Meta-cleavage Pathway Research Articles

Genomic characterization and proteomic analysis of Bacillus amyloliquefaciens in response to lignin

This study examined the lignin degradation characteristics of Bacillus amyloliquefaciens MN-13. Specifically, whole-genome sequencing and comparative proteomic analysis were performed to investigate the responses of the MN-13 strain to lignin. A maximum lignin removal of 38.0 % was achieved after 36 h of inoculation in mineral salt medium with 0.2 g/L alkaline lignin, under the following conditions: the carbon to nitrogen ratio C/N = 1/1; inoculum size 6 %; addition of glucose as an exogenous carbon source. When the MN-13 strain was inoculated into mineral salt medium with and without lignin, respectively, 831 differentially expressed proteins were identified, 404 of which were up-regulated and 427 were down-regulated. Enrichment analysis revealed that up-regulated proteins were associated with microbial metabolism in diverse environment, biosynthesis of amino acids, and pathways related to energy production, including carbon metabolism, pyruvate metabolism, the TCA cycle etc. Genomic analysis revealed that the MN-13 strain possesses many ligninolytic enzymes and aromatics degradation pathway, including benzoate degradation and aminobenzoate degradation etc. Taken together, the proteomic and genomic analyses indicated that the meta-cleavage pathway of catechol, including benzoate degradation, etc., is the main lignin degradation pathway. These findings provide new insight into lignin degradation mediated by B. amyloliquefaciens.

Aerobic phenol degradation using native bacterial consortium via ortho-and meta-cleavage pathways.

Effective bioremediation of a phenol-polluted environment harnesses microorganisms' ability to utilize hazardous compounds as beneficial degraders. In the present study, a consortium consisting of 15 bacterial strains was utilized. The current study aims to monitor the phenol biodegradation pathway. The tested consortium showed effective potential in the bioremediation of phenol-contaminated industrial wastewater. The enzymatic studies conducted brought to light that the bacterial consortium under test was proficient in degrading phenol under aerobic conditions while exhibiting the simultaneous expression of both ortho- and meta-cleavage pathways. It was observed that pheA, pheB, and C12O genes were maximally expressed, and the enzymes responsible for phenol degradation, namely, phenol hydroxylase, catechol 1,2-dioxygenase, and catechol 2,3-dioxygenase, reached maximum activity after 48 h of incubation with a 20-ppm phenol concentration. To gain a deeper understanding of the activation of both ortho- and meta-cleavage pathways involved in phenol degradation, a technique known as differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) was applied. This method allowed for the specific amplification and detection of genes responsible for phenol degradation. The expression levels of these genes determined the extent to which both ortho- and meta-cleavage pathways were activated in response to the presence of phenol.

Bioremediation mechanisms of chlorophenol-Cr(Ⅵ): The role of amines, immobilization, and DEGs in Pseudomonas sp. PC

The combined pollution of p-Chlorophenol (4-CP) and hexavalent chromium [Cr(VI)] is widespread in the environment. However, little information is available on the relationship between the fate of Cr(VI) and its amines during dechlorination, which are dominant in pH value changes. Here, Pseudomonas sp. PC can degrade 4-CP and convert Cr(VI). The removal efficiency of Cr(VI) accords with the first-order reaction kinetics. Notably, the pH value (7.95) of the chlorophenol-Cr(VI) systems increased compared to the 4-CP treatment due to the significant accumulation of amines, especially ethambutol, which was 1.06 folds higher than 4-CP treatment. The pH value of the 4-CP treatment decreased to 5.89. Thus, Cr(III) was more likely to immobilize on the surface of the cell in the form of Cr(OH)3. Interestingly, Catechol 1,2-dioxygenase (catA) was 0.98 folds higher than Catechol 2,3-dioxygenase (dmpB) in chlorophenol-Cr(Ⅵ) systems, indicating that the presence of Cr(Ⅵ) changed the metabolic pathway of Pseudomonas sp. PC for 4-CP from the dmpB meta-cleavage pathway to the catA ortho-cleavage pathway. These conclusions deepen the cognizance of the relationship between the degradation pathway of chlorophenols, the fixation of Cr(VI) and the amines, providing a better basis for the bioremediation application of chlorophenol-Cr(VI) sites.

Stable-isotope probing combined with amplicon sequencing and metagenomics identifies key bacterial benzene degraders under microaerobic conditions

The primary aim of the present study was to reveal the major differences between benzene-degrading bacterial communities evolve under aerobic versus microaerobic conditions and to reveal the diversity of those bacteria, which can relatively quickly degrade benzene even under microaerobic conditions. For this, parallel aerobic and microaerobic microcosms were set up by using groundwater sediment of a BTEX-contaminated site and 13C labelled benzene. The evolved total bacterial communities were first investigated by 16S rRNA gene Illumina amplicon sequencing, followed by a density gradient fractionation of DNA and a separate investigation of “heavy” and “light” DNA fractions. Results shed light on the fact that the availability of oxygen strongly determined the structure of the degrading bacterial communities. While members of the genus Pseudomonas were overwhelmingly dominant under clear aerobic conditions, they were almost completely replaced by members of genera Malikia and Azovibrio in the microaerobic microcosms. Investigation of the density resolved DNA fractions further confirmed the key role of these two latter genera in the microaerobic degradation of benzene. Moreover, analysis of a previously acquired metagenome-assembled Azovibrio genome suggested that benzene was degraded through the meta-cleavage pathway by this bacterium, with the help of a subfamily I.2.I-type catechol 2,3-dioxygenase. Overall, results of the present study implicate that under limited oxygen availability, some potentially microaerophilic bacteria play crucial role in the aerobic degradation of aromatic hydrocarbons.

Widespread and active piezotolerant microorganisms mediate phenolic compound degradation under high hydrostatic pressure in hadal trenches

Phenolic compounds, as well as other aromatic compounds, have been reported to be abundant in hadal trenches. Although high-throughput sequencing studies have hinted at the potential of hadal microbes to degrade these compounds, direct microbiological, genetic and biochemical evidence under in situ pressures remain absent. Here, a microbial consortium and a pure culture of Pseudomonas, newly isolated from Mariana Trench sediments, efficiently degraded phenol under pressures up to 70 and 60 MPa, respectively, with concomitant increase in biomass. By analyzing a high-pressure (70 MPa) culture metatranscriptome, not only was the entire range of metabolic processes under high pressure generated, but also genes encoding complete phenol degradation via ortho- and meta-cleavage pathways were revealed. The isolate of Pseudomonas also contained genes encoding the complete degradation pathway. Six transcribed genes (dmpKLMNOPsed) were functionally identified to encode a multicomponent hydroxylase catalyzing the hydroxylation of phenol and its methylated derivatives by heterogeneous expression. In addition, key catabolic genes identified in the metatranscriptome of the high-pressure cultures and genomes of bacterial isolates were found to be all widely distributed in 22 published hadal microbial metagenomes. At microbiological, genetic, bioinformatics, and biochemical levels, this study found that microorganisms widely found in hadal trenches were able to effectively drive phenolic compound degradation under high hydrostatic pressures. This information will bridge a knowledge gap concerning the microbial aromatics degradation within hadal trenches.

Acidovorax benzenivorans sp. nov., a novel aromatic hydrocarbon-degrading bacterium isolated from a xylene-degrading enrichment culture.

A Gram-stain-negative strain, designated as D2M1T was isolated from xylene-degrading enrichment culture and characterized using a polyphasic approach to determine its taxonomic position. The 16S rRNA gene sequence analysis revealed that strain D2M1T belongs to the genus Acidovorax, with the highest 16S rRNA gene similarity to Acidovorax delafieldii DSM 64T (99.93 %), followed by Acidovorax radicis DSM 23535T (98.77 %) and Acidovorax kalamii MTCC 12652T (98.76 %). The draft genome sequence of strain D2M1T is 5.49 Mb long, and the G+C content of the genome is 64.2 mol%. Orthologous average nucleotide identity and digital DNA-DNA hybridization relatedness values between strain D2M1T and its closest relatives were below the threshold values for species demarcation confirming that strain D2M1T is distinctly separated from its closest relatives. The whole genome analysis of the strain revealed a phenol degradation gene cluster, encoding a multicomponent phenol hydroxylase (mPH) together with a complete meta-cleavage pathway including an I.2.C-type catechol 2,3-dioxygenase (C23O) gene. The strain was able to degrade benzene and ethylbenzene as sole sources of carbon and energy under aerobic and microaerobic conditions. Cells were facultatively aerobic rods and motile with a single polar flagellum. The predominant fatty acids (>10 % of the total) of strain D2M1T were summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), C16 : 0 and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). The major ubiquinone of strain D2M1T was Q8, while the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Based on polyphasic data, it is concluded that strain D2M1T represents a novel species of the genus Acidovorax, for which the name of Acidovorax benzenivorans sp. nov. is proposed. The type strain of the species is strain D2M1T (=DSM 115238T=NCAIM B.02679T).

Biodegradation of Benzene Under Microaerobic Conditions: a Groundwater Microcosm Experiment Combined with a Multiomics Approach and DNA Stable Isotope Probing

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are the most common contaminants of the groundwater and can easily contaminate drinking water sources due to their relatively high water solubility. Among these compounds, benzene is known to have carcinogenic properties and is considered to be persistent under anoxic conditions. Although deep knowledge was acquired both on the aerobic and anaerobic degradation of benzene in the past three decades, the diversity of those bacteria which are able to degrade it under microaerobic conditions, is still unknown. To come over this limitation aerobic and microaerobic benzene-degrading microcosms were established using groundwater sediment of a BTEX-contaminated site and the evolved bacterial communities were investigated through a polyphasic approach including multiomics analysis and DNA stable isotope probing. The obtained results shed light on the fact that the aerobic and microaerobic benzene-degrading bacterial communities were distinctly different. In the aerobic microcosms members of the genus Pseudomonas overwhelmingly dominated the bacterial communities by showing even 40-50% abundance. In contrast, under microaerobic conditions members of the genera Azovibrio and Malikia dominated the communities, while the abundance of Pseudomonas drastically decreased compared to that of was observable in the aerobic microcosms. Results obtained by the analysis of microcosms spiked with 13C6-benzene confirmed these observations. In heavy DNA fractions obtained from the aerobic microcosms the enrichment of Pseudomonas and Rhizobiaceae-related 16S rRNA gene fragments was observable. At the same time, labelled DNA from the microaerobic microcosms contained mostly Azovibrio and Malikia-related sequences, while the abundance of Pseudomonas 16S rRNA gene sequences was below 2%. Besides, by using metagenomic dataset of a previous microaerobic experiment we managed to reconstruct an Azovibrio genome, in which a complete meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons was identified. Overall, it can be concluded that under microaerobic conditions members of Comamonadaceae and Rhodocyclaceae can be the key benzene degraders in contaminated subsurface environments.

Flexible catabolism of monoaromatic hydrocarbons by anaerobic microbiota adapting to oxygen exposure

Microbe-mediated anaerobic degradation is a practical method for remediation of the hazardous monoaromatic hydrocarbons (BTEX, including benzene, toluene, ethylbenzene and xylenes) under electron-deficient contaminated sites. However, how do the anaerobic functional microbes adapt to oxygen exposure and flexibly catabolize BTEX remain poorly understood. We investigated the switches of substrate spectrum and bacterial community upon oxygen perturbation in a nitrate-amended anaerobic toluene-degrading microbiota which was dominated by Aromatoleum species. DNA-stable isotope probing demonstrated that Aromatoleum species was involved in anaerobic mineralization of toluene. Metagenome-assembled genome of Aromatoleum species harbored both the nirBD-type genes for nitrate reduction to ammonium coupled with toluene oxidation and the additional meta-cleavage pathway for aerobic benzene catabolism. Once the anaerobic microbiota was fully exposed to oxygen and benzene, 1.05 ± 0.06% of Diaphorobacter species rapidly replaced Aromatoleum species and flourished to 96.72 ± 0.01%. Diaphorobacter sp. ZM was isolated, which was not only able to utilize benzene as the sole carbon source for aerobic growth and but also innovatively reduce nitrate to ammonium with citrate/lactate/glucose as the carbon source under anaerobic conditions. This study expands our understanding of the adaptive mechanism of microbiota for environmental redox disturbance and provides theoretical guidance for the bioremediation of BTEX-contaminated sites.

Impact of active lifestyle on the primary school children saliva microbiota composition.

Male (114) and female children (8-10 years) belonging to five primary schools in the neighborhoods of Turin were classified as active (A) or sedentary (S) based on PAQ-C-It questionnaire. PCR amplification of salivary DNA targeted the hypervariable V3-V4 regions of the 16S rRNA bacterial genes. DADA2 workflow was used to infer the Amplicon Sequence Variants and the taxonomic assignments; the beta-diversity was obtained by PCoA with the UniFrac method; LEfSe algorithm, threshold at 5%, and Log LDA cutoff at ±0.5 were used to identify differently abundant species in A compared to S saliva sample. Daily food intake was assessed by 3-Days food record. The metabolic potential of microbial communities was assessed by PICRUSt. No significant differences were found in individual's gender distribution (p = 0.411), anthropometry, BMI (p > 0.05), and all diet composition between A and S groups (p > 0.05). Eight species were differently abundant: Prevotella nigrescens (LDA score = -3.76; FDR = 1.5×10-03), Collinsella aerofaciens (LDA score = -3.17; FDR = 7.45×10-03), Simonsiella muelleri (LDA score = -2.96; FDR = 2.76×10-05), Parabacteroides merdae (LDA score = -2.43; FDR = 1.3×10-02) are enriched in the A group; Gemella parahaemolysans, Prevotella aurantiaca (LDA score = -3.9; FDR = 5.27×10-04), Prevotella pallens (LDA score = 4.23; FDR = 1.93×10-02), Neisseria mucosa (LDA score = 4.43; FDR = 1.31×10-02; LDA score = 2.94; FDR = 7.45×10-03) are enriched in the S group. A prevalence of superpathway of fatty acid biosynthesis initiation (E. coli) and catechol degradation II (meta-cleavage pathway) was found in saliva from A compared to S children. Our results showed that active children had an enrichment of species and genera mainly associated with a healthier profile. By contrast, the genera and the species enriched in the sedentary group could be linked to human diseases.

Biodegradation of p-hydroxybenzoic acid in Herbaspirillum aquaticum KLS-1 isolated from tailing soil: Characterization and molecular mechanism

The wide distribution of p-hydroxybenzoic acid (PHBA) in the environments has attracted great concerns due to its potential risks to organisms. Bioremediation is considered a green way to remove PHBA from environment. Here, a new PHBA-degrading bacterium Herbaspirillum aquaticum KLS-1was isolated and its PHBA degradation mechanisms were fully evaluated. Results showed that strain KLS-1 could utilize PHBA as the sole carbon source and completely degrade 500 mg/L PHBA within 18 h. The optimal conditions for bacterial growth and PHBA degradation were pH values of 6.0–8.0, temperatures of 30 °C-35 °C, shaking speed of 180 rpm, Mg2+ concentration of 2.0 mM and Fe2+ concentration of 1.0 mM. Draft genome sequencing and functional gene annotations identified three operons (i.e., pobRA, pcaRHGBD and pcaRIJ) and several free genes possibly participating in PHBA degradation. The key genes pobA, ubiA, fadA, ligK and ubiG involved in the regulation of protocatechuate and ubiquinone (UQ) metabolisms were successfully amplified in strain KLS-1 at mRNA level. Our data suggested that PHBA could be degraded by strain KLS-1 via the protocatechuate ortho-/meta-cleavage pathway and UQ biosynthesis pathway. This study has provided a new PHBA-degrading bacterium for potential bioremediation of PHBA pollution.

Characterization and Expression Analysis of Extradiol and Intradiol Dioxygenase of Phenol-Degrading Haloalkaliphilic Bacterial Isolates

Haloalkophilic bacteria have a potential advantage as a bioremediation organism of high oil-polluted and industrial wastewater. In the current study, Haloalkaliphilic isolates were obtained from Hamralake, Wadi EL-Natrun, Egypt. The phenotype script, biochemical characters, and sequence analysis of bacterial-16S rRNA were used to identify the bacterial isolates; Halomonas HA1 and Marinobacter HA2. These strains required high concentrations of NaCl to ensure bacterial growth, especially Halomonas HA1 strain. Notably, both isolates can degrade phenol at optimal pH values, between 8 and 9, with the ability to grow in pH levels up to 11, like what was seen in the Halomonas HA1 strain. Moreover, both isolates represent two different mechanistic pathways for phenol degradation. Halomonas HA1 exploits the 1,2 phenol meta-cleavage pathway, while Marinobacter HA2 uses the 2,3 ortho-cleavage pathway as indicated by universal primers for 1,2 and 2,3 CTD genes. Interestingly, Marinobacter HA2 isolate eliminated the added phenol within an incubation period of 72 h, while the Halomonas HA1 isolate invested 96 h in degrading 84% of the same amount of phenol. Phylogenetic analysis of these 1,2 CTD (catechol dioxygenase) sequences clearly showed an evolutionary relationship between 1,2 dioxygenases of both Halomonadaceae and Pseudomonadaceae. In comparison, 2,3 CTD of Marinobacter HA2 shared the main domains of the closely related species. Furthermore, semi-quantitative RT-PCR analysis proved the constitutive expression pattern of both dioxygenase genes. These findings provide new isolates of Halomonas sp. and Marinobacter sp. that can degrade phenol at high salt and pH conditions via two independent mechanisms.

Biodegradation kinetics and metabolism of Benzo(a)fluorene by Pseudomonas strains isolated from refinery effluent

The final sinkers of polyaromatic hydrocarbons are water sources, where they undergo bioaccumulation and biomagnification, leading to adverse mutagenic, carcinogenic, and teratogenic effects on exposure in flora, fauna, and humans. Two indigenous strains, Pseudomonas sp. WDE11 and Pseudomonas sp. WD23, isolated from refinery effluent, degraded over 97.5% of benzo(a)fluorene (10 mg/L) in 7 days. On growth at concentration dependent amounts (50 mg/L and 100 mg/L), the degradation reduced to approximately 90% and 80% respectively in 56 days. Degradation kinetics was concentration dependent, as degradation followed first-order and second-order kinetics for 50 mg/L and 100 mg/L respectively. The half-life for degradation of benzo(a)fluorene ranged between 11.64 - 12.26 days and 13.11–14.5 days for strains WDE11 and WD23 respectively. The values of Andrew-Haldane kinetic parameters i.e. μmax, Ks, and Ki were 0.306 day−1, 11.11 mg/L, and 120.41 mg/L for strain WDE11 respectively, while for strain WD23, the respective values were 0.312 day−1, 9.97 mg/L, and 152 mg/L. Degradation metabolites were identified by their MS patterns as 3,4-dihydroxy fluorene, 2-(1-oxo-2,3-dihydro-1H-inden-2-yl) acetic acid, 3,4-dihydrocoumarin, salicylic acid, catechol, and oxalic acid. Metabolic pathway of degradation constructed, revealed that benzo(a)fluorene was metabolized via the formation of fluorene, further metabolized by salicylate pathway forming catechol. The catechol formed was degraded into simpler metabolites by meta-cleavage pathway, which was validated by catechol 2,3 dioxygenase enzyme activity.

Microaerobic enrichment of benzene-degrading bacteria and description of Ideonella benzenivorans sp. nov., capable of degrading benzene, toluene and ethylbenzene under microaerobic conditions

In the present study, the bacterial community structure of enrichment cultures degrading benzene under microaerobic conditions was investigated through culturing and 16S rRNA gene Illumina amplicon sequencing. Enrichments were dominated by members of the genus Rhodoferax followed by Pseudomonas and Acidovorax. Additionally, a pale amber-coloured, motile, Gram-stain-negative bacterium, designated B7T was isolated from the microaerobic benzene-degrading enrichment cultures and characterized using a polyphasic approach to determine its taxonomic position. The 16S rRNA gene and whole genome-based phylogenetic analyses revealed that strain B7T formed a lineage within the family Comamonadaceae, clustered as a member of the genus Ideonella and most closely related to Ideonella dechloratans CCUG 30977T. The sole respiratory quinone is ubiquinone-8. The major fatty acids are C16:0 and summed feature 3 (C16:1ω7c/iso-C15:0 2-OH). The DNA G + C content of the type strain is 68.8 mol%. The orthologous average nucleotide identity (OrthoANI) and in silico DNA–DNA hybridization (dDDH) relatedness values between strain B7T and closest relatives were below the threshold values for species demarcation. The genome of strain B7T, which is approximately 4.5 Mb, contains a phenol degradation gene cluster, encoding a multicomponent phenol hydroxylase (mPH) together with a complete meta-cleavage pathway including a I.2.C-type catechol 2,3-dioxygenase (C23O) gene. As predicted by the genome, the type strain is involved in aromatic hydrocarbon-degradation: benzene, toluene and ethylbenzene are degraded aerobically and also microaerobically as sole source of carbon and energy. Based on phenotypic characteristics and phylogenetic analysis, strain B7T is a member of the genus Ideonella and represents a novel species for which the name Ideonella benzenivorans sp. nov. is proposed. The type strain of the species is strain B7T (= LMG 32,345T = NCAIM B.02664T).

BIODEGRADATION OF HIGH MOLECULAR WEIGHT POLYCYCLICAROMATIC HYDROCARBON CHRYSENE BY SOIL BACTERIALISOLATES

PAHs (polycyclic aromatic hydrocarbons) are common and dangerous organic pollutants made primarily of carbon and hydrogen. Chrysene is also a potential human carcinogen and a high molecular weight PAH. The current research focuses on chrysene biodegradation, and effective chrysene degrading microbial strains were identified from soil samples taken from the hydrocarbon-contaminated surrounding soil of the Mathura petroleum refinery in Uttar Pradesh, India. The isolated isolates were morphologically examined, and basic biochemical tests were performed. 16sRNA analysis was performed for molecular analysis, and it was discovered that 3AS belongs to Enterobacter sp. and 5AS belongs to Ochrobactrum sp. The biodegradation efficiency of strain 3AS was 60%, while strain 5AS could only digest 55% of chrysene in MSM (Minimal Salt Media). The principal metabolite of chrysene biodegradation discovered in the cultures of strain3AS using the GC-MS (Gas chromatography-mass spectrophotometry) technique, as well assignificant metabolites such as phenol, 2, 5-bis (1,1- dimethyl ethyl), 1-naphthoic acid, 8-methoxy,and 3-nitrophthalic acid. In 5AS cultures, phthalic acid and di-isobutyl ester was detected. Thecatechol-1, 2-dioxygenase gene was not found, but the presence of catechol-2, 3-dioxygenasesuggests that chrysene can be degraded via the meta cleavage pathway.

Structural insights into the substrate specificity of 5-chloro-2-hydroxymuconate tautomerase CnbG

The catechol meta-cleavage pathway is widely involved in the degradation of aromatic compounds, including those halogenated aromatic hydrocarbons and their derivatives. CnbG is a kind of 4-oxalocrotonate tautomerase (4-OT) located in the catechol meta-cleavage pathway, catalyzes the ketonization of cis,cis-5-chloro-2-hydroxymuconate and cis,cis-2-hydroxymuconate to yield 5-chloro-2-oxo-3-hexene-1,6-dioate and 2-oxo-3-hexene-1,6-dioate, and contributes to the degradation of 4-chloronitrobenzene and chlorobenzene in Comamonas testosteroni CNB-1. Yet, the reason why CnbG and those 4-OTs could recognize various substrates is not well explained. Here, we determined the crystal structure of CnbG at resolution of 2.0 Å and identified that the potential substrate pocket involved in four conserved residues, residues Pro1, Arg11, Arg39 and Trp50, but not five conserved residues as those reported in other 4-OTs. We also found the four conserved residues assemble different sequence patterns in different 4-OTs, indicating their potential roles in catalysis and substrate binding. Via molecular docking, we found the 5-chloro group was clamped by two residues and extended to the solvent, indicating a substrate binding mode that could bear the substitution of different groups in the 5-position. Our work extends the knowledge of the substrate specificity of enzymes in the catechol meta-cleavage pathway.

Pseudomonas phenolilytica sp. nov., a novel phenol-degrading bacterium.

This study describes a bacterium strain RBPA9 isolated from a municipality waste dumping area capable of degrading phenol, proposed as a novel species ofPseudomonas. Cells are Gram-negative, rod shaped, aerobic and motile. The genome is 3.92Mb, and the G + C content is 64.64%. The overall genome relatedness indices such as in silico DNA-DNA hybridization (isDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) values were below 70% and 95-96%, respectively. Phylogenetic analysis based on genome-wide core genes and 16S rRNA gene sequences revealed that strain RBPA9 clustered withPseudomonas stutzeriATCC 17588T in both the phylogenetic trees. Maximum growth was recorded at 200mg /L concentration of phenol which was consumed within 24h. A gene cluster of phenol degradation pathway was detected. The quantitative real-time PCR (RT-PCR) demonstrated the expression of all the genes required in the meta-cleavage pathway of phenol in RBPA9. Our results reveal that strain RBPA9 represents a novel species for whichPseudomonas phenolilyticasp. nov. is proposed. The type strain is RBPA9T (= TBRC 15231T = NBRC 115284T).

Effect of mixing intensity on biodegradation of phenol in a moving bed biofilm reactor: Process optimization and external mass transfer study

In this work, an effort has been made to design the process variables and to analyse the impact of mixing intensity on mass transfer diffusion in a moving bed biofilm reactor (MBBR). A lab-scale MBBR, filled with Bacillus cereus GS2 IIT (BHU) immobilized-polyethylene biocarriers, was employed to optimize the process variables, including mixing intensity (60–140 rpm), phenol concentration (50–200 mg/L), and hydraulic retention time (HRT) (4–24 h) using response surface methodology. The optimum phenol removal of 87.64 % was found at 100 rpm of mixing intensity, 200 mg/L of phenol concentration, and 24 h of HRT. The higher mixing intensity improved the substrate diffusion between the liquid phase and the surface of the biofilm. The external mass transfer coefficients were found in the range of 1.431 × 10-5-1.845 × 10-5 m/s. Moreover, the detection of catechol and 2-hydroxymuconic semialdehyde revealed that the Bacillus sp. followed the meta-cleavage pathway during the biodegradation of phenol.

Degradation of Diuron by a Bacterial Mixture and Shifts in the Bacterial Community During Bioremediation of Contaminated Soil.

Diuron, a phenylurea herbicide, has been extensively applied in controlling a wide range of weeds in several crops. In the current study, a mixed culture of three bacterial strains, i.e., Bacillus subtilis DU1, Acinetobacter baumannii DU, and Pseudomonas sp. DUK, isolated from sugarcane soil, completely degraded diuron and 3,4-DCA in liquid media at 20mg L-1 within 48h. During diuron degradation, a few metabolites (DCPMU, DCPU, and 3,4-DCA) were produced. Further determination of ring-cleavage pathways demonstrated that Acinetobacter baumannii DU and Pseudomonas fluorescens DUK degraded diuron and 3,4-DCA via ortho-cleavage. In contrast, Bacillus subtilis DU transformed these compounds via meta-cleavage pathways. Moreover, diuron caused a significant shift in the bacterial community in soil without diuron history. The augmentation of mountain soil with the isolated bacteria resulted in nearly three times higher degradation rate of diuron than the degradation by indigenous microorganisms. This study provides important information on in situ diuron bioremediation from contaminated sites by bioaugmentation with a mixed bacterial culture.

Metagenomic insights into bacterial communities’ structures in polycyclic aromatic hydrocarbons degrading consortia

Assembly of bacterial communities under xenobiotic stress, such as in the presence of various PAHs and their metabolic activities in form of consortia, are less studied. Eight bacterial consortia were developed from PAHs-contaminated sediments in minimal media, using various combinations of naphthalene, phenanthrene, fluoranthene and pyrene. Among eight, competency of consortium ACDMRT-8 was notable, it showed 88.5%, 85.8%, 58.0% and 60.3% degradation of naphthalene, phenanthrene, fluoranthene and pyrene (concentration of total-PAHs 800 ppm, each PAH 200 ppm) within 9 d at room temperature, respectively. After 40 enrichment cycles, bacterial communities were characterized using high-throughput 16S rRNA gene amplicon sequencing of all eight consortia. Though bacterial communities in all consortia were pre-dominated by Proteobacteria, individual diversity varied depending upon enrichment conditions and combinations of PAHs provided. Pseudomonas sp. were abundant in four consortia, which favored all-four PAHs degradation. Bordetella, Chelatococcus and Azoarcus were abundantly found in other consortia with changes in temperature and PAHs. The radical changes in bacterial community at genus level were well reflected in beta-diversity analysis, where four consortia were clustered together, while another group contained three consortia and single consortium remained separated from other two clusters. Dissimilarity analysis using SIMPER suggested that Pseudomonas, Azoarcus, Bordetella, Chelatococcus and Chelativorans formed top 5 abundant genera within eight consortia enriched for PAH biodegradation. Molecular ecological network amongst dominant 17 genera from eight consortia was constructed. The functional competence in form of hydrocarbon degrading genes and pathways were predicted using PICRUSt2 tool and has indicated abundance of ortho-cleavage over meta-cleavage pathways.

Analysis of Phenol Biodegradation in Antibiotic and Heavy Metal Resistant Acinetobacter lwoffii NL1.

Phenol is a common environmental contaminant. The purpose of this study was to isolate phenol-degrading microorganisms from wastewater in the sections of the Chinese Medicine Manufactory. The phenol-degrading Acinetobacter lwoffii NL1 was identified based on a combination of biochemical characteristics and 16S rRNA genes. To analyze the molecular mechanism, the whole genome of A. lwoffii NL1 was sequenced, yielding 3499 genes on one circular chromosome and three plasmids. Enzyme activity analysis showed that A. lwoffii NL1 degraded phenol via the ortho-cleavage rather than the meta-cleavage pathway. Key genes encoding phenol hydroxylase and catechol 1,2-dioxygenase were located on a megaplasmid (pNL1) and were found to be separated by mobile genetic elements; their function was validated by heterologous expression in Escherichia coli and quantitative real-time PCR. A. lwoffii NL1 could degrade 0.5 g/L phenol within 12 h and tolerate a maximum of 1.1 g/L phenol, and showed resistance against multiple antibiotics and heavy metal ions. Overall, this study shows that A. lwoffii NL1 can be potentially used for efficient phenol degradation in heavy metal wastewater treatment.