Preparation of extracts of culture liquids for gas-chromatographic determination of non-acidic fermentation products

A simplified extraction method of high efficiency has been worked out to permit the determination of small amounts of alcohols present in fermentation solutions. Methods of extracting are described for large and small amounts of culture media. The proposed method has been successfully applied to known amounts of alcohol in bacterial fermentation solutions. Clostridium acetobutylicum andZymomonas mobilis were used for this investigation. The method is described in details with theClostridium culture.     

Simultaneous determination of volatile and non-volatile acidic fermentation products of anaerobes by capillary gas chromatography

A method previously used for the analysis of organic acids in silage has been applied to the detection and quantification of acidic fermentation products (C₁ to C₆ volatile fatty acids, lactic and succinic acid) of rumen bacteria and anaerobic fungi grown in pure culture. The acids were converted to tertiary butyldimethylsilyl derivatives prior to separation on a 30 m DB1 capillary gas chromatographic column. The quantitative recoveries of formic and succinic acids were found to be comparable to the recoveries of other acids reported in the original study. The quantitative recovery of lactic acid was found to be dependent on storage of the samples at ambient temperature for at least 24 h following derivatization. The simultaneous determination of a wide range of volatile and non-volatile acidic products is an important feature of this method.     

Isolation from a methanogenic ferulate degrading consortium of an anaerobe that converts methoxyl groups of aromatic acids to volatile fatty acids

An anaerobic, non-motile, rod shaped bacterium is described which cleaves the phenylether bonds of methoxylated aromatic substrates to give the corresponding hydroxy aromatic derivatives and mixed volatile fatty acids, chain length, C₁, C₂ and C₄. The bacterium was isolated from an anaerobic digestor fed with contents from a wood fiber to alcohol fermentation plant, using anaerobic rolltube medium with ferulate as the carbon and energy source. Moles fatty acid produced per 100 mole of methoxyl group of aromatic substrate fermented were approximately: acetate, 14; butyrate, 18; and formate, 15. For the fermentation of equimolar amounts of methoxylated aromatic compounds, growth yields were proportional to the number of methoxylated groups per molecule, and the amount of cells per methoxyl group did not alter when phenylacrylate derivatives were used as substrates. The organism was unable to reduce the side-chain double bond of phenylacrylate derivatives. Coculture of the bacterium on ferulate with Methanospirillum hungatei, or Desulfovibrio in the presence of SO ₄ ⁼ resulted in no nett production of formate, and small quantities of methane and sulfide were produced respectively. The isolate utilized glucose, fructose, and lactate, but not methanol or H₂–CO₂ as growth substrates. Lactate, butyrate, acetate, formate and small quantities of H₂ were produced from glucose fermentation. No reduction of SO ₄ ⁼ or NO ₃ ^- occurred during fermentation of glucose or methoxylated aromatics and no growth occurred in the presence of oxygen.     

Tandem mass spectrometry for on-line fermentation monitoring

Summary Membrane introduction mass spectrometry has been employed for on-line determination of the major products and volatile metabolites ofBacillus polymyxa fermentation. Samples were introduced into the mass spectrometer via a direct insertion membrane probe in which the aqueous solution flowed past a membrane located in the ion source of the mass spectrometer. Concentrations of the products 2,3-butanediol, acetoin, ethanol and acetic acid in fermentation broth were measured by tandem mass spectrometry after permeation through the membrane and ionization by chemical ionization. External standards were employed for quantification and a large linear response range was available for each of the major products observed. Dissolved CO₂ and O₂, as well as CO₂ in the off gases, were also monitored on-line by mass spectrometry. The use of tandem mass spectrometry has allowed the identification of products that were not previously known to be present in measurable amounts.     

Microbiological Studies of Coli-aerogenes Bacteria

α-Ketoglutarate was obtained in a very small amount by the oxidative fermentation of acetate with either a growing culture or the washed cells of Escherichia coli. This microorganism was also observed to accumulate a considerable amount of α-ketoglutarate as the oxidation-product of C₄-dicarboxylic acids such as succinate, fumarate, malate and oxalacetate. The addition of acetate to the reaction mixtures containing either C₃- or C₄-acids brought about an increase in the yield of α-ketoglutarate. The bacteria of coli-aerogenes revealed an ability of oxidizing tricarboxylic acids under suitable conditions, but there was no noticeable production of α-ketoglutarate. The formation of glyoxylate was observed to occur during the degradation of citrate by the bacteria of coli-aerogenes. Finally, a cyclic mechanism of aerobic carbon-metabolism in the bacteria was propounded and discussed.     

Über die Bildung von flüchtigen Gärungsnebenprodukten durch Milchsäurebakterien

Zusammenfassung Die Bildung von flüchtigen Gärungsnebenprodukten durch Milchsäurebakterien wurde gaschromatographisch durch Analyse des Dampfraumes über den Kulturlösungen und Analyse von Ätherextrakten untersucht. Da von den meisten Substanzen nur sehr geringe Mengen gebildet werden, war es wegen des Vorkommens störender Substanzen in den Medien erforderlich, zur Kultur der Organismen synthetische Nährlösungen zu verwenden. In den Kulturlösungen der 3 homofermentativen Milchsäurebakterien Lactobacillus plantarum, Pediococcus cerevisiae und P. pentosaceus wurden nur Acetaldehyd, Acetoin, Diacetyl, sowie Spuren von 2- oder 3-Methyl-1-butanol, Isobutanol und 2 nicht identifizierte Substanzen gefunden. Bei den heterofermentativen Arten Lactobacillus brevis und Leuconostoc oenos wurde (neben Äthanol) zusätzlich die Bildung von sehr geringen Mengen von Propanol, i-Propanol, Essigsäureäthylester, n-Hexanol, 2,3-Butandiol, n-Octanol, n-Nonanol (oder Phenylacetaldehyd) und einiger weiterer Verbindungen nachgewiesen.

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On the formation of volatile by-products of fermentation by lactic acid bacteria

Summary The formation of volatile by-products of fermentation by several strains of lactic acid bacteria was investigated by gas liquid chromatography. Since only very small amounts of volatile compounds were formed, the synthetic media used for the growth of the bacteria had to be stripped by vacuum distillation from substances interfering with the analysis. The culture solutions were analysed by gas chromatography using both the head-space-technique and extraction with ethyl ether. The homofermentative species Lactobacillus plantarum, Pediococcus pentosaceus, and P. cerevisiae were found to form small amounts of acetaldehyde, acetoin, diacetyl and traces of 2- or 3-methyl-1-butanol, isobutanol and two compounds that were not identified. In the culture solutions of the heterofermentative species L. brevis and Leuconostoc oenos a greater number of substances could be detected. These bacteria formed, besides ethanol and the products of the homofermentative organisms, small amounts of propanol, isopropanol, ethylacetate, n-hexanol, 2,3-butandiol, n-octanol and a few unidentified compounds.

     

Olfactory responses of aquatic and terrestrial tiger salamanders to airborne and waterborne stimuli

Summary Electro-olfactograms (EOGs) were used to assess olfactory responding by aquatic larval and terrestrial adult tiger salamanders (Ambystoma tigrinum) to airborne volatile compounds, and volatile and non-volatile compounds in aqueous solution. Both forms of salamander showed saturation effects to presentations of airborne stimuli (Fig. 2). Saturation was not observed, however, to stimulus presentations in aqueous solution (Figs. 2, 3). When threshold values and concentration-response curve parameters were compared, non-volatile amino acids in solution were more potent stimuli for larvae while airborne volatiles were more potent stimuli for adults (Tables 1, 2). We infer that metamorphosis in the tiger salamander is accompanied by changes in olfactory response characteristics, due possibly to changes in receptor population, changes in perireceptor properties (e.g. mucus) or to changes in stimulus access.Abbreviations EOG electro-olfactogram - PPM (ppm) parts per million     

不同碳源和生长因子条件下粒毛盘菌代谢产物初步研究

采用GC-MS法对一种粒毛盘菌(Lachnum sp.)在不同碳源、生长因子条件下发酵代谢产物的挥发性成分组成与差异进行分析。结果显示,不同碳源和生长因子条件下产生的代谢产物不同,主要包括有机酸、胺类、烷烃类、酯类、醇类、吡咯等物质。分别以20 g/L的葡萄糖、蔗糖、淀粉为碳源的发酵液中检测到的挥发性代谢产物为7、7、10种;添加1 mg/L的V_C、V_(B1)、甘氨酸、色氨酸作为不同生长因子的发酵液中检测到的挥发性代谢产物分别为6、7、7、12种。结果显示粒毛盘菌YM406发酵代谢产物具有丰富的多样性,并且在不同的培养条件下产生的代谢产物存在一定的差异。     

Autochthonous fermentation starters for the industrial production of Negroamaro wines

The aim of the present study was to establish a new procedure for the oenological selection of Saccharomyces cerevisiae strains isolated from natural must fermentations of an important Italian grape cultivar, denoted as “Negroamaro”. For this purpose, 108 S. cerevisiae strains were selected as they did not produce H₂S and then assayed by microfermentation tests. The adopted procedure made it possible to identify 10 strains that were low producers of acetic acid and hydrogen sulphide and showed that they completed sugar consumption during fermentation. These strains were characterized for their specific oenological and technological properties and, two of them, strains 6993 and 6920, are good candidates as industrial starter cultures. A novel protocol was set up for their biomass production and they were employed for industrial-scale fermentation in two industrial cellars. The two strains successfully dominated the fermentation process and contributed to increasing the wines’ organoleptic quality. The proposed procedure could be very effective for selecting “company-specific” yeast strains, ideal for the production of typical regional wines. “Winery” starter cultures could be produced on request in a small plant just before or during the vintage season and distributed as a fresh liquid concentrate culture.     

Rock-degrading endophytic bacteria in cacti

A plant–bacterium association of the cardon cactus (Pachycereus pringlei) and endophytic bacteria promotes establishment of seedlings and growth on igneous rocks without soil. These bacteria weather several rock types and minerals, unbind significant amounts of useful minerals for plants from the rocks, fix in vitro N₂, produce volatile and non-volatile organic acids, and reduce rock particle size to form mineral soil. This study revealed the presence of large populations of culturable endophytic bacteria inside the seeds extracted from wild plants, from seeds extracted from the guano of bats feeding on cactus fruit, in seedlings growing from these seeds, in the pulp of fruit, and in small, mature wild plants, and are comparable in size to populations of endophytic populations in some agricultural crops. The dominant culturable endophytes were isolates of the genera Bacillus spp., Klebsiella spp., Staphylococcus spp., and Pseudomonas spp. Based on partial sequencing of the 16s rRNA gene, the isolated strains had low similarity to known strains in these genera. However, these strains have higher molecular similarity among endophytes obtained from seeds, endophytes from roots, and some bacterial strains from the rhizoplane. Seedlings developed from seeds with endophytes contain the similar species of endophytes in their shoots, possibly derived from the seeds. This study shows the involvement of endophytic bacteria in rock weathering by cacti in a hot, subtropical desert and their possible contribution to primary colonization of barren rock. This study proposes that cacti capable of acquiring diverse populations of endophytes may give them an evolutionary advantage to gain a foothold on highly uncompromising terrain.     

Determination of Steam-Volatile Organic Acids in Fermentation Media by Gas-Liquid Chromatography

Five gas chromatographic liquid phases (25% Carbowax 20 M plus 4% H₃PO₄, 17.5% dioctyl sebacate plus 7.5% sebacic acid, 17.5% dioctyl sebacate plus 7.5% docosanoic acid, 5% Tween 80, and 20% LAC-296 [poly (diethylene glycol adipate)] plus 2% H₃PO₄) were studied with respect to their utility in the separation and quantitation of steam-volatile organic acids commonly produced in fermentation. Optimal operating conditions and column stability for routine analysis were established. An Aerograph Hy-Fi gas chromatograph was used for all work, except the studies with Tween 80 in which an Aerograph A-90-C was employed. Chromatographic traces are presented of volatile fatty acid analyses with each of the liquid phases. Complete separation of all isomers of the fatty acids from C₂ to C₅ was accomplished by the Carbowax 20 M plus H₃PO₄, dioctyl sebacate plus sebacic acid, and dioctyl sebacate plus docosanoic acid columns. The latter two liquid phases were extremely unstable and proved to be unsatisfactory for analysis of aqueous samples. A column of Carbowax 20 M + H₃PO₄ separated steam-volatile organic acids completely. The volatile fatty acid isomers were separated by 5% Tween 80 somewhat less completely, and the peak shapes were not as sharp and symmetrical as that desired for good quantitative work. LAC-296 (20%) plus 2% H₃PO₄ proved to be the most satisfactory of the liquid phases for routine analysis of deproteinated in vitro rumen fermentation media. The column has been used for routine analysis of rumen fermentation fluid and in vitro rumen incubation fluid. All the organic acids from C₂ to C₅, except isobutyric, could be quantitated with this column. Stability of the column with the aqueous solutions was extremely good. The standard deviation of the analysis of each volatile acid component in a fermentation fluid was less than 0.5 molar per cent. The short-chain organic acids (C₂ to C₅) were shown to be extremely stable in aqueous solution for as long as 6 months after preparation for gas chromatographic analysis by protein precipitation with metaphosphoric acid-H₂SO₄ and refrigeration at 4 C in stoppered tubes.     

Biocatalytic reduction of short‐chain carboxylic acids into their corresponding alcohols with syngas fermentation

Short‐chain carboxylic acids generated by various mixed‐ or pure‐culture fermentation processes have been considered valuable precursors for production of bioalcohols. While conversion of carboxylic acids into alcohols is routinely performed with catalytic hydrogenation or with strong chemical reducing agents, here, a biological conversion route was explored. The potential of carboxydotrophic bacteria, such as Clostridium ljungdahlii and Clostridium ragsdalei, as biocatalysts for conversion of short‐chain carboxylic acids into alcohols, using syngas as a source of electrons and energy is demonstrated. Acetic acid, propionic acid, n‐butyric acid, isobutyric acid, n‐valeric acid, and n‐caproic acid were converted into their corresponding alcohols. Furthermore, biomass yields and fermentation stoichiometry from the experimental data were modeled to determine how much metabolic energy C. ljungdahlii generated during syngas fermentation. An ATP yield of 0.4–0.5 mol of ATP per mol CO consumed was calculated in the presence of hydrogen. The ratio of protons pumped across the cell membrane versus electrons transferred from ferredoxin to NAD⁺ via the Rnf complex is suggested to be 1.0. Based on these results, we provide suggestions how n‐butyric acid to n‐butanol conversion via syngas fermentation can be further improved. Biotechnol. Bioeng. 2013; 110: 1066–1077. © 2012 Wiley Periodicals, Inc.     

Alkali treatment and fermentation of straw for animal feed

The feed value of annual ryegrass straw was improved by treatment with various concentrations of NaOH or NH₃ followed by fermentation of the treated straw with a mixed culture of Cellulomonas sp. and Alcaligenes faecalis. Laboratory feeding trials with voles showed that NaOH or NH₃ treatment considerably increased the feed efficiency of straw, but apparently gave a poorly palatable product. Fermentation tended to decrease the in vitro rumen digestibility (IVRD) of alkali-treated straw. The fermentations were carried out aerobically on a semisolid straw matrix having 11–86% moisture. Treatment by both NaOH and NH₃ increased the IVRD of straw. NH₃ also increased the nitrogen content in straw. The optimum condition for alkaline treatment of the straw was 4–6% NaOH for 1 hr or with 3% NH₃ for four weeks at room temperature. A minimum of 63% moisture was needed for significant fermentation of the straw. The combined effects of NaOH treatment and fermentation more than doubled crude protein, doubled crude fat, and increased IVRD by 75%. The NH₃ plus fermentation treatment tripled crude protein, doubled crude fat, and increased IVRD by 60%. Acetic acid was the main volatile fatty acid in the fermented straw.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

An unstructured kinetic model to study NaCl effect on volatile ester fermentation by <Emphasis Type="Italic">Candida etchellsii</Emphasis> for soy sauce production

Salt-tolerant aromatic yeast is an important microorganism arising from the solid state fermentation of soy sauce. The fermentation kinetics of volatile esters by Candida etchellsii was studied in a batch system. The data obtained from the fermentation were used for determining the kinetic parameters of the model. Batch experimental results at four NaCl levels (180, 200, 220, and 240 g/L) were used to formulate the parameter estimation model. The kinetic parameters of the model were optimized by specifically designed Runge-Kutta Genetic Algorithms (GA). The resulting mathematical model for volatile ester production, cell growth and glucose consumption simulates the experimental data well. The resulting new model was capable of explaining the behavior of volatile ester fermentation. The optimized parameters (μ_o, X _max, K _i, α, β, Y _X/S, m, and Y _P/S) were characterized by a correlation of functions assuming salinity dependence. The kinetic models optimized by GA describe the batch fermentation process adequately, as demonstrated by our experimental results.     

Production of volatiles by degradation of lipids during manufacture of black tea

During manufacture of black tea, lipids are degraded to volatile constituents. Cis-3-hexenal was present in appreciable amounts in the various parts of fresh shoots and decreased in the second leaves during manufacture. There was a simultaneous increase in trans-2-hexenal. Linalol and methyl salicylate also increased appreciably during rolling and fermentation. Most of the volatiles were lost during the firing process. The above trend was borne out by the ‘potential’ of the leaves for the production of volatiles as indicated by the increased amounts of volatiles produced by homogenizing the tissue in water against controls homogenized in 0.1 N acid. The C₆-aldehydes present in the headspace of withered shoots increased significantly following mechanical damage. The major fatty acids of the lipids in the various parts of the shoots were linolenic, linoleic, palmitic, oleic and stearic acids. The ratio of linoleic to linolenic acid in the stems was much higher than that of the leaves or buds and this was reflected in its higher 'potential for formation of hexanal. During withering and rolling of the second leaves, the unsaturated fatty acids showed substantial losses compared with the saturated acids. It is suggested that the enzymic breakdown of membrane lipids initiate the formation of volatile carbonyl compounds which are partly responsible for the flavour of black tea.     

Effect of glyphosate contaminated feed on rumen fermentation parameters and in sacco degradation of grass hay and corn grain

Four rumen fistulated wethers were used to investigate the effect of glyphosate contaminated feed on rumen fermentation. The rations were based on corn silage, urea and a vitamin-mineral premix, either in the absence or presence of 0.77?g glyphosate per kg DM. Furthermore, rations were fed either with or without aromatic amino acid supplementation. During four periods of 28 days, sheep received each of the four dietary treatments according to a Latin square. After 14 days of adaptation rumen fermentation parameters (pH, ammonia, volatile fatty acids) were measured on day 15 over a five-hour period after the morning feeding. The remaining 13 days served for in sacco degradation studies with grass hay and corn grain. Ammonia (NH₃) and pH of rumen fluid were within the normal range for all dietary treatments (NH₃: 9.1 – 32.3?mmol·l^???1, pH: 6.2 – 6.7). Neither rumen fermentation parameters nor in sacco DM and NDF degradation of incubated feedstuffs were significantly affected by glyphosate, with or without aromatic amino acid supplementation. Kinetic profiles of the in sacco dry matter and NDF degradation of grass hay were almost identical for the dietary treatments.     

Fatty acids in green algae cultivated on a pilot-plant scale

Fatty acids fromChlorella vulgaris, Scenedesmus obliquus var.acutus and from a mixed culture of the two strains, Melnik, were converted to methyl esters, separated by gas chromatography, and identified by means of standards. The spectrum of fatty acids included both saturated and unsaturated acids (with odd and even numbers of carbon atoms) from C₁₂ to C₂₂. Fatty acids C_16:0, C_18:0 and C_20:3 were the major components in all cultures. Pure strains differed from the mixed culture in the production of C_18:1, C_12:0 and C_19:2 acids; the first of these was present in higher amounts in pure cultures only, the latter two being found in the mixed culture. The level of lipids was lower as compared to the literature data and their extractability was affected by the manner of preparation of algae and extraction conditions.     

Enzymic formation of carbonyls from linoleic acid in leaves of Phaseolus vulgaris

Homogenization of Phaseolus vulgaris leaves at acid pH results in the evolution of hexanal, cis-3- and trans-2-hexenal. With cell-free extracts of leaves, linoleic and linolenic acids are enzymically converted to their hydroperoxides (predominantly the 13-hydroperoxide isomers) and to hexanal or hexenal respectively. Activity was highest in young, dark-green leaves and was stimulated by Triton X-100. Oleic acid is not a substrate for these reactions. Both 9- and 13-hydroperoxides were cleaved to carbonyl fragments and are proposed as intermediates in the formation of volatile aldehydes and non-volatile ω-oxoacids in P. vulgaris leaves. Properties of the enzyme systems are described.