that influences the response to a slow decrease in oxygen tension, since almost no ethanol formation was found under these conditions.

Aerobiosis↗

Wood adhesives prepared from lucerne fiber fermentation residues of Ruminococcus albus and Clostridium thermocellum.

Fermentation residues (consisting of incompletely fermented fiber, adherent bacterial cells, and a glycocalyx material that enhanced bacterial adherence) were obtained by growing the anaerobic cellulolytic bacteria Ruminococcus albus 7 or Clostridium thermocellum ATCC 27405 on a fibrous fraction derived from lucerne (Medicago sativa L.). The dried residue was able to serve as an effective co-adhesive for phenol-formaldehyde (PF) bonding of aspen veneer sheets to one another. Testing of the resulting plywood panels revealed that the adhesive, formulated to contain 30% of its total dry weight as fermentation residue, displayed shear strength and wood failure values under both wet and dry conditions that were comparable with those of industry standards for PF that contained much smaller amounts of fillers or extenders. By contrast, PF adhesives prepared with 30% of dry weight as either unfermented lucerne fiber or conventional fillers or extenders rather than as fermentation residues, displayed poor performance, particularly under wet conditions.

Adhesives↗