Assimilation spectrum of the yeastCandida utilis 49 used for producing fodder yeast from synthetic ethanol

Oxidizing and assimilating ability of the yeastCandida utilis 49 was tested with 21 different low-boiling organic compounds which come as components of raw synthetic ethanol. The highest yields of yeast dry weight were obtained with ethanol (72.0%), propanol (48.2%), ethyl acetate (43.4%) and acetic acid (34.2%). To a minor extent, the yeast was capable of utilizing also 2-propanol, butanol and 2-butanol; it oxidized most of the compounds tested.     

Comparative Volatile Profiles in Soy Sauce According to Inoculated Microorganisms

We compared the volatile profiles in soy sauce according to inoculation with Tetragenococcus halophilus and/or Zygosaccharomyces rouxii. Totals of 107 and 81 volatiles were respectively identified by using solid-phase microextraction and solvent extraction. The various volatile compounds identified included acids, aldehydes, esters, ketones, furans and furan derivatives, and phenols. The major volatiles in the samples treated with T. halophilus were acetic acid, formic acid, benzaldehyde, methyl acetate, ethyl 2-hydroxypropanoate, 2-hydroxy-3-methyl-2-cyclopenten-1-one, and 4-hydroxy-3-methoxybenzaldehyde, while those in the samples inoculated with Z. rouxii were mainly ethanol, acetaldehyde, ethyl propanoate, 2/3-methylbutanol, 1-butanol, 2-phenylethanol, ethyl 2-methylpropanoate, and 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone. The results indicate that T. halophilus produced significant acid compounds and could affect the Z. rouxii activity, supporting the notion that yeasts and lactic acid bacteria respectively have different metabolic pathways of alcoholic fermentation and lactic acid fermentation, and produce different dominant volatile compounds in soy sauce.     

Exhaust gas purification using immobilised monocultures (biocatalysts)

Summary The paper is concerned with the purification of exhaust gases using biocatalysts in a trickle bed reactor. Substance specific strains (monocultures) which were, for example, immobilised on activated carbon served as biocatalyst. Technically important solvents and substances such as aldehydes, methyl ethyl ketone and ethyl acetate were used as pollutants. Their concentration was about 5–40 ppm in the exhaust gas to be purified. The experiments show that with suitable bacterial, strains space velocities of about k ^*=1500 h^-1 can be obtained at a conversion of 90%. The mass transfer through the liquid film around the activated carbon grains seems to be rate determining.     

Investigations of aneuploidy-inducing chemical combinations in Saccharomyces cerevisiae

For several years we have been investigating combinations of chemicals for their ability to induce aneuploidy. Earlier published results indicated that combinations of certain chemicals showed a potentiation effect while other combinations did not. We have continued to explore this phenomenon and report additional findings in this communication. Combinations of ethyl acetate and methyl ethyl ketone showed a potentiation effect as did 1-methyl-2-pyrrolidinone-nocodazole combinations. Combinations that did not show a potentiation effect were 2-pyrrolidinone-nocodazole and 1-methyl-2-pyrrolidinone-ethyl acetate. We also found that nocodazole, which is a potent inducer of aneuploidy in yeast extract-peptone-dextrose (YEPD) medium but not in synthetic complete (SC) medium, showed a potentiation effect with ethyl acetate in SC medium. This effect in SC medium is similar to that previously reported for nocodazole with ethyl acetate in YEPD medium. When nocodazole was dissolved in 1-methyl-2-pyrrolidinone as a concentrated stock solution, a potentiation effect occurred even at low concentrations of the solvent.     

Several new substrates for Desulfovibrio vulgaris strain Marburg and a spontaneous mutant from it

The ability of Desulfovibrio vulgaris strain Marburg (DSM 2119) to oxidize alcohols was surveyed in the presence and absence of hydrogen-scavenging anaerobes, Acetobacterium woodii and Methanospirillum hungatei. In the presence of sulfate, D. vulgaris grew not only on ethanol, 1-propanol, and 1-butanol, but also on isobutanol, 1-pentanol, ethyleneglycol, and 1,3-propanediol. Metabolism of these alcohols was simple oxidation to the corresponding acids, except with the last two substrates: ethyleneglycol was oxidized to glycolate plus acetate, 1,3-propanediol to 3-hydroxypropionate plus acetate. Experimental evidence was obtained, suggesting that 2-methoxyethanol was not utilized by all the cells of strain marburg, but by a spontaneous mutant. 2-Methoxyethanol was oxidized to methoxyacetate by the mutant. Co-culture of strain Marburg plus A. woodii grew on ethanol, 1-propanol, 1-butanol, and 1,3-propanediol in the absence of sulfate. Co-culture of strain Marburg plus M. hungatei grew on ethanol, 1-propanol, and 1-butanol, but not on ethyleneglycol and 1,3-propanediol, Co-culture of the mutant plus A. woodii or M. hungatei did not grow on 2-methoxyethanol.     

Neutral Constituents of Volatiles in Cultured Broth of Sporobolomyces odorus

Neutral constituents of volatiles in the ether extract of cultured broth of Sporobolomyces odorus AHU 3246 were analyzed by gas chromatography-mass spectrometry and other methods.

Identified compounds were as follows: Methyl, ethyl, isobutyl, n-butyl, isoamyl, n-amyl, benzyl, and β-phenylethyl alcohol; formaldehyde, acetaldehyde, benzaldehyde, phenyl-acetaldehyde, acetone, and methyl ethyl ketone; ethyl formate, ethyl acetate, and di-n-butyl phthalate; γ-decalactone (4-decanolide) and 4-hydroxy-cis-dodecenoic acid γ-lactone (cis-6-dodecen-4-olide). Di-n-butyl phthalate and parts of methyl, ethyl, and n-butyl alcohol and ethyl acetate were thought to be contaminants. γ-Lactones produced by the yeast were determined by GLC.

Although nine strains of six species of carotenoid pigment accumulating yeasts were cultured under the same conditions, neither flavorful smelling nor γ-lactone production detected in their cultured broths.     

Aneuploidy-inducing chemicals in yeast evaluated by the micronucleus test

Chinese hamsters were exposed to acetone, methyl ethyl ketone, ethyl acetate and 2-methoxy ethyl acetate, known to be strong inducers of aneuploidy in the yeast Saccharomyces cerevisiae. All solvents yielded negative results in the micronucleus test, whereas the vinca alkaloid vindesine--used as a positive control substance--proved to act as a spindle poison in mammals in vivo.     

Recovery of poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) from Ralstonia eutropha cultures with non‐halogenated solvents

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA‐based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate‐co‐hydroxyhexanoate) (P(HB‐co‐HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non‐halogenated solvents. Several non‐halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17–30 mol%) remained completely in solution, while polymer with a lower HHx content (11–16 mol%) formed a gel‐like phase. All PHA in solution could be precipitated by addition of threefold volumes of n‐hexane or n‐heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non‐halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA. Biotechnol. Bioeng. 2013; 110: 461–470. © 2012 Wiley Periodicals, Inc.     

Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cerevisiae

A diploid yeast strain D61.M was used to study induction of mitotic chromosomal malsegregation, mitotic recombination and point mutation. Several ketones (including acetone and methyl ethyl ketone) and some organic acid esters (including the methyl, ethyl and 2-methoxyethyl esters of acetic acid) and acetonitrile strongly induced aneuploidy but not recombination or point mutation. Only diethyl ketone induced low levels of recombination and point mutation in addition to aneuploidy. Related compounds were weak inducers of aneuploidy: methyl n-propyl ketone, the methyl esters of propionic and butyric acid, acetic acid esters of n- and iso-propanol and ethyl propionate. No mutagenicity was found with n-butyl and isoamyl acetate, ethyl formate, acetyl acetone (2,5-dipentanone) and dioxane. Methyl isopropyl ketone induced only some recombination and point mutation but no aneuploidy. Efficient induction was only observed with a treatment protocol in which growing cells were exposed to the chemicals during a growth period of 4 h at 28 degrees C followed by incubation in ice for more than 90 min, usually overnight for 16-17 h. Aneuploid cells could be detected in such cultures during a subsequent incubation at growth temperature if the chemical was still present. Detailed analysis showed that there was a high incidence of multiple events of chromosomal malsegregation. It is proposed that the mutagenic agents act directly on tubulin during growth so that labile microtubules are formed which dissociate in the cold. When cells are brought back to temperatures above the level critical for reassembly of tubulin and allowed to grow, faulty microtubules are formed.     

Induction of chromosome loss by mixtures of organic solvents including neurotoxins

Twenty-three aprotic polar solvents - 3 nitriles, 8 organic esters, 10 ketones and 2 lactones - and LiCl were tested in combination with propionitrile alone or a mixture of ethyl acetate and propionitrile for the induction of mitotic chromosome loss in the D61.M strain of the yeast Saccharomyces cerevisiae. Propionitrile and ethyl acetate are very potent inducers of chromosome loss. Mixtures of propionitrile and ethyl acetate induced chromosome loss at much higher frequencies than was observed with the pure chemicals. To test the potentiating effects of propionitrile or mixtures of propionitrile with ethyl acetate on other chemicals, they were used in concentrations that were at or below the level for induction of chromosome loss. Twenty chemicals when tested in pure form were negative or only marginally active in the test for chromosome loss. Except for amyl propionate and benzyl acetate, the same chemicals showed strong induction in combination treatments with the potentiating chemicals. All the ketones including the neurotoxic methyl ethyl ketone, 2-hexanone and 2.5-hexanedione induced high frequencies of chromosome loss. Only methyl ethyl ketone is capable of inducing high levels of chromosome loss when tested in the pure form at much higher concentrations. 1-Methyl-2-pyrrolidinone and gamma-valerolactone had previously been shown to induce chromosome loss only when the treatment at a growth-supporting temperature was interrupted by a cold shock within a narrow range of low temperatures which prevented growth. Both gave very strong induction in combination treatment performed at a continuous growth-supporting temperature. LiCl is a weak inducer of chromosome loss: strong induction can be achieved in combination treatments.     

The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae

MMS induced mitotic recombination but not mitotic chromosome loss when tested in pure form in strain D61.M of Saccharomyces cerevisiae, confirming previous results of Albertini (1991), whereas in Aspergillus nidulans it also induced chromosomal malsegregation in addition to mitotic recombination (Käfer, 1988). However, induction of mitotic chromosome loss was observed in combination with strong inducers of chromosome loss such as the aprotic polar solvents ethyl acetate and to a lesser extent methyl ethyl ketone but not with γ-valerolactone and propionitrile. In addition to this, 4 solvents, dimethyl formamide, dimethyl sulfoxide, dioxane and pyridine, enhanced the MMS-induced mitotic recombination in strain D61.M. An enhancement of MMS-induced mitotic recombination and reverse mutation could be demonstrated for ethyl acetate and γ-valerolactone in yeast strain D7.     

Antimicrobial volatile organic compounds affect morphogenesis-related enzymes in Guignardia citricarpa,causal agent of citrus black spot

Although non-volatile substances toxic to plant pathogenic microorganisms have been extensively studied over the years, few studies have focused on microbial volatile organic compounds (VOCs). The VOCs produced by the yeast Saccharomyces cerevisiae strain CR-1, used in fermentative processes for fuel ethanol production, are able to inhibit the vegetative development of the fungus Guignardia citricarpa, causal agent of the disease citrus black spot. How microbial VOCs affect the development of fungi is not known. Thus, the objective of the present work was to study the effect of the artificial mixture of VOCs identified from S. cerevisiae on intracellular enzymes involved in the mycelial morphogenesis in G. citricarpa. The phytopathogenic fungus was exposed to artificial mixture of VOCs constituted by alcohols (ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol and phenylethyl alcohol) and esters (ethyl acetate and ethyl octanoate) in the proportions naturally found in the atmosphere produced by the yeast. The VOCs inhibited considerably the mycelial development and interfered negatively with the production of the morphogenesis-related enzymes. After 72 h of exposure to the VOCs the laccase and tyrosinase activities decreased 46 and 32%, respectively, however, the effect on the chitinase and β-1,3-glucanase activities was lower, 17 and 13% of inhibition, respectively. Therefore, the exposure of the fungus to the antimicrobial volatiles can influence both fungal mycelial growth rate and activity of enzymes implicated in morphogenesis. This knowledge is important to understand the microbial interactions mediated by VOCs in nature and to develop new strategies to control plant pathogens as G. citricarpa in postharvest.     

2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

2-Butanol and its chemical precursor butanone (methyl ethyl ketone – MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B₁₂-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions.     

Continous ethyl acetate production by Kluyveromyces fragilis on whey permeate

The synthesis of ethyl acetate by Kluyveromyces fragilis on diluted whey permeate was studied. Ethanol, lactose and O₂ are the direct precursors for ethyl acetate synthesis by this yeast. Ethyl acetate production is affected by many parameters, particularly the carbon/nitrogen (C/N) ratio, Tween 80 and iron. Ethyl acetate synthesis is optimum for C/N = 45. Tween 80 lowered slightly the level of ethyl acetate whereas iron completely stopped ethyl acetate production. The level of ethanol in the feed, the dissolved O₂ (DO) and dilution rate (D) were also optimised. Thus at D = 0.24 h ^–1, for 4 g/l of ethanol in the feed and 40% DO, the productivity of ethyl acetate was optimal (0.7 g/l per hour). Correspondence to: A. Miclo     

Effects of chemical combinations on the induction of aneuploidy in Saccharomyces cerevisiae

Nocodazole, ethyl acetate, acetone and methyl ethyl ketone all are known to induce aneuploidy. Treatment of yeast strain D61.M with mixtures containing ineffective low levels of nocodazole and ineffective low levels of these solvents was highly effective in inducing aneuploidy. Ineffective low levels of nocodazole mixed with ineffective low levels of methyl 2-benzimidazolecarbamate also gave elevated frequencies of aneuploidy. Dimethyl formamide, a solvent that does not induce aneuploidy, mixed with low levels of nocodazole gave no increase in aneuploidy frequency above those levels seen in controls.     

Lipogenesis from ketone bodies in the perfused rat liver: effects of acetate and ethanol

The interactions between acetate or ethanol metabolism, lipogenesis, and ketone body utilization have been studied in isolated livers from fed rats perfused with 15 mM glucose and 10 mM acetate or ethanol. The contribution of acetate to ketogenesis is constant; on the other hand, the contribution of ethanol to ketogenesis increases with time, presumably because of the accumulation of acetate in the perfusate. Ketogenesis is decreased in the presence of ethanol (but not acetate), while ketone body utilization is not affected by ethanol or acetate. Acetate contributes one third and ethanol contributes one half of the carbon incorporated into fatty acids and 3-beta-hydroxysterols. Only a small fraction (less than 5%) of the incorporation of acetate or ethanol into fatty acids and sterols occurs via transient incorporation into ketone bodies.     

Synthesis of methyl ketones by metabolically engineered Escherichia coli

Methyl ketones are a group of highly reduced platform chemicals with widespread applications in the fragrance, flavor and pharmacological industries. Current methods for the industrial production of methyl ketones include oxidation of hydrocarbons, but recent advances in the characterization of methyl ketone synthases from wild tomato have sparked interest towards the development of microbial platforms for the industrial production of methyl ketones. A functional methyl ketone biosynthetic pathway was constructed in Escherichia coli by over-expressing two genes from Solanum habrochaites: shmks2, encoding a 3-ketoacyl-ACP thioesterase, and shmks1, encoding a beta-decarboxylase. These enzymes enabled methyl ketone synthesis from 3-ketoacyl-ACP, an intermediate in the fatty acid biosynthetic cycle. The production of 2-nonanone, 2-undecanone, and 2-tridecanone by MG1655 pTH-shmks2-shmks1 was initially detected by nuclear magnetic resonance and gas chromatography–mass spectrometry analyses at levels close to 6?mg/L. The deletion of major fermentative pathways leading to ethanol (adhE), lactate (ldhA), and acetate (pta, poxB) production allowed for the carbon flux to be redirected towards methyl ketone production, doubling total methyl ketone concentration. Variations in methyl ketone production observed under different working volumes in flask experiments led to a more detailed analysis of the effects of oxygen availability on methyl ketone concentration in order to determine optimal levels of oxygen. The methyl ketone concentration achieved with MG1655 ?adhE ?ldhA ?poxB ?pta pTrcHis2A-shmks2-shmks1, the best performer strain in this study, was approximately 500?mg/L, the highest reported for an engineered microorganism. Through the establishment of optimal operating conditions and by executing rational metabolic engineering strategies, we were able to increase methyl ketone concentrations by almost 75-fold from the initial confirmatory levels.     

Growth of Candida utilis on ethanol and isopropanol

Candida utilis grew on ehtanol and an ethanol-isopropanol-water (22:2:1 vols) mixture but not on isopropanol alone. Acetone accumulated in all cultures containing isopropranol but its presence in the alcohol mixture did not lower growth rate or yield significantly when compared with growth experiments on ethanol alone. Growth rate and yield declined at ethanol concentrations greater than 1% (v/v) and 0.3% (v/v) respectively. In a 0.3% (v/v) alcohol mixture, acetate was found only during the exponential growth phase. In a 3% (v/v) mixture, acetate and ethyl acetate accumulated during growth whereas acetaldehyde was present only during the exponential growth phase.     

铁筷子提取物对肿瘤细胞增殖及COX-2 mRNA表达的抑制作用

研究不同铁筷子提取物对肿瘤细胞增殖及 COX-2 mRNA 表达的抑制作用。以铁筷子醇总提取物(TKZ1)、正丁醇萃取部位(TKZ2)、乙酸乙酯萃取部位(TKZ3)分别作用于 DU145、PC3、HeLa、HT-29、HepG2等肿瘤细胞,应用噻唑蓝实验(MTT 法)计算其对细胞增殖的抑制作用,应用荧光定量 PCR 技术检测TKZ1、TKZ2、TKZ3处理后的各肿瘤细胞中 COX-2 mRNA 的表达情况。结果表明：TKZ1、TKZ2、TKZ3均能显著抑制多种肿瘤细胞的增殖,与阴性对照组比较,其可以在 mRNA 水平上抑制 COX-2的表达,且呈明显的量效关系。说明铁筷子提取物对体外肿瘤细胞的增殖具有显著的抑制作用,其抗瘤机制可能与抑制肿瘤细胞中 COX-2 mRNA 的表达有关。