Acetic Acid Tolerance Research Articles

Membrane lipid physiology and toxin catabolism underlie ethanol and acetic acid tolerance inDrosophila melanogaster

Drosophila melanogaster has evolved the ability to tolerate and utilize high levels of ethanol and acetic acid encountered in its rotting-fruit niche. Investigation of this phenomenon has focused on ethanol catabolism, particularly by the enzyme alcohol dehydrogenase. Here we report that survival under ethanol and acetic acid stress in D. melanogaster from high- and low-latitude populations is an integrated consequence of toxin catabolism and alteration of physical properties of cellular membranes by ethanol. Metabolic detoxification contributed to differences in ethanol tolerance between populations and acclimation temperatures via changes in both alcohol dehydrogenase and acetyl-CoA synthetase mRNA expression and enzyme activity. Independent of changes in ethanol catabolism, rapid thermal shifts that change membrane fluidity had dramatic effects on ethanol tolerance. Cold temperature treatments upregulated phospholipid metabolism genes and enhanced acetic acid tolerance, consistent with the predicted effects of restoring membrane fluidity. Phospholipase D was expressed at high levels in all treatments that conferred enhanced ethanol tolerance, suggesting that this lipid-mediated signaling enzyme may enhance tolerance by sequestering ethanol in membranes as phophatidylethanol. These results reveal new candidate genes underlying toxin tolerance and membrane adaptation to temperature in Drosophila and provide insight into how interactions between these phenotypes may underlie the maintenance of latitudinal clines in ethanol tolerance.

A Gene Encoding Phosphatidylethanolamine N-Methyltransferase from Acetobacter aceti and Some Properties of its Disruptant

Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced. One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate.

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200-250 degrees C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4cents/gal of ethanol. This work examined the growth and fermentation performance of xylose-utilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated as the "adapted" strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30 h, followed by CP4:pZB5 at 48 h, with corresponding volumetric productivities of 1.4 and 0.89 g/ (L x h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to ferment arabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.

Induction of acetic acid tolerance and trehalose accumulation by added and produced ethanol in Saccharomyces cerevisiae

Both added (49.6 g/l) and produced ethanol (46.2 g/l) caused an increase in the acetic acid tolerance of Saccharomyces cerevisiaegrown in an anaerobic chemostat; added ethanol, however, to a less extent than produced ethanol. The ethanol induced acetic acid tolerance of the cells was linked with an accumulation of trehalose within the cells. These results indicate that trehalose plays a role in the ethanol induced acetic acid tolerance of S. cerevisiae.

Ethanol and acetic acid tolerance in free and immobilized cells of Saccharomyces cerevisiae and Acetobacter aceti

Saccharomyces cerevisiae and Acetobacter aceti cells were immobilized by entrapment in Ca-alginate or by adsorption on to preformed cellulose beads and were treated with 0-20% (v/v) ethanol and 0-10% (v/v) acetic acid. At 20% (v/v) ethanol, lethal for free yeast cells, 62-72% of the immobilized cells survived. In 10% (v/v) acetic acid, free and adsorbed Acetobacter aceti cells ceased to grow but 69% of entrapped cells survived. Cells released from the carrier showed an intermediate survival (20-60%).

ADAPTATION TO FERMENTING RESOURCES IN DROSOPHILA MELANOGASTER: ETHANOL AND ACETIC ACID TOLERANCES SHARE A COMMON GENETIC BASIS.

Ethanol and acetic acid tolerances were compared in a French, highly tolerant population, and in a Congolese, very sensitive population. For both tolerances, chromosome substitutions demonstrated a major effect on chromosome 3, a lesser effect on chromosome 2, and no effect on chromosome 1, except in interactions. Directional selection experiments led to significant increases of tolerance to both toxics. Of greater interest, a strong correlated response was observed in each line: increased ethanol tolerance was accompanied by higher acetic acid tolerance and vice versa. A high genetic correlation (average value r = 0.77) was found between the two traits. These data suggest that alcohol dehydrogenase (ADH) activity does not play a major role in explaining the physiological differences known between Afrotropical and European populations. The metabolic flux permitting the detoxification of ethanol and acetic acid seems to be mainly controlled by acetyl-coA synthetase (ACS) at least in adult flies. Acetic acid adaptation could be as important as ethanol adaptation in the ecology of Drosophila melanogaster and other Drosophila species.

The effect of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae

The effects of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. Cells grown in the presence of acetic acid at a defined specific growth rate showed a higher acetic acid tolerance and a slightly lower trehalose content. Cells grown at a low specific growth rate showed a lower energy demand, a higher acetic acid tolerance, and a higher trehalose content. These results indicate that trehalose plays a growth rate dependent role in the tolerance of S. cerevisiae to acetic acid.

Acetic acid tolerance in Drosophila is a prerequisite for ethanol tolerance

AbstractAcetic acid tolerance compared with ethanol tolerance of Drosophila simulans and six Drosophila melanogaster strains shows a curvilinear relation with apparent asymptotic hyperbolic profile. The upper limit of acetic acid tolerance is lower than that for ethanol. We compared strains which had pairwise identical alcohol dehydrogenase (ADH) coding regions but different genetic backgrounds. A positive regression existed for ethanol tolerance on ADH activity. Adh‐null mutants with very low ethanol tolerances had appreciable acetic acid tolerances and as a consequence did not fit the curve. ADH‐F and ADH‐S strains selected for high ethanol tolerances had the ability to tolerate high ethanol concentrations even after selection had been relaxed for several years. These selected lines tolerated higher acetic acid concentrations than the non‐selected original strains. We propose that intake of high concentrations of ethanol and oxidation into acetic acid induces esterification of ethanol and acetic acid into ethylacetate. This cannot take place after the intake of acetic acid only, which also gives a lower energy yield.

Parallel selection of ethanol and acetic-acid tolerance in Drosophila melanogaster populations from India

Summary - Nine Indian geographical populations of Drosophila melanogaster, collected along the 20°N latitudinal range, revealed a significant clinal variation at the alcohol dehydrogenase (Adh) locus, Adh allelic frequency increasing significantly with latitude (0.036 ! 0.004 for 1° latitude; genetic divergence F = 0.25). Patterns of ethanol and acetic-acid tolerance in adult individuals revealed significant genetic divergence. Parallel patterns of latitudinal ethanol tolerance (10 to 15%) and acetic-acid tolerance (3.7 to 13.2%) were observed in adult individuals from the 9 geographical populations. Thus, the northern and southern populations revealed divergence in the patterns of resource utilisation. The parallel latitudinal genetic divergence at the Adh locus and for ethanol and acetic-acid tolerance in Indian populations of Drosophila melanogaster could be explained by balancing natural selection varying spatially along the north-south axis of the Indian subcontinent. ADH polymorphism / ethanol tolerance / acetic-acid tolerance / latitudinal clines / Indian populations / Drosophila melanogaster

Ethanol and acetic-acid tolerance in Indian geographical populations of Drosophila immigrans

Summary - Indian geographical populations of Drosophila immigrans revealed a lack of allozymic variation at the alcohol dehydrogenase locus. Latitudinal clines of ethanol tolerance (1.5-4.2%) and acetic-acid tolerance (2.9-4.9%) were observed in adult individuals of 4 geographical populations of Drosophila immigrans. Thus, both ethanol and acetic-acid tolerance decreased towards the equator. The parallel patterns of utilisation of ethanol and acetic acid seem to be correlated with the concentrations of these 2 metabolites in natural food resources. Thus, intra-specific differences for ethanol and acetic-acid tolerance could be adaptively maintained by spatially varying fermenting habitats along the north-south axis of the Indian sub-continent. ethanol tolerance / acetic-acid tolerance / clinal variation / geographical population / Drosophila immigrans

Ethanol and acetic-acid tolerance in Indian geographical populations of Drosophila immigrans

Ethanol and acetic-acid tolerance in Indian geographical populations of Drosophila immigrans

Parallel selection of ethanol and acetic-acid tolerance in Drosophila melanogaster populations from India

Parallel selection of ethanol and acetic-acid tolerance in Drosophila melanogaster populations from India

Adaptation to alcoholic fermentation in Drosophila: a parallel selection imposed by environmental ethanol and acetic acid.

Besides ethanol, acetic acid is produced in naturally fermenting sweet resources and is a significant environmental stress for fruit-breeding Drosophila populations and species. Although not related to the presence of an active alcohol dehydrogenase, adult acetic acid tolerance was found to correlate with ethanol tolerance when sensitive (Afrotropical) and resistant (European) natural populations of Drosophila melanogaster were compared. The same correlation was found when comparing various Drosophila species. Tolerance to acetic acid also correlated with the tolerance to longer aliphatic acids of three, four, or five carbons but did not correlate with the tolerance to inorganic acids (i.e., hydrochloric and sulfuric acids). These observations suggest that acetic acid is detoxified by the conversion of acetate into acetyl-CoA, a metabolic step also involved in ethanol detoxification. Future investigations on the adaptation of Drosophila to fermenting resources should consider selective effects of both ethanol and acetic acid.

Isolation of acetic acid-tolerant Baker’s yeast variants in a turbidostat

A commercial baker’s yeast was subjected to selection in a continuous turbidostat cultivation with increasing concentration of acetic acid. The final acetic acid content in fresh medium was 0.6% or 0.8% v/v. Two of seven selected variants were stable over 15 sequential shake flask cultivations without selection pressure. After laboratory scale production of baker’s yeast, one of the variants also showed increased acetic acid tolerance in sour dough. The overall raising power (mL CO2/h) in sour dough was improved 36%.

Development of increased acetic acid tolerance in anaerobic homoacetogens through induced mutagenesis and continuous selection

Effective methods for large scale mutagenesis of strictly anaerobic, thermophilic microorganisms were developed. Mutagenesis and selection were carried out under highly reduced conditions without using plating techniques. Ultraviolet light (u.v.) or ethyl methanesulphonate (EMS) produced similar improvements in acetic acid tolerance of Clostridium thermoaceticum. An additive effect was observed when u.v. and EMS were applied simultaneously. N-Methyl- N-nitro- N-nitrosoguanidine was ineffective.

Tolerance of acetate, propionate and sorbate by Saccharomyces cerevisiae and Torulopsis holmii

Several strains of Saccharomyces cerevisiae and one of Torulopsis holmii were investigated with regard to their tolerance of acetate, propionate and sorbate. The S. cerevisiae strains had poor acetic acid tolerance in sour dough pH 4·3, the dough raising power decreasing on average by 67% in the presence of 0·34% sodium acetate, while T. holmii was not affected by this concentration. Propionate (0·16%) and sorbate (0·08%) were more inhibitory than acetate, inhibiting the dough raising power even in normal dough at pH 5·9. Torulopsis holmii also had a good tolerance of propionate and sorbate and seems suitable for raising sour doughs.

Acetic acid tolerance in wood- and litter-decomposing Hymenomycetes

Acetic acid tolerance in wood- and litter-decomposing Hymenomycetes