Designing Knowledge-Based Bioremediation Strategies Using Metagenomics.

Functional capacities for bioremediation are governed by metabolic mechanisms of inhabiting microbial communities at polluted niches. Process fluctuations lead to stress scenarios where microbes evolve continuously to adapt to sustain the harsh conditions. The biological wastewater treatment (WWT) process harbors the potential of these catabolic microbes for the degradation of organic molecules. In a typical biological WWT or soil bioremediation process, several microbial species coexist which code for enzymes that degrade complex compounds.High throughput DNA sequencing techniques for microbiome analysis in bioremediation processes have led to a powerful paradigm revealing the significance of metabolic functions and microbial diversity. The present chapter describes techniques in taxonomy and functional gene analysis for understanding bioremediation potential and novel strategies built on in silico analysis for the improvisation of existing aerobic wastewater treatment methods. Methods explaining comparative metagenomics by Metagenome Analysis server (MG-RAST) are described with successful case studies by focusing on industrial wastewaters and soil bioremediation studies.

Characterization of MxiE- and H-NS-Dependent Expression of ipaH7.8, ospC1, yccE, and yfdF in Shigella flexneri.

Shigella flexneri uses a type 3 secretion system (T3SS) apparatus to inject virulence effector proteins into the host cell cytosol. Upon host cell contact, MxiE, an S. flexneri AraC-like transcriptional regulator, is required for the expression of a subset of T3SS effector genes encoded on the large virulence plasmid. Here, we defined the MxiE regulon using RNA-seq. We identified virulence plasmid- and chromosome-encoded genes that are activated in response to type 3 secretion in a MxiE-dependent manner. Bioinformatic analysis revealed that similar to previously known MxiE-dependent genes, chromosome-encoded genes yccE and yfdF contain a regulatory element known as the MxiE box, which is required for their MxiE-dependent expression. The significant AT enrichment of MxiE-dependent genes suggested the involvement of H-NS. Using a dominant negative H-NS system, we demonstrate that H-NS silences the expression of MxiE-dependent genes located on the virulence plasmid (ipaH7.8 and ospC1) and the chromosome (yccE and yfdF). Furthermore, we show that MxiE is no longer required for the expression of ipaH7.8, ospC1, yccE, and yfdF when H-NS silencing is relieved. Finally, we show that the H-NS anti-silencer VirB is not required for ipaH7.8 and yccE expression upon MxiE/IpgC overexpression. Based on these genetic studies, we propose a model of MxiE-dependent gene regulation in which MxiE counteracts H-NS-mediated silencing. IMPORTANCE The expression of horizontally acquired genes, including virulence genes, is subject to complex regulation involving xenogeneic silencing proteins, and counter-silencing mechanisms. The pathogenic properties of Shigella flexneri mainly rely on the acquisition of the type 3 secretion system (T3SS) and cognate effector proteins, whose expression is repressed by the xenogeneic silencing protein H-NS. Based on previous studies, releasing H-NS-mediated silencing mainly relies on two mechanisms involving (i) a temperature shift leading to the release of H-NS at the virF promoter, and (ii) the virulence factor VirB, which dislodges H-NS upon binding to specific motifs upstream of virulence genes, including those encoding the T3SS. In this study, we provide genetic evidence supporting the notion that, in addition to VirB, the AraC family member MxiE also contributes to releasing H-NS-mediated silencing in S. flexneri.

Characterisation of novel microbial strains Proteus mirabilis and Bordetella avium for heavy metal bioremediation and dye degradation.

The entitled study focuses on exploring the microbial diversity and its applicability in the remediation of metal contaminated soil using microbes, which is a reliable and cost effective technique. Tungsten enriched soil of Kuhi-Agargaon-Khobna region (Nagpur, India) were analysed by XRF method to detect heavy metals. The traditional microbiological techniques were used to isolate tungsten tolerant microbes. Applicability of these microbes in bioremediation and Azo dye degradation was mainly studied. The two novel bacterial strains, Proteus mirabilis (RS2K) and Bordetella avium (RS3K), were isolated and identified to show the tolerance to tungsten, using 16S rDNA and phylogenetic analysis. These novel strains have also shown the tolerance to other metallic salts viz., (sodium) tungsten, tungstic acid, ammonium metaparatungstate, mercuric chloride, cobalt chloride and azo dye. These microbes were found to accumulate tungsten intracellularly as confirmed through ICP-MS and SEM-EDS analyses. Microbes exhibited well-equipped cellular mechanisms for metal tolerance to survive in heavy metal-laden ecology. Current study contains substantial potential in bioleaching of heavy metals and green mining along with Nano bioremediation for heavy metal pollution.

Genome Sequences of Five Indian Canine Rabies Virus Isolates Obtained Using Oxford Nanopore Technologies Sequencing.

We report five canine rabies virus genome sequences from India that were obtained from brain samples using Oxford Nanopore Technologies sequencing. The sequences will facilitate understanding of the evolution and transmission of rabies.

From gut to mud: dissemination of antimicrobial resistance between animal and agricultural niches.

With increasing reports on antimicrobial resistance (AMR) in humans, animals and the environment, we are at risk of returning to a pre-antibiotic era. Therefore, AMR is recognized as one of the major global health threats of this century. Antibiotics are used extensively in farming systems to treat and prevent infections in food animals or to increase their growth. Besides the risk of a transfer of AMR between the human and the animal sector, there is another yet largely overlooked sector in the One Health triad. Human-dominated ecosystems such as agricultural soils are a major sink for antibiotics and AMR originating from livestock farming. This review summarizes current knowledge on the prevalence of AMR at the interface of animal and agricultural production and discusses the potential implications for human health. Soil resistomes are augmented by the application of manure from treated livestock. Subsequent transfer of AMR into plant microbiomes may likely play a critical role in human exposure to antibiotic resistance in the environment. Based on the knowledge that is currently available we advocate that more attention should be paid to the role of environmental resistomes in the AMR crisis.

Atrazine Bioremediation and Its Influence on Soil Microbial Diversity by Metagenomics Analysis.

Pesticide accumulation in agricultural soils is an environmental concern, often addressed through distinct bioremediation strategies. This study has tried to analyze various soil bioremediation options viz., biostimulation, bioaugmentation, and natural attenuation in terms of efficiency and the response of autochthonous microbial flora by using atrazine as a model contaminant. Soil mesocosms were established with 100 kg of soil simulating the field conditions. The soil previously exposed to the herbicide was used for the bioaugmentation strategy undertaken in this study. We have tried to analyze how the microbial community responds to a foreign compound, both in terms of taxonomic and functional capacities? To answer this, we have analyzed metagenome of the mesocosms at a time point when 90% atrazine was degraded. Bioaugmentation for bioremediation proved to be efficient with a DT90 value of 15.48 ± 0.79 days, in comparison to the natural attenuation where the DT90 value was observed to be 41.20 ± 1.95 days. Metagenomic analysis revealed the abundance of orders Erysipelotrichales, Selemonadales, Clostridiales, and Thermoanaerobacterales exclusively in SBS mesocosm. Besides Pseudomonas, bacterial genera such as Achromobacter, Xanthomonas, Stenotrophomonas, and Cupriavidus have emerged as the dominant members in various bioremediation strategies tested in this study. Inclusive results suggest that inherent microbial flora adjust their community and metabolic machinery upon exposure to the pollutant. The site under pollutant stress showed efficient microbial communities to bio-remediate the newly polluted terrestrial ecologies in relatively less time and by economic means.

Metagenomic Insights in Activated Biomass Treating Industrial Wastewater at Different DO Levels.

Dissolved oxygen (DO) is an imperative parameter of the activated sludge process (ASP) for wastewater bioremediation. The effect of DO on microbial communities and corresponding metabolic functions in wastewater bioremediation was investigated using next-generation analysis techniques in this study. Illumina-based whole genome sequencing was applied to analyze the composition of the microbial community along with their functional diversity in activated sludge systems operating at three different DO levels. Activated biomass was collected from lab-scale reactors maintained at 1, 2, and 4 mg/L DO levels. Metagenomes were sequenced on an Illumina platform and analyzed using various tools. Results revealed that Proteobacteria phylum and Pseudomonas, Nitrobacter, Thauera, and Alicyclipilus genera were abundant in all reactor samples. Despite distinct DO levels, the microbial communities were conserved and consisted of a common population forming the core group governing the metabolic functions. However, higher diversity was observed at functional level indicating that microbes evolve and adapt to serve their role in a typical ASP. Metabolic pathway related to benzoate dominated at 1 mg/L DO level, while pathways for degradation of aromatic compounds like phenol, toluene, and biphenyl via central metabolic pathway were found dominating at 4 mg/L DO level. Pathways corresponding to homogentisate, naphthalene, cresol, and salicylate degradation enriched at 2 mg/L DO level.

Decoding microbial community intelligence through metagenomics for efficient wastewater treatment.

Activated sludge, a microbial ecosystem at industrial wastewater treatment plants, is an active collection of diverse gene pool that creates the intelligence required for coexistence at the cost of pollutants. This study has analyzed one such ecosystem from a site treating wastewater pooled from over 200 different industries. The metagenomics approach used could predict the degradative pathways of more than 30 dominating molecules commonly found in wastewater. Results were extended to design a bioremediation strategy using 4-methylphenol, 2-chlorobenzoate, and 4-chlorobenzoate as target compounds. Catabolic potential required to degrade four aromatic families, namely benzoate family, PAH family, phenol family, and PCB family, was mapped. Results demonstrated a network of diverse genera, where a few phylotypes were seen to contain diverse catabolic capacities and were seen to be present in multiple networks. The study highlights the importance of looking more closely at the microbial community of activated sludge to harness its latent potential. Conventionally treated as a black box, the activated biomass does not perform at its full potential. Metagenomics allows a clearer insight into the complex pathways operating at the site and the detailed documentation of genes allows the activated biomass to be used as a bioresource.

Genome Sequencing and Analysis of Strains Bacillus sp. AKBS9 and Acinetobacter sp. AKBS16 for Biosurfactant Production and Bioremediation.

Microbial genomics facilitates the analysis of microbial attributes, which can be applied in bioremediation of pollutants and oil recovery process. The biosurfactants derived from microbes can replace the chemical surfactants, which are ecologically detrimental. The aim of this work was to study the genetic organization responsible for biodegradation of aromatics and biosurfactant production in potential microbes isolated from polluted soil. Bacterial isolates were tested for biosurfactant production, wherein Bacillus sp. AKBS9 and Acinetobacter sp. AKBS16 exhibited highest biosurfactant production potential. Whole genome sequencing and annotations revealed the occurrence of sfp and NPRS gene in the Bacillibactin biosynthetic gene cluster in AKBS9 strain and emulsan biosynthetic gene cluster in AKBS16 strain for biosurfactant production. Various aromatic compound ring cleaving oxygenases scavenging organic molecules could be annotated for strain AKBS16 using RAST annotations.

Microbial population shift caused by sulfamethoxazole in engineered-Soil Aquifer Treatment (e-SAT) system.

The engineered-Soil Aquifer Treatment (e-SAT) system was exploited for the biological degradation of Sulfamethoxazole (SMX) which is known to bio-accumulate in the environment. The fate of SMX in soil column was studied through laboratory simulation for a period of 90 days. About 20 ppm SMX concentration could be removed in four consecutive cycles in e-SAT. To understand the microbial community change and biological degradation of SMX in e-SAT system, metagenomic analysis was performed for the soil samples before (A-EBD) and after SMX exposure (B-EBD) in the e-SAT. Four bacterial phyla were found to be present in both the samples, with sample B-EBD showing increased abundance for Actinobacteria, Bacteroidetes, Firmicutes and decreased Proteobacterial abundance compared to A-EBD. The unclassified bacteria were found to be abundant in B-EBD compared to A-EBD. At class level, classes such as Bacilli, Negativicutes, Deltaproteobacteria, and Bacteroidia emerged in sample B-EBD owing to SMX treatment, while Burkholderiales and Nitrosomonadales appeared to be dominant at order level after SMX treatment. Furthermore, in response to SMX treatment, the family Nitrosomonadaceae appeared to be dominant. Pseudomonas was the most dominating bacterial genus in A-EBD whereas Cupriavidus dominated in sample B-EBD. Additionally, the sulfur oxidizing bacteria were enriched in the B-EBD sample, signifying efficient electron transfer and hence organic molecule degradation in the e-SAT system. Results of this study offer new insights into understanding of microbial community shift during the biodegradation of SMX.

Treatment of Common Effluent Treatment Plant Wastewater in a Sequential Anoxic-Oxic Batch Reactor by Developed Bacterial Consortium VN11.

A laboratory-scale anoxic-oxic sequential reactor system was seeded with acclimatized mixed microbial consortium for the treatment of common effluent treatment plant (CETP) wastewater having 7000-7400 mg L(-1) of COD and 3000-3400 mg L(-1) of BOD. Initially, CETP wastewater was treated under anoxic reactor at 5000 mg L(-1) of MLSS concentrations, 5.26 ± 0.27 kg COD m(-3) day(-1) of organic loading rate (OLR) and 36 h of hydraulic retention time (HRT). Further, the effluent of anoxic reactor was treated in oxic reactor with an OLR of 6.6 ± 0.31 kg COD m(-3) day(-1) and 18 h HRT. Maximum color and COD removal were found to be 72 and 85 % at total HRT of 2.25 days under anoxic-oxic sequential reactor at 37 °C and pH 7.0. The UV-VIS, FTIR, NMR and GCMS studies showed that majority of peaks observed in untreated wastewater were either shifted or disappeared after sequential treatment. Phytotoxicity study with the seeds of Vigna radiata and Triticum aestivum showed more sensitivity toward the CETP wastewater, while the products obtained after sequential treatment does not have any inhibitory effects. The results demonstrated that the anoxic-oxic reactor fed with bacterial consortium VN11 could bring about efficient bioremediation of industrial wastewaters.

Shifts in Microbial Community and Its Correlation with Degradative Efficiency in a Wastewater Treatment Plant.

A wastewater treatment plant controls the level of pollution reaching the environment. Yet, despite being the most common aerobic route for treatment of wastewater, the activated sludge process is not utilized to its full potential. This is mainly due to the lack of knowledge base correlating the microbial community in the activated sludge to its degradative performance. In this study, the activated biomass at the treatment site was monitored for five consecutive months. Even though operational parameters were kept constant, the microbial community was observed to change after 3 months. This shift was seen to correlate with 25 % loss of degradative efficiency. Target oxygenases were monitored at two time points, and results indicated that the dominating pathway operating in the common effluent treatment plant (CETP) is the degradation of chlorinated aromatics. This study demonstrates the change in degradative efficiency in a CETP with the change in microbial community and analyzes the parameters influencing the microbial community of activated sludge.

Comparative metagenomics demonstrating different degradative capacity of activated biomass treating hydrocarbon contaminated wastewater.

This study demonstrates the diverse degradative capacity of activated biomass, when exposed to different levels of total dissolved solids (TDS) using a comparative metagenomics approach. The biomass was collected at two time points to examine seasonal variations. Four metagenomes were sequenced on Illumina Miseq platform and analysed using MG-RAST. STAMP tool was used to analyse statistically significant differences amongst different attributes of metagenomes. Metabolic pathways related to degradation of aromatics via the central and peripheral pathways were found to be dominant in low TDS metagenome, while pathways corresponding to central carbohydrate metabolism, nitrogen, organic acids were predominant in high TDS sample. Seasonal variation was seen to affect catabolic gene abundance as well as diversity of the microbial community. Degradation of model compounds using activated sludge demonstrated efficient utilisation of single aromatic ring compounds in both samples but cyclic compounds were not efficiently utilised by biomass exposed to high TDS.

Metagenomic analysis of oxygenases from activated sludge.

Oxygenases play a key role in degradation of the aromatic compounds in the wastewater. This study explores the oxygenase coding gene sequences from the metagenome of activated biomass. Based on these results, the catabolic capacity of the activated sludge was assessed towards degradation of naphthalene, anthracene, phenol, biphenyl and o-toluidine. Oxygenases found in this study were compared with oxygenases from three other metagenome datasets. Results demonstrate that despite different geographical locations and source, many genes coding for oxygenases were common between treatment plants. 1, 2 Homogentisate dioxygenase and phenylacetate CoA oxygenases were present in all four metagenomes. Metagenomics provides a vast amount of data that needs to be mined with specific targets to harness the potential of the microbial world.