Analytical differentiation of wine fermentations using pure and mixed yeast cultures

Summary Thirty-three fermentations of Pedro Ximénez grapes, collected in three degrees of ripeness, were carried out by inoculation with three types of inoculum: pure cultures ofSaccharomyces cerevisiae races and ofTorulaspora delbrueckii, indigenous yeasts, and mixed cultures of indigenous yeasts enriched with the pure cultures. By means of variance analysis 21 compounds were determined whose final concentrations in the wines significantly depended on the musts, the inocula or both. Eleven products that depended significantly on the inocula were subjected to a discriminant analysis in which most of the pure cultures gathered in a discriminant space area different from that occupied by the indigenous yeasts. The centroids corresponding to most of the mixed cultures were shifted to the central area of the discriminant space, moved away from their corresponding pure cultures and approached the indigenous yeasts. The results show a high similarity between the fermentations carried out with mixed cultures with the addedS. cerevisiae races and those fermentations carried out with the indigenous yeasts, with regard to those compounds which were significantly dependent on the inocula.     

A Clinical Study of the Role of Enterococci as Sole Agents of Wound and Tissue Infection

Patients who had enterococci isolated from wounds or tissues were identified from laboratory records. The charts of patients with pure cultures of enterococci were reviewed to determine the degree of clinically significant infection. We found that the frequency of infections in patients with pure cultures of enterococci was not significantly different from the frequency of infections in a control series of patients with negative cultures, but that it was significantly different from the frequency of infections in a series of patients with pure cultures of Staphylococcus aureus. Our conclusion that enterococci are not by themselves significant pathogens in wound or tissue infections is supported by a few experimental studies of other authors.     

Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors.

Glutamine synthetase (GS, EC 6.3.1.2.) has long been considered as a protein specific for astrocytes in the brain, but recently GS immunoreactivity has been reported in oligodendrocytes both in mixed primary glial cell cultures and in vivo. We have investigated its expression and regulation in "pure" oligodendrocyte cultures. "Pure" oligodendrocyte secondary cultures were derived from newborn rat brain primary cultures enriched in oligodendrocytes as described by Besnard et al. (1987) and were grown in chemically defined medium. These cultures contain more than 90% galactocerebroside-positive oligodendrocytes and produce "myelin" membranes (Fressinaud et al., 1990) after 6-10 days in subcultures (30-35 days, total time in culture). The presence of GS in oligodendrocytes from both primary glial cell cultures and "pure" oligodendrocyte cultures was confirmed by double immunostaining with a rabbit antisheep GS and guinea pig antirat brain myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase. In "pure" oligodendrocyte cultures, about half of cells were labeled with anti-GS antibody. Furthermore, on the immunoblot performed with a rabbit antisheep GS, the GS protein in "pure" oligodendrocyte secondary cultures was visualized as a single band with an apparent molecular mass of about 43 kDa. In contrast, two protein bands for GS were observed in cultured astrocytes. On the immunoblot performed with a rabbit antichick GS, two immunopositive protein bands were observed: a major one migrating as the purified adult chick brain GS and a minor one with a lower molecular mass. Two similar immunoreactive bands were also observed in pure rat astrocyte cultures. Compared to pure rat astrocyte cultures, "pure" oligodendrocyte cultures of the same age displayed an unexpectedly high GS specific activity that could not be explained by astrocytic contamination of the cultures (less than 5%). As for cultured astrocytes, treatment of oligodendrocyte cultures with dibutyryl-adenosine 3':5'-cyclic monophosphate, triiodothyronine, or hydrocortisone increased significantly GS specific activity. Interestingly, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor that increase the GS activity in astrocytes do not affect this activity in oligodendrocytes. Thus we confirm the finding of Warringa et al. (1988) that GS is also expressed in oligodendrocytes. We show that its activity is regulated similarly in astrocytes and oligodendrocytes by hormones, but that it is regulated differently by growth factors in these two cell types.     

Chloroform degradation in methanogenic methanol enrichment cultures and by Methanosarcina barkeri 227.

The effects of methanol addition and consumption on chloroform degradation rate and product distribution in methanogenic methanol enrichment cultures and in cultures of Methanosarcina barkeri 227 were investigated. Degradation of chloroform with initial concentrations up to 27.3 microM in enrichment cultures and 4.8 microM in pure cultures was stimulated by the addition of methanol. However, methanol consumption was inhibited by as little as 2.5 microM chloroform in enrichment cultures and 0.8 microM chloroform in pure cultures, suggesting that the presence of methanol, not its exact concentration or consumption rate, was the most significant variable affecting chloroform degradation rate. Methanol addition also significantly increased the number of moles of dichloromethane produced per mole of chloroform consumed. In enrichment cultures, the number of moles of dichloromethane produced per mole of chloroform consumed ranged from 0.7 (methanol consumption essentially uninhibited) to 0.35 (methanol consumption significantly inhibited) to less than 0.2 (methanol not added to the culture). In pure cultures, the number of moles of dichloromethane produced per mole of chloroform consumed was 0.47 when methanol was added and 0.24 when no methanol was added. Studies with [14C]chloroform in both enrichment and pure cultures confirmed that methanol metabolism stimulated dichloromethane production compared with CO2 production. The results indicate that while the addition of methanol significantly stimulated chloroform degradation in both methanogenic methanol enrichment cultures and cultures of M. barkeri 227, the prospects for use of methanol as a growth substrate for anaerobic chloroform-degrading systems may be limited unless the increased production of undesirable chloroform degradation products and the inhibition of methanol consumption can be mitigated.     

Mixed cultures of Serratia marcescens and Kluyveromyces fragilis for simultaneous protease production and COD removal of whey

AIMS: The aim of this study was to investigate the behaviour of a Serratia marcescens-Kluyveromyces fragilis mixed culture in whey, with the objective of proteases production and organic waste reduction. METHODS AND RESULTS: Discontinuous aerobic fermentations in whey were carried out using individual pure cultures and mixed cultures of S. marcescens and K. fragilis. Cell growth, protease production, lactose and proteins consumption and COD/TOC reduction were monitored. Lactose and protein content of the fermenting medium was almost depleted in the mixed cultures, achieving a reduction in the organic content much higher than in both pure cultures. Interestingly, proteolytic activity in the mixed cultures was similar to that obtained for S. marcescens in pure culture. In addition, protease stability was increased in the mixed cultures. Kinetic models were developed fitting well with the experimental results. CONCLUSIONS: Mixed cultures were found to maintain the achievements of each individual fermentation, yielding a high and stable production of proteases and a significant reduction of COD/TOC. SIGNIFICANCE AND IMPACT OF THE STUDY: Mixed cultures tested in this work have shown a synergistic effect with possible industrial applications. These results lead to a gain in the chain value for enzyme production with an environmentally friendly operation.     

Comparing the Dehalogenase Gene Pool in Cultivated α-Halocarboxylic Acid-Degrading Bacteria with the Environmental Metagene Pool

Culture-dependent and culture-independent approaches were used to determine the relationship between the dehalogenase gene pool in bacteria enriched and isolated on 2,2-dichloropropionic acid (22DCPA) and the environmental metagene pool (the collective gene pool of both the culturable and uncultured microbes) from which they were isolated. The dehalogenases in the pure-cultures isolates, which were able to degrade 22DCPA, were similar to previously described group I and II dehalogenases. Significantly, the majority of the dehalogenases isolated from activated sludge by degenerate PCR with primers specific for α-halocarboxylic acid dehalogenases were not closely related to the dehalogenases in any isolate. Furthermore, the dehalogenases found in the pure cultures predominated in the enrichments but were a minor component of the community used to inoculate the batch cultures. Phylogenetic analysis of the dehalogenase sequences isolated by degenerate PCR showed that the diversity of the group II deh gene was greater than that of the group I deh gene. Direct plating of the activated sludge onto minimal media supplemented with 22DCPA resulted in biomass and DNA from which dehalogenases were amplified. Analysis of the sequences revealed that they were much more closely related to the sequences found in the community used to start the enrichments. However, no pure cultures were obtained with this isolation method, and thus no pure cultures were available for identification. In this study we examined the link between genes found in pure cultures with the metagene pool from which they were isolated. The results show that there is a large bias introduced by culturing, not just in the bacteria isolated but also the degradative genes that they contain. Moreover, our findings serve as a caveat for studies involving the culturing of pure cultures of bacteria and conclusions which are drawn from analysis of these organisms.     

A note on the fermentation of pectin by pure strains and combined cultures of rumen bacteria

Bacteroides ruminicola, pure or combined with Selenomonas ruminantium, and Lachnospira multiparus, pure or combined with Succinivibrio dextrinosolvens, were grown on a medium with pectin as energy source. There was a difference in fermentation products between the pure and combined cultures and efficiency of substrate utilization was better with the combined cultures.     

Selective elimination of fibroblasts from cultures of normal human melanocytes

The main obstacle to establishing pure normal human melanocytes in vitro is contamination of the cultures by fibroblasts. The obstacle can be overcome by selective destruction of fibroblasts with geneticin ( G418 sulfate). Treatment of mixed cultures with this drug at a concentration of 100 micrograms/ml for two days results in pure cultures of normal human melanocytes.     

Comparing the dehalogenase gene pool in cultivated alpha-halocarboxylic acid-degrading bacteria with the environmental metagene pool

Culture-dependent and culture-independent approaches were used to determine the relationship between the dehalogenase gene pool in bacteria enriched and isolated on 2,2-dichloropropionic acid (22DCPA) and the environmental metagene pool (the collective gene pool of both the culturable and uncultured microbes) from which they were isolated. The dehalogenases in the pure-cultures isolates, which were able to degrade 22DCPA, were similar to previously described group I and II dehalogenases. Significantly, the majority of the dehalogenases isolated from activated sludge by degenerate PCR with primers specific for alpha-halocarboxylic acid dehalogenases were not closely related to the dehalogenases in any isolate. Furthermore, the dehalogenases found in the pure cultures predominated in the enrichments but were a minor component of the community used to inoculate the batch cultures. Phylogenetic analysis of the dehalogenase sequences isolated by degenerate PCR showed that the diversity of the group II deh gene was greater than that of the group I deh gene. Direct plating of the activated sludge onto minimal media supplemented with 22DCPA resulted in biomass and DNA from which dehalogenases were amplified. Analysis of the sequences revealed that they were much more closely related to the sequences found in the community used to start the enrichments. However, no pure cultures were obtained with this isolation method, and thus no pure cultures were available for identification. In this study we examined the link between genes found in pure cultures with the metagene pool from which they were isolated. The results show that there is a large bias introduced by culturing, not just in the bacteria isolated but also the degradative genes that they contain. Moreover, our findings serve as a caveat for studies involving the culturing of pure cultures of bacteria and conclusions which are drawn from analysis of these organisms.     

Studies on an acetate-fermenting strain of Methanosarcina.

An acetate-fermenting strain of Methanosarcina was isolated from an acetate enrichment culture inoculated with anaerobic sludge from a waste treatment digestor. In pure culture, this organism fermented acetate in the absence of added hydrogen at rates comparable in magnitude to those found in digestor systems. This rate was significantly higher than previously obtained for pure cultures of this genus. Mineral components of yeast extract were highly stimulatory for cultures growing on methanol. Comparable stimulation was not observed for cultures growing on acetate. Labeling studies indicated that acetate was converted to methane and CO2 as predicted by previous studies on mixed cultures. Total oxidation or reduction of acetate was not the mechanism of conversion of acetate to methane by the pure culture. The ability of this strain to form colonies or to produce methane from acetate was apparently influenced by the choice of substrate and conditions used for growing the inoculum.     

Mineralization of the s-triazine ring of atrazine by stable bacterial mixed cultures.

Enrichment cultures containing atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) at a concentration of 100 ppm (0.46 mM) as a sole nitrogen source were obtained from soils exposed to repeated spills of atrazine, alachlor, and metolachlor. Bacterial growth occurred concomitantly with formation of metabolites from atrazine and subsequent biosynthesis of protein. When ring-labeled [14C]atrazine was used, 80% or more of the s-triazine ring carbon atoms were liberated as 14CO2. Hydroxyatrazine may be an intermediate in the atrazine mineralization pathway. More than 200 pure cultures isolated from the enrichment cultures failed to utilize atrazine as a nitrogen source. Mixing pure cultures restored atrazine-mineralizing activity. Repeated transfer of the mixed cultures led to increased rates of atrazine metabolism. The rate of atrazine degradation, even at the elevated concentrations used, far exceeded the rates previously reported in soils, waters, and mixed and pure cultures of bacteria.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

Reactive oxygen species and induction of lignin peroxidase in Phanerochaete chrysosporium

We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O(2)) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O(2) gas. The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O(2) (oxygenated cultures) than in cultures grown with atmospheric air. Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O(2) concentrations and in cultures grown with atmospheric air. These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O(2) is at least partially mediated by the intracellular ROS.     

Synthesis of myosin heavy and light chains in muscle cultures

The weight ratio of myosin/actin, the myosin heavy chain content as the percentage of total protein (wt/wt), and the kinds of myosin light chains were determined in (a) standard muscle cultures, (b) pure myotube cultures, and (c) fibroblast cultures. Cells for these cultures were obtained from the breast of 11-day chick embryos. Standard cultures contain, in addition to myotubes, large numbers of replicating mononucleated cells. By killing these replicating cells with cytosine arabinoside, pure myotube cultures were obtained. The myosin/actin ratio (wt/wt) for pure myotube, standard muscle, and fibroblast cultures average 3.1, 1.9, and 1.1 respectively. By day 7, myosin in myotube cultures represents a minimum of 7% of the total protein, but about 3% in standard cultures and less than 1.5% in fibroblasts cultures. Myosin from standard cultures contains light chain LC1, LC2, and LC3, with a relative stoichiometry of the molarity of 1.0:1.9:0.5 and mol wt of 25,000, 18,000 and 16,000 daltons, identical to those in adult fast muscle. Myosin from pure myotubes exhibits light chains LC1 and LC2, with a molar ratio of 1.5:1.6. Myosin from fibroblast cultures possesses two light chains with a stoichiometry of 1.8:1.8 and mol wt of 20,000 and 16,000 daltons. Clearly, the faster migrating light chain, LC3, found in standard cultures is synthesized not by the myotubes but ty the mononucleated cells. In myotubes, both the assembly of the sarcomeres and the interaction between thick and thin filaments required for spontaneous contraction occur in the absence of light chain LC3. One set of structural genes for the myosin light and heavy chains appears to be active in mononucleated cells, whereas another set appears to be active in multinucleated myotubes.     

Biofilter performance and characterization of a biocatalyst degrading alkylbenzene gases

A biofilter treating alkylbenzene vapors was characterized for its optimal running conditions and kinetic parame-ters. Kinetics of the continuous biofilter were compared to batch kinetic data obtained with biofilm samples as well as with defined microbial consortia and with pure culture isolates from the biofilter. Both bacteria and fungi were present in the bioreactor. Five strains were isolated. Two bacteria, Bacillus and Pseudomonas, were shown to be dominant, as well as a Trichosporon strain which could, however, hardly grow on alkylbenzenes in pure culture. The remaining two strains were most often overgrown by the other three organisms in liquid phase batch cultures μ _max, K_S, K_I values and biodegradation rates were calculated and compared for the difterent mixed and pure cultures. Since filter bed acidification was observed during biofiltration studies reaching a pH of about 4, experiments were also undertaken to study the influence of pH on performance of the different cultures. Biodegradation and growth were possible in all cases, over the pH range 3.5–7.0 at appreciable rates, both with mixed cultures and with pure bacterial cultures. Under certain conditions, microbial activity was even observed in the presence of alkylbenzenes down to pH 2.5 with mixed cultures, which is quite unusual and explains the ability of the present biocatalyst to remove alkylbenzenes with high efficiency in biofilters under acidic conditions.     

Reactive Oxygen Species and Induction of Lignin Peroxidase in Phanerochaete chrysosporium

We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O₂) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O₂ gas. The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O₂ (oxygenated cultures) than in cultures grown with atmospheric air. Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O₂ concentrations and in cultures grown with atmospheric air. These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O₂ is at least partially mediated by the intracellular ROS.     

The microbial degradation of benzothiazoles

The biodegradation of benzothiazoles by pure and mixed microbial cultures derived from activated sludge has been studied. The degradation of 2-aminobenzothiazole (ABT) by both pure and mixed bacterial cultures has been demonstrated for the first time. ABT is degraded to give high yields of ammonia and sulphate (87 and 100 %, respectively of the theoretical yield). We also report for the first time the isolation of a pure bacterial culture PA, thought to be a strain of Rhodococcus , capable of growing on benzothiazole (BTH) itself as a sole carbon, nitrogen and energy source. Evidence is presented to suggest that this organism degrades BTH via the meta-cleavage pathway. The Rhodococcus PA degrades BTH but only releases a small proportion (5%) of the sulphur as sulphate. Mixed cultures containing this organism released ca 100% of the sulphur as sulphate, suggesting that other members of the consortium catalyse conversion of a sulphur-containing intermediate to sulphate. 2-Mercaptobenzothiazole (MBT) could not act as a growth substrate for any of the cultures studied but some could cause biotransformation of MBT to some extent. Attempts to obtain cultures degrading ABT and BTH from polluted river water were unsuccessful.     

Thermodynamics of hydrogen metabolism in methanogenic cocultures degrading ethanol or lactate

Pure cultures of Desulfovibrio vulgaris or Pelobacter acetylenicus do not grow with lactate or ethanol, respectively, under obligately proton- reducing conditions. However, a small part of these substrates was oxidized and molecular hydrogen was produced up to 4.2 and 3.2 kPa, respectively. During growth in syntrophic methanogenic cocultures with Methanospirillum hungatei as partner, maximum hydrogen partial pressures were significantly lower (0.7 to 2.5 kPa) than in the corresponding pure cultures. Calculation of Gibbs free energies for the prevailing culture conditions showed that H₂ partial pressures were kept in a range at which both, H₂-producing and H₂-consuming reactions, were thermodynamically permissive in pure as well as in syntrophic mixed cultures.     

Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation.

Titanium dioxide in the anatase crystalline form was used as a photocatalyst to generate hydroxyl radicals in a flowthrough water reactor. Experiments were performed on pure cultures of Escherichia coli in dechlorinated tap water and a surface water sample to evaluate the disinfection capabilities of the reactor. In water devoid of significant amounts of inorganic-radical scavengers, rapid cell death was observed with both pure cultures and members of the indigenous flora in a natural water sample.     

Dynamic flux balance modeling of microbial co-cultures for efficient batch fermentation of glucose and xylose mixtures

Sequential uptake of pentose and hexose sugars that compose lignocellulosic biomass limits the ability of pure microbial cultures to efficiently produce value-added bioproducts. In this work, we used dynamic flux balance modeling to examine the capability of mixed cultures of substrate-selective microbes to improve the utilization of glucose/xylose mixtures and to convert these mixed substrates into products. Co-culture simulations of Escherichia coli strains ALS1008 and ZSC113, engineered for glucose and xylose only uptake respectively, indicated that improvements in batch substrate consumption observed in previous experimental studies resulted primarily from an increase in ZSC113 xylose uptake relative to wild-type E. coli. The E. coli strain ZSC113 engineered for the elimination of glucose uptake was computationally co-cultured with wild-type Saccharomyces cerevisiae, which can only metabolize glucose, to determine if the co-culture was capable of enhanced ethanol production compared to pure cultures of wild-type E. coli and the S. cerevisiae strain RWB218 engineered for combined glucose and xylose uptake. Under the simplifying assumption that both microbes grow optimally under common environmental conditions, optimization of the strain inoculum and the aerobic to anaerobic switching time produced an almost twofold increase in ethanol productivity over the pure cultures. To examine the effect of reduced strain growth rates at non-optimal pH and temperature values, a break even analysis was performed to determine possible reductions in individual strain substrate uptake rates that resulted in the same predicted ethanol productivity as the best pure culture.