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.

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.

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.