André Lipski

Diversity and Activity of Methanotrophic Bacteria in Different Upland Soils

ABSTRACT Samples from diverse upland soils that oxidize atmospheric methane were characterized with regard to methane oxidation activity and the community composition of methanotrophic bacteria (MB). MB were identified on the basis of the detection and comparative sequence analysis of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. MB commonly detected in soils were closely related to Methylocaldum spp., Methylosinus spp., Methylocystis spp., or the “forest sequence cluster” (USC α), which has previously been detected in upland soils and is related to pmoA sequences of type II MB (Alphaproteobacteria). As well, a novel group of sequences distantly related (<75% derived amino acid identity) to those of known type I MB (Gammaproteobacteria) was often detected. This novel “upland soil cluster γ” (USC γ) was significantly more likely to be detected in soils with pH values of greater than 6.0 than in more acidic soils. To identify active MB, four selected soils were incubated with 13CH4 at low mixing ratios (<50 ppm of volume), and extracted methylated phospholipid fatty acids (PLFAs) were analyzed by gas chromatography-online combustion isotope ratio mass spectrometry. Incorporation of 13C into PLFAs characteristic for methanotrophic Gammaproteobacteria was observed in all soils in which USC γ sequences were detected, suggesting that the bacteria possessing these sequences were active methanotrophs. A pattern of labeled PLFAs typical for methanotrophic Alphaproteobacteria was obtained for a sample in which only USC α sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.

Characterization of N2O-producing Xanthomonas-like isolates from biofilters as Stenotrophomonas nitritireducens sp. nov., Luteimonas mephitis gen. nov., sp. nov. and Pseudoxanthomonas broegbernensis gen. nov., sp. nov.

A group of yellow-pigmented isolates from ammonia-supplied biofilters showed an unusual denitrification reaction. All strains reduced nitrite but not nitrate without production of nitrogen (N2). The only product found was nitrous oxide (N2O). The strains were divided into two clusters and one separate strain by their fatty acid profiles, which were similar to the fatty acid profiles of the genera Xanthomonas and Stenotrophomonas. Analyses of the 165 rDNA sequences showed that these clusters and the separate strain form three independent lines within the Xanthomonas branch of the Proteobacteria. The evolutionary distances of the isolates to members of the related genera Xanthomonas, Stenotrophomonas and Xylella calculated by the 16S rDNA sequences led to the proposal of two new genera and three new species, Stenotrophomonas nitritireducens sp. nov., Luteimonas mephitis gen. nov., sp. nov. and Pseudoxanthomonas broegbernensis gen. nov., sp. nov. The type strains are Stenotrophomonas nitritireducens L2T (= DSM 12575T), Luteimonas mephitis B1953/27.1T (= DSM 12574T) and Pseudoxanthomonas broegbernensis B1616/1T (= DSM 12573T).

Cultivation of a novel cold-adapted nitrite oxidizing betaproteobacterium from the Siberian Arctic

Permafrost-affected soils of the Siberian Arctic were investigated with regard to identification of nitrite oxidizing bacteria active at low temperature. Analysis of the fatty acid profiles of enrichment cultures grown at 4°C, 10°C and 17°C revealed a pattern that was different from that of known nitrite oxidizers but was similar to fatty acid profiles of Betaproteobacteria. Electron microscopy of two enrichment cultures grown at 10°C showed prevalent cells with a conspicuous ultrastructure. Sequence analysis of the 16S rRNA genes allocated the organisms to a so far uncultivated cluster of the Betaproteobacteria, with Gallionella ferruginea as next related taxonomically described organism. The results demonstrate that a novel genus of chemolithoautotrophic nitrite oxidizing bacteria is present in polygonal tundra soils and can be enriched at low temperatures up to 17°C. Cloned sequences with high sequence similarities were previously reported from mesophilic habitats like activated sludge and therefore an involvement of this taxon in nitrite oxidation in nonarctic habitats is suggested. The presented culture will provide an opportunity to correlate nitrification with nonidentified environmental clones in moderate habitats and give insights into mechanisms of cold adaptation. We propose provisional classification of the novel nitrite oxidizing bacterium as ‘Candidatus Nitrotoga arctica’.

Pseudonocardia asaccharolytica sp. nov. and Pseudonocardia sulfidoxydans sp. nov., two new dimethyl disulfide-degrading actinomycetes and emended description of the genus Pseudonocardia

Seven bacterial strains capable of oxidizing methyl sulfides were isolated from experimental biofilters filled with tree-bark compost. The isolates could be divided into two groups according to their method of methyl sulfide degradation. Four isolates could use only dimethyl disulfide as the sole source of energy and three strains were able to use dimethyl sulfide and dimethyl disulfide. Oxidation of the methyl sulfides by both groups led to the stoichiometric formation of sulfate. Chemotaxonomic, morphological, physiological and phylogenetic properties identified all isolates as members of the genus Pseudonocardia. The absence of phosphatidylcholine from the polar lipid pattern, as well as results of 16S rDNA analyses, led to the proposal of two new species, Pseudonocardia asaccharolytica sp. nov. and Pseudonocardia sulfidoxydans sp. nov. The type strains are P. asaccharolytica DSM 44247T and P. sulfidoxydans DSM 44248T. With respect to the characteristic polar lipid pattern and the ability to oxidize sulfides, an emended description of the genus Pseudonocardia is proposed.

The impact of different soil parameters on the community structure of dominant bacteria from nine different soils located on Livingston Island, South Shetland Archipelago, Antarctica.

Microorganisms inhabit very different soil habitats in the ice-free areas of Antarctica, playing a major role in nutrient cycling in cold environments. We studied the soil characteristics and the dominant bacterial composition from nine different soil profiles located on Livingston Island (maritime Antarctica). The total carbon (TC) and total nitrogen (TN) values were high for the vegetated soils, decreasing with depth, whereas the values for the mineral soils were generally low. Soil pH was more acidic for moss-covered soils and neutral to alkaline for mineral soils. Numbers of culturable heterotrophic bacteria were higher at vegetated sites, but significant numbers were also detectable in carbon-depleted soils. Patterns of denaturing gradient gel electrophoresis (DGGE) revealed a highly heterogeneous picture throughout the soil profiles. Subsequent sequencing of DGGE bands revealed in total 252 sequences that could be assigned to 114 operational taxonomic units, showing the dominance of members of the Bacteroidetes and Acidobacteria. The results of phospholipid fatty acid analysis showed a lack of unsaturated fatty acids for most of the samples. Samples with a prevalence of unsaturated over saturated fatty acids were restricted to several surface samples. Statistical analysis showed that the dominant soil bacterial community composition is most affected by TC and TN contents and soil physical factors such as grain size and moisture, but not pH.

Isolation and characterization of a moderately thermophilic nitrite‐oxidizing bacterium from a geothermal spring

Geothermal environments are a suitable habitat for nitrifying microorganisms. Conventional and molecular techniques indicated that chemolithoautotrophic nitrite-oxidizing bacteria affiliated with the genus Nitrospira are widespread in environments with elevated temperatures up to 55 °C in Asia, Europe, and Australia. However, until now, no thermophilic pure cultures of Nitrospira were available, and the physiology of these bacteria was mostly uncharacterized. Here, we report on the isolation and characterization of a novel thermophilic Nitrospira strain from a microbial mat of the terrestrial geothermal spring Gorjachinsk (pH 8.6; temperature 48 °C) from the Baikal rift zone (Russia). Based on phenotypic properties, chemotaxonomic data, and 16S rRNA gene phylogeny, the isolate was assigned to the genus Nitrospira as a representative of a novel species, for which the name Nitrospira calida is proposed. A highly similar 16S rRNA gene sequence (99.6% similarity) was detected in a Garga spring enrichment grown at 46 °C, whereas three further thermophilic Nitrospira enrichments from the Garga spring and from a Kamchatka Peninsula (Russia) terrestrial hot spring could be clearly distinguished from N. calida (93.6-96.1% 16S rRNA gene sequence similarity). The findings confirmed that Nitrospira drive nitrite oxidation in moderate thermophilic habitats and also indicated an unexpected diversity of heat-adapted Nitrospira in geothermal hot springs.

Identification of heterotrophic bacteria isolated from ammonia-supplied experimental biofilters

Summary Laboratory scale biofilters were supplied with an artificial waste gas containing ammonia and an ammonia/dimethyldisulfide mixture. 62 bacterial strains were isolated from the filter material, differentiated and identified by chemotaxonomic and physiological methods. The taxonomic groups were compared with known bacterial strains originating from biofilters which were supplied with an ammonia containing complex industrial waste gas. The different effects of a defined artificial waste gas and a complex multicomponent waste gas on the culturable population of biofilters are discussed.

Application of rRNA-targeted oligonucleotide probes in biotechnology

Abstract. Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.

Chryseobacterium ureilyticum sp. nov., Chryseobacterium gambrini sp. nov., Chryseobacterium pallidum sp. nov. and Chryseobacterium molle sp. nov., isolated from beer-bottling plants

Four Gram-negative, rod-shaped, non-spore-forming and non-motile bacterial strains were isolated from surfaces and biofilms associated with beer-bottling plants. Based on their 16S rRNA gene sequences these isolates were allocated to the genus Chryseobacterium. The sequence similarities of the isolates to the next most closely related type strains of this genus ranged from 96.4 to 98.3%. The presence of menaquinone MK-6 and predominant fatty acids 15:0 iso, 17:1 iso cis9, 15:0 iso 2-OH and 17:0 iso 3-OH supported the affiliation of these strains to the genus. The results of DNA-DNA hybridization, biochemical tests and chemotaxonomic properties allowed genotypic and phenotypic differentiation of the strains from the next most closely related Chryseobacterium species with validly published names. Therefore, the isolates represent four novel species for which the names Chryseobacterium ureilyticum (type strain F-Fue-04IIIaaaa(T)=DSM 18017(T)=CCUG 52546(T)), Chryseobacterium gambrini (type strain 5-1St1a(T)=DSM 18014(T)=CCUG 52549(T)), Chryseobacterium pallidum (type strain 26-3St2b(T)=DSM 18015(T)=CCUG 52548(T)) and Chryseobacterium molle (type strain DW3(T)=DSM 18016(T)=CCUG 52547(T)) are proposed.

Fatty Acid Profiles of Nitrite-oxidizing Bacteria Reflect theirPhylogenetic Heterogeneity

Summary The fatty acid profiles of all described species of the nitrite-oxidizing genera Nitrobacter , Nitrococcus , Nitrospina and Nitrospira were analyzed. The four genera had distinct profiles, which can be used for the differentiation and allocation of new isolates to these genera. The genus Nitrobacter is characterized by vaccenic acid as the main compound with up to 92% of the fatty acids and the absence of hydroxy fatty acids. The genus Nitrococcus showed cis -9-hexadecenoic acid, hexadecanoic acid and vaccenic acid as main parts. Small amounts of 3-hydroxy-dodecanoic acid were detected. The genus Nitrospina possessed tetradecanoic acid and cis -9-hexadecenoic acid as main compounds, also 3-hydroxy-hexadecanoic acid was detected for this genus. The genus Nitrospira showed a pattern with more variations among the two described species. These organisms are characterized by the cis -7 and cis -11-isomers of hexadecenoic acid. For Nitrospira moscoviensis a specific new fatty acid was found, which represented the major constituent in the fatty acid profiles of autotrophically grown cultures. It was identified as 11-methyl-hexadecanoic acid. Since this compound is not known for other bacterial taxa, it represents a potential lipid marker for the detection of Nitrospira moscoviensis relatives in enrichment cultures and environmental samples. A cluster analysis of the fatty acid profiles is in accordance with 16S rRNA sequence-based phylogeny of the nitrite-oxidizing bacteria.