From oil spills to barley growth - oil-degrading soil bacteria and their promoting effects.

Heavy contamination of soils by crude oil is omnipresent in areas of oil recovery and exploitation. Bioremediation by indigenous plants in cooperation with hydrocarbon degrading microorganisms is an economically and ecologically feasible means to reclaim contaminated soils. To study the effects of indigenous soil bacteria capable of utilizing oil hydrocarbons on biomass production of plants growing in oil-contaminated soils eight bacterial strains were isolated from contaminated soils in Kazakhstan and characterized for their abilities to degrade oil components. Four of them, identified as species of Gordonia and Rhodococcus turned out to be effective degraders. They produced a variety of organic acids from oil components, of which 59 were identified and 7 of them are hitherto unknown acidic oil metabolites. One of them, Rhodococcus erythropolis SBUG 2054, utilized more than 140 oil components. Inoculating barley seeds together with different combinations of these bacterial strains restored normal growth of the plants on contaminated soils, demonstrating the power of this approach for bioremediation. Furthermore, we suggest that the plant promoting effect of these bacteria is not only due to the elimination of toxic oil hydrocarbons but possibly also to the accumulation of a variety of organic acids which modulate the barley's rhizosphere environment.

Paenibacillus solani sp. nov., isolated from potato rhizosphere soil.

A novel Gram-stain-positive, endospore-forming bacterium, designated FJAT-22460T, was isolated from a soil sample of a potato field in Xinjiang Autonomous Region, China. Cells were rods that were catalase-positive and motile by peritrichous flagella. The strain was found to grow at temperatures ranging from 10 to 40 °C (optimum 30 °C) and at pH 5.0-12.0 (optimum pH 7) with 0-5 % (w/v) NaCl (optimum 0 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain FJAT-22460T belonged to the genus Paenibacillus and exhibited 16S rRNA gene sequence similarities of 97.3, 97.2, 97.2 and 97.0 % with Paenibacillus glucanolyticus DSM 5162T, Paenibacillus lautus DSM 3035T, Paenibacillus lactis MB 1871T and Paenibacillus chibensis JCM 9905T, respectively. DNA-DNA relatedness of strain FJAT-22460T with Paenibacillus glucanolyticusDSM 5162T and Paenibacillus lautus DSM 3035T was 62.6 % and 33.3 %, respectively, lower than the 70 % accepted for species delineation. The menaquinone was identified as MK-7. The major fatty acids detected were anteiso-C15 : 0 (51.4 %), iso-C15 : 0 (5.3 %), C16 : 0 (12.1 %), iso-C16 : 0 (10.7 %) and anteiso-C17 : 0 (6.9 %). The DNA G+C content was determined to be 50.9 mol%. Phenotypic, chemotaxonomic and genotypic properties clearly indicated that isolate FJAT-22460T represents a novel species within the genus Paenibacillus, for which the name Paenibacillus solani sp. nov. is proposed. The type strain is FJAT-22460T (=DSM 100999T=CCTCC AB 2015207T).

Bacillus solani sp. nov., isolated from rhizosphere soil of a potato field.

A novel Gram-stain-positive, endospore-forming bacterium, designated strain FJAT-18043T, was isolated from a soil sample of a potato field in Xinjiang Autonomous Region, China. Cells were rods that were catalase-positive and motile by peritrichous flagella. The strain grew at 20-45 °C (optimum 35 °C), at pH 6.0-10.0 (optimum pH 9) and with 0-10 % (w/v) NaCl (optimum 0 %). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain FJAT-18043T belonged to the genus Bacillus and exhibited similarities of 97.7, 97.6, 97.2 and 97.2 % with Bacillus eiseniae A1-2T, Bacillus horneckiae DSM 23495T, Bacillus gottheilii WCC 4585T and Bacillus purgationiresistens DS22T, respectively. DNA-DNA relatedness between strain FJAT-18043T and B. eiseniae A1-2 T was lower than 70 % (36.1 %). The menaquinone was identified as MK-7 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids detected were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0, C16 : 0 and iso-C14 : 0. The DNA G+C content was 48.8 mol%. Phenotypic, chemotaxonomic and genotypic properties clearly indicated that isolate FJAT-18043T represents a novel species within the genus Bacillus, for which the name Bacillus solani sp. nov. is proposed. The type strain is FJAT-18043T ( = DSM 29501T = CCTCC AB 2014277T).

Enrichment of aliphatic, alicyclic and aromatic acids by oil-degrading bacteria isolated from the rhizosphere of plants growing in oil-contaminated soil from Kazakhstan.

Three microbial strains were isolated from the rhizosphere of alfalfa (Medicago sativa), grass mixture (Festuca rubra, 75 %; Lolium perenne, 20 %; Poa pratensis, 10 %), and rape (Brassica napus) on the basis of their high capacity to use crude oil as the sole carbon and energy source. These isolates used an unusually wide spectrum of hydrocarbons as substrates (more than 80), including n-alkanes with chain lengths ranging from C12 to C32, monomethyl- and monoethyl-substituted alkanes (C12-C23), n-alkylcyclo alkanes with alkyl chain lengths from 4 to 18 carbon atoms, as well as substituted monoaromatic and diaromatic hydrocarbons. These three strains were identified as Gordonia rubripertincta and Rhodococcus sp. SBUG 1968. During their transformation of this wide range of hydrocarbon substrates, a very large number of aliphatic, alicyclic, and aromatic acids was detected, 44 of them were identified by GC/MS analyses, and 4 of them are described as metabolites for the first time. Inoculation of plant seeds with these highly potent bacteria had a beneficial effect on shoot and root development of plants which were grown on oil-contaminated sand.

Eoetvoesia caeni gen. nov., sp. nov., isolated from an activated sludge system treating coke plant effluent.

A novel bacterium, PB3-7B(T), was isolated on phenol-supplemented inorganic growth medium from a laboratory-scale wastewater purification system that treated coke plant effluent. 16S rRNA gene sequence analysis revealed that strain PB3-7B(T) belonged to the family Alcaligenaceae and showed the highest pairwise sequence similarity to Parapusillimonas granuli Ch07(T) (97.5%), Candidimonas bauzanensis BZ59(T) (97.3%) and Pusillimonas noertemannii BN9(T) (97.2%). Strain PB3-7B(T) was rod-shaped, motile and oxidase- and catalase-positive. The predominant fatty acids were C(16 : 0), C(17 : 0) cyclo, C(19 : 0) cyclo ω8c and C(14 : 0) 3-OH, and the major respiratory quinone was Q-8. The G+C content of the genomic DNA of strain PB3-7B(T) was 59.7 mol%. The novel bacterium can be distinguished from closely related type strains based on its urease activity and the capacity for assimilation of glycerol and amygdalin. On the basis of the phenotypic, chemotaxonomic and molecular data, strain PB3-7B(T) is considered to represent a new genus and species, for which the name Eoetvoesia caeni gen. nov., sp. nov. is proposed. The type strain of Eoetvoesia caeni is PB3-7B(T) ( = DSM 25520(T) = NCAIM B 02512(T)).

Hephaestia caeni gen. nov., sp. nov., a novel member of the family Sphingomonadaceae isolated from activated sludge.

A Gram-staining-negative, rod-shaped and motile bacterium, designated strain ERB1-3(T), was isolated from a laboratory-scale activated sludge system treating coke plant effluent using thiocyanate-supplemented growth medium. Strain ERB1-3(T) was oxidase-positive and weakly catalase-positive. The predominant fatty acids were C18:1ω7c (35.6 %) and C17:1ω6c (29.2%), and the major respiratory quinone was Q-10. Polar lipids were dominated by sphingoglycolipid and phosphatidylglycerol. Major polyamines were spermidine and sym-homospermidine. The G+C content of the genomic DNA of strain ERB1-3(T) was 66.4 mol%. Based on the 16S rRNA gene, strain ERB1-3(T) exhibited the highest sequence similarity values to Sphingomonas sanxanigenens DSM 19645(T) (96.1%), Sphingobium scionense DSM 19371(T) (95.1%) and Stakelama pacifica LMG 24686(T) (94.8%) within the family Sphingomonadaceae. The novel isolate had some unique chemotaxonomic features that differentiated it from these closely related strains, contained much more C17 : 1ω6c, C15 : 0 2-OH, C17:0 and C17:1ω8c fatty acids and possessed diphosphatidylglycerol only in trace amounts. On the basis of the phenotypic, chemotaxonomic and molecular data, strain ERB1-3(T) is considered to represent a novel genus and species, for which the name Hephaestia caeni gen. nov., sp. nov. is proposed. The type strain is ERB1-3(T) ( = DSM 25527(T) = NCAIM B 02511(T)).

The effect of easily degradable substrate feeding on the community structure of laboratory-scale wastewater sludge digesters.

The effect of several easily degradable substrates, such as protein, starch and sunflower oil was investigated on the bacterial community of a laboratory-scale biogas model system. Besides measuring gas yield, Denaturing Gradient Gel Electrophoresis (DGGE), Phospholipids Fatty Acid Analysis (PLFA) for Bacteria and T-RFLP analysis of the mcrA gene for Archaea were used. The community of the examined biogas reactors adapted to the new substrates through a robust physiological reaction followed by moderate community abundance shifts. Gas yield data clearly demonstrated the physiological adaptation to substrate shifts. Statistical analysis of DNA and chemotaxonomic biomarkers revealed community abundance changes. Sequences gained from DGGE bands showed the dominance of the phyla Bacteroidetes and the presence of Firmicutes (Clostridia) and Thermotogae. This was supported by the detection of large amounts of branched 15-carbon non-hydroxy fatty acids in PLFA profiles, as common PLFA markers of the Bacteroidetes group. Minor abundance ratios changes were observed in the case of Archaea in accordance with changes of the fed substrates.

Reclassification of Leifsonia ginsengi (Qiu et al. 2007) as Herbiconiux ginsengi gen. nov., comb. nov. and description of Herbiconiux solani sp. nov., an actinobacterium associated with the phyllosphere of Solanum tuberosum L.

In the context of studying the effects of transgenic fructan-producing potatoes on the community structure of phyllosphere bacteria, a group of strains closely related to the species Leifsonia ginsengi was isolated. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the new isolates and L. ginsengi DSM 19088(T) formed a lineage at the genus level and this finding was supported by chemotaxonomic characterization. The peptidoglycan type of the representative isolate, K134/01(T), and L. ginsengi DSM 19088(T) was B2γ, with d- and l-diaminobutyric acid as the diagnostic diamino acid and glycine, alanine and threo-3-hydroxyglutamic acid. The almost-complete substitution of glutamic acid by threo-3-hydroxyglutamic acid supported the differentiation of the new strains from recognized species of the genus Leifsonia. Furthermore, the detection of substantial amounts of the fatty acid cyclohexyl-C(17 : 0) in the new isolates and L. ginsengi DSM 19088(T) was a prominent chemotaxonomic feature for a clear demarcation of these strains from all genera of the family Microbacteriaceae that display the B2γ cell-wall type. Comparative phylogenetic and phenotypic analyses of the isolates and L. ginsengi DSM 19088(T) revealed the separate species status of the isolates. On the basis of these results, it is proposed that L. ginsengi should be classified as the type species of a novel genus, Herbiconiux gen. nov., with the name Herbiconiux ginsengi gen. nov., comb. nov. (type strain wged11(T) = CGMCC 4.3491(T) = JCM 13908(T) = DSM 19088(T) = NBRC 104580(T)). The phyllosphere isolates are assigned to a novel species, Herbiconiux solani sp. nov. (type strain K134/01(T) = DSM 19813(T) = LMG 24387(T) = NBRC 106740(T)).

Bacillus beijingensis sp. nov. and Bacillus ginsengi sp. nov., isolated from ginseng root.

Four alkaligenous, moderately halotolerant strains, designated ge09, ge10(T), ge14(T) and ge15, were isolated from the internal tissue of ginseng root and their taxonomic positions were investigated by using a polyphasic approach. Cells of the four strains were Gram-positive-staining, non-motile, short rods. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains ge09 and ge10(T) formed one cluster and strains ge14(T) and ge15 formed another separate cluster within the genus Bacillus. 16S rRNA gene sequence similarities with type strains of other Bacillus species were less than 97 %. Levels of DNA-DNA relatedness among the four strains showed that strains ge09 and ge10(T) and strains ge14(T) and ge15 belonged to two separate species; the mean level of DNA-DNA relatedness between ge10(T) and ge14(T) was only 28.7 %. Their phenotypic and physiological properties supported the view that the two strains represent two different novel species of the genus Bacillus. The DNA G+C contents of strains ge10(T) and ge14(T) were 49.9 and 49.6 mol%, respectively. Strains ge10(T) and ge14(T) showed the peptidoglycan type A4alpha l-Lys-d-Glu. The lipids present in strains ge10(T) and ge14(T) were diphosphatidylglycerol, phosphatidylglycerol, a minor amount of phosphatidylcholine and two unknown phospholipids. Their predominant respiratory quinone was MK-7. The fatty acid profiles of the four novel strains contained large quantities of branched and saturated fatty acids. The predominant cellular fatty acids were iso-C(15 : 0) (42.5 %), anteiso-C(15 : 0) (22.2 %), anteiso-C(17 : 0) (7.3 %) and C(16 : 1)omega7c alcohol (5.7 %) in ge10(T) and iso-C(15 : 0) (50.7 %) and anteiso-C(15 : 0) (20.1 %) in ge14(T). On the basis of their phenotypic properties and phylogenetic distinctiveness, two novel species of the genus Bacillus are proposed, Bacillus beijingensis sp. nov. (type strain ge10(T) =DSM 19037(T) =CGMCC 1.6762(T)) and Bacillus ginsengi sp. nov. (type strain ge14(T) =DSM 19038(T) =CGMCC 1.6763(T)).

Agrococcus versicolor sp. nov., an actinobacterium associated with the phyllosphere of potato plants.

The taxonomic position of a group of actinobacterial strains isolated from the phyllosphere of potato plants was investigated by using a polyphasic approach. Although the similarity values for their 16S rRNA gene sequences suggested an intermediate position between Microbacterium and Agrococcus, the phylogenetic tree demonstrated a clear clustering of the representative strain, K 114/01(T), within the genus Agrococcus. The presence of 2,4-diaminobutyric acid as the diagnostic diamino acid in the cell-wall peptidoglycan of strain K 114/01(T) substantiated the affiliation to the genus Agrococcus. An analysis performed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry revealed highly similar spectral patterns for the isolated strains, which, together with their conformity regarding a multitude of phenotypic features, supported their affiliation to the same species. Differences in several physiological features, peptidoglycan and menaquinone composition and whole-cell fatty acid profiles enabled discrimination of the phyllosphere isolates with respect to recognized Agrococcus species. As the 16S rRNA gene sequence similarity values were below 97 %, the strains isolated from the phyllosphere of potato plants represent a novel species of the genus Agrococcus, for which the name Agrococcus versicolor sp. nov. is proposed. The type strain is K 114/01(T) (=DSM 19812(T) =LMG 24386(T)).

Leucobacter tardus sp. nov., isolated from the phyllosphere of Solanum tuberosum L.

In the course of studying the composition of the microbial community in the phyllosphere of potato plants, a Gram-positive bacterium, strain K 70/01(T), was isolated. On the basis of 16S rRNA gene sequence analysis, it was shown that the investigated isolate represents a novel species in the genus Leucobacter; the closest phylogenetic neighbour was Leucobacter chromiireducens subsp. chromiireducens, with a sequence similarity of 96.8 % to the type strain. Chemotaxonomic characteristics supported affiliation to the genus Leucobacter. Strain K 70/01(T) displayed the diagnostic diamino acid 2,4-diaminobutyric acid in the cell-wall peptidoglycan. The major fatty acids were anteiso-C(15 : 0,) anteiso-C(17 : 0) and iso-C(16 : 0), which are typical of the genus. The quinone system consisted of the menaquinones MK-10, MK-11 and MK-9. Differences in several physiological features and the absence of the cell-wall amino acids gamma-aminobutyric acid and threonine enabled the isolate to be differentiated from all recognized Leucobacter species. Based on these phylogenetic and phenotypic results, it can be concluded that the isolate from the phyllosphere of potato plants represents a novel species, for which the name Leucobacter tardus sp. nov. is proposed. The type strain is K 70/01(T) (=DSM 19811(T) =LMG 24388(T)).

Nocardioides oleivorans sp. nov., a novel crude-oil-degrading bacterium.

The crude-oil-degrading strain BAS3(T) represents a novel Nocardioides species, according to a taxonomic study. The 16S rRNA gene sequence of strain BAS3(T) was most similar to that of Nocardioides ganghwensis (IMSNU 14028(T); 99% similarity), but the DNA-DNA relatedness to this type strain was only 32%. The physiological properties of strain BAS3(T) differ from those of N. ganghwensis (IMSNU 14028(T)) and other species of Nocardioides. The diamino acid in the cell-wall peptidoglycan of strain BAS3(T) is LL-diaminopimelic acid and the major menaquinone is MK-8(H(4)). The name Nocardioides oleivorans sp. nov. is proposed for the novel Nocardioides species, since its type strain, BAS3(T) (=DSM 16090(T)=NCIMB 14004(T)), is able to degrade crude oil.

Microbacterium oleivorans sp. nov. and Microbacterium hydrocarbonoxydans sp. nov., novel crude-oil-degrading Gram-positive bacteria.

A taxonomic study of two crude-oil-degrading, Gram-positive bacterial strains, designated BAS69(T) and BNP48(T), revealed that they represent two novel Microbacterium species. 16S rRNA gene sequence similarity to their closest phylogenetic neighbours was 98.5 % for BAS69(T) (Microbacterium paraoxydans DSM 15019(T) and Microbacterium saperdae DSM 20169(T)) and 99 % for BNP48(T) (Microbacterium luteolum DSM 20143(T)). Levels of DNA-DNA relatedness to the closest phylogenetic neighbours of both strains were between 11 and 38 %. According to phylogenetic analysis, the two strains are distinguishable from all recognized species of Microbacterium. Morphological and physiological characteristics of strains BAS69(T) and BNP48(T) were different from those of phylogenetically closely related Microbacterium species. The diamino acid in the cell-wall peptidoglycan of BAS69(T) is lysine and of BNP48(T) is ornithine. The major menaquinones are MK-11 and MK-12 for both strains. Based on their ability to degrade crude oil, the name Microbacterium oleivorans sp. nov. is proposed for strain BAS69(T) (=DSM 16091(T)=NCIMB 14003(T)) and Microbacterium hydrocarbonoxydans is proposed for strain BNP48(T) (=DSM 16089(T)=NCIMB 14002(T)).

Mycobacterium psychrotolerans sp. nov., isolated from pond water near a uranium mine.

An acid-fast, rapidly growing, psychrotolerant short rod was isolated from pond water near a uranium mine. Phylogenetic analysis of the 16S rRNA gene sequence grouped this strain with the rapidly growing mycobacteria. The 16S rRNA gene sequence of isolate WA101T showed highest similarity to that of Mycobacterium sphagni DSM 44076T; however, DNA-DNA relatedness between the two strains was less than 30 %. Chemotaxonomic analyses, which included fatty acid and mycolic acid patterns, confirmed the classification of strain WA101T in the genus Mycobacterium. Physiological data, including antibiotic resistance, NaCl tolerance, carbon sources, temperature growth range and enzyme activities, were also determined. Based on the genotypic and phenotypic results it is proposed that isolate WA101T represents a novel Mycobacterium species. The name Mycobacterium psychrotolerans sp. nov. is proposed, with type strain WA101T (= DSM 44697T = LMG 21953T).

Erysipelothrix inopinata sp. nov., isolated in the course of sterile filtration of vegetable peptone broth, and description of Erysipelotrichaceae fam. nov.

Based on 16S rRNA gene sequence comparison, an isolate that was detected in sterile-filtered vegetable broth was classified as a novel member of the Erysipelothrix line of descent of the Firmicutes. Strain MF-EP02T resembles members of the two species of Erysipelothrix with validly published names, Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum, in morphology, fatty acid composition, lack of menaquinones in aerobically and anaerobically grown cultures, DNA G+C content and peptidoglycan amino acid composition. Distinct differences in physiological characteristics, however, support the allocation of this isolate to a novel species of the genus Erysipelothrix, for which the name Erysipelothrix inopinata sp. nov. (type strain, MF-EP02T=DSM 15511T=CIP 107935T) is proposed. Members of the Erysipelothrix line of descent are included in the family Erysipelotrichaceae fam. nov.

Streptomonospora alba sp. nov., a novel halophilic actinomycete, and emended description of the genus Streptomonospora Cui et al. 2001.

A halophilic actinomycete, strain YIM 90003(T), was isolated from a soil sample collected from Xinjiang Province, China, by using starch-casein agar with a salt concentration of 20 % (w/v), pH 7.0. The strain grew well on most media tested. No diffusible pigment was produced. Aerial mycelium and substrate mycelium were well developed on most media. The aerial mycelium formed short spore chains, bearing non-motile, straight to flexuous spores with wrinkled surfaces. The cell walls of strain YIM 90003(T) contained meso-diaminopimelic acid as the diagnostic diamino acid. Cell-wall hydrolysates contained galactose and arabinose. Menaquinone composition varied with the medium used for cell cultivation; on glucose-yeast extract medium supplemented with 10 % NaCl, the major menaquinone was MK-9(H(4)), while, on vitamin-enriched ISP 2 medium, the major menaquinones were MK-10(H(2)), MK-9(H(8)) and MK-10(H(4)). Phospholipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, diphosphatidyl glycerol, methylphosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and an unidentified phospholipid. 16S rRNA gene sequence analysis showed Streptomonospora salina as the closest phylogenetic neighbour. On the basis of these analyses, strain YIM 90003(T) is a member of the genus Streptomonospora, though its properties do not match the generic description fully with respect to the menaquinone composition and peptidoglycan amino acid. Analyses of mechanically disrupted cell walls of the type species, Streptomonospora salina DSM 44593(T), and strain YIM 90003(T), purified by tryptic digestion and subsequent SDS treatment, revealed the exclusive presence of meso-diaminopimelic acid as the diagnostic diamino acid of peptidoglycan. Thus, the genus description of Streptomonospora, indicating the presence of several amino acids usually not found in the peptidoglycan moiety, is therefore emended. DNA-DNA hybridization and comparison of physiological and chemotaxonomic characteristics demonstrated strain YIM 90003(T) to be different from Streptomonospora salina. The name Streptomonospora alba sp. nov. is proposed, with strain YIM 90003(T) (=CCTCC AA001013(T)=DSM 44588(T)) as the type strain.

Reclassification of Desulfotomaculum auripigmentum as Desulfosporosinus auripigmenti corrig., comb. nov.

The species Desulfotomaculum auripigmentum is reclassified as Desulfosporosinus auripigmenti corrig., comb. nov. on the basis of morphological and physiological traits, phylogenetic position and chemotaxonomic properties. Characteristics supplementary to those provided in the original description reveal that the type strain, DSM 13351(T) (=ATCC 700205(T)), forms oval, subterminal to terminal spores, possesses LL-diaminopimelic acid and contains MK-7 as the predominant menaquinone, while the whole-cell methanolysate contains even-carbon, straight-chain saturated and mono-unsaturated fatty acids and 1,1-dimethylacetals as major components. DNA-DNA reassociation values below 30 % for Desulfosporosinus orientis DSM 765(T) and Desulfosporosinus meridiei DSM 13257(T) demonstrate that strain DSM 13351(T) shows sufficient genomic differences to maintain its species status. Lack of motility, a smaller cell diameter and the ability to use malate and glycerol as electron donors and fumarate and arsenate as electron acceptors are the main properties that differentiate Desulfosporosinus auripigmenti from the other two species of the genus.