Genomic Insights into the Landfill Microbial Community: Denitrifying Activity Supporting One-Carbon Utilization.

In spite of the developments in understanding of denitrifying methylotrophy in the recent years, challenges still exist in unravelling the overall biochemistry of nitrate-dependent methane oxidation in novel or poorly characterized/not-yet-cultured bacteria. In the present study, landfill site was mined for novel C1-carbon-metabolizing bacteria which can use nitrate/nitrite as an electron acceptor. A high-throughput rapid plate assay identified three bacterial isolates with eminent ability for nitrate-dependent methane metabolism under anaerobic conditions. Taxonomic identification by whole-genome sequence-based overall genome relatedness indices accurately assigned the isolates AAK_M13, AAK_M29, and AAK_M39 at the species level to Enterobacter cloacae, Bacillus subtilis, and Bacillus halotolerans, respectively. Several genes encoding sub-components involved in alcohol utilization and denitrification pathways, such as adh, fdh, fdo, nar, nir, and nor, were identified in all the genomes. Though no gene clusters encoding MMO/AMO were annotated, sequencing of PCR amplicons revealed similarity with pMMO/AMO gene using translated nucleotide sequence of strains AAK_M29 and AAK_M39, while strain AAK_M13 showed similarity with XRE family transcriptional regulator. This suggests the horizontal gene transfer and/or presence of a truncated version of a housekeeping enzyme encoded by genes exhibiting partial sequence similarity with pMMO genes that mimicked its function at greenhouse gas emission sites. Owing to lack of conclusive evidence for presence of methane metabolism genes in the selected isolates, further experiment was performed to validate their nitrate-dependent methane oxidation capacities. Bacillus subtilis AAK_M29, Bacillus halotolerans AAK_M39, and Enterobacter cloacae AAK_M13 could oxidize 60%, 75%, and 85% of the added methane respectively accompanied by high nitrate reduction (56-62%) thus supporting the correlation between these two activities. The remarkable ability of these isolates for nitrate-dependent methane metabolism has highlighted their role in ecological contribution and biotechnological potential to serve as methane and nitrate sinks in the landfill sites.

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.

Two-Dimensional Cell Separation: a High-Throughput Approach to Enhance the Culturability of Bacterial Cells from Environmental Samples.

Culture-independent sequence data from various environmental samples have revealed an immense microbial diversity of environmental, clinical, and industrial importance that has not yet been cultured. Cultivation is imperative to validate findings emerging from cultivation-independent molecular data and exploit the isolated organisms for biotechnological purposes. Efforts have been made to boost the cultivability of microbes from environmental samples by use of a range of techniques and instrumentation. The manuscript presents a novel yet simple and innovative approach to improving the cultivability of natural microorganisms without sophisticated instrumentation. By employing gradient centrifugation combined with serial dilution ("two-dimensional cell separation"), significantly higher numbers of genera (>2-fold higher) and species (>3-fold higher) were isolated from environmental samples, including soil, anaerobic sludge, and landfill leachate, than from using serial dilution alone. This simple and robust protocol can be modified for any environment and culture medium and provides access to untapped microbial diversity. IMPORTANCE In the manuscript, we have developed a novel yet simple and innovative approach to improving the cultivability of natural microorganisms without sophisticated instrumentation. The method used gradient centrifugation combined with serial dilution (two-dimensional cell separation) to improve taxum recovery from samples. This simple and robust protocol can be modified for any environment and culture medium and provides access to untapped microbial diversity. This approach can be incorporated with less labor and complexity in laboratories with minimal instrumentation. As cultivation is a workflow that is well suited to lower-resource microbiology labs, we believe improvements in cultivability can increase opportunities for scientific collaborations between low-resource labs and groups focused on high-resource cultivation-independent methodologies.

A comprehensive review on current status and future perspectives of microbial volatile fatty acids production as platform chemicals.

Volatile fatty acids (VFA), the secondary metabolite of microbial fermentation, are used in a wide range of industries for production of commercially valuable chemicals. In this review, the fermentative production of VFAs by both pure as well mixed microbial cultures is highlighted along with the strategies for enhancing the VFA production through innovations in existing approaches. Role of conventionally applied tools for the optimization of operational parameters such as pH, temperature, retention time, organic loading rate, and headspace pressure has been discussed. Furthermore, a comparative assessment of above strategies on VFA production has been done with alternate developments such as co-fermentation, substrate pre-treatment, and in situ removal from fermented broth. The review also highlights the applications of different bioreactor geometries in the optimum production of VFAs and how metagenomic tools could provide a detailed insight into the microbial communities and their functional attributes that could be subjected to metabolic engineering for the efficient production of VFAs.

Dissolved oxygen-mediated enrichment of quorum-sensing phenomenon in the bacterial community to combat oxidative stress.

Microbial community with their plasticity follows a course of changes that allow adaptation and survival in a particular habitat. In this study perturbations in microbial flora dwelling in two reactors with phenol as a carbon source under the limiting nitrogen and phosphorus conditions were monitored for 3 months with alterations of dissolved oxygen (DO). With the time, the shift in diversity and abundance of bacteria were observed with simultaneous increase in biofilm-forming bacteria like Pseudomonas, Escherichia, etc. Functional level screening revealed that the abundance of core metabolic genes were not much altered, however, the regulated level of increase in quorum sensing genes (acyl-homoserine lactone), biofilm-forming genes, catalase and ferroxidase enzymes at high DO suggest the survival mechanism of the community. This study sheds light on survival route followed by the bacterial community with abiotic stress, such as an increase in DO.

Annotation and De Novo Sequence Characterization of Extracellular ß-Fructofuranosidase from Penicillium chrysogenum Strain HKF42.

The genome of a fungal strain Penicillium chrysogenum strain HKF42, which can grow on 20% sucrose has been annotated for 7595 protein coding sequences. On mining of CAZymes, we could annotate a ß-fructofuranosidase gene responsible for fructo-oligosaccharides (FOS) synthesis which is a known prebiotic. The enzyme activity was demonstrated and validated with the generation of FOS as kestose and nystose.

In silico characterization of broad range proteases produced by Serratia marcescens EGD-HP20.

In the present study, Serratia marcescens EGD-HP20 strain was demonstrated to utilize poultry waste comprising of both white non-melanized and dark/brown melanized poultry feathers. The potential of the isolate to hydrolyze diverse keratinous wastes was further corroborated by comparative genomics which indicated the presence of genes for broad substrate specific proteases including metallo-proteases, serine endoprotease, dipeptidase, oligopeptidase, etc. Multiple gene sequence alignments of above genes showed 99-100% sequence identities with that of closely related strains of S. marcescens. The secondary structure, 3D structures and energy models suggested the stable nature of all the observed enzymes. Comparative genomics and hydrolysis of mixed feather waste indicated that the above potential of the isolate was associated with synergistic action of various types of proteases.

Draft genome sequence of Penicillium chrysogenum strain HKF2, a fungus with potential for production of prebiotic synthesizing enzymes.

In this study, we have characterized a novel set of extracellular enzymes produced by Penicillium chrysogenum strain HKF2. A draft genome data of 31.5 Mbp was generated and annotation suggested a total of 11,243 protein-coding genes out of which 609 were CAZymes, majority of which were found to have homology with Penicillium rubens, Penicillium chrysogenum followed by Penicillium expansum and Penicillium roqueforti. The prominent CAZyme genes identified in the draft genome encoded for enzymes involved in the production of prebiotics such as inulo-oligosaccharides and fructo-oligosaccharides. Corresponding enzyme assay indicated that the isolate possessed the potential to produce 11.8 and 3.8 U/mL of ß-fructofuranosidase and inulinase, respectively. This study highlights the significance of Effluent Treatment Plants as novel and under-explored niche for isolation of fungi having the potential for production of prebiotics synthesizing enzymes.

Genome Annotation and Validation of Keratin-Hydrolyzing Proteolytic Enzymes from Serratia marcescens EGD-HP20.

Stabilization and utilization of poultry waste demand efficient biodegradation either by mixture of enzymes or by microbial system that can produce different types of protein-hydrolyzing enzymes. For utilization of this keratinous biomass, in the present study, genome was sequenced and annotated for a bacterium having multiple enzymatic options for hydrolysis of different soluble and insoluble protein fractions of poultry waste. Among the soluble protein substrates, optimum production of enzyme and soluble protein was observed in case of casein, whereas among the insoluble protein substrates, maximum production of enzyme was achieved when broken nails were used. Conditions for enhanced enzyme activity with concurrent degradation of keratin-rich poultry feather waste to protein-rich hydrolysate were optimized for different growth parameters. The bacterium grew well and highest protease production occurred in 144 h at mesophilic temperature (30 °C) and alkaline condition (pH 8-10) with enzyme activities of 134 and 168 U/mL, respectively.

Diverse Metabolic Capacities of Fungi for Bioremediation.

Bioremediation refers to cost-effective and environment-friendly method for converting the toxic, recalcitrant pollutants into environmentally benign products through the action of various biological treatments. Fungi play a major role in bioremediation owing to their robust morphology and diverse metabolic capacity. The review focuses on different fungal groups from a variety of habitats with their role in bioremediation of different toxic and recalcitrant compounds; persistent organic pollutants, textile dyes, effluents from textile, bleached kraft pulp, leather tanning industries, petroleum, polyaromatic hydrocarbons, pharmaceuticals and personal care products, and pesticides. Bioremediation of toxic organics by fungi is the most sustainable and green route for cleanup of contaminated sites and we discuss the multiple modes employed by fungi for detoxification of different toxic and recalcitrant compounds including prominent fungal enzymes viz., catalases, laccases, peroxidases and cyrochrome P450 monooxygeneses. We have also discussed the recent advances in enzyme engineering and genomics and research being carried out to trace the less understood bioremediation pathways.

Mining of hemicellulose and lignin degrading genes from differentially enriched methane producing microbial community.

Study creates a scenario for enrichment and selection of ligno-hemicellulose degrading genotypes with anaerobic bioreactor as a model using rice straw, vegetable waste and food waste as substrates. Relative discrimination analysis showed that the hydrolytic pathways and associated microbial communities for ligno-hemicellulose degradation were dominatingly colonized with rice straw as substrate. The dominating bacteria were Caldicellulosiruptor, Fervidobacterium, Cytophaga, Ruminococcus, Thermotoga associated with hemicellulose degradation and Burkholderia, Pandorea, Sphingomonas, Spirochaeta, Pseudomonas for lignocellulose hydrolysis. This was further supported by the abundance of anaerobic aromatic compound degrading genes along with genes for xylanase and xylosidase in rice straw enriched community. The metagenome analysis data was validated by evaluation of the biochemical methane potential for these substrates. Food waste being most amenable substrate yielded 1410mL of biogas/gVS added whereas, biogas yield of 1160mL/gVS and 1080mL/gVS was observed in presence of vegetable waste and rice straw respectively.

Biomethanation of vegetable market waste in an anaerobic baffled reactor: Effect of effluent recirculation and carbon mass balance analysis.

In the present study, feasibility of biomethanation of vegetable market waste in a 4-chambered anaerobic baffled reactor (ABR) was investigated at 30d hydraulic retention time and organic loading rate of 0.5gVS/L/d for one year. Indicators of process stability viz., butyrate/acetate and propionate/acetate ratios were consistent with phase separation in the different chambers, which remained unaltered even during recirculation of effluent. Chemical oxygen demand (COD) and volatile solids (VS) removal efficiencies were observed to be consistently high (above 90%). Corresponding biogas and methane yields of 0.7-0.8L/g VS added/d and 0.42-52L/g VS added/d respectively were among the highest reported in case of AD of vegetable waste in an ABR. Process efficiency of the ABR for vegetable waste methanation, which is indicated by carbon recovery factor showed that, nearly 96.7% of the input carbon considered for mass balance was accounted for in the product.

Identification and monitoring of nitrification and denitrification genes in Klebsiella pneumoniae EGD-HP19-C for its ability to perform heterotrophic nitrification and aerobic denitrification.

Microbes capable of performing heterotrophic nitrification and aerobic denitrification simultaneously have application in nitrogen level management in effluent treatment plants. Klebsiella pneumoniae EGD-HP19-C is a metabolically versatile bacterium capable of utilising NH3-N, NO2-N and NO3-N as sole sources of nitrogen. The annotation was done for the genes involved in N-assimilation and N-dissimilation pathways from the draft genome sequences of this bacterium (NCBI GenBank accession no. AUTW02000000.1). The sequence data also suggested possible existence of plasmid associated with this bacterium. Multiple gene sequence alignments of glutamine synthetase (gln), hydroxylamine reductase (har), nitrite reductase (nir), nitric oxide reductase (nor), assimilatory nitrate reductase (nas) and respiratory nitrate reductase (nar) genes from EGD-HP19-C genome were performed to compare sequence identities with that of closely related bacterial species. The metabolic pathways were mapped using KAAS and 3D structures for representative enzyme sub-units were also elucidated. The study suggested that the organism, though it has incomplete nitrification and denitrification pathways still removes the inorganic nitrogen content from the system via ammonification reaction.

Shifts in microbial community in response to dissolved oxygen levels in activated sludge.

This study evaluates the degradative efficiency of activated biomass collected from a Common Effluent Treatment Plant (CETP) under three different dissolved oxygen (DO) levels, 1, 2 and 4mgl(-1). The change in bacterial diversity with reference to DO levels was also analyzed. Results demonstrate that degradative efficiency was the highest, when the reactor was maintained at 4mgl(-1) DO, but amplicon library analysis showed a greater diversity of bacteria in the reactor maintained at 2mgl(-1) DO. Bacteria belonging to the order Desulfuromonadales, Entomoplasmatales, Pasteurellales, Thermales and Chloroflexales have only been detected in this reactor. Ammonia and nitrate levels in all three reactors indicated efficient nitrification process. Results of this study offer new insights into understanding the performance of activated biomass vis-à-vis microbial diversity and degradative efficiency with reference to DO. This information would be useful in improving the efficiency of any wastewater treatment plant.

Salicylic-acid-mediated enhanced biological treatment of wastewater.

Activated sludge represents a microbial community which is responsible for reduction in pollution load from wastewaters and whose performance depends upon the composition and the expression of degradative capacity. In the present study, the role of salicylic acid (SA) has been evaluated for acclimatization of activated sludge collected from a combined effluent treatment plant followed by analysis of the physiological performance and microbial community of the sludge. The biodegradative capacity of the acclimatized activated sludge was further evaluated for improvement in efficiency of chemical oxygen demand (COD) removal from wastewater samples collected from industries manufacturing bulk drugs and dyes and dye intermediates (wastewater 1) and from dye industry (wastewater 2). An increase in COD removal efficiency from 50% to 58% and from 78% to 82% was observed for wastewater 1 and wastewater 2, respectively. Microbial community analysis data showed selective enrichment and change in composition due to acclimatization by SA, with 50% of the clones showing sequence homology to unidentified and uncultured bacteria. This was demonstrated by analysis of partial 16S rDNA sequence data generated from dominating clones representing the metagenome which also showed the appearance of a unique population of clones after acclimatization, which was distinct from those obtained before acclimatization and clustered away from the dominating population.

Utilization of molasses spentwash for production of bioplastics by waste activated sludge.

Present study describes the treatment of molasses spentwash and its use as a potential low cost substrate for production of biopolymer polyhydroxybutyrate (PHB) by waste activated sludge. Fluorescence microscopy revealed the presence of PHB granules in sludge biomass which was further confirmed by fourier transform-infra-red spectroscopy (FT-IR) and (13)C nuclear magnetic resonance (NMR). The processing of molasses spentwash was carried out for attaining different ratios of carbon and nitrogen (C:N). Highest chemical oxygen demand (COD) removal and PHB accumulation of 60% and 31% respectively was achieved with raw molasses spentwash containing inorganic nitrogen (C:N ratio=28) followed by COD removal of 52% and PHB accumulation of 28% for filtered molasses containing inorganic nitrogen (C:N ratio=29). PHB production yield (Y(p/s)) was highest (0.184 g g(-1) COD consumed) for deproteinized spentwash supplemented with nitrogen. In contrast, the substrate consumption and product formation were higher in case of raw spentwash. Though COD removal was lowest from deproteinized spentwash, evaluation of kinetic parameters suggested higher rates of conversion of available carbon to biomass and PHB. Thus the process provided dual benefit of conversion of two wastes viz. waste activated sludge and molasses spentwash into value-added product-PHB.

Processing of poultry feathers by alkaline keratin hydrolyzing enzyme from Serratia sp. HPC 1383.

The present study describes the production and characterization of a feather hydrolyzing enzyme by Serratia sp. HPC 1383 isolated from tannery sludge, which was identified by the ability to form clear zones around colonies on milk agar plates. The proteolytic activity was expressed in terms of the micromoles of tyrosine released from substrate casein per ml per min (U/mL min). Induction of the inoculum with protein was essential to stimulate higher activity of the enzyme, with 0.03% feathermeal in the inoculum resulting in increased enzyme activity (45U/mL) that further increased to 90U/mL when 3d old inoculum was used. The highest enzyme activity, 130U/mL, was observed in the presence of 0.2% yeast extract. The optimum assay temperature and pH for the enzyme were found to be 60 degrees C and 10.0, respectively. The enzyme had a half-life of 10min at 60 degrees C, which improved slightly to 18min in presence of 1mM Ca(2+). Inhibition of the enzyme by phenylmethyl sulfonyl fluoride (PMSF) indicated that the enzyme was a serine protease. The enzyme was also partially inhibited (39%) by the reducing agent beta-mercaptoethanol and by divalent metal ions such as Zn(2+) (41% inhibition). However, Ca(2+) and Fe(2+) resulted in increases in enzyme activity of 15% and 26%, respectively. The kinetic constants of the keratinase were found to be 3.84 microM (K(m)) and 108.7 microM/mLmin (V(max)). These results suggest that this extracellular keratinase may be a useful alternative and eco-friendly route for handling the abundant amount of waste feathers or for applications in other industrial processes.

Simultaneous nitrification and denitrification by diverse Diaphorobacter sp.

Eight bacterial isolates closely related to Diaphorobacter sp. were isolated from activated biomass surviving on wastewater laden with dyes and nitro-substituted chemicals and were identified by 16S rDNA sequence analysis. The isolates showed sequence similarity of 99-100% to other Diaphorobacter strains such as ZY 2006b, F2, NA5, PCA039, D. nitroreducens KSP4, and KSP3 and 98-99% sequence homology to D. nitroreducens NA10B (type strain JCM 11421). Neighbor-joining tree revealed that all the eight strains formed tight cluster together and also showed close clustering with other Diaphorobacter strains. Isolates demonstrated the ability to perform simultaneous nitrification and denitrification under aerobic conditions. Strains HPC 805, 815, 821, and 856 gave highest chemical oxygen demand removal (85-93%) and ammonia removal (92-96%), which correlated well with higher growth rates of the cultures. Simultaneously, complete removal of nitrate supplied in the medium in presence of ammonium and acetate (electron donor) was observed in addition to aerobic nitrite release from ammonium. Thus, the above strains showed ability to perform partial nitrification followed by further aerobic removal of common intermediate nitrite, which indicated their potential application in treatment systems for treatment of high-nitrogen-containing wastewaters.