Isolation and characterization of indigenous thermophilic bacteria active in natural attenuation of bio-hazardous petrochemical pollutants

Common petrochemical compounds, such as homocyclic polyaromatic hydrocarbons and heterocyclic NOS-polyaromatics (NOS-compounds), were used as the sole carbon and energy source to enrich indigenous bacteria harboring the catabolic ability to degrade these compounds from petroleum-contaminated soils from Kuwait. Chemical analysis of the extracted soil materials revealed residual amounts of oil (<5% w/w), presumably of heavy oil fractions with elevated S-content. Aerobic culturable mesophilic polyaromatic hydrocarbon- and NOS-degraders were abundant in these soils, whereas their moderately thermophilic counterparts constituted only a minor fraction. Glucose stimulated the growth of mesophiles and drastically suppressed the number of thermophiles. 16S rDNA was amplified by PCR from nine of the purified thermophilic strains, using primers specific for eubacteria. Sequencing of 900 bp of the 16S rDNA and database homology search tentatively aligned these isolates to low G+C Gram positive bacteria of the family Bacillaceae. Electron microscopy characterization revealed endospore-forming bacilli varying in size, with well-structured cell walls. Gas chromatography and mass spectrometry (GC/MS) analysis revealed a versatile catabolic ability of the pure and mixed cultures to degrade all tested compounds. The metabolism of the offered substrates does not involve co-metabolism, since all pure cultures consumed the offered substrates completely.     

Molecular analysis and development of 16S rRNA oligonucleotide probes to characterize a diclofop-methyl-degrading biofilm consortium

Genomic DNA from nine individual bacteria, isolated from a diclofop-methyl-degrading biofilm consortium, was extracted for genetic characterization. The degradation of diclofop-methyl produces metabolites that are known intermediates or substrates for bacteria that degrade a variety of chlorinated aromatic compounds. Accordingly, oligonucleotide primers were designed from specific catabolic genes for chlorinated organic degradation pathways, and tested by PCR to determine if these genes are involved in diclofop-methyl degradation. DNA homology between the PCR products and the known catabolic genes investigated by Southern hybridization analysis and by sequencing, suggested that novel catabolic genes are functioning in the isolates. Specific fluorescent oligonucleotides were designed for two of the isolates, following 16S rDNA sequencing and identification of each of the isolates. These probes were successfully used for fluorescent in situ hybridization (FISH) studies of the two isolates in the biofilm consortium.     

The frequency and characteristics of highly thermophilic bacteria in cool soil environments

Following enrichment at 70 degrees C and 80 degrees C, five highly thermophilic aerobic eubacteria have been isolated from cool soil environments. These organisms show a temperature range for growth of 40-80 degrees C and have optimal and very high growth rates around 70 degrees C with generation times less than 30 min. All isolates are narrow rods, which stain Gram-negative, but have a Gram-positive cell wall structure and only one of five isolates is a spore former. All cultures contain a small proportion of previously unreported extremely long flexuous rods, which can be seen to divide eventually. Biochemical testing of five strains reveals a significant ability to utilize alkanes and some aromatic hydrocarbons. Using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of 16S rDNA the five strains were differentiated into three categories, which paralleled the biochemical results. 16S rDNA sequences showed high similarity with thermophilic Bacillus species now reclassified as Geobacillus. These bacteria are present in high numbers in apparently all soils and the question is raised of how these organisms, which are apparently unable to grow at the temperatures experienced in these cool soils, are so prominent.     

Identification and Phylogenetic analysis of thermophilic sulfate-reducing bacteria in oil field samples by 16S rDNA gene cloning and sequencing

Thermophilic sulfate-reducing bacteria (SRB) have been recognized as an important source of hydrogen sulfide (H2S) in hydrocarbon reservoirs and in production systems. Four thermophilic SRB enrichment cultures from three different oil field samples (sandstone core, drilling mud, and production water) were investigated using 16S rDNA sequence comparative analysis. In total, 15 different clones were identified. We found spore-forming, low G+C content, thermophilic, sulfate-reducing Desulfotomaculum-related sequences present in all oil field samples, and additionally a clone originating from sandstone core which was assigned to the mesophilic Desulfomicrobium group. Furthermore, three clones related to Gram-positive, non-sulfate-reducing Thermoanaerobacter species and four clones close to Clostridium thermocopriae were found in enrichment cultures from sandstone core and from production water, respectively. In addition, the deeply rooted lineage of two of the clones suggested previously undescribed, Gram-positive, low G+C content, thermophilic, obligately anaerobic bacteria present in production water. Such thermophilic, non-sulfate-reducing microorganisms may play an important ecological role alongside SRB in oil field environments.     

Metabolic and Genetic Diversity of Mesophilic and Thermophilic Bacteria Isolated from Composted Municipal Sludge on Poly-ε-caprolactones

Thirty mesophilic and thermophilic bacteria were isolated from thermobiotically digested sewage sludge in culture medium supplemented with poly-ε-caprolactone (PCL). The ability of each purified isolate to degrade PCL and to produce polymer-degrading extracellular enzymes was assessed. Isolates were characterized based on random amplified polymorphic DNA (RAPD), 16S rDNA sequence-based phylogenetic affiliation and carbohydrate-based nutritional versatility. Mesophilic isolates with ability to degrade PCL were attributed to the genera Acinetobacter, Burkholderia, Pseudomonas, and Staphylococcus. Thermophilic isolates were members of the genus Bacillus. Despite the restricted phylogenetic and genotypic diversity observed for thermophiles, their metabolic versatility and wide range of growth temperatures suggest an important activity of these organisms during the whole composting process.     

Rarity associated with specific ecological niches in the bacterial world: the 'Synergistes' example

The 'Synergistes' group, which apparently represents an as yet unnamed division of the bacteria, was explored in 93 anaerobic environments (guts, soils, digestors, etc.). From 16S rDNA gene-targeted polymerase chain reaction (PCR) assays, this group appeared to be present in 90% of the anaerobic microbial ecosystems analysed. The phylogeny of 103 16S rDNA sequences from 30 ecosystems showed a strong link between 16S rDNA sequences and given ecosystems. 'Synergistes' 16S rDNA sequences from animal sources (termites, guinea pigs, pigs, birds, etc.) formed clustered phylogenetical groups. 'Synergistes' groups were also associated either with anaerobic digestors and soils or with thermophilic conditions. Sequences available from the DNA database were consistent with the results. These results show the wide diversity of the 'Synergistes' division as well as the specific ecological niche of each 16S rDNA sequences.     

Biodegradation of Crude Oil by Thermophilic Bacteria Isolated from a Volcano Island

One-hundred and fifty different thermophilic bacteria isolated from a volcanic island were screened for detection of an alkane hydroxylase gene using degenerated primers developed to amplify genes related to the Pseudomonas putida and Pseudomonas oleovorans alkane hydroxylases. Ten isolates carrying the alkJ gene were further characterized by 16s rDNA gene sequencing. Nine out of ten isolates were phylogenetically affiliated with Geobacillus species and one isolate with Bacillus species. These isolates were able to grow in liquid cultures with crude oil as the sole carbon source and were found to degrade long chain crude oil alkanes in a range between 46.64% and 87.68%. Results indicated that indigenous thermophilic hydrocarbon degraders of Bacillus and Geobacillus species are of special significance as they could be efficiently used for bioremediation of oil-polluted soil and composting processes.     

Species Diversity and Substrate Utilization Patterns of Thermophilic Bacterial Communities in Hot Aerobic Poultry and Cattle Manure Composts

This study investigated the species diversity and substrate utilization patterns of culturable thermophilic bacterial communities in hot aerobic poultry and cattle manure composts by coupling 16S rDNA analysis with Biolog data. Based on the phylogenetic relationships of 16S rDNA sequences, 34 thermophilic (grown at 60 degrees C) bacteria isolated during aerobic composting of poultry manure and cattle manure were classified as Bacillus licheniformis, B. atrophaeus, Geobacillus stearothermophilus, G. thermodenitrificans, Brevibacillus thermoruber, Ureibacillus terrenus, U. thermosphaericus, and Paenibacillus cookii. In this study, B. atrophaeus, Br. thermoruber, and P. cookii were recorded for the first time in hot compost. Physiological profiles of these bacteria, obtained from the Biolog Gram-positive (GP) microplate system, were subjected to principal component analysis (PCA). All isolates were categorized into eight different PCA groups based on their substrate utilization patterns. The bacterial community from poultry manure compost comprised more divergent species (21 isolates, seven species) and utilized more diverse substrates (eight PCA groups) than that from cattle manure compost (13 isolates, five species, and four PCA groups). Many thermophilic bacteria isolated in this study could use a variety of carboxylic acids. Isolate B110 (from poultry manure compost), which is 97.6% similar to U. terrenus in its 16S rDNA sequence, possesses particularly high activity in utilizing a broad spectrum of substrates. This isolate may have potential applications in industry.     

Utilization of Alkylbenzenes during Anaerobic Growth of Pure Cultures of Denitrifying Bacteria on Crude Oil

Four pure cultures of denitrifying bacteria, which had previously been isolated on defined alkylbenzenes, were capable of anaerobic growth with crude oil as the only source of organic substrates. Chemical analyses after growth revealed that the known growth substrates toluene, ethylbenzene, and m-xylene were selectively consumed from the oil. o-Xylene and p-xylene, which as pure compounds did not support growth, were consumed to a lesser extent.     

Antibacterial Potential of 2,4-Di-tert-Butylphenol and Calixarene-Based Prodrugs from Thermophilic Bacillus licheniformis Isolated in Algerian Hot Spring

The present investigation reports the isolation, molecular identification and structure elucidation of antibacterial produced by two thermophilic spore-forming bacteria from hot spring (98?°C) of Guelma (Algeria). Morphological, biochemical and physiological characteristics were carried out. The molecular identification by 16S rRNA and 16-23S rRNA ITS-PCR sequencing identified the thermophilic strains as Bacillus licheniformis with 99% of similarity with GenBank accession numbers KX100031 and KX100032. Phenotypic characterization has mentioned several differences between thermophilic isolates and Bacillus licheniformis ATCC 14580. The ability of the thermophilic spore- forming bacteria to produce antibacterial compounds against two multidrug resistance bacteria Pseudomonas aeruginosa (NR_0754828.1) and Staphylococcus aureus (NR_075000.1) in pure and mixed culture was investigated by Radial Diffusion Assay at 55?°C. Structural elucidation of actives compounds was carried out using gas chromatography–mass spectrometry analyses. Antibacterial potency of the thermophilic isolates might be due to the association between two phenolic compounds: 2,4-Di-tert-butyl-phenol as principal active compound and p-tert-butylcalix[4]arene as prodrugs comparing between gas chromatography–mass spectrometry analysis of pure and mixed extract. To the best of our knowledge, this is the first report showing production of p-tert-butylcalix[4]arene and 2,4-Di-tert-butyl-phenol as extremolytes compounds from thermophilic Bacillus licheniformis at 55?°C.     

嗜热厌氧纤维素分解菌的分离、鉴定及其酶学特性

【目的】分离高效降解纤维素的嗜热厌氧菌,通过与嗜热产乙醇菌株联合培养的方式,为生产纤维素乙醇提供微生物资源。【方法】利用厌氧分离技术从降解纤维素的马粪富集物中分离到一株嗜热厌氧细菌HCp。采用形态学观察、生理生化鉴定、结合16S rDNA序列的系统发育学分析确定该菌株的分类地位,利用DNS酶活分析方法测定此分离菌株的酶学性质。【结果】分离菌株HCp革兰氏染色阴性,直杆,细胞单个或成对出现,菌体大小为(0.35-0.50)μm×(2.42-6.40)μm,严格厌氧,形成芽胞,能运动,对新霉素有一定的抗性。此菌能利用滤纸纤维素、纤维素粉、微晶纤维素、脱脂棉和水稻秸秆、明胶等,还可以利用葡萄糖、纤维二糖、木糖、木聚糖、果糖、蔗糖、核糖、半乳糖、麦芽糖、山梨糖、海藻糖、蜜二糖、甘露糖等。该菌株在pH6.5-8.5、温度35-70℃、盐浓度0%-1.0%范围内利用纤维素生长,最适pH为6.85,最适温度为60℃,最适NaCl浓度为0.2%,最佳生长条件下,在10 d内滤纸纤维素降解率可达90.40%。在HCp的纤维小体中,滤纸酶、羧甲基纤维素酶、β-葡萄糖苷酶、木聚糖酶的最适作用温度分别为70℃、70℃、70℃、60℃,并且羧甲基纤维素酶具有较高的热稳定性。部分长度的16S rDNA序列分析表明,分离菌株HCp与Acetivibrio cellulolyticus、A.cellulosolvens相似性为97.5%。【结论】分离菌株HCp是从马粪富集物中分离到的一株嗜热厌氧细菌,该菌具有较强的降解纤维素能力,生长温度范围广,酶的热稳定性好,纤维素底物利用广泛等特性,为纤维素降解产乙醇提供了良好的材料。     

Thermophilic biofiltration of benzene and toluene

In the current studies, we characterized the degradation of a hot mixture of benzene and toluene (BT) gases by a thermophilic biofilter using polyurethane as packing material and high-temperature compost as a microbial source. We also examined the effect of supplementing the biofilter with yeast extract (YE). We found that YE substantially enhanced microbial activity in the thermophilic biofilter. The degrading activity of the biofilter supplied with YE was stable during long-term operation (approximately 100 d) without accumulating excess biomass. The maximum elimination capacity (1,650 g x m(-3) h(-1)) in the biofilter supplemented with YE was 3.5 times higher than that in the biofilter without YE (470 g g x m(-3) h(-1)). At similar retention times, the capacity to eliminate BT for the YE-supplemented biofilter was higher than for previously reported mesophilic biofilters. Thus, thermophilic biofiltration can be used to degrade hydrophobic compounds such as a BT mixture. Finally, 16S rDNA polymerase chain reaction-DGGE (PCR-DGGE) fingerprinting revealed that the thermophilic bacteria in the biofilter included Rubrobacter sp. and Mycobacterium sp.     

Aromatic hydrocarbon-degrading bacteria from soil near Scott Base, Antarctica

Hydrocarbons persist in Antarctic soils when fuel oils such as JP8 jet fuel are spilled. For clean-up of hydrocarbon-contaminated soils in Antarctica, bioremediation has been proposed using hydrocarbon-degrading microbes indigenous to Antarctic soils. A number of alkane-degrading bacteria have been isolated previously from Antarctic soils. In this paper we describe the direct isolation of aromatic hydrocarbon-degrading bacteria from oil-contaminated Antarctic soil. Isolates that grew on JP8 jet fuel were characterised for their ability to degrade aromatic and aliphatic hydrocarbons and for growth at a range of temperatures. All isolates were gram-negative, oxidase-positive, rod-shaped bacteria. Representative strains were identified using 16S rDNA sequence analysis as either Sphingomonas spp. or Pseudomonas spp. Aromatic-degrading bacteria from Antarctic soils were psychrotolerant and appear similar to those found worldwide. Accepted: 27 September 1999     

Degradation of crude oil by an arctic microbial consortium

The ability of a psychrotolerant microbial consortium to degrade crude oil at low temperatures was investigated. The enriched arctic microbial community was also tested for its ability to utilize various hydrocarbons, such as long-chain alkanes (n-C₂₄ to n-C₃₄), pristane, (methyl-)naphthalenes, and xylenes, as sole carbon and energy sources. Except for o-xylene and methylnaphthalenes, all tested compounds were metabolized under conditions that are typical for contaminated marine liquid sites, namely at pH 6–9 and at 4–27°C. By applying molecular biological techniques (16S rDNA sequencing, DGGE) nine strains could be identified in the consortium. Five of these strains could be isolated in pure cultures. The involved strains were closely related to the following genera: Pseudoalteromonas (two species), Pseudomonas (two species), Shewanella (two species), Marinobacter (one species), Psychrobacter (one species), and Agreia (one species). Interestingly, the five isolated strains in different combinations were unable to degrade crude oil or its components significantly, indicating the importance of the four unculturable microorganisms in the degradation of single or of complex mixtures of hydrocarbons. The obtained mixed culture showed obvious advantages including stability of the consortium, wide range adaptability for crude oil degradation, and strong degradation ability of crude oil.     

Possible pathways for destruction of polyaromatic hydrocarbons by some oil-degrading bacteria isolated from plant endosphere and rhizosphere

Six strains of oil-degrading bacteria isolated from the endosphere and rhizosphere of plants growing on oil polluted soils of the Irkutsk region were studied to determine the pathways for biodestruction of polyaromatic oil hydrocarbons. All strains were able to efficiently degrade polyaromatic hydrocarbons with the formation of pyrocatechin as a final product; strains 90, 108, and 112 additionally formed protocathechuic acid. The culture broth of the studied strains contained ferulic, n-coumaric, n-oxybenzoic, vanillic, and lilac acids, which probably represent metabolites of cinnamic alcohol, cinnamic aldehyde, and benzoic acid presenting in oil and metabolized by bacteria.     

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang oil field (P. R. China)

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 (H₂ + CO₂) and from acetate was established, and this result was confirmed by radioisotope methods involving NaH¹⁴CO₃ and ¹⁴CH₃COONa. 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 H₂-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.     

16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils

Boreal soils have been suspected reservoirs of infectious environmental mycobacteria. Detection of these bacteria in the environment is hampered by their slow growth. We applied a quantitative sandwich hybridization approach for direct detection of mycobacterial 16S rRNA in soil without a nucleic acid amplification step. The numbers of mycobacterial 16S rRNA molecules found in the soil indicated the presence of up to 10(7) to 10(8) mycobacterial cells per gram of soil. These numbers exceed by factor of 10 to 100 x the previous estimates of mycobacteria in soil based on culture methods. When real-time PCR with mycobacteria targeting primers was used to estimate the number of 16S rDNA copies in soil, one copy of 16S rDNA was detected per 10(4) copies of 16S rRNA. This is close to the number of 16S rRNA molecules detected per cell by the same method in laboratory pure cultures of M. chlorophenolicum. Therefore a major part of the mycobacterial DNA in the studied soils may thus have represented metabolically active cells. The 16S rRNA sandwich hybridization method described in this paper offers a culture independent solution for tracking environmental reservoirs of viable and potentially infectious mycobacteria.     

A halotolerant Alcanivorax sp. strain with potential application in saline soil remediation

Biodegradation of petroleum compounds in saline environments seems intricate and needs more attention. In this study, tetracosane was used to enrich alkane-degrading bacteria from oil-contaminated saline soils. Among the isolates, strain Qtet3, with the highest 16s rRNA gene sequence similarity to Alcanivorax dieselolei B-5^T, was able to grow at a wide range of NaCl concentrations and was shown by GC analysis to degrade more than 90% of tetracosane in 10 days. This strain has at least two alkB genes and could grow on crude oil and diesel fuel, and utilize various pure aliphatic hydrocarbon substrates (from C₁₂ to C₃₄). Highly hydrophobic cell surfaces and lack of significant surface tension reduction in the media suggest that the main mechanism of the cells for accessing substrate is to attach directly to hydrocarbon particles. Application of this strain for remediating crude oil-contaminated soils irrigated with defined saline water demonstrated that this halotolerant bacterium could survive and grow in saline soils irrigated with NaCl solutions up to 5% w/v, with the highest hydrocarbon degradation of 26.1% observed at 2.5% NaCl. This strain is promising for future industrial applications especially in bioremediation of saline soils and wastes.     

The Bioremediation Potential of Hydrocarbonoclastic Bacteria Isolated From a Mangrove Contaminated by Petroleum Hydrocarbons on the Cilacap Coast,Indonesia

The main purpose of the study was to isolate strains of bacteria capable of degrading hydrocarbons from contaminated mangroves and to investigate the ability of the isolated bacteria to degrade total petroleum hydrocarbons (TPH) in a microcosm model of an oily sludge. The potential use of these bacteria strains as environmental clean-up agents was tested by culturing them with six different polyaromatic hydrocarbon (PAH) compounds (phenothiazine, fluorene, fluoranthene, dibenzothiophene, phenanthrene, and pyrene). Six viable and culturable bacteria were isolated, and the 16S rDNA sequence for each was amplified using the primers 9F and 1510R. Sequence results were compared using the National Center for Biotechnology Information (NCBI) BLAST program and, combined with phenotypic and phylogenetic data, were used to identify three strains that belonged to the Bacillus genus and were most closely related (98–99%) to Bacillus aquimaris, Bacillus megaterium, and Bacillus pumilus. The other three strains were closely related (98–100%) to Flexibacteraceae bacterium, Halobacilus trueperi, and Rhodobacteraceae bacterium. Two isolates, BA-PZN and BM-PFFP, which were related to Bacillus aquimaris and Bacillus megaterium, respectively, were further characterized and showed great potential for the removal of more complex hydrocarbon compounds in the oily microcosm model.     

Effect of nitrite on a thermophilic, methanogenic consortium from an oil storage tank

Samples from an oil storage tank (resident temperature 40 to 60 °C), which experienced unwanted periodic odorous gas emissions, contained up to 2,400/ml of thermophilic, lactate-utilizing, sulfate-reducing bacteria. Significant methane production was also evident. Enrichments on acetate gave sheathed filaments characteristic of the acetotrophic methanogen Methanosaeta thermophila of which the presence was confirmed by determining the PCR-amplified 16S rDNA sequence. 16S rDNA analysis of enrichments, grown on lactate- and sulfate-containing media, indicated the presence of bacteria related to Garciella nitratireducens, Clostridium sp. and Acinetobacter sp. These sulfidogenic enrichments typically produced sulfide to a maximum concentration of 5–7 mM in media containing excess lactate and 10 mM sulfate or thiosulfate. Both the production of sulfide and the consumption of acetate by the enrichment cultures were inhibited by low concentrations of nitrite (0.5–1.0 mM). Hence, addition of nitrite may be an effective way to prevent odorous gas emissions from the storage tank.