Potential of camel rumen derived Bacillus subtilis and Bacillus velezensis strains for application in plant biomass hydrolysis.

Rumen inhabiting Bacillus species possesses a high genetic potential for plant biomass hydrolysis and conversion to value-added products. In view of the same, five camel rumen-derived Bacillus strains, namely B. subtilis CRN 1, B. velezensis CRN 2, B. subtilis CRN 7, B. subtilis CRN 11, and B. velezensis CRN 23 were initially assayed for diverse hydrolytic activities, followed by genome mining to unravel the potential applications. CRN 1 and CRN 7 showed the highest endoglucanase activity with 0.4 U/ml, while CRN 23 showed high ß-xylosidase activity of 0.36 U/ml. The comprehensive genomic insights of strains resolve taxonomic identity, clusters of an orthologous gene, pan-genome dynamics, and metabolic features. Annotation of Carbohydrate active enzymes (CAZymes) reveals the presence of diverse glycoside hydrolases (GH) GH1, GH5, GH43, and GH30, which are solely responsible for the effective breakdown of complex bonds in plant polysaccharides. Further, protein modeling and ligand docking of annotated endoglucanases showed an affinity for cellotrioside, cellobioside, and ß-glucoside. The finding indicates the flexibility of Bacillus-derived endoglucanase activity on diverse cellulosic substrates. The presence of the butyrate synthesis gene in the CRN 1 strain depicts its key role in the production of important short-chain fatty acids essential for healthy rumen development. Similarly, antimicrobial peptides such as bacilysin and non-ribosomal peptides (NRPS) synthesized by the Bacillus strains were also annotated in the genome. The findings clearly define the role of Bacillus sp. inside the camel rumen and its potential application in various plant biomass utilizing industry and animal health research sectors.

Pangenome-driven insights into nitrogen metabolic characteristics of Citrobacter portucalensis strain AAK_AS5 associated with wastewater nitrogen removal.

Nitrogen metabolism in the genus Citrobacter is very poorly studied despite its several implications in wastewater treatment. In the current study, Citrobacter portucalensis strain AAK_AS5 was assessed for remediation of simulated wastewater supplemented with different inorganic nitrogen sources. Combination of (NH4)2SO4 with KNO3 was the most preferred for achieving high growth density followed by (NH4)2SO4 and KNO3 alone. This was in agreement with highest ammonical nitrogen removal of 92.9% in the presence of combined nitrogen sources and the corresponding nitrate nitrogen removal of 93% in the presence of KNO3. Furthermore, these removal capacities were validated by investigating the uniqueness and the spread of metabolic features through pan-genomic approach that revealed the largest number of unique genes (2097) and accessory genes (705) in strain AAK_AS5. Of the total 44 different types of nitrogen metabolism-related genes, 39 genes were associated with the core genome, while 5 genes such as gltI, nasA, nasR, nrtA, and ntrC uniquely belonged to the accessory genome. Strain AAK_AS5 possessed three major nitrate removal pathways viz., assimilatory and dissimilatory nitrate reduction to ammonia (ANRA & DNRA), and denitrification; however, the absence of nitrification was compensated by ammonia assimilation catalyzed by gene products of the GDH and GS-GOGAT pathways. narGHIJ encoding the respiratory nitrate reductase was commonly identified in all the studied genomes, while genes such as nirK, norB, and nosZ were uniquely present in the strain AAK_AS5 only. A markedly different genetic content and metabolic diversity between the strains reflected their adaptive evolution in the environment thus highlighting the significance of C. portucalensis AAK_AS5 for potential application in nitrogen removal from wastewater.

In Silico Genomic Characterization of Bacillus velezensis Strain AAK_S6 for Secondary Metabolite and Biocontrol Potential.

This study reports the draft genome sequence of Bacillus velezensis strain AAK_S6 as a valuable biocontrol agent with high genetic potential to harbor broad-spectrum secondary metabolite producing capacity. A genome data of 4,430,946 bp were generated with a GC content of 46.4% that comprised a total of 4861 genes including a total of 4757 coding sequences (CDS), 104 rRNAs, 85 tRNAs and 80 pseudo-genes. Based on the overall genome-based relatedness indices (OGRI), the strain AAK_S6 has been reassigned to its correct taxonomic position. The strain shared > 99% OrthoANI, > 98% ANIb, > 99% ANIm, > 0.9900 TETRA, > 93% dDDH and 0.08% GC content difference with model strains B. velezensis FZB42T and B. velezensis NRRL B-41580T thus delineating them as closely related species. The genome was mined for strain-specific secondary metabolites that revealed 20 gene clusters for the biosynthesis of several cyclic lipopeptides, saccharides, polyketides along with bacilysin. Thus, the comparative genome analysis of strain AAK_S6 with members of the genus Bacillus by phylogenomic approach revealed that the genomes were almost similar genetically and contained the core genome for B. velezensis. Genomic data strongly supported that the strain AAK_S6 represented an excellent potential candidate for the production of secondary metabolites that could serve as a basis for developing new biocontrol agents, plant growth promoters, and microbial fertilizers.

Social Media Data Mining of Antitobacco Campaign Messages: Machine Learning Analysis of Facebook Posts.

BACKGROUND: Social media platforms provide a valuable source of public health information, as one-third of US adults seek specific health information online. Many antitobacco campaigns have recognized such trends among youth and have shifted their advertising time and effort toward digital platforms. Timely evidence is needed to inform the adaptation of antitobacco campaigns to changing social media platforms. OBJECTIVE: In this study, we conducted a content analysis of major antitobacco campaigns on Facebook using machine learning and natural language processing (NLP) methods, as well as a traditional approach, to investigate the factors that may influence effective antismoking information dissemination and user engagement. METHODS: We collected 3515 posts and 28,125 associated comments from 7 large national and local antitobacco campaigns on Facebook between 2018 and 2021, including the Real Cost, Truth, CDC Tobacco Free (formally known as Tips from Former Smokers, where "CDC" refers to the Centers for Disease Control and Prevention), the Tobacco Prevention Toolkit, Behind the Haze VA, the Campaign for Tobacco-Free Kids, and Smoke Free US campaigns. NLP methods were used for content analysis, including parsimonious rule-based models for sentiment analysis and topic modeling. Logistic regression models were fitted to examine the relationship of antismoking message-framing strategies and viewer responses and engagement. RESULTS: We found that large campaigns from government and nonprofit organizations had more user engagements compared to local and smaller campaigns. Facebook users were more likely to engage in negatively framed campaign posts. Negative posts tended to receive more negative comments (odds ratio [OR] 1.40, 95% CI 1.20-1.65). Positively framed posts generated more negative comments (OR 1.41, 95% CI 1.19-1.66) as well as positive comments (OR 1.29, 95% CI 1.13-1.48). Our content analysis and topic modeling uncovered that the most popular campaign posts tended to be informational (ie, providing new information), where the key phrases included talking about harmful chemicals (n=43, 43%) as well as the risk to pets (n=17, 17%). CONCLUSIONS: Facebook users tend to engage more in antitobacco educational campaigns that are framed negatively. The most popular campaign posts are those providing new information, with key phrases and topics discussing harmful chemicals and risks of secondhand smoke for pets. Educational campaign designers can use such insights to increase the reach of antismoking campaigns and promote behavioral changes.

Phylogenomic analysis of Citrobacter sp. strain AAK_AS5 and its metabolic capabilities to support nitrogen removal behavior.

Despite the ubiquity of the genus Citrobacter in clinical, industrial, and environmental scenarios, a large number of Citrobacter strains have not been explored at the genome-scale level. In this study, accurate taxonomic assignment of strain AAK_AS5 isolated from activated sludge was achieved by in-silico genomic comparison using Overall Genome-based Relatedness Indices (ANI(OAT): 97.55%, ANIb:97.28%, and ANIm: 97.83%) that indicated its closest identity to the related strain Citrobacter portucalensis A60T . Results were consistent with a digital DNA-DNA hybridization value of 80% with C. portucalensis A60T which was greater than the species boundary value >70% for delineating closely related bacterial species. Gene mining through Kyoto Encyclopedia of Genes and Genomes (KEGG), and annotation using rapid annotation subsystem technology (RAST) revealed the notable gene contents for nitrogen metabolism and other pathways associated with nitrate/nitrite ammonification (28 genes), ammonia assimilation (22 genes), and denitrification pathways (14 genes). Furthermore, the strain AAK_AS5 also exhibited a high soluble chemical oxygen demand (sCOD), NH4 + -N, and NO3 - -N removal efficiency of 91.4%, 90%, and 93.6%, respectively thus validating its genetic capability for utilizing both (NH4 )2 SO4 and KNO3 as the nitrogen source. The study provided deeper insights into the phylogenomics and the genetic potential of Citrobacter, sp. strain AAK AS5 associated with nitrogen metabolism thus signifying the potential application of the isolate for treating nitrogen-rich wastewaters.

Managing gene expression in Pseudomonas simiae EGD-AQ6 for chloroaromatic compound degradation.

Pseudomonas simiae EGD-AQ6 is capable of utilizing chloroaromatic compound i.e., 2-4-D efficiently in its biofilm phenotype. The differential accumulation of intermediate 4-chlorocatechol rates were significant in planktonic and biofilm phenotypes, as well as in the  increased biofilm adapted cell numbers. Interestingly, response surface analysis demonstrated the combined positive effects of 2-4-D degradation and 4-CCA accumulation rates and the gene expression profiles, with significant up-regulation of degradative and biofilm genes, and greater participation of pellicle genes in the biofilm phenotypes than their planktonic counterparts, thereby revealing a phenotype variation. It positively validated the physiological data. Furthermore, the sequence similarity of the 2-4-D catabolic and biofilm-forming proteins (pel ABCDEFG and pga ABCD), which are responsible for building carbohydrate rich extracellular matrix, were significant with the respective organisms. This is the first study, which endorses this strain to be unique in efficient chloro-aromatic degradation through phenotype variation, thereby proving a potential candidate in the improvement of bioremediation technologies.

Mining the landfill soil metagenome for denitrifying methanotrophic taxa and validation of methane oxidation in microcosm.

In the present study, the microbial community residing at different depths of the landfill was characterized to assess their roles in serving as a methane sink. Physico-chemical characterization revealed the characteristic signatures of anaerobic degradation of organic matter in the bottom soil (50-60 cm) and, active process of aerobic denitrification in the top soil (0-10 cm). This was also reflected from the higher abundance of bacterial domain in the top soil metagenome represented by dominant phyla Proteobacteria and Actinobacteria which are prime decomposers of organic matter in landfill soils. The multiple fold higher relative abundances of the two most abundant genera; Streptomyces and Intrasporangium in the top soil depicted greater denitrifying taxa in top soil than the bottom soil. Amongst the aerobic methanotrophs, the genera Methylomonas, Methylococcus, Methylocella, and Methylacidiphilum were abundantly found in the top soil metagenome that were essential for oxidizing methane generated in the landfill. On the other hand, the dominance of archaeal domain represented by Methanosarcina and Methanoculleus in the bottom soil highlighted the complete anaerobic digestion of organic components via acetoclasty, carboxydotrophy, hydrogenotrophy, methylotrophy. Functional characterization revealed a higher abundance of methane monooxygenase gene in the top soil and methyl coenzyme M reductase gene in the bottom soil that correlated with the higher relative abundance of aerobic methanotrophs in the top soil while methane generation being the active process in the highly anaerobic bottom soil in the landfill. The activity dependent abundance of endogenous microbial communities in the different zones of the landfill was further validated by microcosm studies in serum bottles which established the ability of the methanotrophic community for methane metabolism in the top soil and their potential to serve as sink for methane. The study provides a better understanding about the methanotrophs in correlation with their endogenous environment, so that these bacteria can be used in resolving the environmental issues related to methane and nitrogen management at landfill site.

Unique pool of carbohydrate-degrading enzymes in novel bacteria assembled from cow and buffalo rumen metagenomes.

Reconstruction of genomes from environmental metagenomes offers an excellent prospect for studying the metabolic potential of organisms resilient to isolation in laboratory conditions. Here, we assembled 12 high-quality metagenome-assembled genomes (MAGs) with an estimated completion of ≥ 90% from cow and buffalo rumen metagenomes. Average nucleotide identity (ANI) score-based screening with an existing database suggests the novelty of these genomes. Gene prediction led to the identification of 30,359 protein-encoding genes (PEGs) across 12 genomes, of which only 44.8% were annotated against a specific functional attribute. Further analysis revealed the presence of 985 carbohydrate-active enzymes (CAZymes) from more than 50 glycoside hydrolase families, of which 90% do not have a proper match in the CAZy database. Genome mining revealed the presence of a high frequency of plant biomass deconstructing genes in Bacteroidetes MAGs compared to Firmicutes. The results strongly indicate that the rumen chamber harbors high numbers of deeply branched and as-yet uncultured microbes that encode novel CAZymes, candidates for prospective usage in plant biomass-hydrolyzing and biofuels industries. KEY POINTS: • Genome binning plays a crucial role in revealing the metabolic potential of uncultivable microbes. • Assembled 12 novel genomes from cow and buffalo rumen metagenome datasets. • High frequency of plant biomass deconstructing genes identified in Bacteroidetes MAGs.

Differential colonization and functioning of microbial community in response to phosphate levels.

Microbes play a major role in phosphate cycling and regulate its availability in various environments. The metagenomic study highlights the microbial community divergence and interplay of phosphate metabolism functional genes in response to phosphate rich (100 mgL-1), limiting (25 mgL-1), and stressed (5 mgL-1) conditions at lab-scale bioreactor. Total five core phyla were found responsive toward different phosphate (Pi) levels. However, major variations were observed in Proteobacteria and Actinobacteria with 33-81% and 5-56% relative abundance, respectively. Canonical correspondence analysis reflects the colonization of Sinorhizobium (0.8-4%), Mesorhizobium (1-4%), Rhizobium (0.5-3%) in rich condition whereas, Pseudomonas (1-2%), Rhodococcus (0.2-2%), Flavobacterium (0.2-1%) and Streptomyces (0.3-4%) colonized in limiting and stress condition. The functional profiling demonstrates that Pi limiting and stress condition subjected biomass were characterized by abundant PQQ-Glucose dehydrogenase, alkaline phosphatase, 5'-nucleotidase, and phospholipases C genes. The finding implies that the major abundant genera belonging to phosphate solubilization enriched in limiting/stressed conditions decide the functional turnover by modulating the metabolic flexibility for Pi cycling. The study gives a better insight into intrinsic ecological responsiveness mediated by microbial communities in different Pi conditions that would help to design the microbiome according to the soil phosphate condition. Furthermore, this information assists in sustainably maintaining the ecological balance by omitting excessive chemical fertilizers and eutrophication.

Genomic characterization of denitrifying methylotrophic Pseudomonas aeruginosa strain AAK/M5 isolated from municipal solid waste landfill soil.

Municipal landfills are known for methane production and a source of nitrate pollution leading to various environmental issues. Therefore, this niche was selected for the isolation of one-carbon (C1) utilizing bacteria with denitrifying capacities using anaerobic enrichment on nitrate mineral salt medium supplemented with methanol as carbon source. Eight axenic cultures were isolated of which, isolate AAK/M5 demonstrated the highest methanol removal (73.28%) in terms of soluble chemical oxygen demand and methane removal (41.27%) at the expense of total nitrate removal of 100% and 33% respectively. The whole genome characterization with phylogenomic approach suggested that the strain AAK/M5 could be assigned to Pseudomonas aeruginosa with close neighbours as type strains DVT779, AES1M, W60856, and LES400. The circular genome annotation showed the presence of complete set of genes essential for methanol utilization and complete denitrification process. The study demonstrates the potential of P. aeruginosa strain AAK/M5 in catalysing methane oxidation thus serving as a methane sink vis-à-vis utilization of nitrate. Considering the existence of such bacteria at landfill site, the study highlights the need to develop strategies for their enrichment and designing of efficient catabolic activity for such environments.

Genomic insight for algicidal activity in Rhizobium strain AQ_MP.

Occurrence of Harmful Algal Blooms (HABs) creates a threat to aquatic ecosystem affecting the existing flora and fauna. Hence, the mitigation of HABs through an eco-friendly approach remains a challenge for environmentalists. The present study provides the genomic insights of Rhizobium strain AQ_MP, an environmental isolate that showed the capability of degrading Microcystis aeruginosa (Cyanobacteria) through lytic mechanisms. Genome sequence analysis of Rhizobium strain AQ_MP unraveled the algal lytic features and toxin degradative pathways in it. Functional genes of CAZymes such as glycosyltransferases (GT), glycoside hydrolases (GH), polysaccharide lyases (PL) which supports algal polysaccharide degradation (lysis) were present in Rhizobium strain AQ_MP. Genome analysis also clarified the presence of the glutathione metabolic pathway, which is the biological detoxification pathway responsible for toxin degradation. The conserved region mlrC, a microcystin toxin-degrading gene was also annotated in the genome. The study illustrated that Rhizobium strain AQ_MP harbored a wide range of mechanisms for the lysis of Microcystis aeruginosa cells and its toxin degradation. In future, this study finds promiscuity for employing Rhizobium strain AQ_MP species for bioremediation, based on its physiological and genomic analysis.

Unraveling the camel rumen microbiome through metaculturomics approach for agriculture waste hydrolytic potential.

Cellulose is the most abundant natural polymer present on Earth in the form of agriculture waste. Hydrolysis of agriculture waste for simple fermentable reducing sugars is the bottleneck in the area of biofuel generation and other value-added products. The present study aims to utilize the camel rumen as a bioreactor for potent cellulolytic and hemicellulolytic bacteria by altering the feed types with varying cellulosic concentrations. A total of 6716 bacterial cultures were subjected to three layers of screening, where plate zymography and chromophoric substrate screening served as primary screening method for cellulolytic and hemicellulolytic potential. The potential isolates were genetically grouped using RAPD, and 51 representative isolates from each group were subjected to molecular identification through 16S rDNA sequencing, followed by quantification of various cellulolytic and hemicellulolytic enzymes. Out of 51 potent isolates, 5 isolates had high endoglucanase activity ranging from 0.3 to 0.48 U/ml. The selected five key isolates identified as Pseudomonas, Paenibacillus, Citrobacter, Bacillus subtilis, and Enterobacter were employed for hydrolyzing the various agriculture residues and resulted in approximately 0.4 mg/ml of reducing sugar. Furthermore, the metaculturomics approach was implemented to deduce the total cultured diversity through 16S rRNA amplicon library sequencing. The metaculturomics data revealed the dominance of proteobacteria and unidentified bacterial population in all four feed types, which indicates the possibility of culturing novel cellulose-deconstructing bacteria. Moreover, the presence of diverse hydrolytic enzymes in cultured isolates supports the usage of these bacteria in bio-processing of agriculture waste residues and obtaining the biofuels and other value-added products.

Isolation, purification and characterization of a novel esterase from camel rumen metagenome.

Bacterial esterases are gaining the importance in pharmaceuticals and agrochemical industries due to their excellent biocatalytic properties and a wide range of applications. In the present study, a novel gene encoding an esterase (designated as Est-CR) was identified from shotgun metagenomic sequencing data of camel rumen (Camelus dromedarius) liquor. The open reading frame consisted of 1,224bp, which showed 84.03% sequence identity to Bacteroidales bacterium, corresponding to a protein of 407 amino acids and has a catalytic domain belonging to an esterase. Est-CR belonged to family V with GLSMG domain. The purified enzyme with a molecular mass of 62.64 kDa was checked on SDS-PAGE, and its expression was confirmed by western blotting. The enzyme was active and stable over a broad range of temperature (35-65 °C), displayed the maximum activity at 50 °C and pH 7.0. Individually all metal ions inhibited the enzyme activity, while in combination, K2+, Ca2+, Mg2+ and Mn2+ metal ions enhanced the enzyme activity. The detergents strongly inhibited the activity, while EDTA (10 mM) increased the activity of the Est-CR enzyme. The enzyme showed specificity to short-chain substrates and displayed an optimum activity against butyrate ester. This novel enzyme might serve as a promising candidate to meet some harsh industrial processes enzymatic needs.

Integrated Genomic and Functional Characterization of the Anti-diabetic Potential of Arthrobacter sp. SW1.

For decades, bacterial natural products have served as valuable resources for developing novel drugs to treat several human diseases. Recent advancements in the integrative approach of using genomic and functional tools have proved beneficial in obtaining a comprehensive understanding of these biomolecules. This study presents an in-depth characterization of the anti-diabetic activity exhibited by a bacterial isolate SW1, isolated from an effluent treatment plant. As a primary screening, we assessed the isolate for its potential to inhibit alpha-amylase and alpha-glucosidase enzymes. Upon confirmation, we further utilized LC-MS, ESI-MS/MS, and NMR spectroscopy to identify and characterize the biomolecule. These efforts were coupled with the genomic assessment of the biosynthetic gene cluster involved in the anti-diabetic compound production. Our investigation discovered that the isolate SW1 inhibited both α-amylase and α-glucosidase activity. The chemical analysis suggested the production of acarbose, an anti-diabetic biomolecule, which was further confirmed by the presence of biosynthetic gene cluster "acb" in the genome. Our in-depth chemical characterization and genome mining approach revealed the potential of bacteria from an unconventional niche, an effluent treatment plant. To the best of our knowledge, it is one of the first few reports of acarbose production from the genus Arthrobacter.

Mobility of antibiotic resistance and its co-occurrence with metal resistance in pathogens under oxidative stress.

The bacterial communities are challenged with oxidative stress during their exposure to bactericidal antibiotics, metals, and different levels of dissolved oxygen (DO) encountered in diverse environmental habitats. The frequency of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) co-selection is increased by selective pressure posed by oxidative stress. Hence, study of resistance acquisition is important from an evolutionary perspective. To understand the dependence of oxidative stress on the dissemination of ARGs and MRGs through a pathogenic bacterial population, 12 metagenomes belonging to gut, water and soil habitats were evaluated. The metagenome-wide analysis showed the chicken gut to pose the most diverse pool of ARGs (30.4 ppm) and pathogenic bacteria (Simpson diversity = 0.98). The most common types of resistances found in all the environmental samples were efflux pumps (13.22 ppm) and genes conferring resistance to vancomycin (12.4 ppm), tetracycline (12.1 ppm), or beta-lactam (9.4 ppm) antibiotics. Additionally, limiting DO level in soil was observed to increase the abundance of excision nucleases (uvrA and uvrB), DNA polymerase (polA), catalases (katG), and other oxidative stress response genes (OSGs). This was further evident from major variations occurred in antibiotic efflux genes due to the effect of DO concentration on two human pathogens, namely Salmonella enterica and Shigella sonnei found in all the selected habitats. In conclusion, the microbial community, when challenged with oxidative stress caused by environmental variations in oxygen level, tends to accumulate higher amounts of ARGs with increased dissemination potential through triggering non-lethal mutagenesis. Furthermore, the genetic linkage or co-occurrence of ARGs and MRGs provides evidence for selecting ARGs under high concentrations of heavy metals.

Myco-remediation of Chlorinated Pesticides: Insights Into Fungal Metabolic System.

Synthetic chemicals including organochlorine pesticides pose environment and health hazard due to persistent and bio-accumulation property. Majority of them are recognized as endocrine disruptors. Fungi are ubiquitous in nature and employs efficient enzymatic machinery for the biotransformation and degradation of toxic, recalcitrant pollutants. This review critically discusses the organochlorine biotransformation process mediated by fungi and highlights the role of enzymatic system responsible for biotransformation, especially distribution of dehalogenase homologs among fungal classes. It also explores the potential use of fungal derived biomaterial, mainly chitosan as an adsorbing biomaterial for pesticides and heavy metals removal. Further, prospects of employing fungus to over-come the existing bioremediation limitations are discussed. The study highlights the potential scope of utilizing fungi for initial biotransformation purposes, preceding final biodegradation by bacterial species under environmental conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00940-8.

Unfolding microbial community intelligence in aerobic and anaerobic biodegradation processes using metagenomics.

Environmental factors and available nutrients influence microbial communities, and with that, there exists a dynamic shift in community structure and hierarchy in wastewater treatment systems. Of the various factors, the availability and gradient of oxygen selectively enrich a typical microbial community and also form the community stratification which could be established through metagenomics studies. In recent years, metagenomics with various sets of bioinformatics tools has assisted in exploration and better insight into the organization and relation of the taxonomical and functional composition and associate physiological intelligence of the microbial communities. The microbial communities, under defined conditions acquire a typical hierarchy with flexible but active network of the metabolic route, which ensures the survival needs of every member residing in that community and their abundance. This knowledge of community functional organization defines the rule in designing and improving biodegradation processes in case of both aerobic and anaerobic systems.

Exploring the eukaryotic diversity in rumen of Indian camel (Camelus dromedarius) using 18S rRNA amplicon sequencing.

In addition to a wide variety of anaerobic and facultative anaerobic bacteria, camel rumen also harbors a diverse of eukaryotic organisms. In the present study, the eukaryotic communities of camel rumen were characterized using 18S rRNA amplicon sequencing. Metagenomic DNA was isolated from rumen samples of fourteen adult Bikaneri and Kachchhi breeds of camel fed different diets containing Jowar, Bajra, Maize, and Guar. Illumina sequencing generated 27,161,904 number of reads corresponding to 1543 total operational taxonomic units (OTUs). Taxonomic classification of community metagenome sequences from all the samples revealed the presence of 92 genera belonging to 16 different divisions, out of which Ciliophora (73%), Fungi (13%) and Streptophyta (9%) were found to be the most dominant. Notably, the abundance of Ciliophora was significantly higher in the case of Guar feed, while Fungi was significantly higher in the case of Maize feed, indicating the influence of cellulose and hemicellulose content of feedstuff on the composition of eukaryotes. The results suggest that the camel rumen eukaryotes are highly dynamic and depend on the type of diet given to the animal. Pearson's correlation analysis suggested the ciliate protozoa and fungi were negatively correlated with each other. To the best of our knowledge, this is first systematic study to characterize camel rumen eukaryotes, which has provided newer information regarding eukaryotic diversity patterns amongst camel fed on different diets.

Metagenomic Insight Towards Vanillin-Mediated Membrane Biofouling Prevention: In Silico Docking Validation.

Biofouling leads to water quality deterioration and higher maintenance cost for cleaning of membranes. The present study has demonstrated the application of a biomolecule (vanillin) in scrubbing and destabilizing biofilms of drinking water reverse osmosis (RO) membrane module in lab scale reactor set-up. Reverse osmosis membrane reactor was connected with tap water supply and subjected with optimal concentration of vanillin. The pressure drop was delayed by 17-20 days as compared to control reactor. Real-time PCR analysis of metagenome indicated the reduced copy number of functional biofilm-associated genes (bdlA, lasI, pgaC) in treated membrane. SEM and metagenome analysis revealed that the sticky biofilm communities shifted to loosely bound emboli after vanillin treatment. Metagenome sequence analysis revealed the inhibitory activity against major biofouling biota like members of Proteobacteria, Acidobacteria, Acnitobacteria, Bacteroidetes, Candidatus, Nitrospira, and Firmicutes. Biofouled membrane metagenome sequence was also compared with real-life (brackish water, waste water, domestic drinking water) biofouled membrane communities. In silico docking of vanillin to receptor proteins and chemical configuration simulation along with other phenolic derivatives were performed, which suggested that the autoiducer signal capability of vanillin was effective against representative broad spectrum biofilm population. Vanillin exhibited the quorum-quenching mode of action by virtue of docking towards similar amino acid (Thr 131, Ilu 214) responsible of autoinducer signal anchoring in the transcriptional regulatory proteins.

Assembling a genome for novel nitrogen-fixing bacteria with capabilities for utilization of aromatic hydrocarbons.

Metagenome from refinery wastewater treatment plant running under nitrogen stress was analyzed for mining of novel aromatic hydrocarbon-degrading bacteria. The sequence data were assembled using metaspade followed by binning using the Metabat tool to assemble genome; where coverage and depth were calculated using bowtie and samtools. The analysis picked a novel genome belonging to family Bradyrhizobiaceae, identified based on 16S rDNA gene which was supported by CheckM and Kraken analysis. Using RAST, the assembled genome showed the capabilities for nitrogen fixation with the utilization of multiple hydrocarbon substrates with 14 different types of oxygenases as mapped by Minpath. An additional genetic feature like genes for stress and resistance towards heavy metals and antibiotic suggested that the genome has gone through the rigorous process of adaptation. If such bacteria could be cultivated then it will open the broad window of bioremediation strategies under nitrogen stress environment.