Reductive Dehalogenation of Trichloroacetic Acid by Trichlorobacter thiogenes gen. nov., sp. nov.

A bacterium able to grow via reductive dechlorination of trichloroacetate was isolated from anaerobic soil enrichments. The isolate, designated strain K1, is a member of the δ proteobacteria and is related to other known sulfur and ferric iron reducers. In anaerobic mineral media supplemented with acetate and trichloroacetate, its doubling time was 6 h. Alternative electron donor and acceptors were acetoin and sulfur or fumarate, respectively. Trichloroacetate dehalogenation activity was constitutively present, and the dechlorination product was dichloroacetate and chloride. Trichloroacetate conversion seemed to be coupled to a novel sulfur-sulfide redox cycle, which shuttled electrons from acetate oxidation to trichloroacetate reduction. In view of its unique physiological characteristics, the name Trichlorobacter thiogenes is suggested for strain K1.     

Reductive dehalogenation of trichloroacetic acid by Trichlorobacter thiogenes gen. nov., sp. nov

A bacterium able to grow via reductive dechlorination of trichloroacetate was isolated from anaerobic soil enrichments. The isolate, designated strain K1, is a member of the delta proteobacteria and is related to other known sulfur and ferric iron reducers. In anaerobic mineral media supplemented with acetate and trichloroacetate, its doubling time was 6 h. Alternative electron donor and acceptors were acetoin and sulfur or fumarate, respectively. Trichloroacetate dehalogenation activity was constitutively present, and the dechlorination product was dichloroacetate and chloride. Trichloroacetate conversion seemed to be coupled to a novel sulfur-sulfide redox cycle, which shuttled electrons from acetate oxidation to trichloroacetate reduction. In view of its unique physiological characteristics, the name Trichlorobacter thiogenes is suggested for strain K1.     

Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp.

A new enzyme, DL-2-haloacid dehalogenase, was isolated and purified to homogeneity from the cells of Pseudomonas sp. strain 113. This enzyme catalyzed non-stereospecific dehalogenation of both of the optical isomers of 2-chloropropionate through an SN2 type of reaction; L- and D-lactates were formed from D- and L-2-chloropropionates, respectively. The enzyme acted on 2-halogenated aliphatic carboxylic acids whose carbon chain lengths were less than five. It also dehalogenated trichloroacetate to form oxalate and showed maximum activity at pH 9.5. The Michaelis constants for substrates were as follows: 5.0 mM for monochloroacetate, 1.1 mM for L-2-chloropropionate, and 4.8 mM for D-2-chloropropionate. DL-2-Haloacid dehalogenase was inhibited by HgCl2, ZnSO4, and MnSO4, but was not affected by thiol reagents, such as p-chloromercuribenzoate and iodoacetamide. This enzyme had a molecular weight of about 68,000 and appeared to be composed of two subunits identical in molecular weight.     

Nutrition of Acetobacter rancens S3 and F11 Isolated from Tanks for Vinegar Production

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain Fl1. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.     

RESPONSES OF THE ACIDOPHILIC ALGA EUGLENA MUTABILIS (EUGLENOPHYCEAE) TO CARBON ENRICHMENT AT pH 31

A defined medium (MAM) simulating acid mine drainage waters was developed which supported reproducible growth rates of three axenic strains of Euglena mutabilis Schmitz. Growth responses to various pHs and carbon sources were examined under defined culture conditions. A lab strain and two 5eld isolates, tested over pH range 1.5-9.0, grew best under acidic conditions (pH < 5.5) with highest growth rates at pH 3-4. Photoauxotrophic growth rates of all strains at pH 3 were improved significantly over unstirred batch controls by bubbling with air and even more by enrichment with 5% CO₂ in air. These results confirmed inorganic carbon limitation in batch culture. Organic carbon substrates were tested as possible carbon supplements in batch culture at pH 3. None of the strains survived in the dark on any of the twenty organic sources added. In the light, the lab strain exhibited some photoheterotrophic growth potential on glucose, sucrose, ethanol, and amino acids but growth was inhibited by acetate. Field strains showed little or no growth improvement with any organic substrate addition. Under simultaneous enrichment with acetate and 5% CO₂ acetate continued to be inhibitory. Simultaneous enrichment with glucose and 5% CO₂ gave higher yields of the lab strain than with CO₂ alone but did not enhance growth of the field strain. We conclude that E. mutabilis is an acidophilic photoauxotroph which appears unable to use organic carbon supplements for growth even under conditions of carbon limitation.     

Stimulation of the growth of leuconostoc oenos by tomato juice

Summary Tomato juice contains a growth factor (TJF) active for some strains of Leuconostoc oenos but not required by other lactic acid bacteria. TJF does not appear to be identical with any known growth factor, is of limited distribution in natural products and is destroyed by the growth of most strains of Leuconostoc oenos and by Lactobacillus plantarum and Pediococcus cerevisiae.TJF is essential for the growth of Leuconostoc oenos NCDO 1674 when organic acids are in a medium with an initial pH of 4.8 and incubated at 22° C and also with an initial pH of 6.0 and incubated at 30° C. Under the first conditions only and in the absence of organic acids Tween 80 is essential for growth if tomato juice is absent.     

Antibiotic effect of linolenic acid fromChlorococcum strain HS-101 andDunaliella primolecta on methicillin-resistantStaphylococcus aureus

Methanol extracts fromChlorococcum strain HS-101 andDunaliella primolecta strongly inhibited the growth of a strain of methicillin-resistantStaphylococcus aureus (MRSA), which is causing serious problems in Japanese hospitals. So that the anti-MRSA substance(s) could be purified and identified, the growth medium was improved for antibiotic production. When the two strains were cultured in their improved media, antibiotic production byChlorococcum strain HS-101 was 1.8-fold that in the standard BG-11 medium, and production byD. primolecta was 2.3-fold. The activity pattern of fractions eluted by silica-gel or gel-permeation chromatography suggested that both strains produced two antibiotic substances. Identification of the purified substances by NMR and GC-MS showed that one of the active substances in both strains was-linolenic acid. Ten fatty acids from other sources were tested, and it was found that unsaturated fatty acids had antibiotic activity against MRSA, with the highest activity that of -linolenic acid.     

Tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans: substrate specificity of the native enzyme and its corrinoid cofactor

The substrate specificity of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivoransand its corrinoid cofactor were studied. Besides reduced methyl viologen, titanium(III) citrate could serve as electron donor for reductive dehalogenation of tetrachloroethene (PCE) and trichloroethene to cis-1,2-dichloroethene. In addition to chlorinated ethenes, chlorinated propenes were reductively dechlorinated solely by the native enzyme. trans-1,3-Dichloropropene, 1,1,3-trichloropropene and 2,3-dichloropropene were reduced to a mixture of mono-chloropropenes, 1,1-dichloropropene, and 2-chloropropene, respectively. Other halogenated compounds that were rapidly reduced by the enzyme were also dehalogenated abiotically by the heat-inactivated enzyme and by commercially available cyanocobalamin. The rate of this abiotic reaction was dependent on the number and type of halogen substituents and on the type of catalyst. The corrinoid cofactor purified from the tetrachloroethene dehalogenase of D. multivorans exhibited an activity about 50-fold higher than that of cyanocobalamin (vitamin B(12)) with trichloroacetate as electron acceptor, indicating that the corrinoid cofactor of the PCE dehalogenase is not cyanocobalamin. Corrinoids catalyzed the rapid dehalogenation of trichloroacetic acid. The rate was proportional to the amount of, e.g. cyanocobalamin; therefore, the reductive dehalogenation assay can be used for the sensitive and rapid quantification of this cofactor.     

Influence of Phosphate on the Growth and Nodulation Characteristics of Rhizobium trifolii

The growth and nodulating characteristics of Rhizobium trifolii 6 and 36 differed under different external phosphate conditions. Under growth conditions designed to deplete the internal phosphate content of the rhizobia, strain 6 maintained a generation time of 5 h during the exponential phase over two cycles of growth in phosphate-depleted medium. In contrast, the generation time of strain 36 was extended from 3.5 to 9.8 h over two cycles of phosphate-depleted growth, although the organism eventually achieved the same cell density and cellular phosphate content as that of strain 6 at stationary phase. Phosphate-depleted strain 6 required 0.51 ± 0.08 μM phosphate to commence proliferation, whereas phosphate-depleted strain 36 required 0.89 ± 0.04 μM phosphate under the same conditions. Phosphate-depleted strain 6 maintained viability when exposed to external phosphate concentrations subcritical for growth to occur, whereas phosphate-depleted strain 36 lost viability within 48 h when exposed to medium containing phosphate at concentrations subcritical for growth. Phosphate-depleted strain 36 was inferior to phosphate-depleted strain 6 at nodulating subterranean clover (Trifolium subterraneum L. cv. Mt. Barker) by taking 2 to 4 days longer to develop nodules in phosphatedepleted plant grown medium at pH 5.5. Nodulation by phosphate-depleted strain 36 was accelerated either by including phosphate in the plant growth medium at pH 5.5 or by raising the solution pH of phosphate-depleted plant growth medium to pH 6.5. External phosphate and pH effects were not observed on the nodulating capabilities of phosphate-depleted strain 6 or on luxury phosphate-grown cells of either strain. Phosphatedepleted strains 6 and 36 proliferated to a similar extent on the rhizoplanes even under stringently low external P_i concentrations. The phosphatase activities of both phosphate-depleted strains were significantly (P = 0.05) higher at pH 6.5 than at pH 5.5, and the activity of strain 6 was significantly higher (P = 0.05) than that of strain 36 at pH 5.5 and 5.0.     

Ketogluconate formation by Gluconobacter species

Summary When G. oxydans ATCC 621-H was grown in batch culture in a complex medium with glucose, ketogluconates were produced when the pH in the culture was maintained at 5.5. Without pH control gluconate was the only product of glucose oxidation, but at pH 5.5 the gluconate so produced was further oxidized to ketogluconates. Production of ketogluconates started when glucose was almost completely exhausted. It was shown that the actual glucose and gluconate concentrations in the culture do not determine the onset of ketogluconate formation during growth. Both 2 and 5 ketogluconate were produced. Addition of CaCO₃ to the medium favored the production of 5 ketogluconate. However, under these conditions minor quantities of 2 ketogluconate were also formed. The sequential production of gluconate and ketogluconates from glucose was not only restricted to G. oxydans ATCC 621-H. A number of G. oxydans strains when grown under standard conditions in a pH controlled batch culture, all produced ketogluconates from glucose via an intermediate accumulation of gluconate. Although the ratios of the ketogluconates produced varied from strain to strain, all strains produced both 2 and 5 ketogluconate.     

Cellular Envelopes and Tolerance to Acid pH in Mesorhizobium loti

Changes in the cell envelopes in response to acidity were studied in two strains of Mesorhizobium loti differing in their tolerance to pH. When the less acid-tolerant strain LL22 was grown at pH 5.5, membrane phosphatidylglycerol decreased and phosphatidylcholine increased, compared with cells grown at pH 7.0. On the other hand, when the more acid-tolerant strain LL56 was grown at pH 5.5, phosphatidylglycerol, phosphatidylethanolamine, and lysophospholipid decreased 25%, 39%, and 51% respectively, while phosphatidyl-N-methylethanolamine and cardiolipin increased 26% and 65% respectively compared with cells grown at pH 7.0. The longest-chain fatty acids (19:0 cy and 20:0) increased in both strains at pH 5.5, while in LL56 these fatty acids increased still further at pH 4.0. Variations in other wall and membrane properties such as cell hydrophobicity, lypopolysaccharides, and protein composition of the outer membrane in relation to acid pH are also discussed. Received: 22 December 1998 / Accepted: 2 February 1999     

Biodegradation of chlorinated alkanes and their commercial mixtures by Pseudomonas sp. strain 273

The biodegradation of chlorinated alkanes was studied under oxic conditions with the objective of identifying favorable and unfavorable intramolecular chlorination sequences with respect to the enzymes studied. Several dehalogenating bacterial strains were screened for their ability to degrade middle-chain polychlorinated alkanes as well as a commercial mixture. Of the organisms tested, the most promising was Pseudomonas sp. strain 273, which possesses an oxygenolytic dehalogenase. The effects of carbon chain length (C₆–C₁₆), halogen position, and overall chlorine content (14–61% w/w) were examined using both commercially available compounds and molecules synthesized in our laboratory. The effects of co-substrates, solvents, and inducing agents were also studied. The results with pure chlorinated alkanes showed that the relative positions of the chlorine atoms strongly influenced the total amount of dehalogenation achieved. The greatest dehalogenation yields were associated with terminally chlorinated alkanes. The α- and α,ω-chlorinated compounds yielded similar results. Vicinal chlorination had the most dramatic impact on degradation. When present on both ends or at the center of the molecule, no dehalogenation was detected. Although partial dehalogenation of 1,2-dichlorodecane was observed, it was likely due to a combination of β-oxidation and an abiotic mechanism. Cereclor S52 was appreciably dehalogenated in shake flasks only when 1,10-dichlorodecane was present as a co-substrate and after increasing the oil surface area through mechanical emulsification, demonstrating the importance of abiotic factors in degrading commercial polychlorinated alkane mixtures.     

A nutritional study of eight strains of Gymnoascus reessii

Summary Eight strains ofGymnoascus reessii representing several morphological variants were grown in media which contained a variety of carbon and nitrogen sources in order to determine whether there was a correlation between morphological variation and physiological characteristics. Seven strains were similar in their assimilatory abilities, while one strain, 0-309 (NRRL 3612), was consistently dissimilar from the others. The defined medium which permitted the most growth of all eight isolates contained glycine as the nitrogen source and glucose as the carbon source. Other good, defined sources of carbon and nitrogen included soluble starch, maltose, KNO₃ and NaNO₃. Peptone and casamino acids were effective nitrogen sources also. Seven strains grew better with added growth factors although they did not have an absolute requirement for such factors. The other strain, 0-309, appeared to have a growth factor deficiency. Seven of the eight strains were basically similar in their nutritional characteristics. Only strain 0-309 (NRRL 3612) consistently demonstrated sufficient differences so that it could possibly be considered to be a variety ofG. reessii.     

Hydrolytic dehalogenation of 4-chlorobenzoic acid by an Acinetobacter sp

Acinetobacter sp. strain ST-1, isolated from garden soil, can mineralize 4-chlorobenzoic acid (4-CBA). The bacterium degrades 4-CBA, starting with dehalogenation to yield 4-hydroxybenzoic acid (4-HBA) under both aerobic and anaerobic conditions, suggesting that the dehalogenating enzyme in the strain is not an oxygenase; the enzyme may catalyze halide hydrolysis. To identify the oxygen source of the C(4)-hydroxy groups in the dehalogenation step, we used H(2)(18)O as the solvent under anaerobic conditions. When resting cells were incubated in the presence of 4-CBA and H(2)(18)O under a nitrogen gas stream, the hydroxy group on the aromatic nucleus of the 4-HBA produced was derived from water, not from molecular oxygen. This dehalogenation was hydrolytic, because analysis of the mass spectrum of the trimethylsilyl derivative of one of the metabolites, (18)O-labeled 4-HBA, showed that 80% of the C4-hydroxy groups were labeled with (18)O. Hydrolytic dehalogenation of 4-CBA in intact cells has not been reported earlier. To identify substrate specificity, we next examined the ability of the strain to dehalogenate 4-CBA analogues and dichlorobenzoic acids. The results of metabolite analysis by high-pressure liquid chromatography showed that the strain dehalogenated 4-bromobenzoic acid and 4-iodobenzoic acid, yielding 4-HBA, suggesting that these compounds could be further degraded and mineralized by the strain via the beta-ketoadipate pathway, as occurs with 4-CBA. This strain, however, did not dehalogenate 4-fluorobenzoic acid, 2- and 3-chlorobenzoic acids, or 2,4-, 3,4-, and 3,5-dichlorobenzoic acids during 4 days of incubation, implying that the dehalogenating enzyme of the strain has high substrate specificity.     

Environmental optimization for bioconversion of triolein into 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFAs), originally found in small amount mainly from plant systems, are well known to have special properties such as higher viscosity and reactivity compared with other normal fatty acids. Recently, various microbial strains were tested to produce HFAs from different unsaturated fatty acids. Among those microbial strains tested, Pseudomonas aeruginosa PR3 are well known to utilize various unsaturated fatty acids to produce mono-, di-, and tri-HFAs. Previously, we reported that strain PR3 could utilize triolein as a substrate for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) via the induction of lipase activity (Chang et al., Appl Microbiol Biotechnol, 74:301–306, 2007). In this study, we focused on the development of the optimal environmental conditions for DOD production from triolein by PR3. Optimal initial medium pH and incubation temperature were pH 8.0 and 25°C, respectively. Magnesium ion was essentially required for DOD production. Optimal inoculum size, time for substrate addition, and substrate concentration were 1%, 12 to 24 h, and 300 mg, respectively.     

Isolation and survey of novel fluoroacetate-degrading bacteria belonging to the phylum Synergistetes

Microbial dehalogenation of chlorinated compounds in anaerobic environments is well known, but the degradation of fluorinated compounds under similar conditions has rarely been described. Here, we report on the isolation of a bovine rumen bacterium that metabolizes fluoroacetate under anaerobic conditions, the mode of degradation and its presence in gut ecosystems. The bacterium was identified using 16S rRNA gene sequence analysis as belonging to the phylum Synergistetes and was designated strain MFA1. Growth was stimulated by amino acids with greater quantities of amino acids metabolized in the presence of fluoroacetate, but sugars were not fermented. Acetate, formate, propionate, isobutryate, isovalerate, ornithine and H(2) were end products of amino acid metabolism. Acetate was the primary end product of fluoroacetate dehalogenation, and the amount produced correlated with the stoichiometric release of fluoride which was confirmed using fluorine nuclear magnetic resonance ((19) F NMR) spectroscopy. Hydrogen and formate produced in situ were consumed during dehalogenation. The growth characteristics of strain MFA1 indicated that the bacterium may gain energy via reductive dehalogenation. This is the first study to identify a bacterium that can anaerobically dehalogenate fluoroacetate. Nested 16S rRNA gene-specific PCR assays detected the bacterium at low numbers in the gut of several herbivore species.     

一株产纤维素酶菌株的分离、鉴定及产酶特性

【目的】筛选并鉴定一株产纤维素酶的菌株,初步探究该菌的产酶特性,为综合利用纤维素筛选菌源。【方法】在常温条件下,采用滤纸培养基对菌种富集,采用CMC-Na初筛纤维素降解菌,采用LB培养基分离纯化菌株,经形态学、生理生化特征试验、16S r RNA基因序列测定等分析筛选菌株的系统分类地位。单因素试验确定培养时间、培养温度、初始p H及Na Cl浓度对筛选菌株产酶活力的影响。【结果】从腐烂的玉米秸秆中分离出一株在常温下产纤维素酶细菌KZ-2,根据菌落形态特征、生理生化特征鉴定以及16S r RNA基因序列分析,初步鉴定KZ-2为肠杆菌(Enterobacter sp.),为潜在新种。产酶条件实验显示:该菌使用产酶发酵培养基120 h产酶量达到最大值,在25–35°C、初始p H 4.5–5.5、Na Cl浓度1.0%–2.0%范围内为最佳产酶条件,在最适条件下酶活可达80.93 U/m L。该菌株所产纤维素酶最适反应p H为7.0,最适反应温度为50°C。【结论】KZ-2是一株具有降解纤维素能力的细菌,在常温下即可分泌纤维素酶,并且该菌株为潜在新种,具有潜在的开发价值。     

Physiological and morphological acid resistance ofCandida boidinii

The growth ofCandida boidinii strain 2 in a methanol-limited chemostat at a dilution rate of 0.1/h and a low extracellular pH (2.8–4.0) is characterized by a maximum yield coefficient referred to the methanol consumedY _S of 0.4 g/g and a maximum cell content of nitrogenous compounds of 60%. The cell proteins are rich in essential amino acids. At pH<2.6 or >4.0 the cell concentration decreases due to lower growth rate, accompanied by increased metabolic quotientsQ _S,Q _CO2 andQ _form, and increased activities of dissimilating dehydrogenases. The activity of alcohol oxidase (AO) in intact cells (0.54 IU/mg protein) was unaffected by pH 2.8–3.8 although in a cell-free extract the AO activity decreased at these low pH values after a 10-min incubation. The lower AO activity in cells at pH<2.8 and pH>3.8 brought about increased residual methanol levels in the medium, and also an increased level of riboflavin phosphate, arising probably by the release of FAD from active AO. Catalase activity was completely pH-independent. Cell morphology also showed no changes at pH 2.8–4.2, formation of cell chains being observed only at pH<2.8. However, the ultrastructure of cells grown in the chemostat at pH 2.6, however, did not evince any changes as compared with cells grown, at higher pH apart from a lag in cytokinesis. These findings, which point to acid resistance of strain 2, make it possible to produce biomass from methanol, with a high content of valuable proteins and AO, under nonsterile conditions.