Growth of Syntrophomonas wolfei in pure culture on crotonate

A Synthrophomonas wolfei-Methanospirillum hungatei coculture was adapted to catabolize crotonate. S. wolfei was then isolated in axenic culture using agar spread plates and roll tubes with crotonate as the sole energy source. S. wolfei catabolized crotonate via a disproportionation mechanism similar to that of some Clostridium species. Growth on crotonate was very slow (specific growth rate of 0.029 h^–1) but the conversion of energy into cell material was very efficient with cell yields of 14.6 g (dry wt.) per mol of crotonate. S. wolfei alone did not catabolize butyrate, but butyrate was stoichiometrically degraded to acetate and presumably methane when S. wolfei was reassociated with M. hungatei. S. wolfei-M. hungatei cocultures accumulated some butyrate during growth on crotonate indicating that protons were not the sole electron acceptors used for crotonate oxidation by the coculture.     

Syntrophobacter pfennigii sp. nov., new syntrophically propionate-oxidizing anaerobe growing in pure culture with propionate and sulfate

A new strain of syntrophically propionate-oxidizing fermenting bacteria, strain KoProp1, was isolated from anoxic sludge of a municipal sewage plant. It oxidized propionate or lactate in cooperation with the hydrogen- and formate-utilizingMethanospirillum hungatei and grew as well in pure culture without a syntrophic partner with propionate or lactate plus sulfate as energy source. In all cases, the substrates were oxidized stoichiometrically to acetate and CO₂, with concomitant formation of methane or sulfide. Cells formed gas vesicles in the late growth phase and contained cytochromesb andc, a menaquinone-7, and desulforubidin, but no desulfoviridin. Enzyme measurements in cell-free extracts indicated that propionate was oxidized through the methylmalonyl CoA pathway. Protein pattern analysis by SDS-PAGE of cell-free extracts showed that strain KoProp1 differs significantly fromSyntrophobacter wolinii and from the propionate-oxidizing sulfate reducerDesulfobulbus propionicus. 16S rRNA sequence analysis revealed a significant resemblance toS. wolinii allowing the assignment of strain KoProp1 to the genusSyntrophobacter as a new species,S. pfennigii.     

Fermentation of phenoxyethanol to phenol and acetate by a homoacetogenic bacterium

A strictly anaerobic gram-positive, rod-shaped bacterium, strain LuPhet1, was isolated from sewage sludge with phenoxyethanol as sole carbon and energy source, and was assigned to the genus Acetobacterium. The new isolate fermented the alkylaryl ether compound phenoxyethanol stoichiometrically to phenol and acetate, whereas phenoxyacetic acid was not degraded. In cell-free extracts of strain LuPhet1, cleavage of the ether linkage was shown, and acetaldehyde was detected as reaction product. Coenzyme A-dependent acetaldehyde: acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results indicate that the ether linkage of phenoxyethanol is cleaved by a shift of the hydroxyl group to the subterminal carbon atom, analogous to a corrinoid-dependent diol dehydratase reaction, to form an unstable hemiacetal that releases phenol and acetaldehyde. Obviously, phenoxyethanol is degraded by the same strategy as in anaerobic degradation of the alkyl ether polyethylene glycol.     

Fermentation of mandelate to benzoate and acetate by a homoacetogenic bacterium

A strictly anaerobic, Gram-positive, rod-shaped bacterium, strain AmMan1, was isolated from freshwater sediment with mandelate (-hydroxy-phenylacetate) as sole carbon and energy source, and was assigned to the genus Acetobacterium. Only the d-enantiomer of mandelate was degraded, and was fermented to acetate and benzoate. Non-aromatic growth substrates (pyruvate, lactate, malate, glycerol, ethylene glycol, and H₂/CO₂) were fermented to acetate as sole product. Methoxylated aromatics were demethoxylated to the corresponding phenols. The guanine-plus-cytosine content of the DNA was 36.5±1.5%. Carbon monoxide dehydrogenase, dichlorophenol indophenol-reducing lactate dehydrogenase, NAD-dependent mandelate dehydrogenase, phosphate acetyl transferase, acetate kinase, and pyruvate- or phenylglyoxylate-dependent benzylviologen reductase were measured in mandelate-and/or lactate-grown cells, respectively. A pathway of the homoacetogenic fermentation of mandelate is suggested as another example of incomplete substrate oxidation by homoacetogenic bacteria.     

合成气厌氧发酵生产有机酸和醇的研究进展

合成气来自于煤、石油、生物质和有机废物的气化,其主要成份为CO、H2和CO2。研究发现某些厌氧菌能利用合成气生成乙醇、乙酸、丁醇和丁酸等燃料和化学品。由于生物转化所具有的优势,合成气厌氧发酵被认为是一项极具潜力和竞争力的技术,在生物质及有机废物的利用方面将发挥重要作用。对厌氧发酵合成气生产有机酸和醇的研究进展,包括利用合成气产有机酸和醇的微生物,合成气发酵的代谢途径和关键酶（一氧化碳脱氢酶/乙酰辅酶A合成酶）及用于合成气发酵的反应器等进行了综述,并对该项技术的发展提出了一些建议。     

Fermentation of glutamate and other compounds by Acidaminobacter hydrogenoformans gen. nov. sp. nov., an obligate anaerobe isolated from black mud. Studies with pure cultures and mixed cultures with sulfate-reducing and methanogenic bacteria

From mud from the Ems-Dollard estuary (The Netherlands) an L-glutamate-fermenting bacterium was isolated. The isolated strain glu 65 is Gram-negative, rodshaped, obligately anaerobic, non-sporeforming and does not contain cytochromes. The G+C content of its DNA is 48 mol percent.Pure cultures of strain glu 65 grew slowly on glutamate (_max 0.06 h^-1) and formed acetate, CO₂, formate and hydrogen, and minor amounts of propionate. A more rapid fermentation of glutamate was achieved in mixed cultures with sulfate-reducing bacteria (Desulfovibrio HL21 or Desulfobulbus propionicus) or methanogens (Methanospirillum hungatei or Methanobrevibacter arboriphilicus AZ). In mixed culture with Desulfovibrio HL21 a _max of 0.10 h^-1 was observed. With Desulfovibrio or the methanogens propionate was a major product (up to 0.47 mol per mol glutamate) in addition to acetate.Extracts of glutamate-grown cells possessed high activities of 3-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate, and very high activities of NAD⁺-dependent glutamate dehydrogenase, an enzyme most likely involved in the pathway to propionate.The following other substrates allowed reasonable to good growth in pure culture: histidine, -ketoglutarate, serine, cysteine, glycine, adenine, pyruvate, oxaloacetate and citrate. Utilization in mixed cultures was demonstrated for: glutamine, arginine, ornithine, threonine, lysine, alanine, valine, leucine and isoleucine (with Desulfovibrio HL21) and malate (with Methanospirillum).The shift in the fermentation of glutamate and the syntrophic utilization of the above substrates are explained in terms of interspecies hydrogen transfer.Strain glu 65 is described as the type strain of Acidaminobacter hydrogenoformans gen. nov. sp. nov.     

Degradation of hydroquinone,gentisate, and benzoate by a fermenting bacterium in pure or defined mixed culture

From a methanogenic fixed-bed reactor fed with hydroquinone as sole energy and carbon source, a rodshaped bacterium was isolated in pure culture which could degrade hydroquinone and gentisate (2,5-dihydroxybenzoate). In syntrophic coculture with either Desulfovibrio vulgaris or Methanospirillum hungatei, also benzoate could be degraded. Other substrates such as sugars, fatty acids, alcohols, and cyclohexane derivatives were not degraded. Sulfate, sulfite, or nitrate were not used as external electron acceptor. The isolate was a Gram-negative, non-motile, nonsporeforming strict anaerobe; the guanine-plus-cytosine content of the DNA was 53.2±1.0 mol%. In pure culture, hydroquinone was degraded to acetate and benzoate, probably via an intermediate carboxylation. In syntrophic mixed cultures, all three substrates were converted completely to acetate. Phenol was never detected as a fermentation product.     

Metabolism of position-labelled glucose in anoxic methanogenic paddy soil and lake sediment

Abstract Turnover times of radioactive glucose were shorter in paddy soil (4–16 min) than in Lake Constance sediment (18–62 min). In the paddy soil, 65–75% of the radioactive glucose was converted to soluble metabolites. In the sediment, only about 25% of the radioactive glucose was converted to soluble metabolites, the rest to particulate material. In anoxic paddy soil, the degradation pattern of position-labelled glucose was largely consistent with glucose degradation via the Embden-Meyerhof-Parnas (EMP) pathway followed by methanogenic acetate cleavage: CO₂ mainly originated from C-3,4, whereas CH₄ mainly originated from C-1 and C-6 of glucose. Acetate-carbon originated from C-1, C-2 and C-6 rather than from C-3,4 of glucose. In both paddy soil and Lake Constance sediment acetate and CO₂ were the most important early metabolites of radioactive glucose. Other early products included propionate, ethanol/butyrate, succinate, and lactate, but accounted each for less than 1–8% of the glucose utilized. The labelling of propionate by [3,4-¹⁴C]glucose suggests that it was mainly produced from glucose or lactate rather than from ethanol. Isopropanol and caproate were also detectable in paddy soil, but were not produced from radioactive glucose. Chloroform inhibited methanogenesis, inhibited the further degradation of radioactive acetate and resulted in the accumulation of H₂, however, did not inhibit glucose degradation. Since acetate was the main soluble fermentation product of glucose and was produced at a relatively high molar acetate: CO₂ ratio (2.5:1), homoacetogenesis appeared to be the most important glucose fermentation pathway.     

Fermentation of glycerol by Clostridium pasteurianum--batch and continuous culture studies

The fermentation of glycerol by Clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3-propanediol, butyric and acetic acids and ethanol. More than 60 g/l glycerol was utilized, and up to 17 g/l butanol was produced. Fed-batch cultures did not offer an advantage. When molecular nitrogen was used as a nitrogen source, the fermentation time was prolonged by a factor of 1.5. Fermentations at constant pH values between 4.5 and 7.5 did not reveal significant differences in product formation except for an increase in the ethanol content starting at pH 6.5. Chemostat cultures also yielded predominantly n-butanol, but in some fermentations, the 1,3-propanediol fraction was relatively high. The pH auxostat cultures, which were operated at a glycerol excess, contained 1,3-propanediol as the main product. As a whole, the fermentations were characterized by a certain variability in product formation under seemingly equal or slightly varied conditions. It appears that the regulation of the numerous fermentation pathways occurring in this organism is not very strict. Journal of Industrial Microbiology & Biotechnology (2001) 27, 18–26. Received 25 September 2000/ Accepted in revised form 07 April 2001     

厌氧发酵产氢细菌的筛选及其产氢优化

本研究以河底泥为来源, 使用产氢培养基进行初筛, 再利用小管产氢试验进行复筛, 得到5株产氢能力较好的菌株。对产氢量最高的菌株FML-C1进行16S rDNA序列分析, 鉴定为阴沟肠杆菌, 确定了其分类地位。培养基优化采用Plackett-Burman试验设计筛选出影响产氢的3个主要因素: 葡萄糖、缓冲液和还原剂。利用最陡爬坡路径逼近最大响应区, 采用中心复合试验设计(CCD)及响应面分析法(RSM)进行回归分析, 建立产氢培养基优化的二次模型。模型求解产氢最佳培养基为葡萄糖21.5 g/L、缓冲液 13.6 mL/L 和还原剂10.0 mL/L, 最大理论产氢量2367.83 mL/L。5批验证试验结果平均值与预测值接近, 表明该模型与实际情况拟合良好, 实际最大产氢量2347.40 mL/L, 较优化前产氢量提高127.42%。     

一株琥珀酸产生菌的筛选及鉴定

从牛的瘤胃中筛选获得一株能发酵生产琥珀酸的兼性厌氧菌。对其进行生理生化特性鉴定及16S rRNA基因分析。该菌株短杆状,无鞭毛,革兰氏染色阴性,V-P反应阴性,能发酵多种糖类产酸;其16S rRNA基因与琥珀酸放线杆菌的同源性高达99．8%,认为属于琥珀酸放线杆菌(Actinobacillus succinogenes),并将其命名为琥珀酸放线杆菌(Actinobacillus succinogenes)SW0580,保藏号CGMCC 1593。初步发酵试验表明该菌能发酵60g/L葡萄糖产生25．8g/L的丁二酸。     

Glycerol and propanediols degradation by Desulfovibrio alcoholovorans in pure culture in the presence of sulfate, or in syntrophic association with Methanospirillum hungatei

Abstract In a mineral medium containing sulfate as terminal electron acceptor, the sulfate-reducing bacterium Desulfovibrio alcoholovorans oxidized stoichiometrically 1 mol glycerol to 1 mol acetate and 1 mol 1,3-propanediol to 1 mol acetate with the concomitant reduction of 0.75 and 1 mol sulfate, respectively; 1 mol 1,2-propanediol was degraded to 0.8 mol acetate and 0.1 mol proprionate, with the reduction of approximately 1 mol sulfate. The maximum specific growth rates (μ_max in h⁻¹) were 0.22, 0.086 and 0.09 with glycerol, 1,3-propanediol and 1,2-propanediol, respectively. The growth yields were 12.7 g, 11.1 g and 7.2 g dry weight/mol 1,3-propanediol, glycerol and 1,2-propanediol degraded, respectively. The growth yields and maximum specific growth rates of the H₂-transferring associations were also calculated. In the absense of sulfate, all these reduced substrates were degraded to acids and methane when D. alcoholovorans was cocultured with Methanospirillum hungatei . Changes in the metabolic pathway were observed in the degradation of 1,2- and 1,3-propanediol. The metabolic efficiency of D. alcoholovorans to degrade glycerol, 1.2- and 1,3-propanediol is discussed.     

Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes

In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte–keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.     

Fermentation of isoleucine and arginine by pure and syntrophic cultures of Clostridium sporogenes

Abstract The fermentation of isoleucine, arginine and isoleucine + arginine by pure and syntrophic cultures of Clostridium sporogenes was investigated. Growth of C. sporogenes on isoleucine, if any, was poor, but some isoleucine was fermented to 2-methylbutyrate and hydrogen. In syntrophic cultures with Methanobacterium formicicum or Methanosarcina barkeri growth was better, and isoleucine was completely fermented, the hydrogen being used for methane production. Pure cultures of C. sporogenes grew on arginine and produced 5-aminovalerate, ornithine and acetate. The reducing equivalents for 5-aminovalerate production from intermediarily formed proline were provided by oxidative conversion of arginine to acetate and by oxidative metabolism of some amino acids present in the yeast extract. However, when isoleucine was available together with arginine in syntrophic cultures of C. sporogenes and M. formicicum , the reducing equivalents for arginine fermentation came mainly from the oxidation of isoleucine (Stickland reaction), and the hydrogen produced in excess served for the reduction of CO₂ to methane.     

Biochemistry of microbial polyvinyl alcohol degradation

Effect of minor chemical structures such as 1,2-diol content, ethylene content, tacticity, a degree of polymerization, and a degree of saponification of the main chain on biodegradability of polyvinyl alcohol (PVA) is summarized. Most PVA-degraders are Gram-negative bacteria belonging to the Pseudomonads and Sphingomonads, but Gram-positive bacteria also have PVA-degrading abilities. Several examples show symbiotic degradation of PVA by different mechanisms. Penicillium sp. is the only reported eukaryotic degrader. A vinyl alcohol oligomer-utilizing fungus, Geotrichum fermentans WF9101, has also been reported. Lignolytic fungi have displayed non-specific degradation of PVA. Extensive published studies have established a two-step process for the biodegradation of PVA. Some bacteria excrete extracellular PVA oxidase to yield oxidized PVA, which is partly under spontaneous depolymerization and is further metabolized by the second step enzyme (hydrolase). On the other hand, PVA (whole and depolymerized to some extent) must be taken up into the periplasmic space of some Gram-negative bacteria, where PVA is oxidized by PVA dehydrogenase, coupled to a respiratory chain. The complete pva operon was identified in Sphingopyxis sp. 113P3. Anaerobic biodegradability of PVA has also been suggested.     

Fermentation of methanethiol and dimethylsulfide by a newly isolated methanogenic bacterium

From dilution series in defined mineral medium, a marine iregular coccoid methanogenic bacterium (strain MTP4) was isolated that was able to grow on methanethiol as sole source of energy. The strain also grew on dimethylsulfide, mono-, di-, and trimethylamine, methanol and acetate. On formate the organism produced methane without significant growth. Optimal growth on MT, with doubling times of about 20 h, occurred at 30°C in marine medium. The isolate required p-aminobenzoate and a further not identified vitamin. Strain MTP4 had a high tolerance to hydrogen sulfide but was very sensitive to mechanical forces or addition of detergents such as Triton X-100 or sodium dodecylsulfate. Methanethiol was fermented by strain MTP4 according to the following equation:

Measurement and regulation of the culture reduction state in Clostridium acetobutylicum

With a constant glucose feed concentration, the change in the continuous culture dillution rate resulted in an altered fermentation profile and the cellular NADH content. The cultures growing at high dillution rates demonstrated an oxidative metabolism low NADH and butanol concentrations. The low specific NADH flourescence (F/X) at high butanol production rates suggested that a rapid regeneration of NADH to NAD is essential for a high solventogenic culture activity. The culture florescence and butanol concentration remained constant in the solventogenic dilution rate range of D = 0.05-0.2 h(-1) with an inverse relationship between the specific flourescence (F/X) and the specific butanol production rate, q(B). Flourometric NADH observations were confirmed by enzymatic NADH determination. The stiochiometric "Fermentation Equation" was used to check the experimental data consistency and to investigate the role of the available biosynthetic and reduction energy on the culture metabolic activities under different growth conditions. The butanol concentration in the broth was stabilized in a fed-batch process when the culture NADH fluorescence was being controlled through the addition of fresh medium.     

Effects of thymol and diphenyliodonium chloride against Campylobacter spp. during pure and mixed culture in vitro

Aims: To determine if the purported deaminase inhibitors diphenyliodonium chloride (DIC) and thymol reduce the growth and survivability of Campylobacter. Methods and Results: Growth rates of Campylobacter jejuni and Camp. coli were reduced compared to unsupplemented controls during culture in Muellar–Hinton broth supplemented with 0·25 μmol DIC or thymol ml^?1 but not with 0·01 μmol monensin ml^?1 or 1% ethanol. Recovery of Camp. jejuni and Camp. coli was reduced >5 log₁₀ CFU from controls after 24 h pure culture in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Similarly, each test Campylobacter strain was reduced >3 log₁₀ CFU from controls after 24 h mixed culture with porcine faecal microbes in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Treatments with 0·25 μmol thymol ml^?1, 0·01 μmol monensin ml^?1 or 1% ethanol were less effective. Ammonia production during culture or incubation of cell lysates was reduced by 0·25 or 1·0 μmol DIC ml^?1 but only intermittently reduced, if at all, by the other treatments. Conclusions: Diphenyliodonium chloride and thymol reduced growth, survivability and ammonia production of Camp. jejuni and Camp. coli. Significance and Impact of the Study: Results suggest a potential physiological characteristic that may be exploited to develop interventions.