Enrichment and isolation of Acetitomaculum ruminis,gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen

Five strains of acetogenic bacteria were isolated by selective enrichment from the rumen of a mature Hereford crossbred steer fed a typical high forage diet. Suspensions of rumen bacteria, prepared from contents collected 7 h postfeeding, blended and strained through cheesecloth, were incubated in a minimal medium containing 10% clarified rumen fluid under either H₂:CO₂ (80:20) or N₂:CO₂ (80:20) headspace atmosphere. The selection criterion was an increment of acetate in the enrichments incubated under H₂:CO₂. Periodically, the enrichment broths were plated onto agar media and presumed acetogenic bacteria subsequently were screened for acetate production. Selected acetogenic bacteria utilized a pressurized atmosphere of H₂:CO₂ to form acetate in quantities 2 to 8-fold higher than when grown under N₂:CO₂. All presumptive acetogenic isolates were derived from either the 10^-7 or 10^-8 dilutions of rumen contents. All 5 strains were Gram-positive rods, and all utilized formate, glucose and CO. One strain required, and all were stimulated by, rumen fluid. No spores were observed with phase-contast microscopy and two strains were motile. No methane was detected in the headspace of pure cultures grown under either gas phase. The isolation of these bacteria indicates that acetogenic bacteria are inhabitants of the rumen of the bovine fed a typical diet and suggests that they may be participants in the utilization of hydrogen in the rumen ecosystem. Strain 139B (= ATCC 43876) is named Acetitomaculum ruminis gen. nov., sp. nov. and is the type strain of this new species. Portions of this work were presented previously (Greening RC, Leedle JAZ (1987) Abstr Annu Meet Am Soc Microbiol I 131, pp 194)     

A new H2 / CO2-using acetogenic bacterium from the rumen: Description of Ruminococcus schinkii sp. nov.

Abstract Two strains of H2 / CO2-using acetogenic bacteria were isolated from the rumen of suckling lambs. Both strains displayed a coccobacillar morphology and possessed a Gram-positive type cell wall. Numerous organic substrates, including some O-methylated aromatic compounds, were used heterotrophically. 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa. Based on phenotypic and phylogenetic considerations a new species, Ruminococcus schinkii sp. nov., is proposed.     

The immune response to Lactococcus lactis: Implications for its use as a vaccine delivery vehicle

Abstract The development of hydrogenotrophic bacteria in the rumen of lambs was investigated by culture and labeling experiments. ¹⁴CO₂ and ¹³CO₂ incorporation by the rumen microflora of a 24-h-old lamb showed that while there was no labeled methane, double-labeled acetate was formed indicating the presence of hydrogen-dependent acetogenesis. In vitro counts from rumen fluid of 20-h-old lambs confirmed an extensive colonization of acetogenic bacteria while methanogens were absent. Methanogens appeared in the rumen of 30-h-old lambs, and as they developed there was a proportional decrease in the numbers of acetogens, indicating a competition for hydrogen between these two groups. Hydrogen-utilizing sulfate-reducing bacteria, which were established by the 3rd day after birth, did not seem to be affected by this competition.     

Isolation from the rumen of a new acetogenic bacterium phylogenetically closely related to Clostridium difficile

Five strains of filamentous acetogenic bacterium were isolated from high dilutions of ruminal content of newborn lambs. These Gram-positive spore-forming bacteria grew either chemolithotrophically with H2+ CO2 or chemo-organotrophically with glucose, cellobiose, fructose, maltose, mannose and syringic acid. The DNA base composition of the five strains were between 29.1 and 31.3 mol% G + C. Their temperature and pH optimum for growth were 35-40 degrees C and 6.5-7.0, respectively. The full 16S rRNA gene sequence analysis of the reference strain indicated that it was most closely related to Clostridium difficile. The sequence similarity value between the 16S rRNA gene of the reference strain and this pathogenic strain was 99.7%.     

New species of psychrophilic acetogens: Acetobacterium bakii sp. nov., A. paludosum sp. nov., A. fimetarium sp. nov.

Three strains of new acetogenic bacteria were isolated from several low temperature environments. Cells were gram-positive, oval-shaped flagellated rods. The organisms fermented H₂/CO₂, CO, formate, lactate, and several sugars to acetate. Strains Z-4391 and Z-4092 grew in the temperature range from 1 to 30°C with an optimum at 20°C; strain Z-4290 grew in the range from 1 to 35°C with an optimum at 30°C. The DNA G+C content of strains Z-4391, Z-4092, and Z-4290 was 42.1, 41.7, and 45.8 mol% respectively.     

Acetobacterium tundrae sp. nov., a new psychrophilic acetogenic bacterium from tundra soil

A new psychrophilic, anaerobic, acetogenic bacterium from the tundra wetland soil of Polar Ural is described. The organism fermented H2/CO2, formate, methanol, and several sugars to acetate as the sole end-product. The temperature range for growth was 1-30 degrees C with an optimum at 20 degrees C. The bacterium showed no growth at 32 degrees C. Cells were gram-positive, oval-shaped, flagellated rods 0.7-1.l x 1.1-4.0 microm in size when grown at 1-20 degrees C. At 25-30 degrees C, the cell size increased up to 2-3 x 10-15 microm due to a defect in cell division. The DNA G+C content of the organism was 39.2 mol%. Based upon 16S rDNA analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium as a new species, for which the name Acetobacterium tundrae sp. nov. is proposed. The type strain is Z-4493 (=DSM 9173T).     

The Effects on Temperature on Growth in vitro of Pseudomonas syringae and Xanthomonas pruni

Growth rates in vitro of Pseudomonas syringae and Xanthomonas pruni were measured over the temperature range 0–36 °C. The estimated temperature optimum for X. pruni was 31 °C, with a doubling time of 1.53 h. The estimated temperature optimum for P. syringae was 28 °C with a doubling time of 1.27 h, although analysis showed no significant difference in the doubling times over the range 23–33 °C, indicating an unusual plateau at the maximum rate of growth of this organism. P. syringae and related plant pathogenic Pseudomonas spp. grew well at low temperatures, but X. pruni did not. Cultures of P. syringae and X. pruni had a very short lag phase after their incubation temperature was changed from 4 °C to a temperature close to their optimum (29 °C). When the incubation temperature of these organisms was changed from 11.5–29 °C, X. pruni grew without a lag phase at the rate expected for the higher temperature. However, the initial growth rate of P. syringae at the higher temperature was significantly greater than that at which the organism subsequently developed. The ecological significance of these points is discussed. The usefulness of the Arrhenius coefficients as characteristics of these organisms is discussed.     

Isolation and Characterization of an H2-Oxidizing Thermophilic Methanogen

A thermophilic methanogen was isolated from enrichment cultures originally inoculated with sludge from an anaerobic kelp digester (55°C). This isolate exhibited a temperature optimum of 55 to 60°C and a maximum near 70°C. Growth occurred throughout the pH range of 5.5 to 9.0, with optimal growth near pH 7.2. Although 4% salt was present in the isolation medium, salt was not required for optimal growth. The thermophile utilized formate or H₂-CO₂ but not acetate, methanol, or methylamines for growth and methanogenesis. Growth in complex medium was very rapid, and a minimum doubling time of 1.8 h was recorded in media supplemented with rumen fluid. Growth in defined media required the addition of acetate and an unknown factor(s) from digester supernatant, rumen fluid, or Trypticase. Cells in liquid culture were oval to coccoid, 0.7 to 1.8 μm in diameter, often occurring in pairs. The cells were easily lysed upon exposure to oxygen or 0.08 mg of sodium dodecyl sulfate per ml. The isolate was sensitive to tetracycline and chloramphenicol but not penicillin G or cycloserine. The DNA base composition was 59.69 mol% guanine plus cytosine.     

Characterization of aMethanosarcina strain isolated from goat feces,and that grows on H2-CO2 only after adaptation

AMethanosarcina species, designated strain ChGul, was isolated from goat feces; this is the first fully described pure culture ofMethanosarcina obtained from feces. Antigenic fingerprinting suggests that isolate ChGul is a new immunotype. The mol% G + C content of DNA was 42.2%. Strain ChGul grew on methanol, methylamines, and acetate in a minimal salts medium. It grew on H₂-CO₂ only after adaptation. Growth occurred as a milky-white suspension and contained cells mostly in doublets and quadruplets of irregular cocci; many cells contained phase bright spots typical of gas vacuoles. The isolate did not grow on formate, or CO₂ plus isopropanol, ethanol, or acetone as substrates and did not produce methane from formate. The optimum growth temperature was 35–37°C, and optimum pH was 6.2–6.8. ChGul is unusually sensitive to sulfide and has low tolerance for NaCl. Optimal levels of total sulfide and NaCl for growth were 0.5 mM and 20–40 mM, respectively. Since ChGul requires adaptation for growth on H₂-CO₂ and cannot use formate, it may be restricted to methylotropic or acetoclastic methanogenesis in the rumen, a function not observed in previously isolated rumen methanogens that use H₂-CO₂ and formate. Our work suggests that improper NaCl and sulfide concentrations, and cell lysis, may have made isolation of rumenMethanosarcina difficult in the past. It also underscores the need to evaluate feed compositions and media components for most probable number studies, with respect to NaCl and sulfide levels, to understand the role ofMethanosarcina in the rumen.     

Isolation and characterization of an h(2)-oxidizing thermophilic methanogen

A thermophilic methanogen was isolated from enrichment cultures originally inoculated with sludge from an anaerobic kelp digester (55 degrees C). This isolate exhibited a temperature optimum of 55 to 60 degrees C and a maximum near 70 degrees C. Growth occurred throughout the pH range of 5.5 to 9.0, with optimal growth near pH 7.2. Although 4% salt was present in the isolation medium, salt was not required for optimal growth. The thermophile utilized formate or H(2)-CO(2) but not acetate, methanol, or methylamines for growth and methanogenesis. Growth in complex medium was very rapid, and a minimum doubling time of 1.8 h was recorded in media supplemented with rumen fluid. Growth in defined media required the addition of acetate and an unknown factor(s) from digester supernatant, rumen fluid, or Trypticase. Cells in liquid culture were oval to coccoid, 0.7 to 1.8 mum in diameter, often occurring in pairs. The cells were easily lysed upon exposure to oxygen or 0.08 mg of sodium dodecyl sulfate per ml. The isolate was sensitive to tetracycline and chloramphenicol but not penicillin G or cycloserine. The DNA base composition was 59.69 mol% guanine plus cytosine.     

Isolation and characterization of a temperate bacteriophage from a ruminal acetogen

Nine acetogenic bacterial cultures recently isolated from the bovine rumen were tested for phage susceptibility by plaque formation. Both clear plaques and plaques with turbid centers were occasionally seen, but could not be used repeatedly to lyse pure cultures of acetogens, suggesting the possibility of a temperate phage. Five of the nine acetogenic isolates showed a response to mitomycin C induction. Acetogenic isolate H3HH was chosen for further study because it produced the greatest lysogenic response to mitomycin C. The bacteriophage was induced with mitomycin C, examined by transmission electron microscopy, and shown to have a hexagonal head (diameter, 59 m), a long flexible tail (192 m), and a flat collar (diameter, 31 m). The bacteriophage was classified within Bradley's group B. Bacteriophage DNA was determined to contain 36.2 kilobases of linear double-stranded DNA.Journal Paper #14665 of the Agricultural Research Programs of Purdue University.     

Effect of Yeast Extract on Growth and Metabolism of H2-Utilizing Acetogenic Bacteria from the Human Colon

The aim of this work was to determine the effect of yeast extract and of its vitamin contents on autotrophic and heterotrophic growth and metabolism of four acetogenic bacteria from the human colon. Yeast extract exerted a stimulatory effect on autotrophic growth of the colonic acetogens, but concentration of this compound above 1–2 g. L⁻¹ in the medium did not enhance utilization of H₂/CO₂. Vitamins provided by yeast extract were shown to be essential cofactors of the reductive pathway of acetate synthesis except for one Clostridium strain. Yeast extract was also necessary to maintain heterotrophic growth and acetate synthesis from glucose in acetogenic species, except in the Streptococcus strain. In the absence of yeast extract, vitamins could efficiently restore glucose fermentation via acetate. The reductive and oxidative pathways of acetate synthesis might, therefore, depend on vitamin cofactors supplied by yeast extract in most of the human acetogenic bacteria. Non-vitaminic factors appeared also to be involved in the metabolism of some of these acetogenic species. Received: 6 March 1998 / Accepted: 3 April 1998     

Characterization of the psychrotolerant acetogen strain SyrA5 and the emended description of the species Acetobacterium carbinolicum

A psychrotolerant, obligate anaerobic, acetogenic bacterium designated strain SyrA5 was isolated from black anoxic sediment of a brackish fjord. Cells were Gram-positive, non-sporeforming rods. The isolate utilized H(2)/CO(2), CO, fructose, glucose, ethanol, ethylene glycol, glycerol, pyruvate, lactate, betaine and the methyl-groups of several methoxylated benzoic derivatives such as syringate, trimethoxybenzoate and vallinate. The optimum temperature for growth was 29 degrees C, whilst slow growth occurred at 2 degrees C. The strain grew optimally with NaCl concentrations below 2.7% (w/v), but growth occurred up to 4.3% (w/v) NaCl. Growth was observed in the range from pH 5.9 to 8.5, optimum at pH 8. The G+C content was 44.1 mol%. Based upon 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium. Strain SyrA5 shared a 16S rRNA sequence similarity with A. carbinolicum of 100%, a fthfs gene (which codes for the N5,N10 tetrahydrofolate synthetase) sequence identity of 98.5-98.7% (amino acid sequence similarities were 99.4-100%) and a RNA-DNA hybridization homology of 64-68%. Despite a number of phenotypic differences between strain SyrA5 and A. carbinolicum we propose including strain SyrA5 as a subspecies of A. carbinolicum for which we propose the name Acetobacterium carbinolicum subspecies kysingense. The type strain is SyrA5 (=DSM 16427(T), ATCC BAA-990).     

Survival, development and food energy partitioning of nase larvae and early juveniles at different temperatures

Survival was generally high, 94–100%, for newly hatched larvae of the nase Chondrostoma nasus held at 10, 13, 16, 19, 22, 25 and 28° C up to day 66 post-fertilization. The developmental rate decreased with age and increased with temperature. Specific growth rates increased with temperature; within one temperature range growth rate decreased with ontogenetic development. Food consumption and respiration increased with temperature and body size. A temperature increase from 25 to 28° C resulted in slightly reduced survival, minor acceleration of developmental growth and respiration rates, and impeded skeleton formation. Growth efficiency of consumed energy decreased throughout the larval period from 55 to 67% at the first larval stage (L1) to 36–48% at the first juvenile stage (J₁). A similar trend for assimilation efficiency and its utilization for growth was observed. The constant temperatures required by larval nase ranged from a minimum 8–10° C to a maximum 25–28° C. A shift of optimum temperatures, 8–12, 13–16, 15–18, 19 and 22° C for nase spawning, embryonic development, yolk feeding larvae, early externally feeding larvae and, late larvae and juveniles, respectively, paralleled the spring rise in the river water temperature. Larval and juvenile nase show high survival, growth and energy conversion efficiencies compared with other fish species. On the other hand, low survival rates and growth can be attributed to external perturbations; thus, young nase may be considered a good indicator of the environmental and ecological integrity of river systems.     

Cultural conditions for production of glucoamylase from Lactobacillus amylovorus ATCC 33621

Lactobacillus amylovorus ATCC 33621 is an actively amylolytic bacterial strain which produces a cell-bound glucoamylase (EC 3.2.1.3). Conditions of growth and glucoamylase production were investigated using dextrose-free de Man-Rogosa-Sharpe (MRS) medium in a 1.5 I fermenter, with varying dextrin concentration (0.1–1.5% (w/v)), pH (4.5–6.5) and temperature (25–55°C). Cell extracts were prepared by subjecting cells to treatment with a French Pressure cell in order to release intracellular proteins. Glucoamylase activity was then assayed. The effects of pH (4.0–9.0), temperature (15–85°C) and substrate (dextrin and starch, 0–2% w/v) concentration on crude enzyme activity were investigated. Optimal growth was obtained in MRS medium containing 1% (w/v) dextrin, at pH 5.5 and 37°C. Glucoamylase production was maximal at the late logarithmic phase of growth, during 16–18 h. Crude enzyme had a pH optimum of 6.0 and temperature optimum of 60°C. With starch as the substrate, maximal activity was obtained at a concentration of 1.5% (w/v). The effects of ions and inhibitors on glucoamylase activity were also investigated. Enzyme activity was not significantly influenced by Ca²⁺ and EDTA at 1 mmol 1⁻¹ concentration; however Pb²⁺ and Co²⁺ were found to inhibit the activity at concentrations of 1 mmol 1⁻¹. The crude enzyme was found to be thermolabile when glucoamylase activity decreased after about 10 min exposure at 60°C. This property can be exploited in the brewing of low calorie beers where only mild pasteurization treatments are used to inactivate enzymes. The elimination of residual enzyme effect would prevent further maltodextrin degradation and sweetening during long-term storage, thus helping to stabilize the flavour of beer.     

一株新型同型产乙酸菌Clostridium sp.的自养和异养生长特性

【背景】同型产乙酸菌是一类利用乙酰辅酶A途径固定CO_2合成自身细胞物质并生成乙酸、乙醇等代谢产物的厌氧菌群,其分布广泛、种类繁多且代谢多样。深入研究同型产乙酸菌菌株的代谢能力及特性,对探索该种群的生理生化特性及其环境作用至关重要。【目的】研究一株同型产乙酸菌Clostridium sp. BXX的最适培养条件及其自养与异养生长特性。【方法】设置BXX菌株培养温度10-55°C、初始pH 6.0-9.0、NaCl浓度0-2.0%、不同氮源,测定菌体细胞含量和产物生成浓度,确定菌株最适培养条件。研究BXX菌株分别以H_2/CO_2、合成气、CO、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚、甘油、丙酮酸和乳酸为底物时的底物消耗、产物生成、菌体细胞含量和pH等,探究其自养和异养生长特性。【结果】BXX菌株的最适培养温度为30°C,初始pH为7.0,NaCl浓度为1.0%,氮源为酵母粉。BXX菌株能以H2/CO2、合成气、葡萄糖、1,2-丙二醇、甲酸钠、乙二醇甲醚和甘油为底物生长,不能以CO、丙酮酸或乳酸为底物生长。【结论】BXX菌株既能自养生长产乙酸,又能异养生长产乙醇。BXX菌株是乙酸发酵的优良菌种资源,有较好的工业应用潜力。     

Butyrate production in the acetogen Eubacterium limosum is dependent on the carbon and energy source

Eubacterium limosum KIST612 is one of the few acetogenic bacteria that has the genes encoding for butyrate synthesis from acetyl-CoA, and indeed, E. limosum KIST612 is known to produce butyrate from CO but not from H₂ + CO₂. Butyrate production from CO was only seen in bioreactors with cell recycling or in batch cultures with addition of acetate. Here, we present detailed study on growth of E. limosum KIST612 on different carbon and energy sources with the goal, to find other substrates that lead to butyrate formation. Batch fermentations in serum bottles revealed that acetate was the major product under all conditions investigated. Butyrate formation from the C1 compounds carbon dioxide and hydrogen, carbon monoxide or formate was not observed. However, growth on glucose led to butyrate formation, but only in the stationary growth phase. A maximum of 4.3 mM butyrate was observed, corresponding to a butyrate:glucose ratio of 0.21:1 and a butyrate:acetate ratio of 0.14:1. Interestingly, growth on the C1 substrate methanol also led to butyrate formation in the stationary growth phase with a butyrate:methanol ratio of 0.17:1 and a butyrate:acetate ratio of 0.33:1. Since methanol can be produced chemically from carbon dioxide, this offers the possibility for a combined chemical-biochemical production of butyrate from H₂ + CO₂ using this acetogenic biocatalyst. With the advent of genetic methods in acetogens, butanol production from methanol maybe possible as well.     

Dichloromethane as the sole carbon source for an acetogenic mixed culture and isolation of a fermentative, dichloromethane-degrading bacterium.

Dichloromethane (DCM) is utilized by the strictly anaerobic, acetogenic mixed culture DM as a sole source of carbon and energy for growth. Growth with DCM was linear, and cell suspensions of the culture degraded DCM with a specific activity of 0.47 mkat/kg of protein. A mass balance of 2 mol of chloride and 0.42 mol of acetate per mol of DCM was observed. The dehalogenation reaction showed similar specific activities under both anaerobic and aerobic conditions. Radioactivity from [14C]DCM in cell suspensions was recovered largely as 14CO2 (58%), [14C]acetate (23%), and [14C]formate (11%), which subsequently disappeared. This suggested that formate is a major intermediate in the pathway from DCM to acetate. Efforts to isolate from culture DM a pure culture capable of anaerobic growth with DCM were unsuccessful, although overall acetogenesis and the partial reactions are thermodynamically favorable. We then isolated bacterial strains DMA, a strictly anaerobic, gram-positive, endospore-forming rod, and DMB, a strictly anaerobic, gram-negative, endospore-forming homoacetogen, from culture DM. Both strain DMB and Methanospirillum hungatei utilized formate as a source of carbon and energy. Coculture of strain DMA with either M. hungatei or strain DMB in solid medium with DCM as the sole added source of carbon and energy was observed. These data support a tentative scheme for the acetogenic fermentation of DCM involving interspecies formate transfer from strain DMA to the acetogenic bacterium DMB or to the methanogen M. hungatei.     

Diversity of H2/CO2-Utilizing Acetogenic Bacteria from Fecesof Non-Methane-Producing Humans

The purpose of this work was to study H₂/CO₂-utilizing acetogenic population in the colons of non-methane-producing individuals harboring low numbers of methanogenic archaea. Among the 50 H₂-consuming acetogenic strains isolated from four fecal samples and an in vitro semi-continuous culture enrichment, with H₂/CO₂ as sole energy source, 20 were chosen for further studies. All isolates were Gram-positive strict anaerobes. Different morphological types were identified, providing evidence of generic diversity. All acetogenic strains characterized used H₂/CO₂ to form acetate as the sole metabolite, following the stoichiometric equation of reductive acetogenesis. These bacteria were also able to use a variety of organic compounds for growth. The major end product of glucose fermentation was acetate, except for strains of cocci that mainly produced lactate. Yeast extract was not necessary, but was stimulatory for growth and acetogenesis from H₂/CO₂. Received: 28 December 1995 / Accepted: 30 January 1996     

Effects of Temperature on Germination of Peronospora parasitica Conidia and Infection of Brassica oleracea

The effect of temperature on germination of a South African isolate of Peronospora parasitica , and infection of Brassica oleracea was studied. The optimum condition for germination was 20° C at 100% relative humidity. The percentage germination obtained was 80–98% and 70–80% between 15 and 25° C at 100% relative humidity, after a 12 and 6h incubation period, respectively. Optimum temperature for germ tube growth was also 20° C. The temperature range for maximum infection of seedlings of a highly susceptible cabbage cultivar and subsequent disease development in vitro was 15–25° C and 90–100% infection was achieved after 48 h of incubation. At<15°C and 26–30° C infection percentage was decreased to 40–50% and 35–40%, respectively. No disease incidence was recorded at temperatures above 35° C. A scanning electron microscope study of the infection process showed that penetration of cotyledons by germ tubes was mostly via stomata and occasionally directly through the cuticle. Results are discussed in relation to the need for future studies of P. parasitica in South Africa.