Ability to degrade crude oil
n
-alkanes was revealed in new strains of thermophilic bacilli isolated from petroleum reservoirs and a hot spring:
Geobacillus toebii
В-1024,
Geobacillus
sp. 1017, and
...Aeribacillus pallidus
8m3. The strains utilized С
10
–С
30
n
-alkanes (В-1024), С
10
, C
11
, and С
13
–С
19,22
n
-alkanes (1017), and C
11
–C
29
n
-alkanes (8m3). In all three strains, PCR amplification with specific degenerate oligonucleotide primers revealed the
alkB
gene encoding rubredoxin-dependent alkane monooxygenase. In strains В-1024 and 1017, fragments of the genes homologous to the
ladA
gene determining flavin-dependent alkane monooxygenase were also amplified. Nucleotide sequences of these genes were practically identical to those of the genes
ladA
α
B23
,
ladA
β
B23
, and
ladB
B23
, which were revealed previously in
Geobacillus thermoleovorans
strain B23. For the latter strain, activity of respective enzymes in the oxidation of long-chain
n
-alkanes has been shown. Thus, simultaneous presence of the
alkB
and
ladA
genes coding alkane monooxygenases responsible for oxidation of medium-chain and long-chain n-alkanes in thermophilic bacilli was revealed for the first time.
Functional and phylogenetic microbial diversity in formation waters of carbonate low-temperature bed 302 of the Romashkinskoe oil field was studied using microbiological, molecular and radioisotope ...techniques. Cultivable sulfate-reducing prokaryotes (103–106 cells ml−1) predominated in formation water and the process of sulfate reduction was paramount. The rate of sulfate reduction varied within the range 2.28–26.60 μg S2− l−1−day−1. The number of fermentative bacteria varied from 102 to 104 cells ml−1, of methanogens – from 10 to 104 cells ml−1. Methanogenesis was a minor process with the rate not exceeding 0.395 μg CH4 l−1 day−1. 16S rRNA gene clone libraries for Bacteria and Archaea were constructed on the basis of DNA extracted from formation water. From the bacterial clone library, 16S rRNA genes of aerobic organotrophs (genera Pseudomonas, Acinetobacter, ‘Brachymonas’ etc.), anaerobic organotrophs (genus Thauera, Bacteroidetes) and sulfate reducers (Desulfoglaeba, Desulfomicrobium, Desulfovibrio and Desulfobacterales) were retrieved. For the first time, the predominance of nucleotide sequences of the bacterium Desulfoglaeba alkanexedens known to grow on n-alkanes was revealed in the clone library from the carbonate oil reservoir. Archaeal sequences in the library of formation water were not attributable to any recognized methanogens. Both the sulfate-reducing bacterium D. alkanexedens and syntrophic assemblages could be involved in biodegradation of crude oil associated with sulfate reduction in the carbonate petroleum reservoir.
► Microorganisms of carbonate petroleum reservoir (Russia) were studied. ► Fermentative and sulfate-reducing bacteria were predominant cultivated groups. ► Sulfate reduction is a major process in produced water samples. ► 16S rRNA genes of Desulfoglaeba dominate in the clone library of water samples. ► Both sulfidogenic assemblages and sulfate-reducer Desulfoglaeba degrades oil in the oil field.
The paper deals with the phylogenetic and functional microbial diversity in the samples of umbric luvisol contaminated and uncontaminated with hydrocarbons and of urban stratozem. High-throughput ...sequencing of the V3–V4 region of the 16S rRNA gene revealed predomination of bacteria of the phyla
Proteobacteria
,
Actinobacteria
, and
Acidobacteria
in the studied soil samples. Bacteria of the genera
Sphingomonas
,
Bradyrhizobium
, and
Pseudolabrys
in the samples of umbric luvisol and bacteria of the genera
Brevundimonas
,
Stenotrophomonas
, and
Streptomyces
in urbostratozem had the greatest potential functional activity in conversion of nitrogen compounds, degradation of chloroalkanes, chloroalkenes, and benzoate. Strains of
Stenotrophomonas maltophilia
, which degraded oil, petroleum products, and polycyclic aromatic hydrocarbons (PAHs, phenanthrene, anthracene, and pyrene) were isolated from oil-contaminated soils. Under microcosm conditions, microorganisms of umbric luvisol efficiently degraded various components of naphthenic-methane and methane-naphthenic oil. While the content of aromatic components in naphthenic-methane oil degraded by the soil microbial community and by the community enriched with
S. maltophilia
strain P420c was almost the same, in the variant with bioaugmentation the degradation process of these components proceeded 14 days faster. The enrichment of soil with specific degraders of PAHs and aromatic compounds may be appropriate for the soils of northern regions with a low duration of the summer period, which are polluted with oil with a high content of aromatic and asphalt-resinous compounds.
Analysis of complete genomes of thermophilic bacteria of the genus
Geobacillus
, oxidizing
n
-alkanes and crude oil revealed the
ladA
gene and its homologues. In the genomes of some strains, the
ladA
...gene was not detected, although they were capable of growth on
n
-alkanes. Cloning with degenerate primers has previously revealed eight homologues of the
alkB
gene (
alkB
-geo1–
alkB
-geo8) encoding alkane hydroxylases in
Geobacillus.
In the present work, investigation of the
alkB
genes of several new strains of thermophilic, hydrocarbon-oxidizing bacilli was carried out. In the clone libraries obtained using degenerate primers for the
alkB
genes from five strains of the genera
Geobacillus
,
Parageobacillus,
and
Aeribacillus
, mostly the universal homologous genes
alkB
-geo1 and
alkB-geo4
were revealed. Additional PCR amplification with specific primers for each of the eight known
alkB
homologues revealed the universal homologous genes only in some of the studied strains. A correlation was found between the set of the
alkB
-
geo3
–
alkB
-
geo6
homologus genes from
Geobacillus subterraneus
strain K amplified with specific primers and the previously revealed a set of these homologous genes transcripted at different cultivation conditions. This correlation may be due to accumulation of the copies of individual homologues under different cultivation conditions, which results in higher sensitivity of specific primers. The least common homologue,
alkB
-
geo7
, which was not detected in the relevant clone libraries, was found in two strains, indicating the possibility of development of more specific primers for amplification of this homologue in order to reveal hydrocarbon-oxidizing bacteria of related genera
Geobacillus–Parageobacillus
in molecular ecological research.
The goal of the present work was to investigate the physicochemical and radiochemical conditions and the composition of the microbial community in the groundwater of a suspended surface repository ...for radioactive waste (Russia) and to determine the possibility of
groundwater bioremediation by removal of nitrate ions. Groundwater in the repository area (10-m depth) had elevated concentrations of strontium, tritium, nitrate, sulfate, and bicarbonate ions. High-throughput sequencing of the V3-V4/V4 region of the 16S rRNA gene revealed the presence of members of the phyla
(genera
, and uncultured
),
(genera
and
), and
(
Planktophila,
). Canonical correspondence analysis suggested that major contaminant - nitrate, uranium, and sulfate shaped the composition of groundwater microbial community. Groundwater samples contained culturable aerobic organotrophic, as well as anaerobic fermenting, iron-reducing, and denitrifying bacteria. Pure cultures of 33 bacterial strains belonging to 15 genera were isolated. Members of the genera
, and
reduced nitrate to nitrite and/or dinitrogen. Application of specific primers revealed the
and
genes encoding nitrite reductases in bacteria of the genera
, and
. Nitrate reduction by pure bacterial cultures resulted in decreased ambient Eh. Among the organic substrates tested, sodium acetate and milk whey were the best for stimulation of denitrification by the microcosms with groundwater microorganisms. Injection of these substrates into the subterranean horizon (single-well push-pull test) resulted in temporary removal of nitrate ions in the area of the suspended radioactive waste repository and confirmed the possibility for
application of this method for bioremediation.
A novel thermophilic spore-forming strain, Ga(T), was isolated from the Garga hot spring located in the northern part of the Transbaikal region (Russia). Strain Ga(T) was found to be an aerobic, ...Gram-positive, rod-shaped, thermophilic (optimum growth temperature is 60-65 degrees C), chemo-organotrophic bacterium that grows on various sugars, carboxylic acids and hydrocarbons. The G+C content of its DNA is 52.9 mol%. The 16S rRNA gene sequence similarity data show that strain Ga(T) is closely related to members of the genus Geobacillus. Relevant chemotaxonomic data (in particular, the major fatty acid profile of strain Ga(T), which includes iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0 acids) support the assignment of this strain to the genus Geobacillus. The physiological, biochemical and DNA-DNA hybridization studies of strain Ga(T) showed that it differs both genotypically and phenotypically from the recognized Geobacillus species. Based on these data, strain Ga(T) belongs to a novel species, Geobacillus gargensis sp. nov. (type strain, Ga(T)=VKM B-2300(T)=DSM 15378(T)). The analysis of the phenotypic characteristics (additional to those given in the original description) of the type strain of Bacillus vulcani (DSM 13174(T)) showed that they are very similar to the major phenotypic characteristics of the genus Geobacillus. The low DNA-DNA reassociation values of strain DSM 13174(T) with various species of this genus (from 38 to 54 %) clearly demonstrate a sufficient genomic distinction of this strain and its taxonomic status as a species. The physiological characteristics, phylogenetic position and DNA-DNA reassociation values of B. vulcani allow this species to be reclassified as Geobacillus vulcani comb. nov. The main properties that differentiate G. vulcani from the other species of the genus are its ability to produce acids from glycerol, lactose and ribose.
Four thermophilic, spore-forming bacterial strains, DS1
T, DS2, 46 and 49, were isolated from the high-temperature Dagang oilfield, located in China. The strains were identified by using the ...polyphasic taxonomy approach. These were aerobic, gram-positive, rod-shaped, moderately thermophilic (with an optimum growth temperature of 60–65
°C), chemoorganotrophic bacteria capable of growing on various sugars, carboxylic acids and crude oil. Two strains, DS1
T and DS2, were capable of growing on individual saturated hydrocarbons. The G+C content of the DNA of strains DS1
T and DS2 was 54.5 and 53.8
mol%, respectively. The phylogenetic analysis of the 16S rDNA of strains DS1
T and DS2 showed that they form a separate cluster within the genus
Geobacillus. The cellular fatty acids of the isolates were dominated by
iso-15:0,
iso-16:0 and
iso-17:0 acids, which are the typical fatty acids of bacteria from the genus
Geobacillus. The DNA−DNA hybridization study and the comparative analysis of the morphological and chemotaxonomic characteristics of strains DS1
T and DS2 showed that they differ from the previously described
Geobacillus species and belong to a new species, which was called
Geobacillus jurassicus. DS1
T (=VKM B-2301
T,=DSM 15726
T) is the type strain of this species According to both DNA–DNA reassociation studies and 16S rDNA sequence analysis, two other strains, 46 and 49, were assigned to the species
G. stearothermophilus. In this paper, we provide evidence that the new combinations G.
stearothermophilus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and
G. thermodenitrificans may be considered to be valid.
The goal of this work was to study the overall genomic diversity of microorganisms of the Dagang high-temperature oilfield (PRC) and to characterize the metabolically active fraction of these ...populations. At this water-flooded oilfield, the microbial community of formation water from the near-bottom zone of an injection well where the most active microbial processes of oil degradation occur was investigated using molecular, cultural, radiotracer, and physicochemical techniques. The samples of microbial DNA and RNA from back-flushed water were used to obtain the clone libraries for the 16S rRNA gene and cDNA of 16S rRNA, respectively. The DNA-derived clone libraries were found to contain bacterial and archaeal 16S rRNA genes and the
B genes encoding alkane monooxygenases similar to those encoded by
-
and
-
of geobacilli. The 16S rRNA genes of methanogens (
, and
) were predominant in the DNA-derived library of
cloned sequences; among the bacterial sequences, the 16S rRNA genes of members of the genus
were the most numerous. The RNA-derived library contained only bacterial cDNA of the 16S rRNA sequences belonging to metabolically active aerobic organotrophic bacteria (
), as well as of denitrifying (
), fermenting (
), iron-reducing (
), and sulfate- and sulfur-reducing bacteria (
). The presence of the microorganisms of the main functional groups revealed by molecular techniques was confirmed by the results of cultural, radioisotope, and geochemical research. Functioning of the mesophilic and thermophilic branches was shown for the microbial food chain of the near-bottom zone of the injection well, which included the microorganisms of the carbon, sulfur, iron, and nitrogen cycles.
Two facultatively anaerobic, chemoorganoheterotrophic bacterial strains, designated JR1/69-2-13
T
and JR1/69-3-13
T
, were isolated from nitrate- and radionuclide-contaminated groundwater (Ozyorsk ...town, South Urals, Russia). Both strains were found to be motile, Gram-stain negative rod-shaped neutrophilic, psychrotolerant bacteria that grow within the temperature range from 5–10 to 33 °C at 0–3 (0–5)% NaCl (w/v). The major cellular fatty acids were identified as C
16:0
, C
16:1
ω
7
c
, C
18:1
ω
7
c
and C
17:0
cyclo. The major polar lipids were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol and unidentified aminophospholipids. The genomic G + C content of strains JR1/69-2-13
T
and JR1/69-3-13
T
was determined to be 57.2 and 57.9%, respectively. The 16S rRNA gene sequences of the strains showed high similarity between each other (98.6%) and to members of the genera
Pusillimonas
(96.8–98.4%) and
Candidimonas
(97.1–98.0%). The average nucleotide identity and digital DNA–DNA hybridization (dDDH) values among genomes of the new isolates and
Pusillimonas
and
Candidimonas
genomes were below 84.5 and 28.8%, respectively, i.e., below the thresholds for species delineation. Based on the phylogenomic, phenotypic and chemotaxonomic characterisation, we propose assignment of strains JR1/69-3-13
T
(= VKM B-3223
T
= KCTC 62615
T
) and JR1/69-2-13
T
(= VKM B-3222
T
= KCTC 62614
T
) to a new genus
Pollutimonas
as the type strains of two new species,
Pollutimonas subterranea
gen. nov., sp. nov. and
Pollutimonas nitritireducens
sp. nov., respectively. As a result of the taxonomic revision of the genus
Pusillimonas
, three novel genera,
Allopusillimonas
,
Neopusillimonas
, and
Mesopusillimonas
are also proposed; and
Candidimonas bauzanensis
is reclassified as
Pollutimonas bauzanensis
comb. nov. Genome analysis of the new isolates suggested molecular mechanisms of their adaptation to an environment highly polluted with nitrate and radionuclides.
The number of microorganisms of major metabolic groups and the rates of sulfate reduction and methanogenesis processes in the formation waters of the high-temperature horizons of Dagang oil field ...have been determined. Using cultural methods, it was shown that the microbial community contained aerobic bacteria oxidizing crude oil, anaerobic fermentative bacteria, sulfate-reducing bacteria, and methanogens. Using cultural methods, the possibility of methane production from a mixture of hydrogen and carbon dioxide (H sub(2) + CO sub(2)) and from acetate was established, and this result was confirmed by radioisotope methods involving NaH super(14)CO sub(3) and super(14)CH sub(3)COONa. Analysis of enrichment cultures 16S rDNA of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not revealed. Phylotypes of the representatives of Thermococcus spp. were found among archaeal 16S rDNA. 16S rRNA genes of bacterial clones belong to the orders Thermoanaerobacteriales (Thermoanaerobacter, Thermovenabulum, Thermacetogenium, and Coprothermobacter spp.), Thermotogales, Nitrospirales (Thermodesulfovibrio sp.) and Planctomycetales. 16S rDNA of a bacterium capable of oxidizing acetate in the course of syntrophic growth with H sub(2)-utilizing methanogens was found in high-temperature petroleum reservoirs for the first time. These results provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs, indicating that syntrophic processes play an important part in acetate degradation accompanied by methane production.
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