Formation of a methylotrophic denitrifying biocenosis in a system of sewage treatment for nitrates

A methylotrophic denitrifying bioenosis composed of hyphomicrobes and paracocci was isolated from the active ooze in a system of sewage purification from nitrates. The morphological and physiological characteristics of the isolated Hyphomicrobium sp. Z-115 and Paracoccus denitrificans Z-100 and Z-121 strains differed from those of the type strains, which made it difficult to identify them and to isolate them as a pure culture. This should be taken into account while determining the agents operating in such purification systems. The rate of growth, the rate of nitrate reduction and the activity of enzymes involved in methanol assimilation are higher in the anabolic syntrophic bicenosis than in its components in pure culture. A combined culture composed of the collection Hyphomicrobium and Paracoccus strains was neither effective nor stable under the conditions of anaerobic growth with nitrate and methanol. Therefore, the natural biocenosis af the purification system cannot be substituted by an artificial one composed of the collection cultures.     

Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling.

The localization of prominent proteins in intact cells of two methylotrophic bacteria, Hyphomicrobium sp. strain X and bacterium W3A1, was investigated by radiochemical labeling with [14C]isethionyl acetimidate. In bacterium W3A1, trimethylamine dehydrogenase was not labeled by the reagent and is, therefore, an intracellular protein, whereas the periplasmic location of the methylamine and methanol dehydrogenases was evidenced by being readily labeled in intact cells. Similarly, an intracellular location of the trimethylamine and dimethylamine dehydrogenases in Hyphomicrobium sp. strain X was indicated, whereas methanol dehydrogenase was periplasmic.     

亚硝化菌株的筛选及其初步鉴定

从化工废水和城镇污水生物处理反应器水中取样,用斯凯尔曼亚硝化假单孢杆菌培养基及硅胶平板分离得到4株可以利用氨氮生长的菌株,并对其形态和生理生化性状进行分析。初步研究结果表明。它们可能分属硝化菌、丝状菌和假单孢菌。在溶氧浓度大于1mg/L的液体培养基中,以上菌株能够将氨氮转化为NO2^-,基本没有NO3^-,又能将氨氮转化为气态氮,且水中NO2^0和NO3^-不再累积,分别保持在小于9mg/L和小于2．0mg/L的水平。     

Denitrification with Methanol: a Selective Enrichment for Hyphomicrobium Species

Hyphomicrobium species were enriched in media with methanol as sole carbon source under conditions supporting denitrification. Pure cultures of Hyphomicrobium species were isolated which denitrified vigorously with methanol. Hyphomicrobium B522, isolated by aerobic enrichment, was adapted to anaerobic growth and denitrification. Hyphomicrobium B522 and a new isolate were surveyed for anaerobic growth and denitrification on a number of simple organic compounds. Cell suspensions were tested for denitrifying activity. Nitrogen production from nitrate and nitrite and carbon dioxide production from methanol were stoichiometric.     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

Interactions in a mixed bacterial population growing on methane in continuous culture

The growth of a mixed methane-utilizing culture in a continuous flow fermenter has been studied under both methane and oxygen limitation. Small additions of methanol have been shown to inhibit the methane-utilizing moiety in the culture and it has been shown that the Hyphomicrobium sp. in the mixed culture removes any inhibitory methanol. The interaction between the methane-utilizing Pseudomonas sp., and the Hyphomicrobium sp. has been explained and a model of the continuous mixed culture under oxygen limitation has been formulated. Qualitative predictions of transient phenomena by the model have been verified experimentally.     

Synergistic degradation of linuron by a bacterial consortium and isolation of a single linuron-degrading variovorax strain

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.     

L-tyrosine is the precursor of PQQ biosynthesis in Hyphomicrobium X

A method was developed to study amino acids as possible precursors of PQQ biosynthesis. Cultures of Hyphomicrobium X, growing on [13C]methanol, were supplemented with unlabelled amino acids. Uptake and participation in metabolism were determined via gas chromatography/mass spectrometry of derivatized amino acids, obtained from hydrolyzed cellular protein, by measuring their 12C content. Several amino acids appeared to be incorporated into the protein to a significant extent, without degradation or conversion. Among these were the aromatic amino acids, L-tyrosine and L-phenylalanine. Using the same replacement approach, their incorporation into PQQ was determined by 1H- and 13C-NMR spectroscopy of purified PQQ obtained from the culture medium. It appeared that the complete carbon skeleton of tyrosine was present, forming the o-quinone and pyrrole-2-carboxylic acid moieties in PQQ, while phenylalanine was not incorporated at all. Starting with L-tyrosine, possible biosynthetic routes to PQQ are discussed.     

Metabolic characteristics of an aerobe isolated from a methylotrophic methanogenic enrichment culture

An anaerobic methylotrophic methanogenic enrichment culture, with sustained metabolic characteristics, including that of methanation for over a decade, was the choice of the present study on interspecies interactions. Growth and methanation by the enrichment were suppressed in the presence of antibiotics, and no methanogen grown on methanol could be isolated using stringent techniques. The present study confirmed syntrophic metabolic interactions in this enrichment with the isolation of a strain ofPseudomonas sp. The organism had characteristic metabolic versatility in metabolizing a variety of substrates including alcohols, aliphatic acids, amino acids, and sugars. Anaerobic growth was favoured with nitrate in the growth medium. Cells grown anaerobically with methanol, revealed maximal nitrate reductase activity. Constitutive oxidative activity of the membrane system emerged from the high-specific oxygen uptake and nitrate reductase activities of the aerobically and anerobically grown cells respectively. Cells grown anaerobically on various alcohols effectively oxidized methanol in the presence of flavins, cofactor FAD and the methanogenic cofactor F₄₂₀, suggesting a constitutive alcohol oxidizing capacity. In cells grown anaerobically on methanol, the rate of methanol oxidation with F₄₂₀ was three times that of FAD. Efficient utilization of alcohols in the presence of F₄₂₀ is a novel feature of the present study. The results suggest that utilization of methanol by the mixed culture would involve metabolic interactions between thePseudomonas sp. and the methanogen(s). Methylotrophic, methanogenic partnership involving an aerobe is a novel feature hitherto unreported among anaerobic syntrophic associations and is of ecological significance.     

Deoxyribonucleic Acid Base Sequence Homologies of Some Budding and Prosthecate Bacteria

The genetic relatedness of a number of budding and prosthecate bacteria was determined by deoxyribonucleic acid (DNA) homology experiments of the direct binding type. Strains of Hyphomicrobium sp. isolated from aquatic habitats were found to have relatedness values ranging from 9 to 70% with strain "EA-617," a subculture of the Hyphomicrobium isolated by Mevius from river water. Strains obtained from soil enrichments had lower values with EA-617, ranging from 3 to 5%. Very little or no homology was detected between the amino acid-utilizing strain Hyphomicrobium neptunium and other Hyphomicrobium strains, although significant homology was observed with the two Hyphomonas strains examined. No homology could be detected between prosthecate bacteria of the genera Rhodomicrobium, Prosthecomicrobium, Ancalomicrobium, or Caulobacter, and Hyphomicrobium strain EA-617 or H. neptunium LE-670. The grouping of Hyphomicrobium strains by their relatedness values agrees well with a grouping according to the base composition of their DNA species. It is concluded that bacteria possessing cellular extensions represent a widely diverse group of organisms.     

A combination of methods for the on-line determination of alcohols in microbial cultures

Summary A new method for the continuous on-line determination of methanol (range 0.2 to 10 gl^–1) and ethanol (0.2 to 120 gl^–1) is described. The rate limiting step is diffusion of the alcohol through the walls of a silicone tube immersed in the culture broth. A sintered SnO₂ sensor was used instead of a Flame Ionization Detector for alcohol determination. Measurement is not affected by bioreactor aeration or agitation rates, dissolved oxygen, carbon dioxide, ammonia or the concentration of cells in the medium. The assay system was tested in extended batch cultivation of Methylomonas sp. with methanol as the sole carbon source (final biomass concentration, 35 gl^–1). Sensor readings agreed well with simultaneous off-line gas chromatographic methanol determination.     

Growth and enzymological characteristics of a pink-pigmented facultative methylotrophMethylobacterium sp. MB1

Growth characteristics of batch and continuous cultures of the pink facultative methylotrophMethylobacterium sp. MB1 were determined. The response of a chemostat culture to a pulse increase of methanol concentration was studied. Malate, succinate and oxaloacetate additions to the methanol-supplemented medium decreased batch culture growth inhibition by methanol. The carotenoid content in cells grown in a chemostat decreased with increasing growth rate. The key enzyme activities of C₁-metabolism were measured in a chemostat culture at different dilution rates.     

Quantitative and qualitative studies of the bacterial microflora of turbot, Scophthalmus maximus L., gills

Populations of aerobic heterotrophic bacteria, occurring on the gills of healthy turbot, were estimated using a dilution plate technique. From a comparison of 18 media, the highest counts, i.e. 7.0 × l0⁵ g⁻¹, were obtained after incubation at 15–25°C on a specifically formulated medium which contained low quantities of beef extract, casein, tryptone and yeast extract. These bacteria were equated with Asticacaulis sp., Hyphomicrobium sp., Janthinobacterium lividum, Prosthecomicrobium sp., Pseudomonas fluorescens and Vibrio sp. Evidence from scanning electron microscopy pointed to a general lack of intimate colonization of exposed areas of the gill. Instead, micro-organisms colonized protected niches, such as the clefts between lamellae and in secluded areas on the arches.     

Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris

A single-chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol-inducible AOX1 promoter. In high-cell-density cultivation on defined medium, methanol-limited and methanol-saturated conditions were compared. After batch and fed-batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate. Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable. To postpone the onset of limitation, the inlet air was enriched by pure oxygen. Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period. Without additional oxygen supply, the process was robust. Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates; it can thus serve as a sensitive indicator of forthcoming problems. Single-step downstream processing from the culture supernatant by His-affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.     

Over-expression of a hydroxypyruvate reductase in Methylobacterium sp. MB200 enhances glyoxylate accumulation

Methylobacterium sp. MB200 capable of producing glyoxylate from methanol was obtained by enrichment culture using a medium containing methanol as the sole carbon source. A hpr gene that encodes a hydroxypyruvate reductase (HPR) was cloned from this strain and was ligated into the vector pLAFR3 to obtain the recombinant plasmid pLAFRh, which was transferred into M. sp. MB200 to generate an recombinant strain MB201. Homologous expression of hpr under the control of the lacZ promoter led to the enhanced glyoxylate accumulation in cultures of Methylobacterium sp MB201. The yield of glyoxylate reached 14.38 mg/mL, representing nearly a twofold increase when compared with the wild-type strain.     

丝氨酸生产菌的筛选及静息细胞培养系统中L-丝氨酸的合成

从广西隆安县沼气池里的残渣中筛选到一株能以甲醇为唯一碳源生长的MB200菌株。根据常规形态特征、生理生化性状及16S rDNA基因序列分析将其鉴定为甲基杆菌属(M ethylobacteriumsp.)。其最佳生长条件为:温度32℃、pH值8.0、甲醇体积分数1.25%。建立了MB200生成L-丝氨酸的静息细胞培养系统。确定静息细胞培养的条件为:甘氨酸质量浓度为10 g.L-1,甲醇50 g.L-1,菌体质量浓度为30 g.L-1,pH8.9,于摇床250 r.m in-1,32℃静息培养48 h,L-丝氨酸产量为7.2 g.L-1。     

Development of a species-specific gene probe for Hyphomicrobium facilis with the inverse PCR.

A species-specific gene probe for Hyphomicrobium facilis was generated from a transposon Tn5-132 insertion mutant defective in methanol oxidation by the inverse PCR. With this probe, the abundance of H. facilis in a garden soil was determined as a subfraction of the total cultivable hyphomicrobia.     

利用麻风树油驯化筛选高活性脂肪酶产生菌及其催化酯化反应

以1株分解麻风树油的脂肪酶产生菌Pseudomonas sp. LP-1为出发菌株, 通过麻疯树油定向驯化筛选获得1株酶活较高且产酶稳定的菌株P. sp. X-2-45, 其水解酶活为29.79 U/mL, 比原始菌株提高了288%。对P. sp. X-2-45生长与产酶特征、对植物油脂水解能力及在有机相中催化脂肪酸和有机醇间的酯化反应研究发现, 该菌株生长速率和产酶速率明显加快, 培养30 h时生物量和酶活达到最大, 稳定期延长, 培养过程中脂肪酶在培养基中的稳定性提高。以麻疯树油诱导合成的P. sp. X-2-45脂肪酶对麻疯树油的水解能力比原始菌株提高了378%, 说明采用麻风树油定向驯化可提高脂肪酶对相应底物的水解能力。X-2-45脂肪酶可以催化月桂酸与正丁醇、正辛醇、月桂醇和丙三醇之间, 棕榈酸、硬脂酸与甲醇、正辛醇、月桂醇和丙三醇之间, 油酸与甲醇、正丁醇、正辛醇、月桂醇和丙三醇之间发生酯化反应。     

Use of secondary-treated wastewater for the production of Muriellopsis sp.

In this paper, the use of secondary-treated wastewater as the culture medium for the production of Muriellopsis sp. microalgal biomass is analyzed. Using this wastewater, a maximum biomass productivity of 0.5 g?l^?1?day^?1 was measured, it being only 38 % lower than that achieved using the standard culture medium. Due to the low nitrogen content of secondary-treated wastewater, cultures produced in a medium containing a high percentage of it become nitrate-limited, thus the quantum yield reduces by up to 0.38 g?E^?1—this compares to 0.67 g?E^?1 when using a standard culture medium. On the other hand, nitrate limitation enhances the accumulation of lipids and carbohydrates, with values measured at 33 and 66 % dry weight, respectively. It was also demonstrated that secondary-treated wastewater does not have any toxic effect on the growth of Muriellopsis sp. in spite of nitrogen being in the form of ammonium rather than in nitrate. Moreover, the secondary-treated wastewater was depurated when used to produce Muriellopsis sp., with the outlet biological oxygen demand and chemical oxygen demand being lower than at the inlet; the nitrate and phosphate concentrations were zero. Therefore, Muriellopsis sp. production using secondary-treated wastewater allows a reduction in the process cost by decreasing freshwater and fertilizer use, as well as by depurating the water, thus greatly enhancing process sustainability.