Ring-hydroxylating Dioxygenase Genes Research Articles

Assessing the impacts of oil contamination on microbial communities in a Niger Delta soil

Oil spills are a global challenge, contaminating the environment with organics and metals known to elicit toxic effects. Ecosystems within Nigeria's Niger Delta have suffered from prolonged severe spills for many decades but the level of impact on the soil microbial community structure and the potential for contaminant bioremediation remains unclear. Here, we assessed the extent/impact of an oil spill in this area 6 months after the accident on both the soil microbial community/diversity and the distribution of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHDGNα) genes, responsible for encoding enzymes involved in the degradation of PAHs, across the impacted area. Analyses confirmed the presence of oil contamination, including metals such as Cr and Ni, across the whole impacted area and at depth. The contamination impacted on the microbial community composition, resulting in a lower diversity in all contaminated soils. Gamma-, Delta-, Alpha- proteobacteria and Acidobacteriia dominated 16S rRNA gene sequences across the contaminated area, while Ktedonobacteria dominated the non-contaminated soils. The PAH-RHDαGN genes were only detected in the contaminated area, highlighting a clear relationship with the oil contamination/hydrocarbon metabolism. Correlation analysis indicated significant positive relationships between the oil contaminants (organics, Cr and Ni), PAH-RHDαGN gene, and the presence of bacteria/archaea such as Anaerolinea, Spirochaetia Bacteroidia Thermoplasmata, Methanomicrobia, and Methanobacteria indicating that the oil contamination not only impacted the microbial community/diversity present, but that the microbes across the impacted area and at depth were potentially playing an important role in degrading the oil contamination present. These findings provide new insights on the level of oil contamination remaining 6 months after an oil spill, its impacts on indigenous soil microbial communities and their potential for in situ bioremediation within a Niger Delta's ecosystem. It highlights the strength of using a cross-disciplinary approach to assess the extent of oil pollution in a single study.

High expression of ring-hydroxylating dioxygenase genes ensure efficient degradation of p-toluate, phthalate, and terephthalate by Comamonas testosteroni strain 3a2.

Para-toluic acid, a major pollutant in industrial wastewater, is hazardous to human health. It has been demonstrated that Gram-negative bacteria are among the most effective degraders of para-toluic acid. In this study, the ability of Comamonas testosteroni strain 3a2, isolated from a petrochemical industry wastewater, to degrade para-toluic acid was investigated. The effect of different carbon (glucose and ethylene glycol) and nitrogen sources (urea, yeast extract, peptone, NaNO3, NH4NO3) on the biodegradation of para-toluic acid by the isolate 3a2 was evaluated. Furthermore, ring hydroxylating dioxygenase genes were amplified by PCR and their expression was evaluated during the biodegradation of para-toluic acid. The results indicated that strain 3a2 was able to degrade up to 1000mg/L of para-toluic acid after 14h. The highest degradation yield was recorded in the presence of yeast extract as nitrogen source. However, the formation of terephthalic acid and phthalic acid was noted during para-toluic acid degradation by the isolate 3a2. Toluate 1,2-dioxygenase, terephthalate 1,2 dioxygenase, and phthalate 4,5 dioxygenase genes were detected in the genomic DNA of 3a2. The induction of ring hydroxylating dioxygenase genes was proportional to the concentration of each hydrocarbon. This study showed that the isolate 3a2 can produce terephthalate and phthalate during the para-toluic acid biodegradation, which were also degraded after 24h.

Assessment of PAH degradation potential of native species from a coking plant through identifying of the beneficial bacterial community within the rhizosphere soil

Understanding the mechanisms underlying plant–rhizobacteria interactions in field-contaminated soils is crucial for designing effective rhizoremediation strategies. This study aimed to test the ability of four native herb species to remove polycyclic aromatic hydrocarbons (PAHs) and to analyze their associated bacterial community structures and functional genes within the rhizosphere from the abandoned site of a former Shenyang coking plant in China; the bulk soil was collected as control. All four species removed PAHs, of which the rhizosphere of Kochia scoparia had the highest PAH removal rate (almost 30.2%). Although the composition of the bacterial community within the rhizosphere varied among plant species, all plant species could promote the growth of Sphingomonas, Pedomicrobium, Rhodoplanes, Blastoccus, Mycobacterium, Devosia, and Pseudomonas, and their relative abundance positively correlated with the removal rates of PAHs, soil moisture, and total carbon/total nitrogen in the rhizosphere. Moreover, the activities of 1-aminocyclopropane-1 -carboxylic deaminase gene and Gram-negative ring-hydroxylating dioxygenase gene significantly (P < 0.05) increased compared with those in the control, and these activities had a strong positive correlation with the removal rates of PAHs [r = 0.759 (P < 0.01) and 0.87 (P < 0.01), respectively]. The findings of this study indicated that PAHs were the main factor driving the composition of beneficial bacteria in PAH rhizodegradation, and the PAH rhizoremediation of native plants grown in coking plant can be controlled though altering soil properties.

Response of soil bacterial communities to polycyclic aromatic hydrocarbons during the phyto-microbial remediation of a contaminated soil

Rhizo-box experiments were conducted to analyze the phyto-microbial remediation potential of a grass (Lolium multiflorum L.) and a crop (Glycine max L.) combined with exogenous strain (Pseudomonas sp.) for polycyclic aromatic hydrocarbons (PAHs) contaminated soils. The dynamics of bacterial community composition, abundances of 16 S rDNA and ring hydroxylating dioxygenases (RHDα) genes, and removal of PAHs were evaluated and compared on four culture stages (days 0, 10, 20, and 30). The results showed that 8.65%–47.42% of Σ12 PAHs were removed after 30 days of cultivation. Quantitative polymerase chain reaction (qPCR) analysis indicated that treatments with soybean and ryegrass rhizosphere markedly increased the abundances of total bacteria and PAH-degraders, especially facilitated the growth of gram-negative degrading bacteria. Flavobacterium sp. and Pseudomonas sp. were the main and active strains in the control soil. However, the presence of plants and/or exogenous Pseudomonas sp. changed the soil bacterial community structure and modified the bacterial diversity of PAH-degraders. On the whole, this study showed that the high molecular weight PAHs removal efficiency of phyto-microbial remediation with ryegrass was better than those of remediation with soybean. Furthermore, the removals of PAHs strongly coincided with the abundance of PAH-degraders and bacterial community structure.

Assessment of the use of compost stability as an indicator of alkane and aromatic hydrocarbon degrader abundance in green waste composting materials and finished composts for soil bioremediation application

Green waste composting materials and finished composts were collected from different commercial ex situ composting sites all treating source segregated green waste feedstocks. Stability of each material was determined using the standard ORG0020 dynamic respiration test. To assess whether stability could be used as an indicator for the potential suitability of green waste composting materials and finished composts as amendments for soil bioremediation, comparison was made with alkane and aromatic hydrocarbon degrader abundance determined using a quantitative PCR (qPCR) approach. Specifically, primers targeting alkB and, polyaromatic hydrocarbon ring-hydroxylating dioxygenases genes (PAH-RHD) of Gram positive (GP) and Gram negative (GN) populations were used for qPCR analysis. The results showed no direct correction between compost stability and gene abundance. Further, increase in alkB gene abundance was not linked to PAH-RHD gene abundance. The results support the use of qPCR as a tool for screening organic amendments on a site by site basis for soil bioremediation treatment.

Distribution of bacterial polycyclic aromatic hydrocarbon (PAH) ring-hydroxylating dioxygenases genes in oilfield soils and mangrove sediments explored by gene-targeted metagenomics.

PAH ring-hydroxylating dioxygenases (PAH-RHDα) gene, a useful biomarker for PAH-degrading bacteria, has been widely used to examine PAH-degrading bacterial community in different contaminated sites. However, the distribution of PAH-RHDα genes in oilfield soils and mangrove sediments and their relationship with environmental factors still remain largely unclear. In this study, gene-targeted metagenomics was first used to investigate the diversity of PAH-degrading bacterial communities in oilfield soils and mangrove sediments. The results showed that higher diversity of PAH-degrading bacteria in the studied samples was revealed by gene-targeted metagenomics than traditional clone library analysis. Pseudomonas, Burkholderia, Ralstonia, Polymorphum gilvum, Mycobacterium, Sciscionella marina, Rhodococcus, and potential new degraders were prevailed in the oilfield area. For mangrove sediments, novel PAH degraders and Mycobacterium were predominated. The spatial distribution of PAH-RHDα gene was dependent on geographical location and regulated by local environmental variables. PAH content played a key role in shaping PAH-degrading bacterial communities in the studied samples, which would enrich PAH-degrading bacterial population and decrease PAH-degrading bacterial diversity. This work brings a more comprehensive and some new insights into the distribution and biodegradation potential of PAH-degrading bacteria in soil and sediments ecosystems.

Evolution of Functional Bacteria in a Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bioreactor.

In this study a laboratory-scale sequencing batch bioreactor was constructed, using phenanthrene/pyrene as the sole carbon-source, to demonstrate the removal efficiencies of polycyclic aromatic hydrocarbons (PAHs) and the evolution process of a functional bacterial community. The removal rates were: COD, 51.4 to 76.3%; phenanthrene, 63.5 to 92.4%; and pyrene, 65.9 to 90.1%. Quantification analysis indicated that in sludge samples from the phenanthrene/pyrene degradation stage, the contents of 16S rRNA and ring-hydroxylating dioxygenase genes of Gram-positive and Gram-negative bacteria were 10-and 72-, 102- and 1152-, 31- and 905-fold higher, respectively, than those in seed sludge samples. Clone sequencing and evolution analysis showed that genera Comamonas, Pseudomonas, and Mycobacterium were distributed across all samples and were significant PAH-degrading populations, but some genera, such as Polaromonas and Nocardioides, were substantially enriched only when phenanthrene/pyrene was fed. Correspondingly, the functional genes nidA3, phnAc, and ndoC2 were distributed across all samples, whereas nagAc, pdoA2, and pdoA were enriched only when phenanthrene/pyrene was fed.

Comparative genomic analysis of pyrene-degrading Mycobacterium species: Genomic islands and ring-hydroxylating dioxygenases involved in pyrene degradation.

The genome sequences of two pyrene-degrading bacterial strains of Mycobacterium spp. PYR10 and PYR15, isolated from the estuarine wetland of the Han river, South Korea, were determined using the PacBio RS II sequencing platform. The complete genome of strain PYR15 was 6,037,017 bp in length with a GC content of 66.5%, and contained 5,933 protein-coding genes. The genome of strain PYR10 was 5,999,427 bp in length with a GC content of 67.7%, and contained 5,767 protein-coding genes. Based on the average nucleotide identity values, these strains were designated as M. gilvum PYR10 and M. pallens PYR15. A genomic comparison of these pyrene-degrading Mycobacterium strains with pyrene-non-degrading strains revealed that the genomes of pyrene-degrading strains possessed similar repertoires of ringhydroxylating dioxygenases (RHDs), including the pyrenehydroxylating dioxygenases encoded by nidA and nidA3, which could be readily distinguished from those of pyrenenon-degraders. Furthermore, genomic islands, containing catabolic gene clusters, were shared only among the pyrenedegrading Mycobacterium strains and these gene clusters contained RHD genes, including nidAB and nidA3B3. Our genome data should facilitate further studies on the evolution of the polycyclic aromatic hydrocarbon-degradation pathways in the genus Mycobacterium.

Oxidation of benzo[a]pyrene by laccase in soil enhances bound residue formation and reduces disturbance to soil bacterial community composition

Laccases are capable of rapidly oxidizing benzo[a]pyrene. It is thought that the metabolites with an increase in water solubility caused by the oxidation of benzo[a]pyrene may stimulate the subsequent mineralization. However, to date, there has been no experimental evidence to support this. In this study, the fate of benzo[a]pyrene in soil affected by laccase amendment and the resulting soil bacterial responses were investigated. Laccase amendment promoted benzo[a]pyrene dissipation (15.6%) from soil, accompanied by trace mineralization (<0.58 ± 0.02%) and substantial bound residue formation (∼80%). An increase of ∼15% in the bound residue fraction was observed by laccase amendment, which mainly resulted from covalent binding of the residues to humin fraction. During the incubation, the abundance of bacterial 16S rRNA and polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes did not change markedly. In contrast, benzo[a]pyrene treated with laccase resulted in a smaller shift in the bacterial community composition, indicating a reduced disturbance to the soil microbial communities. These results here suggest that benzo[a]pyrene contaminated soil can be detoxified by laccase amendment mainly due to the enhanced bound residue formation to soil organic matter via covalent binding.

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community.

Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation.

Assessing environmental drivers of microbial communities in estuarine soils of the Aconcagua River in Central Chile.

Aconcagua River basin (Central Chile) harbors diverse economic activities such as agriculture, mining and a crude oil refinery. The aim of this study was to assess environmental drivers of microbial communities in Aconcagua River estuarine soils, which may be influenced by anthropogenic activities taking place upstream and by natural processes such as tides and flood runoffs. Physicochemical parameters were measured in floodplain soils along the estuary. Bacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Pseudomonas, Bacillus and Fungi were studied by DGGE fingerprinting of 16S rRNA gene and ribosomal ITS-1 amplified from community DNA. Correlations between environment and communities were assessed by distance-based redundancy analysis. Mainly hydrocarbons, pH and the composed variable copper/arsenic/calcium but in less extent nitrogen and organic matter/phosphorous/magnesium correlated with community structures at different taxonomic levels. Aromatic hydrocarbons degradation potential by bacterial community was studied. Polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenases genes were detected only at upstream sites. Naphthalene dioxygenase ndo genes were heterogeneously distributed along estuary, and related to Pseudomonas, Delftia, Comamonas and Ralstonia. IncP-1 plasmids were mainly present at downstream sites, whereas IncP-7 and IncP-9 plasmids showed a heterogeneous distribution. This study strongly suggests that pH, copper, arsenic and hydrocarbons are main drivers of microbial communities in Aconcagua River estuarine soils.

Identification of benzo[a]pyrene-metabolizing bacteria in forest soils by using DNA-based stable-isotope probing.

DNA-based stable-isotope probing (DNA-SIP) was used in this study to investigate the uncultivated bacteria with benzo[a]pyrene (BaP) metabolism capacities in two Chinese forest soils (Mt. Maoer in Heilongjiang Province and Mt. Baicaowa in Hubei Province). We characterized three different phylotypes with responsibility for BaP degradation, none of which were previously reported as BaP-degrading microorganisms by SIP. In Mt. Maoer soil microcosms, the putative BaP degraders were classified as belonging to the genus Terrimonas (family Chitinophagaceae, order Sphingobacteriales), whereas Burkholderia spp. were the key BaP degraders in Mt. Baicaowa soils. The addition of metabolic salicylate significantly increased BaP degradation efficiency in Mt. Maoer soils, and the BaP-metabolizing bacteria shifted to the microorganisms in the family Oxalobacteraceae (genus unclassified). Meanwhile, salicylate addition did not change either BaP degradation or putative BaP degraders in Mt. Baicaowa. Polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) genes were amplified, sequenced, and quantified in the DNA-SIP (13)C heavy fraction to further confirm the BaP metabolism. By illuminating the microbial diversity and salicylate additive effects on BaP degradation across different soils, the results increased our understanding of BaP natural attenuation and provided a possible approach to enhance the bioremediation of BaP-contaminated soils.

Effects of polycyclic aromatic hydrocarbons on microbial community structure and PAH ring hydroxylating dioxygenase gene abundance in soil.

Development of successful bioremediation strategies for environments contaminated with recalcitrant pollutants requires in-depth knowledge of the microorganisms and microbial processes involved in degradation. The response of soil microbial communities to three polycyclic aromatic hydrocarbons, phenanthrene (3-ring), fluoranthene (4-ring) and benzo(a)pyrene (5-ring), was examined. Profiles of bacterial, archaeal and fungal communities were generated using molecular fingerprinting techniques (TRFLP, ARISA) and multivariate statistical tools were employed to interpret the effect of PAHs on community dynamics and composition. The extent and rate of PAH removal was directly related to the chemical structure, with the 5-ring PAH benzo(a)pyrene degraded more slowly than phenathrene or fluoranthene. Bacterial, archaeal and fungal communities were all significantly affected by PAH amendment, time and their interaction. Based on analysis of clone libraries, Actinobacteria appeared to dominate in fluoranthene amended soil, although they also represented a significant portion of the diversity in phenanthrene amended and unamended soils. In addition there appeared to be more γ-Proteobacteria and less Bacteroidetes in soil amended with either PAH compared to the control. The soil bacterial community clearly possessed the potential to degrade PAHs as evidenced by the abundance of PAH ring hydroxylating (PAH-RHDα) genes from both gram negative (GN) and gram positive (GP) bacteria in PAH-amended and control soils. Although the dioxygenase gene from GP bacteria was less abundant in soil than the gene associated with GN bacteria, significant (p < 0.001) increases in the abundance of the GP PAH-RHDα gene were observed during phenanthrene and fluoranthene degradation, whereas there was no significant difference in the abundance of the GN PAH-RHDα gene during the course of the experiment. Few studies to-date have examined the effect of pollutants on more than one microbial community in soil. The current study provides information on the response of soil bacterial, archaeal and fungal communities during the degradation of three priority pollutants and contributes to a knowledge base that can inform the development of effective bioremediation strategies for contaminated sites.

Microbial community structure during fluoranthene degradation in the presence of plants

To investigate bacterial and fungal community structure during degradation of varying concentrations (0-5000 mg kg(-1) ) of the polycyclic aromatic hydrocarbon (PAH) fluoranthene in the presence or absence of tomato plants. Fluoranthene amended or unamended growing media, with or without a plant, were incubated in pots in a glasshouse for 30 days. Fluoranthene concentration was quantified using GC-FID, while bacterial and fungal community structure was investigated using trflp and arisa, respectively. The abundance of two ring hydroxylating dioxygenase genes was measured using qPCR. More fluoranthene was degraded in the presence of tomato plants at 500 and 5000 mg fluoranthene kg(-1) (P < 0·001), and it had a toxic effect on plant growth. Bacterial and fungal community composition in the rhizosphere was significantly different from that in nonrhizospheric and unplanted samples. The influence of fluoranthene on bacterial communities overcame that of the plant root such that community composition in rhizosphere and planted nonrhizospheric samples was broadly similar when fluoranthene was present. PAH-RHDα-GP genes were more abundant than PAH-RHDα-GN genes in all treatments. PAH-RHDα-GN abundance was unaffected by either PAH concentration or growing medium type, while the abundance of PAH-RHDα-GP was greatest in the rhizosphere. Plants promoted microbial growth and diversity and led to increases in abundance of Gram-positive dioxygenase genes, correlated with increased degradation of fluoranthene. This work contributes to knowledge in the broad area of biodegradation and also provides useful information for the design of future bioremediation strategies.

Comparative molecular analysis of bacterial communities inhabiting pristine and polluted with polycyclic aromatic hydrocarbons Black Sea coastal sediments

Molecular analysis was applied to characterize bacterial community structure in sediment samples collected from pristine site and oil-polluted Black Sea harbor. Amplified Ribosomal DNA Restriction Analysis (ARDRA) revealed a high similarity in the restriction patterns of both samples thus not demonstrating the effect of the pollutant on the structure of the bacterial communities. Constructed 16S rRNA gene libraries gave more detailed assessment of members. Results showed that α- and γ-Proteobacteria were dominant in the oil polluted site, whereas the pristine site was characterized by prevalence of Actinobacteria. The biodegradative potential of the adapted bacterial community in the oil-polluted sediments was demonstrated by the presence of the aromatic ring hydroxylating dioxygenase genes.

Characterization of polycyclic aromatic hydrocarbons degradation and arsenate reduction by a versatile Pseudomonas isolate

A Pseudomonas isolate, designated PAHAs-1, was found capable of reducing arsenate and degrading polycyclic aromatic hydrocarbons (PAHs) independently and simultaneously. This isolate completely reduced 1.5 mM arsenate within 48 h and removed approximately 100% and 50% of 60 mg l−1 phenanthrene and 20 mg l−1 pyrene within 60 h, respectively. Using PAHs as the sole carbon source, however, this isolate showed a slow arsenate reduction rate (4.62 μM h−1). The presence of arsenic affected cell growth and concurrent PAHs removal, depending on PAH species and arsenic concentration. Adding sodium lactate to the medium greatly enhanced the arsenate reduction and pyrene metabolism. The presence of the alpha subunit of the aromatic ring-hydroxylating dioxygenase (ARHD) gene, arsenate reductase (arsC) and arsenite transporter (ACR3(2)) genes supported the dual function of the isolate. The finding of latter two genes indicated that PAHAs-1 possibly reduced arsenate via the known detoxification mechanism. Preliminary data from hydroponic experiment showed that PAHAs-1 degraded the majority of phenanthrene (>60%) and enhanced arsenic uptake by Pteris vittata L. (from 246.7 to 1187.4 mg kg−1 As in the fronds). The versatile isolate PAHAs-1 may have potentials in improving the bioremediation of PAHs and arsenic co-contamination using the plant-microbe integrated strategy.

Community structure and PAH ring-hydroxylating dioxygenase genes of a marine pyrene-degrading microbial consortium

Marine microbial consortium UBF, enriched from a beach polluted by the Prestige oil spill and highly efficient in degrading this heavy fuel, was subcultured in pyrene minimal medium. The pyrene-degrading subpopulation (UBF-Py) mineralized 31 % of pyrene without accumulation of partially oxidized intermediates indicating the cooperation of different microbial components in substrate mineralization. The microbial community composition was characterized by culture dependent and PCR based methods (PCR-DGGE and clone libraries). Molecular analyses showed a highly stable community composed by Alphaproteobacteria (84 %, Breoghania, Thalassospira, Paracoccus, and Martelella) and Actinobacteria (16 %, Gordonia). The members of Thalasosspira and Gordonia were not recovered as pure cultures, but five additional strains, not detected in the molecular analysis, that classified within the genera Novosphingobium, Sphingopyxis, Aurantimonas (Alphaproteobacteria), Alcanivorax (Gammaproteobacteria) and Micrococcus (Actinobacteria), were isolated. None of the isolates degraded pyrene or other PAHs in pure culture. PCR amplification of Gram-positive and Gram-negative dioxygenase genes did not produce results with any of the cultured strains. However, sequences related to the NidA3 pyrene dioxygenase present in mycobacterial strains were detected in UBF-Py consortium, suggesting the representative of Gordonia as the key pyrene degrader, which is consistent with a preeminent role of actinobacteria in pyrene removal in coastal environments affected by marine oil spills.

Gene Cloning and Function Analysis Involved in the Biodegradation of Aromatic Compounds by Bacillus NAPZ

A bacterial strain NAPZ, which utilized polycyclic aromatic hydrocarbons (PAHs) as the sole carbon and energy source for growth, was studied on its apparent characteristics, key gene structure and functions. It was preliminarily identified as a Bacillus sp. according to its physiological characteristics and the phylogenetic property of its 16S rRNA gene sequence. Based on the reported RHD gene sequences, a pair of primers was designed to amplify the RHD gene from the genomic DNA of strain NAPZ by PCR. Then, the RHD gene was cloned and sequenced. Based on this sequence and its related sequences in the GenBank database, a multiple alignment was conducted then a phylogenetic tree was constructed with DNAMAN and MEGA software. The analysis revealed that strain NAPZ oxidized PAHs via ring hydroxylating dioxygenase (RHD) pathway. The study provided experimental and theoretical support for revealing the structure and function of key genes as well as the PAHs-degrading pathways in strain NAPZ.

Microbial community adaptation to quaternary ammonium biocides as revealed by metagenomics

Quaternary ammonium compounds (QACs) represent widely used cationic biocides that persist in natural environments. Although microbial degradation, sensitivity and resistance to QACs have been extensively documented, a quantitative understanding of how whole communities adapt to QAC exposure remain elusive. To gain insights into these issues, we exposed a microbial community from a contaminated river sediment to varied levels of benzalkonium chlorides (BACs, a family of QACs) for 3 years. Comparative metagenomic analysis showed that the BAC-fed communities were dramatically decreased in phylogenetic diversity compared with the control (no BAC exposure), resulting presumably from BAC toxicity, and dominated by Pseudomonas species (> 50% of the total). Time-course metagenomics revealed that community adaptation occurred primarily via selective enrichment of BAC-degrading Pseudomonas populations, particularly P. nitroreducens, and secondarily via amino acid substitutions and horizontal transfer of a few selected genes in the Pseudomonas populations, including a gene encoding a PAS/PAC sensor protein and ring-hydroxylating dioxygenase genes. P. nitroreducens isolates were reproducibly recoverable from communities after prolonged periods of no-BAC exposure, suggesting that they are robust BAC-degraders. Our study provides new insights into the mechanisms and tempo of microbial community adaptation to QAC exposure and has implications for treating QACs in biological engineered systems.

Bacterial polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenases in the sediments from the Pearl River estuary, China

Bacterial community compositions were characterized using denaturing gradient gel electrophoresis analysis of bacterial 16S rRNA gene in the sediments of the Pearl River estuary. Sequencing analyses of the excised bands indicated that Gram-negative bacteria, especially Gammaproteobacteria, were dominant in the Pearl River estuary. The diversity of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) gene in this estuary was then assessed by clone library analysis. The phylogenetic analyses showed that all PAH-RHD gene sequences of Gram-negative bacteria (PAH-RHD[GN]) were closely related to the nagAc gene described for Ralstonia sp. U2 or nahAc gene for Pseudomonas sp. 9816-4, while the PAH-RHD gene sequences of Gram-positive bacteria (PAH-RHD[GP]) at sampling site A1 showed high sequence similarity to the nidA gene from Mycobacterium species. Meanwhile, molecular diversity of the two functional genes was higher at the upstream of this region, while lower at the downstream. Redundancy analysis indicated that environmental factors, such as NH₄--N, ∑PAHs, pH, SiO₃--Si, and water depth, affected the distribution of the PAH-RHD[GN] gene in the Pearl River estuary.