Pathway of Propionate Oxidation by a Syntrophic Culture of Smithella propionica and Methanospirillum hungatei

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by ¹³C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-¹³C]propionate was converted to [2-¹³C]acetate, with no [1-¹³C]acetate formed. Butyrate from [3-¹³C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-¹³C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-¹³C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-¹³C-labeled propionate yielded both [1-¹³C]acetate and [2-¹³C]acetate. When ¹³C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, ¹³C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.     

Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H₂-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [¹³C]propionate (<10 mM) and the oxidation of ~30 μmol of ¹³C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were ¹³C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” ¹³C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of ¹³C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [¹³C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.     

A Proposed Pathway for Catabolism of Propionate in Methanogenic Cocultures

A metabolic pathway for the catabolism of propionate is proposed. This pathway incorporates a transcarboxylation reaction involving propionyl coenzyme A and oxaloacetate and a carboxylation of pyruvate to regenerate oxaloacetate. Data indicated that the proposed pathway is reversible. The proposed pathway and its apparent reversibility provide a reasonable explanation of observations obtained from metabolism of labeled substrate.     

Biochemical Evidence for Formate Transfer in Syntrophic Propionate-Oxidizing Cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei

The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H₂ lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H₂ lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.     

Separation of Syntrophomonas wolfei from Methanospirillum hungatii in Syntrophic Cocultures by Using Percoll Gradients

Percoll gradient centrifugation effectively separated Syntrophomonas wolfei cells from Methanospirillum hungatii cells, resulting in a 70- to 80-fold enrichment of S. wolfei cells relative to M. hungatii cells. The separated S. wolfei cells were viable. Gram quantities of cellular protein which was enzymatically active and had low levels of contamination by the methanogenic cofactor, factor₄₂₀, were obtained.     

Syntrophic Degradation of Cadaverine by a Defined Methanogenic Coculture

A novel, strictly anaerobic, cadaverine-oxidizing, defined coculture was isolated from an anoxic freshwater sediment sample. The coculture oxidized cadaverine (1,5-diaminopentane) with sulfate as the electron acceptor. The sulfate-reducing partner could be replaced by a hydrogenotrophic methanogenic partner. The defined coculture fermented cadaverine to acetate, butyrate, and glutarate plus sulfide or methane. The key enzymes involved in cadaverine degradation were identified in cell extracts. A pathway of cadaverine fermentation via 5-aminovaleraldehyde and crotonyl-coenzyme A with subsequent dismutation to acetate and butyrate is suggested. Comparative 16S rRNA gene analysis indicated that the fermenting part of the coculture belongs to the subphylum Firmicutes but that this part is distant from any described genus. The closest known relative was Clostridium aminobutyricum, with 95% similarity.Cadaverine is a biogenic primary aliphatic amine. Together with other biogenic amines, like putrescine or spermidine, it is formed during oxygen-limited decomposition of protein-rich organic matter by decarboxylation of amino acids or by amination of aldehydes and ketones (8, 27, 30, 42, 53, 54). These putrid-smelling and, at higher concentrations (100 to 400 mg per kg), often toxic compounds play a major role in food microbiology, e.g., as flavoring constituents in the ripening of cheese or as contaminants of fish and meat products, wine, and beer (24, 29, 49).Little is known about the degradation of primary amines. Mono- and diamine oxidases of higher organisms and bacteria (23, 41, 64) initiate aerobic degradation, leading to the respective formation of aldehyde, ammonia, and hydrogen peroxide as products (28). Alternatively, in a putrescine-degrading mutant of Escherichia coli, putrescine is degraded by a putrescine-2-oxoglutarate transaminase and a subsequent dehydrogenase to form 4-aminobutyrate, which is further metabolized via succinate (43).Anaerobic degradation of primary amines could follow basically similar pathways. The released reducing equivalents can be disposed of in a manner similar to that described for primary alcohols (9, 15, 16). In the absence of external electron acceptors, such as sulfate or nitrate, incomplete oxidation of cadaverine to fatty acids or dicarboxylic acids could be coupled to syntrophic methane production, homoacetogenesis, or reductive synthesis of long-chain fatty acids (1, 25, 31).In the present study, we describe a new isolate of strictly anaerobic bacteria which oxidizes cadaverine syntrophically with the methanogen Methanospirillum hungatei and forms acetate, butyrate, glutarate, and methane as products. The enzymes involved in the degradation of cadaverine were identified, and a catabolic pathway is proposed.     

13C Nuclear Magnetic Resonance Studies of Propionate Catabolism in Methanogenic Cocultures

Propionate catabolism was monitored in anaerobic cocultures of propionate-degrading and methanogenic bacteria. Metabolism was monitored by use of ¹³C-enriched propionate and succinate. The intermediates identified indicated that the methylmalonyl coenzyme A pathway was used in these cultures. The data also indicated that a transcarboxylation reaction between succinate and propionyl coenzyme A occurred, yielding propionate and methylmalonyl coenzyme A.     

Degradation and Mineralization of High-Molecular-Weight Polycyclic Aromatic Hydrocarbons by Defined Fungal-Bacterial Cocultures

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria (Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus (Penicillium janthinellum VUO 10,201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[a]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[a]pyrene was mineralized to CO₂ by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[a]pyrene mineralization (53% of added [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.     

球形红杆菌W1分批发酵和恒化培养的研究

本文对球形红杆菌Ｗ１的培养方式进行了研究。由分批培养确定了适于常规发酵的培养基及培养条件。恒化培养该菌的最大生长速率Ｐ＝３．８ｇ／ｌ．ｈ;最适稀释率Ｄ＝０．１５０ｈ＾－１;最大比生长速率为０．２０ｈ＾－１;饱和常数Ｋｓ＝２．８２ｇ／ｌ。恒化培养可获得比分批培养高的生长速率。     

Energetics of Growth of a Defined Mixed Culture of Desulfovibrio vulgaris and Methanosarcina barkeri: Interspecies Hydrogen Transfer in Batch and Continuous Cultures

Interspecies hydrogen transfer was studied in Desulfovibrio vulgaris-Methanosarcina barkeri mixed cultures. Experiments were performed under batch and continuous growth culture conditions. Lactate or pyruvate was used as an energy source. In batch culture and after 30 days of simultaneous incubation, these organisms were found to yield 1.5 mol of methane and 1.5 mol of carbon dioxide per mol of lactate fermented. When M. barkeri served as the hydrogen acceptor, growth yields of D. vulgaris were higher compared with those obtained on pyruvate without any electron acceptor other than protons. In continuous culture, all of the carbon derived from the oxidation of lactate was recovered as methane and carbon dioxide, provided the dilution rate was minimal. Increasing the dilution rate induced a gradual accumulation of acetate, causing acetate metabolism to cease at above μ = 0.05 h⁻¹. Under these conditions all of the methane produced originated from carbon dioxide. The growth yields of D. vulgaris were measured when sulfate or M. barkeri was the electron acceptor. Two key observations resulted from the present study. First, although sulfate was substituted by M. barkeri, metabolism of D. vulgaris was only slightly modified. The coculture-fermented lactate produced equimolar quantities of carbon dioxide and methane. Second, acetogenesis and methane formation from acetate were completely separable.     

Influence of Vitamin B12 and Cocultures on the Growth of Dehalococcoides Isolates in Defined Medium

Bacteria belonging to the genus Dehalococcoides play a key role in the complete detoxification of chloroethenes as these organisms are the only microbes known to be capable of dechlorination beyond dichloroethenes to vinyl chloride (VC) and ethene. However, Dehalococcoides strains usually grow slowly with a doubling time of 1 to 2 days and have complex nutritional requirements. Here we describe the growth of Dehalococcoides ethenogenes 195 in a defined mineral salts medium, improved growth of strain 195 when the medium was amended with high concentrations of vitamin B₁₂, and a strategy for maintaining Dehalococcoides strains on lactate by growing them in consortia. Although strain 195 could grow in defined medium spiked with ~0.5 mM trichloroethene (TCE) and 0.001 mg/liter vitamin B₁₂, the TCE dechlorination and cellular growth rates doubled when the vitamin B₁₂ concentration was increased 25-fold to 0.025 mg/liter. In addition, the final ratios of ethene to VC increased when the higher vitamin concentration was used, which reflected the key role that cobalamin plays in dechlorination reactions. No further improvement in dechlorination or growth was observed when the vitamin B₁₂ concentration was increased to more than 0.025 mg/liter. In defined consortia containing strain 195 along with Desulfovibrio desulfuricans and/or Acetobacterium woodii and containing lactate as the electron donor, tetrachloroethene (~0.4 mM) was completely dechlorinated to VC and ethene and there was concomitant growth of Dehalococcoides cells. In the cultures that also contained D. desulfuricans and/or A. woodii, strain 195 cells grew to densities that were 1.5 times greater than the densities obtained when the isolate was grown alone. The ratio of ethene to VC was highest in the presence of A. woodii, an organism that generates cobalamin de novo during metabolism. These findings demonstrate that the growth of D. ethenogenes strain 195 in defined medium can be optimized by providing high concentrations of vitamin B₁₂ and that this strain can be grown to higher densities in cocultures with fermenters that convert lactate to generate the required hydrogen and acetate and that may enhance the availability of vitamin B₁₂.     

Growth and Antithraquinone Biosynthesis by Galium mollugo L. Cells in Batch and Chemostat Culture

The kinetics of growth, nutrient uptake, and anthraquinone biosynthesisby suspension cultures of Galium mollugo L. cells were examinedin batch and continuous (chemostat) culture. In batch culture,although the initial growth rate was constant (minimum doublingtime = 35 h) characteristic changes in cell composition wereobserved during the growth cycle particularly cell dry weight(between 3.9 and 9.2 g/10⁹ cells), cell anthraquinone (22–80mg/10⁹ cells), and cell protein (0.7–1.6 g/10⁹ cells).Using a chemostat steady state growth was established at twodifferent specific growth rates with mean doubling times of40 h and 25 h. Phosphate was established as the growth-limitingnutrient in chemostat culture at a concentration of 11 µgP ml^–1. In steady state growth at a doubling time of 40h the cell composition remained constant although this was differentfrom any cells grown in batch culture. The cell anthraquinonelevel in steady state growth was between 7 and 30 times lowerthan in batch culture. This result raises the question of therelative importance of growth rate and the growth-limiting nutrientin determining accumulation of secondary products by culturedplant cells.     

不同培养方式下dCO_2对黑曲霉发酵产糖化酶的影响

通过在进气中混入部分CO_2气体,在分批培养和恒化培养两种培养方式下研究了不同dCO_2水平对黑曲霉产糖化酶的影响。在分批培养方式下,较高的dCO_2对细胞的生长具有抑制作用,并且随着dCO_2水平的增加,抑制程度加强,但是较高浓度的dCO_2对糖化酶的合成有利。而恒化培养时,低稀释率D_1=0.05/h下,较高的dCO_2对细胞的生长并没有明显的抑制作用,但有利于糖化酶合成。高稀释率D_2=0.08/h下,较高的dCO_2对细胞的生长有明显的抑制作用,并且抑制程度随着dCO_2水平的增加而加大。以上实验结果表明,dCO_2对细胞生长和糖化酶合成的影响不仅和dCO_2水平有关,也和培养方式及细胞所处的特定代谢状态有关。这对于黑曲霉产糖化酶工业放大过程中补料分批发酵中比生长速率的设计具有很好的指导作用。     

Microbial Community Dynamics and Stability during an Ammonia-Induced Shift to Syntrophic Acetate Oxidation

Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, we experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidation. In comparison with control digesters, we observed shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the course of the digesters'' 3-year operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When we perturbed the digesters by increasing the ammonia concentration or temperature, the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidation had allowed the experimental bioreactors to regain stable operation. The emergence of syntrophic acetate oxidation coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. Our 16S rRNA gene analysis also revealed that acetate-fed enrichment experiments resulted in communities that did not represent the bioreactor community. Analysis of shotgun sequencing of community DNA suggests that syntrophic acetate oxidation was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.     

Propionate Oxidation by and Methanol Inhibition of Anaerobic Ammonium-Oxidizing Bacteria

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway and a cost-effective way to remove ammonium from wastewater. Anammox bacteria have been described as obligate chemolithoautotrophs. However, many chemolithoautotrophs (i.e., nitrifiers) can use organic compounds as a supplementary carbon source. In this study, the effect of organic compounds on anammox bacteria was investigated. It was shown that alcohols inhibited anammox bacteria, while organic acids were converted by them. Methanol was the most potent inhibitor, leading to complete and irreversible loss of activity at concentrations as low as 0.5 mM. Of the organic acids acetate and propionate, propionate was consumed at a higher rate (0.8 nmol min⁻¹ mg of protein⁻¹) by Percoll-purified anammox cells. Glucose, formate, and alanine had no effect on the anammox process. It was shown that propionate was oxidized mainly to CO₂, with nitrate and/or nitrite as the electron acceptor. The anammox bacteria carried out propionate oxidation simultaneously with anaerobic ammonium oxidation. In an anammox enrichment culture fed with propionate for 150 days, the relative amounts of anammox cells and denitrifiers did not change significantly over time, indicating that anammox bacteria could compete successfully with heterotrophic denitrifiers for propionate. In conclusion, this study shows that anammox bacteria have a more versatile metabolism than previously assumed.     

复合碳源培养对酒糟厌氧消化液中丙酸互营氧化菌及群落结构的影响

丙酸累积是影响厌氧消化系统稳定性的主要因素,为了考察酒糟厌氧消化过程中间代谢产物的累积情况,以总固体含量(TS)（质量分数） 5%和7%的白酒糟为发酵原料进行了批次试验。结果表明,乙酸(最高浓度33~129 mmol/L)、丙酸(39~61 mmol/L)、丁酸(5~44 mmol/L)和15种氨基酸(0.01~0.3 mmol/L)为主要中间代谢产物。为了探究其中关键的丙酸降解菌群,以酒糟原始沼液JO为植种源,10 mmol/L丙酸和0.1 mmol/L混合氨基酸为复合碳源进行富集培养,获得中温厌氧丙酸-氨基酸培养系JO-AP。高通量测序分析表明,互营丙酸降解菌与厚壁菌门（Firmicutes）的丙酸厌氧降解菌（Pelotomaculum schinkii）近缘,16S rRNA基因相似性100%,占细菌总丰度的16.7%。对比酒糟原始沼液JO、丙酸培养系JO-P及丙酸-氨基酸培养系JO-AP中的主要功能菌群,发现采用单一碳源和复合碳源获得的优势互营丙酸降解菌不同;传统培养与分子生物学技术相结合可以更全面地掌握系统中的微生物群落组成。