Valorization of oily sludge waste using biosurfactant-producing bacteria.

Oily sludge generated by the petroleum industry is not only an environmental hazard, but since it contains crude oil too, it is a valuable resource as well. This study demonstrates a methodology for the valorization of the oily sludge that allows the recovery of oil fractions by the action of microbes producing surface-active metabolites. Two bacterial isolates were used in the study that were producing different biosurfactants, identified via FTIR analysis as well as through genomic mapping of the biosurfactant pathways using RAST, ANTISMASH 7.0, STRING databases. Serratia spp. AKBS12, produced a mono-rhamnolipid, while Acinetobacter spp. AKBS16, produced emulsan. Although recovery efficiency of both biosurfactants was similar, the recovery profile with respect to the class of hydrocarbons differed. The rhamnolipid produced by Serratia spp. AKBS12 extracted mono-chained paraffins and linear alkanes, while emulsan, produced by Acinetobacter spp. AKBS16 could extract heavier paraffins. The extraction procedure is simple and involves mixing the biosurfactant with oily sludge at a temperature of 30 °C with an incubation of 9 days. Sulphuric acid precipitation releases the oil trapped in the oily sludge. The study is the first step in developing user-friendly, innovative technologies that can be linked to the concept of a circular economy.

Catalytic resilience of multicomponent aromatic ring-hydroxylating dioxygenases in Pseudomonas for degradation of polycyclic aromatic hydrocarbons.

Hydrophobic organic compounds, either natural or introduced through anthropogenic activities, pose a serious threat to all spheres of life, including humankind. These hydrophobic compounds are recalcitrant and difficult to degrade by the microbial system; however, microbes have also evolved their metabolic and degradative potential. Pseudomonas species have been reported to have a multipotential role in the biodegradation of aromatic hydrocarbons through aromatic ring-hydroxylating dioxygenases (ARHDs). The structural complexity of different hydrophobic substrates and their chemically inert nature demands the explicit role of evolutionary conserved multicomponent enzyme ARHDs. These enzymes catalyze ring activation and subsequent oxidation by adding two molecular oxygen atoms onto the vicinal carbon of the aromatic nucleus. This critical metabolic step in the aerobic mode of degradation of polycyclic aromatic hydrocarbons (PAHs) catalyzed by ARHDs can also be explored through protein molecular docking studies. Protein data analysis enables an understanding of molecular processes and monitoring complex biodegradation reactions. This review summarizes the molecular characterization of five ARHDs from Pseudomonas species already reported for PAH degradation. Homology modeling for the amino acid sequences encoding the catalytic α-subunit of ARHDs and their docking analyses with PAHs suggested that the enzyme active sites show flexibility around the catalytic pocket for binding of low molecular weight (LMW) and high molecular weight (HMW) PAH substrates (naphthalene, phenanthrene, pyrene, benzo[α]pyrene). The alpha subunit harbours variable catalytic pockets and broader channels, allowing relaxed enzyme specificity toward PAHs. ARHD's ability to accommodate different LMW and HMW PAHs demonstrates its 'plasticity', meeting the catabolic demand of the PAH degraders.

Treatment Options for Municipal Solid Waste by Composting and Its Challenges.

Recovery and recycling of municipal solid waste biodegradable fraction (50-55%) are essential for attaining sustainability and a circular economy. Among organic waste treatment methods, composting is used to recycle organic fractions of waste. However, only 10-12% of municipal solid waste is utilized for composting treatment due to a lack of segregation practices and process challenges, including long process periods, odorous and greenhouse gas emissions, nitrogen loss, and low compost quality, which hinders large-scale practice. The current review paper discusses the challenges of composting treatment and its possible solutions. Various strategies were explored to address these challenges, such as utilizing microbial inoculum, additives, and optimization of physicochemical parameters. It also emphasizes the application of metagenomics for exploring key species. The knowledge about the microbial community and biochemical pathways (genome mining) can be exploited for the improvement of treatment efficiency. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01087-4.

Association of DNA repair gene XPC Ala499Val (rs2228000 C>T) and Lys939Gln (rs2228001 A>C) polymorphisms with the risk of chronic myeloid leukemia: A case-control study in a South Indian population.

BACKGROUND: Xeroderma pigmentosum complementation group C (XPC), a DNA repair protein, plays an important role in the maintenance of genomic integrity and is essential for the nucleotide excision repair pathway. Polymorphisms in the XPC gene may alter DNA repair leading to genetic instability and oncogenesis. The present study aimed to assess the relationship between the XPC Ala499Val (rs2228000 C>T) and Lys939Gln (rs2228001 A>C) non-synonymous polymorphisms and susceptibility to chronic myeloid leukemia (CML) pathogenesis, disease progression and the response to targeted therapeutic regimen, imatinib mesylate. METHODS: This case-control study included 212 cases and 212 controls, and the genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism assays. RESULTS: Our results showed significant association of variant CT (odds ratio = 1.92, 95% confidence interval = 1.21-3.06, p = 0.003) and TT (odds ratio = 2.84, 95% confidence interval = 1.22-6.71, p = 0.007) genotypes in patients with the XPC Ala499Val polymorphism and CML risk. In addition, these genotypes were associated with CML progression to advanced phases (p = 0.006), splenomegaly (p = 0.017) and abnormal lactate dehydrogenase levels (p = 0.03). XPC Lys939Gln was found to correlate with a poor response to therapy, showing borderline significant association with minor cytogenetic response (p = 0.08) and a poor molecular response (p = 0.06). Significant association of the Ala499Val and Lys939Gln polymorphisms with prognosis was observed (Hasford high risk, p = 0.031 and p = 0.019, respectively). Haplotype analysis showed a strong correlation of variant TC haplotype with poor therapy responses (minor cytogenetic response, p = 0.019; poor molecular response, p < 0.0001). CONCLUSIONS: In conclusion, our results suggest that XPC Ala499Val is a high-penetrance CML susceptibility polymorphism. Both polymorphisms studied are considered as genetic markers with respect to assessing disease progression, therapy response and prognosis in CML patients.

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.

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.

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.

Seasonal bacterial community dynamics in a crude oil refinery wastewater treatment plant.

The biological treatment of oil refinery effluents in wastewater treatment plants (WWTPs) relies on specialized bacteria contributing to remove organic load, nitrogen, sulfur, and phosphorus compounds. Knowledge about bacterial dynamics in WWTPs and how they affect the performance of the wastewater treatment is limited, particularly in tropical countries. The bacterial communities from three compartments of an oil refinery WWTP in Uran, India, were assessed using 16S-metabarcoding, in winter and monsoon seasons, upstream (from the surge pond) and downstream the biotower (clarifier and guard pond), to understand the effects of seasonal variations in WWTP's efficiency. The organic load and ammonia levels of the treated wastewater increased by 3- and 9-fold in the monsoon time-point. A decreased abundance and diversity of 47 genera (325 OTUs) comprising ammonia and nitrite oxidizing bacteria (AOB, NOB, denitrifiers) was observed in the monsoon season downstream the biotower, whereas 23 OTUs of Sulfurospirillum, Desulfovibrio, and Bacillus, putatively performing dissimilatory nitrate reduction to ammonia (DNRA), were 3-fold more abundant in the same compartments (DNRA/denitrifiers winter ratio < 0.5 vs. monsoon ratio around 3). The total abundance of reported sulfate- and sulfite-reducing bacteria also increased 250- and 500-fold downstream the biotower, in the monsoon time-point. Bacteria performing DNRA may thus outcompete denitrification in this WWTP, limiting the biodegradation process. The alterations detected in bacterial populations involved in the removal of nitrogen and sulfur species evidenced a reduced quality of the released wastewater and may be good candidates for the following monitoring strategies and optimization of the wastewater treatment.

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.

Antimicrobial activity of Alcaligenes sp. HPC 1271 against multidrug resistant bacteria.

Alcaligenes sp. HPC 1271 demonstrated antibacterial activity against multidrug resistant bacteria, Enterobacter sp., resistant to sulfamethoxazole, ampicillin, azithromycin, and tetracycline, as well as against Serratia sp. GMX1, resistant to the same antibiotics with the addition of netilmicin. The cell-free culture supernatant was analyzed for possible antibacterials by HPLC, and the active fraction was further identified by LC-MS. Results suggest the production of tunicamycin, a nucleoside antibiotic. The draft genome of this bacterial isolate was analyzed, and the 4.2 Mb sequence data revealed six secondary metabolite-producing clusters, identified using antiSMASH platform as ectoine, butyrolactone, phosphonate, terpene, polyketides, and nonribosomal peptide synthase (NRPS). Additionally, the draft genome demonstrated homology to the tunicamycin-producing gene cluster and also defined 30 ORFs linked to protein secretion that could also play a role in the antibacterial activity observed. Gene expression analysis demonstrated that both NRPS and dTDP-glucose 4,6-dehydratase gene clusters are functional and could be involved in antibacterial biosynthesis.

Genomic and functional features of the biosurfactant producing Bacillus sp. AM13.

Genomic studies provide deeper insights into secondary metabolites produced by diverse bacterial communities, residing in various environmental niches. This study aims to understand the potential of a biosurfactant producing Bacillus sp. AM13, isolated from soil. An integrated approach of genomic and chemical analysis was employed to characterize the antibacterial lipopeptide produced by the strain AM13. Genome analysis revealed that strain AM13 harbors a nonribosomal peptide synthetase (NRPS) cluster; highly similar with known biosynthetic gene clusters from surfactin family: lichenysin (85 %) and surfactin (78 %). These findings were substantiated with supplementary experiments of oil displacement assay and surface tension measurements, confirming the biosurfactant production. Further investigation using LCMS approach exhibited similarity of the biomolecule with biosurfactants of the surfactin family. Our consolidated effort of functional genomics provided chemical as well as genetic leads for understanding the biochemical characteristics of the bioactive compound.

s-triazine degrading bacterial isolate Arthrobacter sp. AK-YN10, a candidate for bioaugmentation of atrazine contaminated soil.

The Arthrobacter sp. strain AK-YN10 is an s-triazine pesticide degrading bacterium isolated from a sugarcane field in Central India with history of repeated atrazine use. AK-YN10 was shown to degrade 99 % of atrazine in 30 h from media supplemented with 1000 mg L(-1) of the herbicide. Draft genome sequencing revealed similarity to pAO1, TC1, and TC2 catabolic plasmids of the Arthrobacter taxon. Plasmid profiling analyses revealed the presence of four catabolic plasmids. The trzN, atzB, and atzC atrazine-degrading genes were located on a plasmid of approximately 113 kb.The flagellar operon found in the AK-YN10 draft genome suggests motility, an interesting trait for a bioremediation agent, and was homologous to that of Arthrobacter chlorophenolicus. The multiple s-triazines degradation property of this isolate makes it a good candidate for bioremediation of soils contaminated by s-triazine pesticides.

Bioremediation strategies for removal of residual atrazine in the boreal groundwater zone.

Strategies for bioremediation of atrazine, a pesticide commonly polluting groundwater in low concentrations, were studied in two boreal nonagricultural soils. Atrazine was not mineralized in soil without bioremediation treatments. In biostimulation treatment with molasses, up to 52% of atrazine was mineralized at 10 °C, even though the degradation gene copy numbers did not increase. Incubations with radioactively labeled atrazine followed by microautoradiographic analysis revealed that bioremediation strategies increased the relative proportion of active degraders from 0.3 up to 1.9% of the total bacterial count. These results indicate that atrazine degradation might not solely be facilitated by atzA/trzN-atzB genes. In combined biostimulation treatment using citrate or molasses and augmentation with Pseudomonas citronellolis ADP or Arthrobacter aurescens strain TC1, up to 76% of atrazine was mineralized at 30 °C, and the atrazine degradation gene numbers increased up to 10(7) copies g(-1) soil. Clone libraries from passive samplers in groundwater monitoring wells revealed the presence of phylogenetic groups formerly shown to include atrazine degraders, and the presence of atrazine degradation genes atzA and atzB. These results show that the mineralization of low concentrations of atrazine in the groundwater zone at low temperatures is possible by bioremediation treatments.

Soil mesocosm studies on atrazine bioremediation.

Accumulation of pesticides in the environment causes serious issues of contamination and toxicity. Bioremediation is an ecologically sound method to manage soil pollution, but the bottleneck here, is the successful scale-up of lab-scale experiments to field applications. This study demonstrates pilot-scale bioremediation in tropical soil using atrazine as model pollutant. Mimicking field conditions, three different bioremediation strategies for atrazine degradation were explored. 100 kg soil mesocosms were set-up, with or without atrazine application history. Natural attenuation and enhanced bioremediation were tested, where augmentation with an atrazine degrading consortium demonstrated best pollutant removal. 90% atrazine degradation was observed in six days in soil previously exposed to atrazine, while soil without history of atrazine use, needed 15 days to remove the same amount of amended atrazine. The bacterial consortium comprised of 3 novel bacterial strains with different genetic atrazine degrading potential. The progress of bioremediation was monitored by measuring the levels of atrazine and its intermediate, cyanuric acid. Genes from the atrazine degradation pathway, namely, atzA, atzB, atzD, trzN and trzD were quantified in all mesocosms for 60 days. The highest abundance of all target genes was observed on the 6th day of treatment. trzD was observed in the bioaugmented mesocosms only. The bacterial community profile in all mesocosms was monitored by LH-PCR over a period of two months. Results indicate that the communities changed rapidly after inoculation, but there was no drastic change in microbial community profile after 1 month. Results indicated that efficient bioremediation of atrazine using a microbial consortium could be successfully up-scaled to pilot scale.

Luffa cylindrica (Sponge Gourd) Fibers in Treatment of Greywater: an Aerobic Fixed-Film Reactor Approach.

The need for potable water consumption in urban and suburban regions can be decreased by greywater treatment and its reuse. Utilizing natural fibers may provide sustainable solutions in addressing challenges related to water resource management. In this study, a fixed-film reactor was designed with Luffa cylindrica (an annually occurring fruit) as a bio-carrier. The lab-scale reactors were configured with and without Luffa cylindrica and were run for 90 days in fed-batch mode. Scanning electron microscopy (SEM) was performed to validate biofilm production over time. Monitoring COD, nitrogen, and total phosphate removal allowed for analysis of treatment effectiveness. Results demonstrated the treatment efficiency for the experimental reactor was 70.96%, 97.02%, 92.57%, and 81.20% for COD, nitrogen, phosphate, and anionic surfactant (AS), respectively. 16 s rRNA gene sequencing of bio-carrier and control greywater samples was carried out. Many bacteria known to break down anionic surfactants were observed, and microbial succession was witnessed in the control reactor vs. the experimental reactor samples. The three most prevalent genera in the experimental samples were Chlorobium, Chlorobaculum, and Terrimonas. However, it is crucial to underscore that additional research is essential to solidify our understanding in this domain, with this study laying the fundamental groundwork.

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.

Comparative assessment of soil microbial community in crude oil contaminated sites.

Petroleum refineries generate oily sludge that contains hazardous polycyclic aromatic hydrocarbons (PAH), and hence, its proper disposal is of foremost concern. Analysis of the physicochemical properties and functions of indigenous microbes of the contaminated sites are essential in deciding the strategy for bioremediation. This study analyses both parameters at two geographically distant sites, with different crude oil sources, and compares the metabolic capability of soil bacteria with reference to different contamination sources and the age of the contaminated site. The results indicate that organic carbon and total nitrogen derived from petroleum hydrocarbon negatively affect microbial diversity. Contamination levels vary widely on site, with levels of PAHs ranging from 5.04 to 1.66 × 103 µg kg-1 and 6.20 to 5.64 × 103 µg kg-1 in Assam and Gujarat sites respectively, covering a higher proportion of low molecular weight (LMW) PAHs (fluorene, phenanthrene, pyrene, and anthracene). Functional diversity values were observed to be positively correlated (p < 0.05) with acenaphthylene, fluorene, anthracene, and phenanthrene. Microbial diversity was the highest in fresh oily sludge which decreased upon storage, indicating that immediate bioremediation, soon after its generation, would be beneficial. Improvement in the bio-accessibility of hydrocarbon compounds by the treatment of biosurfactant produced by a (soil isolate/isolate) was demonstrated., with respect to substrate utilization.

Antibiotic resistance in wastewater: Indian scenario.

The surge of Antibiotic Resistant Bacteria (ARB) in the environment is poised to be the next health threat. World Health Organisation's (WHO's) Global Antimicrobial Surveillance System (GLASS) report indicates that developing countries may be at a greater risk. Among various factors, the major driver here could be untreated wastewater and poor sanitation. Bacteria are extremely adaptable to their surroundings and develop Antimicrobial Resistance (AMR) when exposed to antibiotics and other pollutants that cause microbial stress. Thus, untreated domestic wastewater drains could easily become hotspots for the occurrence of ARBs. This study reports surveillance of sewage-carrying drains across four urban cities in India and demonstrated the presence of ARBs in the bacterial community against 7 classes of antibiotics, namely, ß-Lactams, Chloramphenicol, Glycopeptides, Macrolides, Tetracycline, Third Generation Cephalosporin, and Quinolones. Untreated domestic wastewater flowing in target drains was collected twice a month, for a period of six months and the microbial community was subjected to Antibiotic Susceptibility Testing (AST) by plate assays. The zone of inhibition was recorded and interpreted as per the interpretive chart of The Clinical & Laboratory Standards Institute (CLSI) & The European Committee on Antimicrobial Susceptibility Testing (EUCAST). The total number of samples showing resistance against antibiotics was used to define an Antibiotic Resistance Index (ARI), calculated for all 20 sampling sites (drains). Results demonstrated that the highest ARI was observed in Delhi and Mumbai, ranging from 0.81 to 0.92 in Delhi and 0.49-0.56 in Mumbai. This surveillance study reveals the antibiotic resistance pattern of the representative bacterial community in the drains and goes beyond few targeted bacterial species. The alarming presence of antibiotic resistant bacterial community highlights the concern of ARBs being the next looming health threat. This report aims to demonstrates the importance of considering sewage surveillance on routine basis by state authorities.

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method.

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.