Adaptation of Saccharomyces cerevisiae cells to high ethanol concentration and changes in fatty acid composition of membrane and cell size

Background

Microorganisms can adapt to perturbations of the surrounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the culture medium.

Methodology/Principal Findings

First, a laboratory strain of S. cerevisiae was cultivated in medium containing a low ethanol concentration, followed by repetitive cultivations. Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repetitive cultivations, the yeast cells adapted to the high ethanol concentration; the specific growth rate of the adapted yeast strain did not decrease during repetitive cultivation in the medium containing the same ethanol concentration, while that of the non-adapted strain decreased during repetitive cultivation. A comparison of the fatty acid composition of the cell membrane showed that the contents in oleic acid (C_18:1) in ethanol-adapted and non-adapted strains were similar, but the content of palmitic acid (C_16:0) in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol. Moreover, microscopic observation showed that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strain.

Conclusions

Our results suggest that activity of cell growth defined by specific growth rate of the yeast cells adapted to stepwise increase in ethanol concentration did not decrease during repetitive cultivation in high-ethanol-concentration medium. Moreover, fatty acid content of cell membrane and the size of ethanol-adapted yeast cells were changed during adaptation process. Those might be the typical phenotypes of yeast cells adapted to high ethanol concentration. In addition, the difference in sizes of the mother cell between the non-adapted and ethanol strains suggests that the cell size, cell cycle and adaptation to ethanol are thought to be closely correlated.     

Utilization of short chain monocarboxylic acids in an effluent of a petrochemical industry by Acinetobacter calcoaceticus

The aqueous effluent generated by the Fischer--Tropsch process, containing a total of 13 g/L C(2)-C(5) monocarboxylic acids, was investigated as a potential substrate for the production of single-cell protein (SCP). A bacterial isolate, Acinetobacter calcoaceticus, could utilize all the acids in the effluent simultaneously in chemostat cultures, and no residual acids were detected in the culture below a dilution rate of 0.78 h(-1). The critical dilution rate was 1.04 h(-1). The maintenance energy requirement of the cells growing on the monocarboxylic acid mixture was considerably lower than that of cells growing on acetate as the sole carbon source. Enrichment of the effluent with ethanol to increase the biomass concentration was successful and still allowed the simultaneous and efficient utilization of all the carbon sources, but resulted in a decrease of the critical dilution rate by ca. 20%.     

Simultaneous ethanol and bacterial ice nuclei production from sugar beet molasses by a Zymomonas mobilis CP4 mutant expressing the inaZ gene of Pseudomonas syringae in continuous culture

AIM: The aim of this work was to construct a Zymomonas mobilis mutant capable of simultaneous ethanol and ice nuclei production from agricultural by-product such as sugar beet molasses, in steady-state continuous culture. METHODS AND RESULTS: A sucrose-hypertolerant mutant of Z. mobilis strain CP4, named suc40, capable of growing on 40% (w/v) sucrose medium was isolated following N-methyl-N'-nitro-N-nitrosoguanidine treatment. Plasmid pDS3154 carrying the inaZ gene of Pseudomonas syringae was conjugally transferred and expressed in suc40. The potential for simultaneous ethanol and bacterial ice nuclei production was assessed in steady-state continuous cultures over a range of dilution rates from 0.04 to 0.13 h(-1). In addition, the fatty acid and phospholipid profile of the three strains was also investigated. Ethanol production up to 43 g l(-1) was achieved at dilution rates below 0.10 h(-1) in sugar beet molasses. Ice nucleation activity gradually increased with increasing dilution rate and the greatest activity, -3.4 log (ice nuclei per cell), was observed at the highest dilution rate (0.13 h(-1)). Both mutant strains displayed a different fatty acid and phospholipid profile compared with the wild-type strain. CONCLUSIONS: The ability of the mutant and recombinant plasmid-containing strains to grow on high sugar concentrations and in high osmotic pressure environments (molasses) can be attributed to their phospholipid and fatty acid contents. SIGNIFICANCE AND IMPACT OF THE STUDY: Taking into account that sugar beet molasses is a low cost agricultural by-product, the simultaneous ethanol and bacterial ice nucleation production achieved under the studied conditions is considered very promising for industrial applications.     

Saccharomyces Cerevisiae Null Mutants in Glucose Phosphorylation: Metabolism and Invertase Expression

A congenic series of Saccharomyces cerevisiae strains has been constructed which carry, in all combinations, null mutations in the three genes for glucose phosphorylation: HXK1, HXK2 and GLK1, coding hexokinase 1 (also called PI or A), hexokinase 2 (PII or B), and glucokinase, respectively: i.e., eight strains, all of which grow on glucose except for the triple mutant. All or several of the strains were characterized in their steady state batch growth with 0.2% or 2% glucose, in aerobic as well as respiration-inhibited conditions, with respect to growth rate, yield, and ethanol formation. Glucose flux values were generally similar for different strains and conditions, provided they contained either hexokinase 1 or hexokinase 2. And their aerobic growth, as known for wild type, was largely fermentative with ca. 1.5 mol ethanol made per mol glucose used. The strain lacking both hexokinases and containing glucokinase was an exception in having reduced flux, a result fitting with its maximal rate of glucose phosphorylation in vitro. Aerobic growth of even the latter strain was largely fermentative (ca. 1 mol ethanol per mol glucose). Invertase expression was determined for a variety of media. All strains with HXK2 showed repression in growth on glucose and the others did not. Derepression in the wild-type strain occurred at ca. 1 mM glucose. The metabolic data do not support- or disprove-a model with HXK2 having only a secondary role in catabolite repression related to more rapid metabolism.     

Histamine production by wine lactic acid bacteria: isolation of a histamine-producingstrain of Leuconostoc oenos

Populations of Leuconostoc œnos were harvested from wines containing a relatively high concentration of biogenic amines. Cultivation of the biomass in synthetic media and wine showed that it consisted of histamine-producing strains. Histamine levels after culture depended on the quantity of precursor available and on the presence of yeast lees, which certainly enriched the medium in histidine. Ethanol and pH, which control bacterial growth rate and total population, were also significant factors: pH and low ethanol concentration enhanced histamine production.
Strain Leuc. œnos 9204 was isolated and studied since it retained its ability to produce histamine after several transfers. In synthetic medium this strain produced large amounts of histamine especially in the poorest nutritional conditions (no glucose, no L-malic acid). These results clearly demonstrate that Leuc. œnos involvedin wine-making might play a role in biogenic amine production. The vinification method might also influence the final amine concentration in wine.     

Selection of Ethanol-Tolerant Yeast Hybrids in pH-Regulated Continuous Culture

Hybrids between naturally occurring wine yeast strains and laboratory strains were formed as a method of increasing genetic variability to improve the ethanol tolerance of yeast strains. The hybrids were subjected to competition experiments under continuous culture controlled by pH with increasing ethanol concentrations over a wide range to select the fastest-growing strain at any concentration of ethanol. The continuous culture system was obtained by controlling the dilution rate of a chemostat connected to a pH-meter. The nutrient pump of the chemostat was switched on and off in response to the pH of the culture, which was thereby kept near a critical value (pH_c). Under these conditions, when the medium was supplemented with ethanol, the ethanol concentration of the culture increased with each pulse of dilution. A hybrid strain was selected by this procedure that was more tolerant than any of the highly ethanol-tolerant wine yeast strains at any concentration of ethanol and was able to grow at up to 16% (vol/vol) ethanol. This improvement in ethanol tolerance led to an increase in both the ethanol production rate and the total amount of ethanol produced.     

Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis.

Clostridium botulinum B-aphis spores plated on medium containing 4% salt at pH 6.0 yielded colonies at a frequency of ca. 1 in 10(6). A subculture of one of these colonies, designated strain Ba410, was compared with the parent strain, B-aphis, for a variety of traits. After 7 days of incubation at 37 degrees C, strain Ba410 grew in medium containing 7% NaCl, whereas strain B-aphis could not grow in salt concentrations greater than 5%. The strains also differed in cellular and colonial morphology. After exponential growth in the basal medium was completed, lysis of both strains was pH dependent; in media containing salt, lysis of Ba410 cells was pH independent. Strain Ba410 was more proteolytic than strain B-aphis in conditions of low pH and high salt, so that its toxin could be detected by the mouse assay. In a medium containing alanine and cysteine, the germination rate of B-aphis was 0.77% min-1, whereas that of Ba410 was 0.14% min-1; 2% salt inhibited the germination of Ba410 but not B-aphis.     

Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis

Clostridium botulinum B-aphis spores plated on medium containing 4% salt at pH 6.0 yielded colonies at a frequency of ca. 1 in 10(6). A subculture of one of these colonies, designated strain Ba410, was compared with the parent strain, B-aphis, for a variety of traits. After 7 days of incubation at 37 degrees C, strain Ba410 grew in medium containing 7% NaCl, whereas strain B-aphis could not grow in salt concentrations greater than 5%. The strains also differed in cellular and colonial morphology. After exponential growth in the basal medium was completed, lysis of both strains was pH dependent; in media containing salt, lysis of Ba410 cells was pH independent. Strain Ba410 was more proteolytic than strain B-aphis in conditions of low pH and high salt, so that its toxin could be detected by the mouse assay. In a medium containing alanine and cysteine, the germination rate of B-aphis was 0.77% min-1, whereas that of Ba410 was 0.14% min-1; 2% salt inhibited the germination of Ba410 but not B-aphis.     

Genomic stability of Saccharomyces cerevisiae baker's yeasts.

The objective of this study has been to gather data on genomic stability of baker's yeast strains during long-term mitotic growth under restrictive conditions so that comparisons could be made to other studies indicating genomic instability during meiosis. The work describes the analysis of mitotic stability of the nuclear and mitochondrial genomes in the baker's yeast strain V1 during incubation in continuous culture for 190 generations (300 days). The cells were cultured in complete medium containing 2% glucose and 8 to 12% ethanol, as a mutagenic agent specific for mtDNA. The high concentration of ethanol severely limited the growth rate of the cells. DNA samples were monitored for chromosomal pattern, polymorphisms in selected nuclear genes (SUC2, MALIT, ADH1) and mobile genetic elements (Ty1 and Y'), and for RFLPs in mtDNA. The results show that both the nuclear and mitochondrial genomes of grande cells were very stable. However, the frequency of petite mutants in the population varied dramatically during the course of the experiment, reaching as high as 87% petite during the first 27 days of the experiment and declining to 5.8% petite at the end. This decline can be attributed to selection against petite mutants in media containing high concentrations of ethanol. Moreover, when samples and the parental strain were compared at the end of the experiment, no change could be observed in parameters such as their growth rate in different media, capacity to leave doughs, viability in ethanol or frequency of petite mutants. Results therefore indicated that the majority of the cells in the population were very similar to the parental throughout the experiments, with no apparent molecular or phenotypical changes.     

Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus

The availability of a strain of Acinetobacter competent for transformation has made it possible to demonstrate the genetic relatedness of a large variety of gram-negative, oxidase-negative, nonmotile, and aerobic coccobacilli originally classified into eleven different genera. Deoxyribonucleic acid (DNA) species from 265 such strains are capable of transforming stable auxotrophs of the competent Acinetobacter to prototrophy. The compositions of these DNA species vary from 40 to 46.8% guanine plus cytosine. Strains with widely differing phenotypic properties are also included in this collection of acinetobacters. DNA species from all oxidase-positive strains of Moraxella and from a variety of common bacteria are unable to transform the competent Acinetobacter. Although acinetobacters are usually considered to be unable to reduce nitrate to nitrite, six strains known to carry out this reduction have been shown to be authentic acinetobacters since their DNA species readily transform the competent Acinetobacter auxotrophs to prototrophy. In contrast to previous findings that acinetobacters rarely grow with glucose as a sole carbon source, the results of the present study show that 17 of the 265 strains grow readily in a glucosemineral medium, and 48 other strains can mutate spontaneously to grow in such a medium. A second competent strain of Acinetobacter, originally unable to use glucose, d-xylose, or d-ribose as carbon sources, has been transformed for ability to dissimilate these compounds using DNA species from strains that normally grow on these sugars. Although most of the 265 Acinetobacter strains studied were originally grown on complex media when isolated from human sources, only nine of these strains require growth factors in order to grow in a mineral medium containing a single carbon and energy source. A simple transformation assay has been devised for rapid examination of large numbers of strains to determine whether or not they are acinetobacters. This assay, which is suitable for routine diagnostic work, includes a procedure for preparation of crude transforming DNA from a small quantity of bacterial paste. Samples of DNA prepared from Acinetobacter cultures that had died on slants and plates were still able to effect transformation of the competent auxotrophs to prototrophy.     

The Role of Carbon Dioxide as an Essential Nutrient for Six Permanent Strains of Fibroblasts

The results of these studies demonstrate that carbon dioxide is required for the growth and maintenance of strains of fibroblasts derived from human tissues, strains FS4-705 and U12-705, from mouse tissue, strain L-705, and from rabbit tissues, strains RM3-56, RS1-56, and RT-56 in a chemically defined medium containing phosphite buffer in place of bicarbonate and supplemented with dialyzed serum and dialyzed embryo extract. Under these conditions, the cells fail to proliferate at a significant rate and begin to degenerate within 5 to 10 days when the flasks are not stoppered. Sufficient carbon dioxide is produced by the cells to promote growth as indicated by the fact that maximal proliferation is obtained in the same phosphite media when stoppered flasks are employed. With the exception of RS1-56, all the remaining strains tested can be propagated serially in open flasks containing phosphite medium prepared with whole serum and embryo extract. The rate of growth under these conditions, however, is only one-half to one-third that obtained in stoppered flasks containing phosphite medium or the conventional bicarbonate medium.     

一株乙醇降解菌株的分离和鉴定

从湖北农田土壤中筛选得到一株ALDH活性较高的菌株,该菌株在含0．64％乙醇的培养基中生长较佳,且耐受0．9％的乙醛。经菌种形态学和生理生化特征,以及16S rRNA基因序列分析,鉴定该菌株为不动杆（Acinetobacter sp．）。该菌株在乙醇和乙醛解毒研究中有重要价值。     

Production of ethanol from the mixture of beet molasses and cheese whey by a 2-deoxyglucose-resistant mutant of Kluyveromyces marxianus

Fourteen lactose-fermenting strains of Kluyveromyces marxianus , including its anamorph, Candida kefyr , were grown in two media containing 20% (w/v) sugar as either beet molasses or cheese whey. Strain NBRC 1963 of K. marxianus converted sucrose and lactose to ethanol in both media most efficiently. However, ethanol was produced from sucrose and not from lactose by strain NBRC 1963 in the medium containing equal amounts of sugar from beet molasses and cheese whey. The spontaneous mutants resistant to 2-deoxyglucose in the minimal medium composed of galactose as the sole carbon source were isolated from strain NBRC 1963. Among them, strain KD-15 vigorously produced ethanol in the media containing beet molasses, cheese whey, or both. The mutant strain KD-15 was insensitive to catabolite repression, as shown by the observation that β-galactosidase was not repressed in the presence of sucrose from beet molasses.     

Enrichment of linear alkylbenzenesulphonate (LAS) degrading bacteria in continuous culture

Enrichment experiments were carried out in continuous-flow units using a mineral medium with commercial linear alkylbenzenesulphonate (LAS) as the limiting carbon- and energy-source. The mixed bacterial culture originating from the waste water of a detergent plant consisted of five strains belonging to the genus Pseudomonas and two strains each of the genera Achromobacter and Acinetobacter. The cultivation conditions corresponding to dilution rates of 0.025-0.1 h^-1 and LAS concentrations of 20–50 mg/1 were examined. During the experiments the composition of mixed cultures and the kinetics of LAS biodegradation were followed. Continuous-flow enrichment experiments resulted in the selection of six bacterial cultures with different compositions of individual species and capability to utilize LAS. From the original seven strains at lower dilution rates (0.025 and 0.05 h^-1) six were selected, excluding Pseudomonas sp. 3, while at the highest dilution rate (0.1/h^-1) five strains were selected after eliminating Pseudomonas sp. 5 and Achromobacter sp. 1. All enriched mixed cultures were more efficient in primary than in ultimate LAS degradation. Two of the culture strains were able to achieve primary LAS degradation ( Pseudomonas sp. 1 in mineral medium with LAS as the sole carbon- and energy-source and Acinetobacter sp. 3 in medium supplemented by yeast extract and nutrient broth).
None of the strains could degrade LAS completely, which indicates that many types of interactions based on combined metabolic attack as well as those based on provision of specific nutrients, may exist between culture members during the complete LAS bio-oxidation.     

Emulsifier production and microscopical study of emulsions and biofilms formed by the hydrocarbon-utilizing bacteria Acinetobacter calcoaceticus MM5

A bacterial strain was isolated from a sample of contaminated heating oil and identified as a strain of Acinetobacter calcoaceticus, named MM5. The bacterial isolate was able to grow on petroleum derivatives and brought about an emulsification of those compounds. A bioemulsifier was extracted from the culture medium of MM5 strain and partially characterized. This compound was able to emulsify petroleum fuels and both aliphatic and aromatic pure hydrocarbons and was stable over a wide range of temperatures. Studies developed by light, scanning electron and transmission electron microscopy showed that, during the growth on petroleum derivatives, the microorganisms were orientated on the surface of drops enclosed in a skin or membranous polymer produced by the bacteria. These droplets may represent the hydrocarbon/water emulsion of the liquid culture. The growth of A. calcoaceticus MM5 on media containing both hydrocarbon and water-soluble substrates as carbon sources also results in the formation of a film, consisting of amorphous and membranous layers. The bacteria were connected to the biofilm and showed intercellular contacts through cell-surface appendages, forming a complex network. The importance of the biofilms for bacterial adhesion to oil droplets and for its nourishment is discussed.     

Modulation of Biological Functions of Naegleria fowleri Amoebae by Growth Medium

Two strains of Naegleria fowleri amoebae were studied when the amoebae were maintained in the same growth medium or in two different media. A weakly pathogenic strain of N. fowleri , LEE, and a highly pathogenic strain, LEEmpCl, were compared for growth properties, the presence or absence of surface structures termed food cups, cytopathogenicity, cellular locomotion, susceptibility to complement-mediated lysis and immunological relatedness by western immunoblot analysis when grown in Nelson medium or in Cline medium. The two different strains of N. fowleri , LEE and LEEmpCl, were more similar in protein profiles and functional activity when both strains were grown in the same nutritional medium. Differences in growth, proteins synthesized, cytopathogenicity, susceptibility to complement lysis and rate of locomotion were noted when the same strain was grown in different media. Naegleria fowleri grown in Cline medium demonstrated an increased rate of growth, an increase in its rate of locomotion, an increased resistance to complement lysis, and destroyed target nerve cells by contact-dependent lysis. In contrast, the same strain of amoeba grown in Nelson medium showed slower growth, destroyed target cells by trogocytosis, and was less resistant to complement-mediated lysis.     

Polygalacturonase production by Kluyveromyces marxianus: effect of medium composition

A strain of Kluyveromyces marxianus (CCT 3172), isolated from a cocoa fermentation in Brazil, secreted an endopolygalacturonase (PG) when grown under self-induced anaerobic conditions; neither polymethylesterase nor pectate lyase appeared in culture filtrates. Replacing glucose in the medium with sucrose had no effect on PG secretion or ethanol production. Growth in fructose-containing medium retarded secretion of PG and ethanol, but had no effect on growth. Growth and ethanol production in media containing galactose resembled those in fructose-containing medium, although PG secretion was lowered. Growth and PG secretion were considerably retarded in xylose-containing medium, and were similarly affected in media containing different concentrations of glucose. Varying the concentration of ammonium sulphate in media had no effect on growth or PG secretion.     

Selection of mutants of microorganisms utilizing ethanol

Mutants of the bacteria Acinetobacter calcoaceticus 34 and Acinetobacter sp. 172 as well as of the yeast Candida requinyii 316 resistant to acetaldehyde grow better in a medium with ethanol than their parent cultures. In their specific growth rate and alcohol dehydrogenase activity, 28.7-66.7% of such mutants are superior to any clone isolated in a non-selective medium. A medium containing ethanol and acetaldehyde (0.5 to 1.0% by volume) is proposed to select and isolate highly productive mutants.