Some Growth Characteristics of a Hyphomicrobium sp. in Batch Culture

S ummary : A Hyphomicrobium sp. isolated from a mixed culture of methane utilizing micro-organisms grew rapidly with generation times as short as 4·7 h in pure culture with methanol as the carbon substrate. The stoichiometry of oxygen utilization by this micro-organism, as indicated by the cell yield with respect to oxygen varied at low levels of dissolved oxygen in the culture medium. In addition, the initial methanol concentration in the medium affected the maximum specific growth rate of the Hyphomicrobium sp.     

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.     

Population analysis in a denitrifying sand filter: conventional and in situ identification of Paracoccus spp. in methanol-fed biofilms.

The microbial community of a denitrifying sand filter in a municipal wastewater treatment plant was examined by conventional and molecular techniques to identify the bacteria actively involved in the removal of nitrate. In this system, denitrification is carried out as the last step of water treatment by biofilms growing on quartz grains with methanol as a supplemented carbon source. The biofilms are quite irregular, having a median thickness of 13 to 20 microns. Fatty acid analysis of 56 denitrifying isolates indicated the occurrence of Paracoccus spp. in the sand filter. 16S rRNA-targeted probes were designed for this genus and the species cluster Paracoccus denitrificans-Paracoccus versutus and tested for specificity by whole-cell hybridization. Stringency requirements for the probes were adjusted by use of a formamide concentration gradient to achieve complete discrimination of even highly similar target sequences. Whole-cell hybridization confirmed that members of the genus Paracoccus were abundant among the isolates. Twenty-seven of the 56 isolates hybridized with the genus-specific probes. In situ hybridization identified dense aggregates of paracocci in detached biofilms. Probes complementary to the type strains of P. denitrificans and P. versutus did not hybridize to cells in the biofilms, suggesting the presence of a new Paracoccus species in the sand filter. Analysis using confocal laser scanning microscopy detected spherical aggregates of morphologically identical cells exhibiting a uniform fluorescence. Cell quantification was performed after thorough disruption of the biofilms and filtration onto polycarbonate filters. An average of 3.5% of total cell counts corresponded to a Paracoccus sp., whereas in a parallel sand filter with no supplemented methanol, and no measurable denitrification, only very few paracocci (0.07% of cells stained with 4',6-diamidino-2-phenylindole) could be detected. Hyphomicrobium spp. constituted approximately 2% of all cells in the denitrifying unit and could not be detected in the regular sand filter. This clear link between in situ abundance and denitrification suggests an active participation of paracocci and hyphomicrobia in the process. Possible selective advantages favoring the paracocci in this habitat are discussed.     

Halomonas campisalis, an obligatorily alkaliphilic, nitrous oxide-reducing denitrifier with a Mo-cofactor-lacking nitrate reductase

We isolated eight strains of denitrifying bacteria that reduce nitrate and nitrous oxide at pH 10 from Lake Magadi (Kenya). Despite certain differences between the strains, they are similar in terms of G+C content (66.1-68.1 mol %) and DNA-DNA homology (75-92%) and represent different morphotypes of the same species. Based on the results of partial 16S rRNA sequencing, strain Z-7398-2 was most closely related to the Halomonas campisalis isolate from Alkali Lake (USA). The DNA-DNA homology level between the tested strain and the type strain of H. campisalis 4A was 88%. These two strains were also similar phenotypically. However, the culture isolated by us was characterized by peculiar properties, such as obligate alkaliphily, which manifested itself in the culture dependence on carbonates and lack of growth at pH values below 7, a nitrous oxide-reducing capacity, and an unusual nitrate reductase that lacked molybdenum and a Mo cofactor.     

Isolation and partial characterization of a bacteriophage active on Hyphomicrobium sp. WI-926

Isolation of a Hyphomicrobium phage from raw sewage from Athens, Ohio, was achieved by a combination of differential centrifugation, filtration, enrichment in mixed Hyphomicrobium cultures, and purification on individual host strains by subculturing single plaques in soft agar overlayers. Enrichments with water from Lake Erie and Lake Beechwood (Ohio) were unsuccessful. Out of 21 Hyphomicrobium strains and 22 other Gram-negative and Gram-positive bacteria tested, only Hyphomicrobium WI-926 (isolated from a German forest pond) was susceptible. This phage had an isometric head (diameter between opposite apices, 67 nm) and a short (12 nm), noncontractile tail and belongs thus to the morphogroup C1. It contained double-stranded DNA. The single-step growth curve showed a latent period of 9 h, a rise period of 6 h, and a burst size of 35. The various differentiation stages in the host development exhibited different affinities for phage adsorption and development. While all stages allowed phage adsorption, the daughter cells were most efficient. Phage multiplication was limited to daughter cells, and the development of infected swarmer cells was arrested permanently at this stage.     

Dimethylsulfone as a growth substrate for novel methylotrophic species of Hyphomicrobium and Arthrobacter

Dimethylsulfone is a major product of the chemical oxidation in the atmosphere of the principal biogenic sulfur gas, dimethylsulfide, but no studies have been reported on the mechanisms for its microbiological degradation. Three novel strains of bacteria have been isolated from enrichment cultures provided with dimethylsulfone as the only carbon and energy substrate. These are novel facultatively methylotrophic species of Hyphonmicrobium and Arthobacter, capable of growth on a range of one-carbon substrates. Cell-free extracts contained activities of enzymes necessary for a reductive/oxidative pathway for dimethylsulfone degradation: membrane-bound-dimethylsulfone and dimethylsulfoxide reductases, dimethylsulfide monooxygenase, and methanethiol oxidase. Enzymatic evidence is also presented for the subsequent oxidation of formaldehyde by formaldehyde and formate dehydrogenases in the Hyphomicrobium strain and by a dissimilatory ribulose monophosphate cycle in the Arthrobacter strains. The strains also grew on dimethylsulfoxide and dimethylsulfide, and dimethylsulfide-grown bacteria oxidized dimethylsulfide and dimethylsulfoxide but not dimethylsulfone. Formaldehyde assimilation was effected in the Hyphomicrobium strain by the serine pathway, but enzymes of the ribulose monophosphate cycle for formaldehyde assimilation were present in the Arthrobacter strains grown on dimethylsulfone. In contrast, one of the Arthrobacter strains was shown to switch to the serine pathway during growth on methanol. Growth yields on dimethylsulfone and formaldehyde were consistent with the occurrence of the serine pathway in Hyphomicrobium strain S1 and the ribulose monophosphate cycle in Arthrobacter strain TGA, and with the proposed reductive pathway for dimethylsulfone degradation in both.     

Molecular detection and isolation from antarctica of methylotrophic bacteria able to grow with methylated sulfur compounds

This study is the first demonstration that a diverse facultatively methylotrophic microbiota exists in some Antarctic locations. PCR amplification of genes diagnostic for methylotrophs was carried out with bacterial DNA isolated from 14 soil and sediment samples from ten locations on Signy Island, South Orkney Islands, Antarctica. Genes encoding the mxaF of methanol dehydrogenase, the fdxA for Afipia ferredoxin, the msmA of methanesulfonate monooxygenase, and the 16S rRNA gene of Methylobacterium were detected in all samples tested. The mxaF gene sequences corresponded to those of Hyphomicrobium, Methylobacterium, and Methylomonas. Over 30 pure cultures of methylotrophs were isolated on methanesulfonate, dimethylsulfone, or dimethylsulfide from ten Signy Island lakes. Some were identified from 16S rRNA gene sequences (and morphology) as Hyphomicrobium species, strains of Afipia felis, and a methylotrophic Flavobacterium strain. Antarctic environments thus contain diverse methylotrophic bacteria, growing on various C1-substrates, including C1-sulfur compounds.     

DNA-probing indicates the occurrence of denitrification and nitrogen fixation genes in Hyphomicrobium. Distribution of denitrifying and nitrogen fixing isolates of Hyphomicrobium in a sewage treatment plant

Abstract: Twenty-six Hyphomicrobium isolates from the sewage treatment plant and its receiving water body in Plön (Schleswig-Holstein, Germany) and two culture collection strains were screened for the occurrence of genes coding for denitrification enzymes (dissimilatory nitrate, nitrite and nitrous oxide reductases), for dinitrogen fixation (nitrogenase reductase) and for nitrification (ammonia monooxygenase catalyzing the first stage of this process) by DNA-probing. More than one half of the isolates had genes coding for denitrification enzymes. The DNA-DNA hybridization signals obtained with the gene segments correlated with enzyme activity measurements. The DNA of some isolates distinctly hybridized with the nif H probe indicating the occurrence of nitrogenase in the genus Hyphomicrobium . No signal was detected with the gene probe for nitrification. The results show that probes consisting of gene segments can be employed successfully to monitor the occurrence of genes which can show complex expression and in bacteria growing at low rates. The distribution pattern of the denitrification genes indicates that methylotrophic prosthecate bacteria of the sewage treatment plant and its receiving water body occupy different ecological niches.     

Halomonas campisalis,an Obligatorily Alkaliphilic,Nitrous Oxide–Reducing Denitrifier with a Molybdenum Cofactor–Lacking Nitrate Reductase

We isolated eight strains of denitrifying bacteria that reduce nitrate and nitrous oxide at pH 10 from Lake Magadi (Kenya). Despite certain differences between the strains, they are similar in terms of G+C content (66.1–68.1 mol %) and DNA–DNA homology (75–92%) and represent different morphotypes of the same species. On the basis of results of partial 16S rRNA sequencing, strain Z-7398-2 was found to be most closely related to the Halomonas campisalis isolate from the Alkali Lake (USA). The DNA–DNA homology level between the tested strain and the type strain of H. campisalis 4A was 88%. These two strains were also similar phenotypically. However, the culture isolated by us was characterized by peculiar properties, such as obligate alkaliphily, which manifested itself in the culture dependence on carbonates and lack of growth at pH values below 7, a nitrous oxide–reducing capacity, and an unusual nitrate reductase that lacked molybdenum and a molybdenum cofactor.     

Experiments on the microbiology of cellulose decomposition in a municipal sewage plant

Summary Analyses of sewage solids show cellulose to be one of the chief components. Culture counts of cellulolytic bacteria in a primary anaerobic sewage digestor show them to be present in numbers as high as 1 million per ml. The tendency of cellulolytic bacteria to cling to cellulose fibers makes it highly probable that the number of cellulolytic cells is much larger. All 10 cellulolytic strains isolated in pure culture show better growth in solid than in liquid media, and for some of them agar possesses growth promoting properties. For some strains, phytone and trypticase can replace the agar but other strains could not be grown in media containing no agar. Hydrogen, carbon dioxide, ethanol, formic acid, acetic acid, and lactic acid have been identified as fermentation products and glucose shown to be a product of cellulose digestion. Cellobiose, starch, dextrin, and maltose were fermented by 5 tested strains, inulin and esculin by one of them, but none of 17 other carbohydrates, including glucose, were attacked. The rate of cellulose fermentation by a mixed culture of aClostridium sp. and a cellulose decomposer is much greater than the rate by the latter alone. The rate of fermentation by a pure culture is not affected by acetate concentrations up to 5000 parts per million. It is postulated that the rate of fermentation of cellulose may be the factor limiting the rate of sewage fermentation though more evidence regarding rates of fermentation of other constituents of sewage is needed before final conclusions can be drawn. This investigation was supported in part by a research grant from the National Institute of Health, U.S. Public Health Service.     

Aerobic and anaerobic growth of Paracoccus denitrificans on methanol

1. The dye-linked methanol dehydrogenase from Paracoccus denitrificans grown aerobically on methanol has been purified and its properties compared with similar enzymes from other bacteria. It was shown to be specific and to have high affinity for primary alcohols and formaldehyde as substrate, ammonia was the best activator and the enzyme could be linked to reduction of phenazine methosulphate.
2. Paracoccus denitrificans could be grown anaerobically on methanol, using nitrate or nitrite as electron acceptor. The methanol dehydrogenase synthesized under these conditions could not be differentiated from the aerobically-synthesized enzyme.
3. Activities of methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, nitrate reductase and nitrite reductase were measured under aerobic and anaerobic growth conditions.
4. Difference spectra of reduced and oxidized cytochromes in membrane and supernatant fractions of methanol-grown P. denitrificans were measured.
5. From the results of the spectral and enzymatic analyses it has been suggested that anaerobic growth on methanol/nitrate is made possible by reduction of nitrate to nitrite using electrons derived from the pyridine nucleotide-linked dehydrogenations of formaldehyde and formate, the nitrite so produced then functioning as electron acceptor for methanol dehydrogenase via cytochrome c and nitrite reductase.

     

A microcomputer program for establishing and maintaining a database for laboratory culture collections

A computer program that facilitates the creation of a culture collection database has been written for a microcomputer (Apple He with a Z-80 card) using dBASE II® (Ashton-Tate). The Culture Collection Program accommodates up to 250 individual strain records on one 5 1/4" floppy disk. For each strain, information that can be stored includes the name of the micro-organism, culture collection number, antibiotic resistance markers, plasmids, genetic markers, references, growth medium, growth temperature and additional comments. The last date of subculturing can be ascertained and information about the status of the preserved cultures can also be noted. With a menu-driven format which requires no computer programming expertise, the user can readily create new entries, update old ones and search the database for strains with certain common properties.     

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.     

Biodegradation of semiconductor volatile organic compounds by four novel bacterial strains: a kinetic analysis

This study isolated pure microorganisms for further bioreactor applications. Four novel strains of Pseudomonas citronellolis YAIP521, Paracoccus versutus HSAC51, Burkholderia sp. HUEL671, and Pseudomonas aeruginosa JUPG561 were isolated and tested for biodegradation of isopropyl alcohol (IPA), acetone, ethyl lactate (EL), and propylene glycol mono methyl ether acetate (PGMEA), respectively. The maximum biodegradation rates for IPA, acetone, EL, and PGMEA were 5.27, 3.87, 26.86, and 48.93 mg L(-1) h(-1), respectively. The Haldane kinetic parameters determined for these strains when degrading targeted volatile organic compounds were maximum specific growth rate, half-saturation constant, and inhibition constant. The isolated strains have potential application in various bioreactors. The kinetic parameters obtained in this study provide a basis for further bioreactor experiments.     

Hyphomicrobium as a component of the aquatic microflora--morphological and physiological studies on two strains

Aspects of the morphology, metabolism and physiology of two oligocarbophilic strains of Hyphomicrobium (H4K and S5K), isolated from the Plusssee, were studied. Both strains are able to grow on mineral salts media without added organic carbon sources. Strain H4K grows well even in double distilled water. The two strains cannot grow on mineral media in the absence of atmospheric CO2. No growth occurred also in air purified of organic carbon, in spite of the presence of CO2. On the contrary, there is good growth in the presence of some organic compounds and without atmospheric CO2, i.e., heterotrophic metabolism without CO2 assimilation is possible. Growth was enhanced in a methanol atmosphere, and by the addition of yeast extract, methylamine, peptone and glucose. In nutrient solutions containing acetate or formate as carbon source, growth of H4K begins only after an adaptation period of ca. 4 weeks.     

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.     

New bacteriophages active on strains of Hyphomicrobium

Fifty-five lytic bacteriophages isolated from water and soil samples were active on many strains of the genus Hyphomicrobium. The optimal isolation procedure was an adsorption method in which samples from a habitat similar to that of the respective host bacterium were used as the phage inoculum. According to the morphology and nucleic acid type these bacteriophages belonged to different families: Myoviridae (type A1: five phages); Styloviridae (type B1: 33 phages; type B2: eight phages) and Podoviridae (type C1: nine phages). The Styloviridae (type B1) appeared in two morphological variants (tails flexible or rigid). All phages investigated were specific for the genus Hyphomicrobium and were unable to lyse members of other genera of hyphal, budding bacteria (e.g. Hyphomonas, Pedomicrobium, genus D, genus T). The host specificity of 42 phages was tested with 156 Hyphomicrobium strains: 122 strains were lysed by at least one of these phages, but 34 Hyphomicrobium strains were not susceptible. Morphotype B1 phages with identical morphology could be distinguished according to their host-range properties on prophage-containing Hyphomicrobium strains. With regard to differences in morphology and host range, 25 phages were selected for more detailed investigations. From these phages DNA was isolated; the melting transition midpoints (Tm) ranged from 67 to 93 degrees C. The upper and higher values suggested the presence of DNA modifications. Six different adsorption patterns could be distinguished among the Hyphomicrobium phages. Preferred attachment sites were the proximal pole of the mother cell, the hyphal tip, the distal pole of the bud, and the distal pole of the swarmer cell.     

Mixed culture of bacteria utilizing methanol for growth

Summary A mixed culture of bacteria MS1 capable of growth on methanol as its sole carbon-energy source was isolated by continuous culture with a minimum mineral salts medium and without asepsis. This bacterial association is composed of four Gram-negative bacterial strains which we identified. The respective numerical proportions of each microorganism are stable in time. The properties, and specially the stability of the continuous culture process have been studied with respect to substrate yield, biomass and productivity.     

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO(3)(-)-N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO(3)(-)-N mg of MLVSS(-1) h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [(13)C]methanol to biomark the DNA of the denitrifiers. The extracted [(13)C]DNA and [(12)C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [(13)C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [(12)C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [(14)C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.