Effect of molecular hydrogen and carbon dioxide on chemo-organotrophic growth of Acetobacterium woodii and Clostridium aceticum

During growth of Acetobacterium woodii on fructose, glucose or lactate in a medium containing less than 0.04% bicarbonate, molecular hydrogen was evolved up to 0.1 mol per mol of substrate. Under an H₂-atmosphere growth of A. woodii with organic substrates was completely inhibited whereas under an H₂/CO₂-atmosphere rapid growth occurred. Under these conditions H₂+CO₂ and the organic substrate were utilized simultaneously indicating that A. woodii was able to grow mixotrophically. Clostridium aceticum differed from A. woodii in that H₂ was only evolved in the stationary phase, that the inhibition by H₂ was observed at pH 8.5 but not at pH 7.5, anf that in the presence of fructose and H₂+CO₂ only fructose was utilized.The hydrogenase activity of fructose-grown cells of C. aceticum amounted to only 12% of that of H₂+CO₂-grown cells. With A. woodii a corresponding decrease of the activity of this enzyme was not observed.     

Fumarate dissimilation and differential reductant flow by Clostridium formicoaceticum and Clostridium aceticum

Methanol and the O-methyl group of vanillate did not support the growth of Clostridium formicoaceticum in defined medium under CO₂-limited conditions; however, they were growth supportive when fumarate was provided concomitantly. Fumarate alone was not growth supportive under these conditions. Fumarate reduction (dissimilation) to succinate was the predominant electron-accepting, energy-conserving process for methanol-derived reductant under CO₂-limited conditions. However, when both reductant sinks, i.e., fumarate and CO₂, were available, reductant was redirected towards CO₂ in defined medium. In contrast, in undefined medium with both reductant sinks available, C. formicoaceticum simultaneously engaged fumarate dismutation and the concomitant usage of CO₂ and fumarate as reductant sinks. With Clostridium aceticum, fumarate also substituted for CO₂, and H₂ became growth supportive under CO₂-limited conditions. Fumarate dissimilation was the predominant electron-accepting process under CO₂-limited conditions; however, when both reductant sinks were available, H₂-derived reductant was routed towards CO₂, indicating that acetogenesis was the preferred electron-accepting process when reductant flow originated from H₂. Collectively, these findings indicate that fumarate dissimilation, not dismutation, is selectively used under certain conditions and that such usage of fumarate is subject to complex regulation.     

Production of acetic acid by Clostridium thermoaceticum in electrodialysis culture using a fermenter equipped with an electrodialyser

Electrodialysis culture of Clostridium thermoaceticum increased the yield of acetate by its continuous removal. In normal batch cultures without pH control the yield was 4.2 g acetic acid/800 ml, while in pH-controlled culture it was 16.8 g/800 ml. Although electrodialysis cultures gave almost the same yield (15.4 g/800 ml) as that in pH-controlled cultures, sparging CO₂ into the broth in electrodialysis culture increased the amount of acetic acid to 22.3 g/800 ml. CO₂ sparging into normal cultures with or without pH control did not significantly increase the amount of acetate produced but yields, in terms of amounts of glucose consumed, were higher than without sparging. The theoretical yield was almost obtained in pH-controlled, electrodialysis cultures with CO₂ sparging.The authors are with the Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860, Japan     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

Sugar and malic acid utilization and acetic acid formation byLeuconostoc oenos

Leuconostoc oenos ST8, isolated from an Argentinian red wine, utilized glucose, fructose and malic acid and produced acetic acid. Fructose was preferred to glucose as a source of carbohydrate. Malic acid was almost completely degraded in 3 days at 25°C. Acetic acid formation correlated with fructose utilization.     

Bioconversion of acid-hydrolyzed poplar hemicellulose to acetic acid byClostridium thermoaceticum

Summary Clostridium thermoaceticum was used to ferment carbohydrate released from pretreated oat splet xylan and hemicellulose isolated from hybrid poplar. Hydrolysis with dilute sulfuric acid (2.5% (v/v) for oat spelt xylan and 4.0% (v/v) for poplar hemicellulose) at 100°C for 60 min was found to release the highest concentration of fermentable substrate.C. thermoaceticum, when grown in non-pH controlled batch culture at 55°C under a headspace of 100% CO₂, typically produced 14gl^–1 acetic acid during a 48 h fermentation in medium containing 2% xylose. In fed-batch fermentations this organism was able to produce 42gl^–1 acetic acid after 116h when the concentration of xylose was maintained at approximately 2% and the pH was controlled at 7.0.     

Clostridium thermoautotrophicum species novum,a thermophile producing acetate from molecular hydrogen and carbon dioxide

Fourteen strains of a thermophilic, rod-shaped, peritrichously flagellatedClostridium species were isolated from various mud and soil samples. Round to slightly oval spores were formed in terminal position. The isolates were obligate anaerobes and grew chemolithotrophically with H₂ plus CO₂ as well as chemoorganotrophically with fructose, glucose, glycerate, or methanol. Under both conditions, acetate was the only organic fermentation product formed in significant amounts. The pH optimum for growth was 5.7; the marginal temperatures for growth wereT _min, 36°C;T _opt, 56–60°C; andT _max, 69/70°C. The DNA contained 53–55 mol% guanine plus cytosine. the isolated strains form a new clostridial species; the nameClostridium thermoautotrophicum is proposed.     

Thiobacillus plumbophilus spec. nov., a novel galena and hydrogen oxidizer

From an uranium mine three strains of rodshaped, mesophilic, chemolithoautotrophic bacteria were isolated. They grow by oxidation of H₂S, galena (PbS) and H₂. Anglesite (PbSO₄) is formed from galena. No ferrous iron is oxidized by the isolates. They grow between pH 4 and 6.5 at temperatures of about 9 to 41°C (optimum around 27°C). The G+C content of the DNA is around 66 mol %. Based on their ability to oxidize sulfur compounds, the new organisms belong to the genus Thiobacillus. No significant homology with Thiobacillus ferrooxidans and Thiobacillus cuprinus was detected by DNA-DNA hybridization. Therefore the new isolates represent a new species within the genus Thiobacillus. Based on the unusual growth on galena, we name the new species Thiobacillus plumbophilus (type strain Gro 7; DSM 6690).     

Monitoring growth and acetic acid secretion by a thermotolerantBacillus using conduction microcalorimetry

Summary A method is described to determine power of heat-time curves by conduction microcalorimetry in order to monitor the viability and ability of a thermotolerantBacillus strain to secrete acetic acid both during exponential growth and during stationary-phase. In this system secreted acetic acid is neutralized by an insoluble source of lime (dolime) which results in a poor correlation between optical density and culture dry weight. As an alternative, cells and residual dolime were rapidly resuspended in isothermal fresh medium with glucose in a conduction microcalorimeter. Heat evolution was rapid over a period of 200–800 s. Steady state heat evolution rate decreased as a function of culture time and did not correlate with: 1) specific growth rate: 2) viable cell number: 3) glucose consumption rate; or 4) acetic acid secretion rate. Glucose consumption and acetic acid secretion during the stationary growth phase were correlated with specific heat evolution rate. These initial results indicate that this technique may be useful for further development as an on-line flow or stopped-flow method to monitor the physiology of bacilli in response to nutrient depletion or growth inhibition.     

Gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium

Comparison of HaeIII- and HpaII-restriction profiles of PCR-amplified 16S-23S rDNA ITS regions of Gluconacetobacter sp. LMG 1529^T and SKU 1109 with restriction profiles of reference strains of acetic acid bacteria described by Tr?ek and Teuber [34] revealed the same but unique restriction profiles for LMG 1529^T and SKU 1109. Further analyses of nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rDNA ITS sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated both strains to a single phylogenetic cluster well separated from the other species of the genus Gluconacetobacter. DNA–DNA hybridizations confirmed their novel species identity by 73% DNA–DNA relatedness between both strains, and values below the species level (<70%) between SKU 1109 and the type strains of the closest phylogenetic neighbors. The classification of strains LMG 1529^T and SKU 1109 into a single novel species was confirmed also by AFLP and (GTG)₅-PCR DNA fingerprinting data, as well as by phenotypic data. Strains LMG 1529^T and SKU 1109 can be differentiated from their closely related Gluconacetobacter species, Gluconacetobacter entanii and Gluconacetobacter hansenii, by their ability to form 2-keto-d-gluconic acid from d-glucose, their ability to use d-mannitol, d-gluconate and glycerol as carbon source and form acid from d-fructose, and their ability to grow without acetic acid. The major fatty acid of LMG 1529^T and SKU 1109 is C_18:1ω7c (60.2–64.8%). The DNA G + C content of LMG 1529^T and SKU 1109 is 62.5 and 63.3 mol% respectively. The name Gluconacetobacter maltaceti sp. nov. is proposed. The type strain is LMG 1529^T (= NBRC 14815^T = NCIMB 8752^T).     

Effect of growth supplements and whey pretreatment on butyric acid production by Clostridium butyricum

Improved production of butyrate (up to 19 g/l) from whey by Clostridium butyricum was achieved by adding either yeast extract (5 g/l) or biotin (50 g/l). Hydrolysed lactose and proteolysed whey were less effective even with added biotin.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Technical University, Radlinského 9, Bratislava 812 37, Slovakia     

Growth characteristics of Geotrichum ingens on propionic acid and acetic acid in batch and continuous culture

Growth of Geotrichum ingens in batch cultures was completely inhibited by 47 g acetic acid/l or 33 g propionic acid/I. With mixtures of acetic and propionic acids, however, growth only ceased at 55 g/l. Acetic acid inhibited growth linearly, whereas propionic acid inhibited growth non-linearly. In continuous culture, two steady states at each dilution rate were observed at high dilution rates for acetic acid and propionic acid. The highest yield coefficient (0.69 g cells/g substrate) was achieved with propionic acid as substrate. On both substrates and their mixtures, the protein content of the biomass increased when the dilution rate was increased.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa     

Accumulation and incorporation of 185W-tungsten into proteins of Clostridium acidiurici and Clostridium cylindrosporum

Uptake of tungstate by growing cells was unaffected by the presence of molybdate in Clostridium cylindrosporum, whereas in C. acidiurici the accumulation was decreased by molybdate at 10^-6 mol/l tungstate and higher concentrations. The labelling pattern of soluble proteins by ¹⁸⁵W-tungsten indicated after gel chromatography the presence of three different tungstoproteins in both bacteria. Formate dehydrogenase activity always eluted at a maximum of tungsten labelling. The incorporation of tungsten into formate dehydrogenase containing fractions and a possible tungsten-binding-storage protein was independent of the presence of excess molydate pointing to a genuine role for tungstate in these bacteria.     

Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH

Sixty yeast strains were previously screened for their ability to produce acetic acid, in shaken flask batch culture, from either glucose or ethanol. Seven of the strains belonging to the Brettanomyces and Dekkera genera, from the ARS Culture Collection, Peoria, IL, were further evaluated for acetic acid production in bioreactor batch culture at 28 °C, constant aeration (0.75 v/v/m) and pH (6.5). The medium contained either 100 g glucose/l or 35 g ethanol/l as the carbon/energy source. Dekkera intermedia NRRL YB-4553 produced 42.8 and 14.9 g acetic acid/l from the two carbon sources, respectively, after 64.5 h. The optimal pH was determined to be 5.5. When the initial glucose concentration was 150 or 200 g/l, the yeast produced 57.5 and 65.1 g acetic acid/l, respectively.     

Aeration-controlled formation of acetic acid in heterolactic fermentations

Summary Controlled aeration ofLeuconostoc mesenteroides was studied as a possible mechanism for control of the formation of acetic acid a metabolite of major influence on the taste of lactic fermented foods. Fermentations were carried out in small scale in a medium in which growth was limited by the buffer capacity only. Ethanol and acetic acid formed during the fermentation were analyzed by rapid head space gas chromatography, and the ratio of the molar concentrations of these two volatiles quantitatively predicted the balance between the formation of acetic acid and lactic acid. The oxygen concentration during the fermentations decreased rapidly to zero, meaning that oxygen transfer was limited by the volumetric oxygen transfer rate,k ₁ aC ^*. A linear correlation between k₁aC^* and the quantity of acetic acid produced was established, and it is suggested that such oxygenated heterolactic fermentation processes should be analyzed as fed-batch fermentations with oxygen as the limiting substrate. Addition of fructose in limited amounts leads to the formation of one half mole of acetic acid for each mole fructose, thus offering an alternative mechanism for controlling acetic acid formation.     

The internal pH of Acetobacterium wieringae and Acetobacter aceti during growth and production of acetic acid

The intracellular pH was measured in growing Acetobacterium wieringae and Acetobacter aceti with an acid equilibrium distribution method. [¹⁴C]-acetylsalicylic acid, [¹⁴C-benzoic acid and [¹⁴C]-acetic acid were used as pH-indicators. The extracellular pH of Acetobacterium wieringae decreased from 7.0 to 5.0 during growth; accordingly, the intracellular pH changed from 7.1 to 5.5, and a pH between 0.1 and 0.65 (interior more alkaline) was maintained. Corresponding results were obtained for Acetobacter aceti. The external pH and the internal pH decreased in parallel from 6.2 to 3.5 and from 5.8 to 3.9, respectively.This demonstrates that neither the anaerobic nor the aerobic acetogen was able to maintain a large pH in the presence of high concentrations of acetic acid.     

Buffering capacity and membrane H+ conductance of acetic acid bacteria

Summary Buffering capacities and membrane conductance to H⁺ were measure inAcetobacter aceti ATCC 15973 andGluconobacter oxydans ATCC 621 by a pulse technique. In both strains the buffering capacity of intact cells was a significant proportion of the total buffering capacity, but the magnitude of the buffering capacity varied between one species and another. Over the pH range studied, 4.02 to 8.15,Gluconobacter oxydans, which oxidizes sugars and alcohols to acids and accumulates them, showed lower values of buffering capacities and membrane conductance to protons thanAcetobacter aceti, which oxidizes these substrates completely to CO₂ and H₂O.     

Selenium requirement for active xanthine dehydrogenase from Clostridium acidiurici and Clostridium cylindrosporum

The xanthine dehydrogenase of Clostridium acidiurici and C. cylindrosporum was assayed with methyl viologen as acceptor. In C. acidiurici the basal activity level was about 0.3 mol/min x mg of protein. Cells grown on uric acid in the presence of 10^-7 M selenite showed a 14-fold increase in xanthine dehydrogenase activity, which decreased with higher selenite concentrations (10^-5 M). The supplementation with 10^-7 M molybdate or tungstate was without effect. High concentrations of tungstate decreased the xanthine dehydrogenase if selenite was also present. In comparison, high concentrations of molybdate affected only a small decrease in activity level at the optimal concentration for selenite and relieved to some degree the inhibitory effect of 10^-5 M selenite. With hypoxanthine and xanthine as substrates for growth again only the addition of selenite was necessary to show a similar increase in xanthine dehydrogenase activity. C. acidiurici could be grown in a mineral medium. Both xanthine dehydrogenase and formate dehydrogenase exhibited the highest level of activity if selenite and tungstate were present in that medium.In C. cylindrosporum the basal activity level of xanthine dehydrogenase was about 0.95 mol/min x mg of protein. The addition of 10^-7 M selenite to the growth medium increased the activity level about 3-fold, but the highest level (3.7 U/mg) was reached if 10^-7 M molybdate was also added. The presence of tungstate resulted in a decreased enzyme activity.     

Utilization of (E)-2-butenoate (Crotonate) by Clostridium kluyveri and some other Clostridium species

Clostridium La 1 obtained from a Clostridium kluyveri culture was compared with a typical C. kluyvery strain (DSM 555). The former grows on crotonate and is unable to use ethanol-acetate as carbon sources. The latter grows on crotonate only after long adaptation periods. Resting cells of both strains show also pronounced differences in the fermentation of crotonate. This holds even for C. kluyveri grown on crotonate. Besides several other differences the most striking is that there is no hybridization between the DNA of both strains.Crotonate seems not to be a very special carbon source since C. butyricum and C. pasteurianum grow on crotonate medium supplemented by peptone and yeast extract.Non Standard Abbreviations EA-medium ethanol and acetate as carbon source - C-medium crotonate as carbon source - DSM Deutsche Sammlung von Mikroorganismen