Bacterial community shift along with the changes in operational conditions in a membrane-aerated biofilm reactor.

Membrane-aerated biofilm reactor (MABR) is a promising wastewater treatment process. Although bacteria inhabiting the MABR biofilm are important in wastewater treatment, the community composition and its correlation with operating conditions were less clear. A laboratory-scale MABR was designed to investigate the shift of bacterial community through a complete operational process by pyrosequencing the bacterial 16S rRNA genes. From around 19,000 sequences, 175 bacterial genera were retrieved, mainly belonging to Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, and Actinobacteria. A large number of unclassified bacterial sequences were also detected in the biofilm, suggesting a wide variety of uncharacterized species in MABR. Redundancy analysis (RDA) revealed that influent chemical oxygen demand (COD), NH4-N, and NaHCO3 concentrations could exert distinct influences on the composition of the bacterial community. The influent COD and NaHCO3 concentrations stimulated proliferation of denitrification-related species such as Dokdonella, Azospira, Hydrogenophaga, Rhodocyclaceae, and Thauera, while inhibiting the growth of Acidovorax and Sinobacteraceae. Some denitrifying Thermomonas spp. tended to survive in NH4-N-rich environments, while Flavobacterium preferred to inhabit NH4-N-poor or COD-rich environments. Conversely, the influent NH4-N and NaHCO3, to some extent, appeared to be the growth-promoting factors for nitrifying bacteria. Furthermore, the presence of potential aerobic denitrifiers such as Comamonas, Enterobacter, and Aeromonas indicated that MABR could have the capability of simultaneous aerobic and anoxic denitrification particularly during treatment of low-ammonia nitrogen sewage.

Roles of sulfate-reducing bacteria in sustaining the diversity and stability of marine bacterial community.

Microbes play central roles in ocean food webs and global biogeochemical processes. Yet, the information available regarding the highly diverse bacterial communities in these systems is not comprehensive. Here we investigated the diversity, assembly process, and species coexistence frequency of bacterial communities in seawater and sediment across ∼600 km of the eastern Chinese marginal seas using 16S rRNA gene amplicon sequencing. Our analyses showed that compared with seawater, bacterial communities in sediment possessed higher diversity and experienced tight phylogenetic distribution. Neutral model analysis showed that the relative contribution of stochastic processes to the assembly process of bacterial communities in sediment was lower than that in seawater. Functional prediction results showed that sulfate-reducing bacteria (SRB) were enriched in the core bacterial sub-communities. The bacterial diversities of both sediment and seawater were positively associated with the relative abundance of SRB. Co-occurrence analysis showed that bacteria in seawater exhibited a more complex interaction network and closer co-occurrence relationships than those in sediment. The SRB of seawater were centrally located in the network and played an essential role in sustaining the complex network. In addition, further analysis indicated that the SRB of seawater helped maintain the high stability of the bacterial network. Overall, this study provided further comprehensive information regarding the characteristics of bacterial communities in the ocean, and provides new insights into keystone taxa and their roles in sustaining microbial diversity and stability in ocean.

Complete genome sequence of Amycolicicoccus subflavus DQS3-9A1T, an actinomycete isolated from crude oil-polluted soil.

Amycolicicoccus subflavus DQS3-9A1(T), isolated from crude oil-polluted soil in the Daqing Oilfield in China, is a type strain of a newly published novel species in the novel genus Amycolicicoccus. Here we report the complete genome of DQS3-9A1(T)and genes associated with oil-polluted environment.

Thermodynamically favorable reactions shape the archaeal community affecting bacterial community assembly in oil reservoirs.

Microbial community assembly mechanisms are pivotal for understanding the ecological functions of microorganisms in biogeochemical cycling in Earth's ecosystems, yet rarely investigated in the context of deep terrestrial ecology. Here, the microbial communities in the production waters collected from water injection wells and oil production wells across eight oil reservoirs throughout northern China were determined and analyzed by proportional distribution analysis and null model analysis. A 'core' microbiota consisting of three bacterial genera, including Arcobacter, Pseudomonas and Acinetobacter, and eight archaeal genera, including Archaeoglobus, Methanobacterium, Methanothermobacter, unclassified Methanobacteriaceae, Methanomethylovorans, Methanoculleus, Methanosaeta and Methanolinea, was found to be present in all production water samples. Canonical correlation analysis reflected that the core archaea were significantly influenced by temperature and reservoir depth, while the core bacteria were affected by the combined impact of the core archaea and environmental factors. Thermodynamic calculations indicate that bioenergetic constraints are the driving force that governs the enrichment of two core archaeal guilds, aceticlastic methanogens versus hydrogenotrophic methanogens, in low- and high-temperature oil reservoirs, respectively. Collectively, our study indicates that microbial community structures in wells of oil reservoirs are structured by the thermodynamic window of opportunity, through which the core archaeal communities are accommodated directly followed by the deterministic recruiting of core bacterial genera, and then the stochastic selection of some other microbial members from local environments. Our study enhances the understanding of the microbial assembly mechanism in deep terrestrial habitats. Meanwhile, our findings will support the development of functional microbiota used for bioremediation and bioaugmentation in microbial enhanced oil recovery.

Purification of eutrophic water containing chlorpyrifos by aquatic plants and its effects on planktonic bacteria.

In this study, the removal of nutrients and chlorpyrifos as well as shifts of planktonic bacterial communities in constructed microcosms were investigated to evaluate the influence of Phragmites australis, Nymphaea alba, and Myriophyllum verticillatum, and their combination, on the restoration of eutrophic water containing chlorpyrifos. Plant-treated groups showed a higher pollutant removal rate than did no-remediation controls, indicating that treatment with plants is effective at remediation of eutrophic water containing chlorpyrifos. Different plants showed different performance on the remediation of eutrophic water, e.g., P. australis manifested stronger capacity for removal of sediment chlorpyrifos. This finding indicated that an appropriate plant combination is needed to deal with complex wastewater. During the treatments, the planktonic bacterial communities were influenced by the concentrations of nutrients and pollutants. The changes of composition of bacterial communities indicated a strong correlation between the bacterial communities and the concentrations of pollutants. The plants also influenced the planktonic bacterial communities, especially at the early phase of treatments. For example, P. australis increased the abundance of Limnohabitans and Nevskia significantly and decreased the abundance of Devosia, Luteolibacter, Methylibium, and Caulobacter significantly. The abundance of Hydrocarboniphaga significantly increased in N. alba-treated microcosms, whereas in M. verticillatum-treated microcosms, the abundance of Limnohabitans and Bdellovibrio significantly increased. Our results suggest that the planktonic bacterial communities may be altered during phytoremediation, and the functions of the affected bacteria should be concerned.

Crude oil as a microbial seed bank with unexpected functional potentials.

It was widely believed that oil is a harsh habitat for microbes because of its high toxicity and hydrophobicity. However, accumulating evidence has revealed the presence of live microbes in crude oil. Therefore, it's of value to conduct an in-depth investigation on microbial communities in crude oil. To this end, microorganisms in oil and water phases were collected from four oil-well production mixtures in Qinghai Oilfield, China, and analyzed for their taxonomic and functional compositions via pyrosequencing and GeoChip, respectively. Hierarchical clustering of 16S rRNA gene sequences and functional genes clearly separated crude oil and water phases, suggestive of distinct taxonomic and functional gene compositions between crude oil and water phases. Unexpectedly, Pseudomonas dominated oil phase where diverse functional gene groups were identified, which significantly differed from those in the corresponding water phases. Meanwhile, most functional genes were significantly more abundant in oil phase, which was consistent with their important roles in facilitating survival of their host organisms in crude oil. These findings provide strong evidence that crude oil could be a "seed bank" of functional microorganisms with rich functional potentials. This offers novel insights for industrial applications of microbial-enhanced oil recovery and bioremediation of petroleum-polluted environments.

Diverse alkane hydroxylase genes in microorganisms and environments.

AlkB and CYP153 are important alkane hydroxylases responsible for aerobic alkane degradation in bioremediation of oil-polluted environments and microbial enhanced oil recovery. Since their distribution in nature is not clear, we made the investigation among thus-far sequenced 3,979 microbial genomes and 137 metagenomes from terrestrial, freshwater, and marine environments. Hundreds of diverse alkB and CYP153 genes including many novel ones were found in bacterial genomes, whereas none were found in archaeal genomes. Moreover, these genes were detected with different distributional patterns in the terrestrial, freshwater, and marine metagenomes. Hints for horizontal gene transfer, gene duplication, and gene fusion were found, which together are likely responsible for diversifying the alkB and CYP153 genes adapt to the ubiquitous distribution of different alkanes in nature. In addition, different distributions of these genes between bacterial genomes and metagenomes suggested the potentially important roles of unknown or less common alkane degraders in nature.

The genome of the moderate halophile Amycolicicoccus subflavus DQS3-9A1(T) reveals four alkane hydroxylation systems and provides some clues on the genetic basis for its adaptation to a petroleum environment.

The moderate halophile Amycolicicoccus subflavus DQS3-9A1(T) is the type strain of a novel species in the recently described novel genus Amycolicicoccus, which was isolated from oil mud precipitated from oil produced water. The complete genome of A. subflavus DQS3-9A1(T) has been sequenced and is characteristic of harboring the genes for adaption to the harsh petroleum environment with salinity, high osmotic pressure, and poor nutrient levels. Firstly, it characteristically contains four types of alkane hydroxylases, including the integral-membrane non-heme iron monooxygenase (AlkB) and cytochrome P450 CYP153, a long-chain alkane monooxygenase (LadA) and propane monooxygenase. It also accommodates complete pathways for the response to osmotic pressure. Physiological tests proved that the strain could grow on n-alkanes ranging from C10 to C36 and propane as the sole carbon sources, with the differential induction of four kinds of alkane hydroxylase coding genes. In addition, the strain could grow in 1-12% NaCl with the putative genes responsible for osmotic stresses induced as expected. These results reveal the effective adaptation of the strain DQS3-9A1(T) to harsh oil environment and provide a genome platform to investigate the global regulation of different alkane metabolisms in bacteria that are crucially important for petroleum degradation. To our knowledge, this is the first report to describe the co-existence of such four types of alkane hydroxylases in a bacterial strain.

Microbial communities in long-term, water-flooded petroleum reservoirs with different in situ temperatures in the Huabei Oilfield, China.

The distribution of microbial communities in the Menggulin (MGL) and Ba19 blocks in the Huabei Oilfield, China, were studied based on 16S rRNA gene analysis. The dominant microbes showed obvious block-specific characteristics, and the two blocks had substantially different bacterial and archaeal communities. In the moderate-temperature MGL block, the bacteria were mainly Epsilonproteobacteria and Alphaproteobacteria, and the archaea were methanogens belonging to Methanolinea, Methanothermobacter, Methanosaeta, and Methanocella. However, in the high-temperature Ba19 block, the predominant bacteria were Gammaproteobacteria, and the predominant archaea were Methanothermobacter and Methanosaeta. In spite of shared taxa in the blocks, differences among wells in the same block were obvious, especially for bacterial communities in the MGL block. Compared to the bacterial communities, the archaeal communities were much more conserved within blocks and were not affected by the variation in the bacterial communities.

The genome sequence of Polymorphum gilvum SL003B-26A1(T) reveals its genetic basis for crude oil degradation and adaptation to the saline soil.

Polymorphum gilvum SL003B-26A1(T) is the type strain of a novel species in the recently published novel genus Polymorphum isolated from saline soil contaminated with crude oil. It is capable of using crude oil as the sole carbon and energy source and can adapt to saline soil at a temperature of 45°C. The Polymorphum gilvum genome provides a genetic basis for understanding how the strain could degrade crude oil and adapt to a saline environment. Genome analysis revealed the versatility of the strain for emulsifying crude oil, metabolizing aromatic compounds (a characteristic specific to the Polymorphum gilvum genome in comparison with other known genomes of oil-degrading bacteria), as well as possibly metabolizing n-alkanes through the LadA pathway. In addition, COG analysis revealed Polymorphum gilvum SL003B-26A1(T) has significantly higher abundances of the proteins responsible for cell motility, lipid transport and metabolism, and secondary metabolite biosynthesis, transport and catabolism than the average levels found in all other genomes sequenced thus far, but lower abundances of the proteins responsible for carbohydrate transport and metabolism, defense mechanisms, and translation than the average levels. These traits support the adaptability of Polymorphum gilvum to a crude oil-contaminated saline environment. The Polymorphum gilvum genome could serve as a platform for further study of oil-degrading microorganisms for bioremediation and microbial-enhanced oil recovery in harsh saline environments.

Bacteria in crude oil survived autoclaving and stimulated differentially by exogenous bacteria.

Autoclaving of crude oil is often used to evaluate the hydrocarbon-degrading abilities of bacteria. This may be potentially useful for bioaugmentation and microbial enhanced oil recovery (MEOR). However, it is not entirely clear if "endogenous" bacteria (e.g., spores) in/on crude oil survive the autoclaving process, or influence subsequent evaluation of the hydrocarbon-degradation abilities of the "exogenous" bacterial strains. To test this, we inoculated autoclaved crude oil medium with six exogenous bacterial strains (three Dietzia strains, two Acinetobacter strains, and one Pseudomonas strain). The survival of the spore-forming Bacillus and Paenibacillus and the non-spore-forming mesophilic Pseudomonas, Dietzia, Alcaligenes, and Microbacterium was detected using a 16S rRNA gene clone library and terminal restriction fragment length polymorphism (T-RFLP) analysis. However, neither bacteria nor bacterial activity was detected in three controls consisting of non-inoculated autoclaved crude oil medium. These results suggest that detection of endogenous bacteria was stimulated by the six inoculated strains. In addition, inoculation with Acinetobacter spp. stimulated detection of Bacillus, while inoculation with Dietzia spp. and Pseudomonas sp. stimulated the detection of more Pseudomonas. In contrast, similar exogenous bacteria stimulated similar endogenous bacteria at the genus level. Based on these results, special emphasis should be applied to evaluate the influence of bacteria capable of surviving autoclaving on the hydrocarbon-degrading abilities of exogenous bacteria, in particular, with regard to bioaugmentation and MEOR. Bioaugmentation and MEOR technologies could then be developed to more accurately direct the growth of specific endogenous bacteria that may then improve the efficiency of treatment or recovery of crude oil.

Degradation of petroleum hydrocarbons (C6-C40) and crude oil by a novel Dietzia strain.

A novel bacterial strain, DQ12-45-1b, was isolated from the production water of a deep subterranean oil-reservoir. Morphological, physiological and phylogenetic analyses indicated that the strain belonged to the genus Dietzia with both alkB (coding for alkane monooxygenase) and CYP153 (coding for P450 alkane hydroxylase of the cytochrome CYP153 family) genes and their induction detected. It was capable of utilizing a wide range of n-alkanes (C6-C40), aromatic compounds and crude oil as the sole carbon sources for growth. In addition, it preferentially degraded short-chain hydrocarbons (≤C25) in the early cultivation phase and accumulated hydrocarbons with chain-lengths from C23 to C27 during later cultivation stage with crude oil as the sole carbon source. This is the first study to report the different behaviors of a bacterial species toward crude oil degradation as well as a species of Dietzia degrading a wide range of hydrocarbons.

Amycolicicoccus subflavus gen. nov., sp. nov., an actinomycete isolated from a saline soil contaminated by crude oil.

Two novel actinomycetes, designated DQS3-9A1(T) and DQS3-9A2, were isolated from a saline soil contaminated with crude oil in the Shengli Oilfield in China. On the basis of 16S rRNA gene sequence analysis, the two strains were most closely related to Mycobacterium species (92.7-94.9 % similarities), and formed a distinct lineage in the suborder Corynebacterineae . In addition, the major sugars in the cell wall, arabinose and galactose, supported the affiliation of strain DQS3-9A1(T) with members of the family Mycobacteriaceae. However, strain DQS3-9A1(T) did not contain mycolic acids and MK-8 (85.5 %) was the major menaquinone for both isolates. The major cellular fatty acids for strain DQS3-9A1(T) were C(16 : 0) (20.5 %), 10-methyl C(17 : 0) (19.3 %), 10-methyl C(18 : 0) (16.1 %), summed feature 3 (11.4 %), C(15 : 0) (11.3 %), C(17 : 0) (5.0 %) and C(17 : 1)omega8c (5.0 %). The polar lipids of strain DQS3-9A1(T) consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol and an unknown glucosamine-containing phospholipid. These chemotaxonomic data indicated that strain DQS3-9A1(T) differs from the present members of the suborder Corynebacterineae. Therefore, the creation of Amycolicicoccus subflavus gen. nov., sp. nov. is proposed, with DQS3-9A1(T) (=DSM 45089(T)=CGMCC 4.3532(T)) as the type strain.

Filomicrobium insigne sp. nov., isolated from an oil-polluted saline soil.

Strain SLG5B-19(T), isolated from an oil-polluted saline soil in Gudao in the coastal Shengli Oilfield, eastern China, was Gram-negative with monoprosthecae or bipolar prosthecae and buds on the prosthecal tips. Growth occurred at NaCl concentrations between 0 and 7 % (w/v), at temperatures between 4 and 45 degrees C, and at pH 6.0-9.0. Strain SLG5B-19(T) had Q-9 as the major respiratory quinone and unsaturated C(18 : 1)omega7c as the predominant cellular fatty acid. The G+C content of the genomic DNA was 59.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SLG5B-19(T) belonged to a clade with the genera Filomicrobium and Hyphomicrobium in the class Alphaproteobacteria. However, 16S rRNA gene sequence similarities of strain SLG5B-19(T) to the phylogenetically most closely related strains, i.e. the type strains of Filomicrobium fusiforme and Hyphomicrobium zavarzinii, were 95.8 and 94.5 %, respectively. In addition, the 16S rRNA gene sequence of strain SLG5B-19(T) had 24 signature nucleotides that were identical to those of the type strain of F. fusiforme. Based on phylogenetic analysis of 16S rRNA gene sequences, strain SLG5B-19(T) could be allocated to the genus Filomicrobium. However, distinct phenotypic differences were observed between strain SLG5B-19(T) and the type strain of F. fusiforme. It is therefore proposed that strain SLG5B-19(T) represents a novel species in the genus Filomicrobium, Filomicrobium insigne sp. nov. The type strain is SLG5B-19(T) (=CGMCC 1.6497(T)=LMG 23927(T)).

Halomonas daqingensis sp. nov., a moderately halophilic bacterium isolated from an oilfield soil.

A Gram-negative, moderately halophilic, short rod-shaped, aerobic bacterium with peritrichous flagellae, strain DQD2-30(T), was isolated from a soil sample contaminated with crude oil from the Daqing oilfield in Heilongjiang Province, north-eastern China. The novel strain was capable of growth at NaCl concentrations of 1-15 % (w/v) [optimum at 5-10 % (w/v)]. Phylogenetic analyses based on 16S rRNA gene sequences showed that the novel strain belonged to the genus Halomonas in the class Gammaproteobacteria; the highest 16S rRNA gene sequence similarities were with Halomonas desiderata DSM 9502(T) (98.8 %), Halomonas campisalis A4(T) (96.6 %) and Halomonas gudaonensis CGMCC 1.6133(T) (95.1 %). The major cellular fatty acids of strain DQD2-30(T) were C(18 : 1)omega7c (43.97 %), C(19 : 0 )cyclo omega8c (23.37 %) and C(16 : 0) (14.83 %). The predominant respiratory lipoquinone was ubiquinone with nine isoprene units (Q9). The DNA G+C content was 67.0 mol%. The DNA-DNA hybridization values of strain DQD2-30(T) with the most closely related species of the genus Halomonas were 51.8 %, 28.4 % and 23.5 % for H. desiderata, H. campisalis and H. gudaonensis, respectively. Based on these analyses, strain DQD2-30(T )(=CGMCC 1.6443(T)=LMG 23896(T)) is proposed to represent the type strain of a novel species, Halomonas daqingensis sp. nov.

Halomonas shengliensis sp. nov., a moderately halophilic, denitrifying, crude-oil-utilizing bacterium.

A moderately halophilic bacterium, designated strain SL014B-85(T), was isolated from a crude-oil-contaminated saline soil from Shengli oilfield, Shandong Province, China. Cells were Gram-negative, aerobic, short rods with lateral flagella. Growth occurred at NaCl concentrations of 0-15 % (optimum 5-15 %), at 10-42 degrees C (optimum 30 degrees C) and at pH 8.0-9.0 (optimum pH 8.5). The only respiratory quinone was Q9, and the main cellular fatty acids were C(18 : 1)omega7c, C(16 : 0) and C(19 : 0) cyclo omega8c. The G+C content of the DNA was 66.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SL014B-85(T) belonged to the genus Halomonas in the Gammaproteobacteria, with highest sequence similarity of 98.1 and 97.8 % to Halomonas alimentaria DSM 15356(T) and Halomonas ventosae DSM 15911(T), respectively. DNA-DNA relatedness values were below 40 % with members of closely related Halomonas species. Results of phenotypic, biochemical and phylogenetic analyses revealed that strain SL014B-85(T) could be classified as representing a novel species of the genus Halomonas, for which the name Halomonas shengliensis sp. nov. is proposed. The type strain is SL014B-85(T) (=CGMCC 1.6444(T)=LMG 23897(T)).