Advances in Molecular Biology Approaches to Guage Microbial Communities and Bioremediation at Contam...

Jai Godheja, Sudhir K. Shekhar, D.R. Modi

  Open Access OPEN ACCESS  Peer Reviewed PEER-REVIEWED

Advances in Molecular Biology Approaches to Guage Microbial Communities and Bioremediation at Contaminated Sites

Jai Godheja1,, Sudhir K. Shekhar1, D.R. Modi1

1Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow

Abstract

Over the past 40 years, research on the microbial degradation of polycyclic aromatic hydrocarbons (PAHs) has resulted in the isolation of numerous genera of bacteria, fungi and algae capable of degrading PAHs. With the development of biology, molecular techniques such as PCR, fingerprinting technique (mainly DGGE/TGGE), ARDRA, TRFLP, FISH, RISA and gene reporter technique have been intensively applied to gain further insight into the mechanism of PAHs degradation. Further recent developments in moleclar microbial ecology like genotypic profiling, ultrafast genome pyrosequencing, metagenomics, metatranscriptomics, metaproteomics and metabolomics along with bioinformatics tools offer new tools that facilitates molecular analyses of microbial populations at contaminated and bioremediated sites. Information provided by such analyses aids in the evaluation of the effectiveness of bioremediation and the formulation of strategies that might accelerate bioremediation. The potential for the use of molecular methods in toxicological risk assessment and in developing bioremediation strategies is expanding rapidly as new methodologies become available. In this paper we present an overview of some molecular methods we feel have the most potential for use in assessment and monitoring in the field.

Cite this article:

  • Godheja, Jai, Sudhir K. Shekhar, and D.R. Modi. "Advances in Molecular Biology Approaches to Guage Microbial Communities and Bioremediation at Contaminated Sites." International Journal of Environmental Bioremediation & Biodegradation 2.4 (2014): 167-177.
  • Godheja, J. , Shekhar, S. K. , & Modi, D. (2014). Advances in Molecular Biology Approaches to Guage Microbial Communities and Bioremediation at Contaminated Sites. International Journal of Environmental Bioremediation & Biodegradation, 2(4), 167-177.
  • Godheja, Jai, Sudhir K. Shekhar, and D.R. Modi. "Advances in Molecular Biology Approaches to Guage Microbial Communities and Bioremediation at Contaminated Sites." International Journal of Environmental Bioremediation & Biodegradation 2, no. 4 (2014): 167-177.

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1. Introduction

PAHs, a large and diverse group of organic molecules, are known for their adverse and cumulative effect in ubiquitous environment [1]. The non-environmentally friendly nature of PAHs makes difficult its treatment and depletion and thereby PAHs in environment impose sinful effect on the whole ecosystem. Modern molecular techniques provide an exciting opportunity to overcome the requirement for culturing and have greatly increased our understanding of bacterial diversity and functionality during bioremediation of oil-polluted soils. Unfortunately, only a fraction of the microorganisms involved in the biodegradation of pollutants in different ecosystems can currently be cultured using standard laboratory agars and conditions [2-11][2]. Molecular tools are especially useful in bioaugmentation, in which exogenous microorganisms that are introduced to accelerate pollutant biodegradation need to be monitored. To elucidate how microbial communities change due to the effects of environmental disturbances like pollution, investigations need to rely on rapid methods that can characterize cellular constituents such as nucleic acids, proteins (enzymes) and other taxa-specific compounds [12, 13, 14, 15]. These molecules can be extracted directly from the soil without the need for culturing and thus can be used to elucidate the microbial community composition in such polluted environments during bioremediation [16]. To attain expected research goal, molecular techniques were indispensable to compensate the restriction of traditional applications. For example, polymerase chain reaction (PCR) and fingerprinting techniques based on PCR such as 16S rRNA gene library, denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP), terminal restriction fragment length polymorphism (T-RFLP), rRNA intergenic spacer analysis (RISA) were selectively employed in microbial flora and ecology research. Besides above mentioned techniques, other molecular approaches including DNA hybridization such as fluorescence in situ hybridization (FISH) and DNA microarray, gene reporters and biosensors were also frequently used. However, as for PAHs biodegradation investigation, the techniques extensively employed were PCR, fingerprinting technique (mainly DGGE), DNA hybridization technique and gene reporters. The major aim of this article is to review these techniques. Genomics has been instrumental in providing complete genome sequence data of Pseudomonas, Shewanella, Deinococcus, Dehalococcoides and other micro-organisms pertinent to bioremediation. Genomics based recognition of various promoters, genes and degradative pathways have influenced the construction of more efficient contaminant degrading strains for pollution abatement. Cultivation-independent analyses of the overall microbial community structures at contaminated sites using molecular profiling techniques have been instrumental in our understanding of the community dynamics, relative abundance and distribution of micro-organisms actively involved in bioremediation. Metagenomics refers to collective analyses of the overall microbial community genomes of a given environment. Metagenomic analyses have enabled researchers to explore the previously uncultivable microorganisms and exploit their genetic potential in pollutant bioremediation [17]. Recent technological breakthroughs in de novo sequencing of microbial metagenomes based on pyrosequencing have provided rapid and relatively inexpensive methods to generate microbial community profiles simultaneously from different environmental samples [18]. Recently, development of microbial ecological DNA microarrays has enabled researchers to simultaneously analyze thousands of phylogenetic or functional genes in order to characterize microbial communities involved in bioremediation [19]. Metaproteomics approaches utilizing two-dimensional electrophoresis (2-DE), mass spectroscopy (MS) have aided in global analysis of catabolic enzymes involved in microbial biodegradation pathways [20, 21]. One of the upcoming ‘‘-omics” technologies known as metabolomics refers to the colossal analyses of primary and secondary proteinaceous metabolites produced by microbial cells under defined physiological conditions [22]. This review highlights advances in the application of these technologies for studying microbial communities and their functional roles in environmental bioremediation.

2. Polymerase Chain Reaction (PCR)

The screening and cultivation of PAHs degrading microorganisms usually proceed the other steps in studies of PAHs microbial degradation. Once such microorganisms were obtained, identification and categorization of related microorganisms is a frequently confused technical problem. Categorization and identification of newly isolated microorganisms traditionally depend on phenotypic characteristics such as colony and cell morphology as well as biochemical and serological characteristics such as protein and fatty acid pattern profiles. However, it is often time-consuming and need expertise. With the rapid development of molecular biology, modern taxonomy prefer sequencing technologies of molecular markers such as 16S rRNA or 18S rRNA. These technologies allow the identification of colonies isolated from microbial consortia and the establishment of phylogenetic relationships between them [23]. In addition to taxonomy, PCR combined with other approaches could also be used to estimate in situ, how pollution affects the bacterial community structure and composition of sediments. Several PCR techniques such as random amplified polymorphic DNA (RAPD-PCR), arbitrarily primed-PCR (AP-PCR), repetitive extragenic palindromic-PCR (REP-PCR) and enterobacterial intragenic consensus sequence-PCR (ERIC-PCR) could be used to identify species. Besides, bacterial 16S rRNA has become the most commonly used molecular index for its evolutionary distinctive sequence. The 16S rRNA gene is essential as it encodes the small subunit of the prokaryotic ribosome and is therefore present in all prokaryotes. In bacteria, the rRNA genes are transcribed from the ribosomal operon as 30S rRNA precursor molecules and then cleaved by RNase III into 16S, 23S and 5S rRNA molecules [24]. The ribosomal operon size, nucleotide sequences, and secondary structures of the three rRNA genes are conserved within a bacterial species [25]. Since 16S rRNA is the most conserved of these three rRNAs, it has been proposed as an ‘‘evolutionary clock’’, which has led to the reconstruction of the tree of life [26].

Real-time quantitative PCR (also referred to as qPCR) has emerged as a promising tool for rapid, reproducible and accurate estimations of microbial community dynamics or monitoring their catabolic activity during active bioremediation processes [27]. The principle of qPCR assay is based on real-time detection of a reporter molecule whose fluorescence increases as the PCR product accumulates during each amplification cycle. The fluorescence chemistry in the qPCR reactions are either based on hybridization probes (TaqMan-molecular beacons with (FRET) fluorescence resonance energy transfer) or utilize double stranded DNA intercalating dyes SYBR Green along with carboxy-X-rhodamine (ROX) as a passive reference dye. However, to design probes and primers, the ‘‘signature sequences” unique to a particular mirco-organism or a catabolic gene need to be determined by comparison with database sequences using alignment tools. In each qPCR assay a known concentration of standard DNA (usually a linearized plasmid or genomic DNA) is used to prepare standard curves for quantification of unknown target microbial genes. The initial amount of target DNA is inversely proportional to the cycle threshold (CT) value which can be defined as the amplification cycle when the signal of fluorescence in the assay is statistically significant above the baseline level of fluorescence. Based on cycle threshold (CT) values, the relative abundance of specific group of microorganisms in the total microbial community DNA can be quantified by targeting either taxon/species/phylum specific rRNA genes or any other catabolite biomarker genes. A disadvantage of qPCR is optimization of amplification efficiencies and PCR biases in each run for accurate quantification. Nyyssonen et al. 2006 [27] validated a sensitive real-time PCR assay for quantification of naphthalene hydroxylating dioxygenase (nahAc) genes of naphthalene-degrading Proteobacteria within soil samples recovered from large-scale remediation processes. These authors also investigated naphthalene biodegradation using qPCR assay for enumeration of naphthalene dioxygenase genes in soil slurry microcosms. Furthermore, their study reports that qPCR assay is more sensitive than hybridization based analysis in monitoring bioremediation processes. Recently, [28] determined abundance of active bacterial populations of an enriched bacterial consortium-AIE2 during the steady-state condition within continuous bioreactors treating Cr(VI) and azo dye mixtures by calculating 16S rRNA gene copy numbers using qPCR assays. Cébron et al. 2008 [29] developed qPCR assays to determine copy numbers of a functional gene that encodes the alpha subunit of the PAH-ring hydroxylating dioxygenases (PAH-RHDa) within bacterial populations capable of degrading PAHs by aerobic metabolism in soil and sediment samples.

3. Denaturing Gradient Gel Electrophoresis (DGGE)/ Temperature Gradient Gel Electrophoresis (TGGE)

A number of PCR-based genotypic fingerprinting techniques are available for monitoring microbial communities and efficacy of bioremediation processes. Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) are based on the principle of amplifying rRNA or functional gene PCR products from community DNA using primers containing a 50 bp GC-clamp and their separation on polyacrylamide gels having chemical or temperature based denaturing gradients [30]. Many studies have reported use of DGGE in monitoring microbial communities and their functional genes at sites contaminated with anthropogenic pollutants [31], such as microbial community shifts were detected in poly-metal contaminated soils [32] and abundances of dsrB (dissimilatory sulfite reductase b-subunit)-genes were assessed at an in situ metal precipitation site using DGGE fingerprinting technique [33]. However, these commonly used techniques (DGGE or TGGE) have inherent limitations, since they are labor-intensive and often less reproducible in terms of band pattern and intensity detection obtained after electrophoretic separation. These limitations can be addressed by designing improved group-specific primers [34] or by using a variant technique known as denaturing high performance liquid chromatography (dHPLC) that utilizes chromatographic separation instead of electrophoresis [35]. Muhling et al. 2008 [34] designed phylum- and class-specific PCR primers and tested their application in denaturing gradient gel electrophoresis (DGGE) analysis of complex bacterial communities. Wagner et al. 2009 [35] reported application of denaturing high performance liquid chromatography (dHPLC) in analyses of different microbial ecosystems within fermentor sludge, compost and soil samples using an ordinary HPLC system. For environmental or contaminated source samples where microbial diversity is largely unknown [36], DGGE/TGGE technique provides the opportunity for the identification of the microbial population through the excision of selected bands followed by their reamplification, cloning and sequencing that can lead to the phylogenetic affiliation of the ribotypes [37, 38]. DGGE in particular has been widely used for the assessment of microbial community structure in contaminated soil and water in a number of studies [39-44][39].

4. Amplified Ribosomal DNA Restriction Analysis (ARDRA)

In amplified ribosomal DNA restriction analysis, PCR-amplified 16S rRNA fragments are digested or ut at specific sites with restriction enzymes and the resulting digest separated by gel electrophoresis. Different DNA sequences will be cut in different locations and will result in a fingerprint unique to the community being analyzed [45, 46]. Divergence of the community rRNA restriction pattern on a gel is highly influenced by the type of restriction enzyme used [47]. Banding patterns in ARDRA can be used to screen clones or be used to measure bacterial community structure. ARDRA is simple, rapid and cost-effective, and as a result has been used in microbial identification [48, 49, 50] and microbial community studies [51, 52, 53]. In recent studies, ARDRA has been combined with other molecular techniques such as T-RFLP and DGGE to characterize microbial communities from contaminated sources [54, 55, 56]. A major challenge in using ARDRA lies in the interpretation of the fingerprints obtained from complex microbial communities.

5. Terminal-restriction Fragment Length Polymorphism (T-RFLP)

Terminal-restriction fragment length polymorphism is a major modification and improvement of the ARDRA method. The main advancement of T-RFLP over ARDRA lies in the fact that per organism detected, only the terminal restriction fragments (T-RFs) will be detected. Terminal-restriction fragment length polymorphism analysis (T-RFLP) is yet another most popular technique in studying microbial communities, since the last decade of its introduction to microbial ecological research. Main advantage of this profiling technique is its simplicity, automation and provision for accurate analysis of in silico data. T-RFLP analysis involves amplification of small subunit (SSU) rRNA genes from the total microbial community DNA using one or two primers tagged with different fluorescent labels. The resultant mixture of community rRNA amplicons is then digested with one or more four-base cutter restriction enzymes to generate T-RFs that are separated through capillary electrophoresis. Every single T-RF represents community fingerprints of a particular length unique to a particular phylogenetic microbial lineage or operational taxonomic unit (OTU). Based on the polymorphisms present in the SSU rRNA genes, different size of T-RF patterns of the whole microbial community are obtained. The size and relative abundances of these florescent T-RFs can be detected using an automated DNA sequencing instrument. Patterns of T-RF peaks on the output electropherogram can be identified using online database comparison tools. Many online automated fragment length assignment tools such as (taxonomic assignment pipeline) TAP-TRFLP (http://rdp.cme.msu.edu), torast (http://www.torast.de), and MiCA (http://mica.ibest.uidaho.edu), have been developed to perform in silico T-RFLP analysis of 16S rRNA gene sequences available in the databases. Similarly, an online tool known as TRF-CUT (www.arb-home.de) has been developed to predict in silico T-RFs from ARB database using aligned small-subunit rRNA gene or functional gene sequences (e.g. pmoA, nirK, and nifH) [57]. Unlike the above mentioned softwares, another online program TRiFle is available that can simulate and create T-RF datasets using arbitrary sets of DNA sequences from specific targets (e.g. genes involved in any metabolic pathways) or from unpublished sequences [58]. These online predictive tools are useful in choosing appropriate combinations of primers and restriction endonucleases to achieve best resolution up to taxonomic level in T-RFLP analysis. Several web based tools such as phylogenetic assignment tool (PAT), TRUFFLER, APLAUS are available to determine microbial community composition by comparision with T-RFs predicted from an in silico analysis of rRNA database sequences. Notwithstanding few of the limitations of TRFLP, such as overestimation of diversity by ‘pseudo T-RFs’ and non-specific action of restriction enzymes, it has become a valuable profiling technique for rapid and sensitive estimation of temporal and spatial variations in microbial communities [30]. Vázquez et al. 2009 [59] utilized T-RFLP to interpret temporal microbial community dynamics during bioremediation of diesel oil-contaminated antarctic soil. These authors performed a mesocosm study which suggested that nutrient addition (biostimulation) was a main factor in restoration of chronically contaminated Antartic soils, which significantly increased the detection of catechol degradation genes (nahH and catA) at treated plots. One of the advances in T-RFLP analysis is the demonstration of multiplex T-RFLP (M-TRFLP) which is useful in simultaneous profiling of multiple taxonomic groups of micro-organisms (two or four different taxa) within an ecosystem [60]. M-TRFLP analysis was validated using multiple primer sets targeted to bacteria, archaea and fungi in the same PCR reaction to study different microbial taxa in an ecosystem. This multiplex molecular profiling is useful in identifying bio-indicators of pollution, environmental health and to study how differently various microbial taxa respond to environmental stress [60].

The PCR primers used in T-RFLP analysis are fluorescently labelled at the 5′-terminus and the resultant PCR products are visualised and quantified [61] T-RFLP relies on variations in the positions of restriction sites among sequences and the determination of the length of fluorescently labelled terminal restriction fragments by high-resolution gel electrophoresis on an automated DNA sequencer. The electropherogram represents the profile of a microbial community as a series of peaks varying in migration distance. The use of fluorescently tagged primers limits the analysis to only the terminal fragments of the digestion [62]. This simplifies the banding pattern, hence enabling the analysis of complex communities as well as providing information on diversity as each visible band represents a single operational taxonomic unit or ribotype [63].

6. Fluorescent in Situ Hybridization (FISH)

The FISH technique is based on selective hybridization of rRNA targeted fluorescent dye-labeled oligonucleotide probes to the ribosomes of permeabilized microbial cells prefixed on membrane filters or glass slides. The resultant microbial cells stained by the complementary rRNA-targeted probes can be visualized or counted using epifluorescence microscopy, confocal laser scanning microscopy (CLSM), or flow cytometry techniques [64]. Multiple group-specific rRNA probes targeting prokaryotic and eukaryotic microbial taxa can be used in a FISH experiment for simultaneous phylogenetic classification as well as quantification of physiologically active microbial populations in an environmental sample. In the FISH approach it is assumed that actively growing microbes have many ribosomes and should theoretically yield brighter fluorescence signals due to higher rRNA-targeted probehybridizations. However, this assumption does hold true for many microbial cells that are smaller in size, slow growing or starving or containing low cellular rRNA content for example Dehalococcoides [65]. Hence, to over-come these limitations and improve the sensitivity of conventional FISH techniques, two new combinative approaches have been developed, namely (CARDFISH) catalyzed reporter deposition-fluorescence in situ hybridization [65] and (FISH-MAR) fluorescence in situ hybridization-microautoradiography [64]. Microautoradiography (MAR) is a process that relies on uptake of radioactive substrates by growing cells; the radioactivity incorporated into these cells is then visualized using radiation-sensitive photographic emulsions and microscopy. Therefore, coupling of FISH with microautoradiography (FISH-MAR) facilitates both phylogenetic as well as functional identification of substrate-active cells within complex microbial communities. The FISH-MAR technique involves short incubation of the environmental sample with radio-actively labeled substrate, followed by identification of microbial populations using FISH and in-parallel processing of identified microbial cells using radiation-sensitive photographic silver emulsions. Consequently, the silver particles deposited around the actively growing cells are visualized under transmission electron microscopy (TEM) to determine whether the microbe identified using rRNA-targeted probes was functionally or metabolically active in consuming the radio-labeled substrate offered at the time of incubation [64]. FISH-MAR technique has been most commonly used to identify key biodegradative microbial phylotypes within activated sludge systems owing to easy availability of sludge biomass for fixation, staining and hybridization experiments. Recently, [66] investigated the functional Bacteria and Archaea community structures responsible for decomposition process in a full-scale anaerobic sludge digester using FISH-MAR technique. In this study, it was observed that [14C] glucose-degrading microbial communities were dominant in terms of abundance and diversity as compared to fatty acids-[14C] propionate-[14C] butyrate-utilizing microbial communities. Moreover, despite the dominance of Betaproteobacteria in the community structures, members of Chloroflexi, Smithella, Syntrophomonas and Methanosaeta groups were more capable of utilizing radio-labeled sugars and fattyacids [66].

7. DNA Microarray Technologies

Microarrays (‘chips’) containing nucleic acids as probes represent a major advancement in molecular detection technology. They are ideal for the high-throughput study of the sequence diversity of 16S rRNA genes as well as of functional genes in environmental samples. DNA microarray technology is a very powerful taxonomic and functional tool that is widely used to study biological processes, including mixed microbial communities involved in pollutant degradation. This technique is similar to FISH, but provides a means for simultaneous analysis of many genes [67]. DNA microarrays are glass chips fabricated with different types of probes (pre-synthesized PCR products, cDNA, oligonucleotides, and known genomic fragments). These probes are deposited or spotted on the glass surface using metal pins (contact printing) or by ink-jets (non-contact printing). In addition, high-density oligonucleotide microarrays are generated by synthesizing probes at discrete locations using photodeprotection by photomasks/digital mirrors/chemical deprotection and ink-jet printing. Oligonucleotide microarrays are commercially available from several companies (Affymetrix, MWG, Agilent Technologies, GE Healthcare- Amersham Biosciences etc.). The probes ranging from sizes of approximately 25–1000 bps can be used to generate homogeneous microarrays (probes from a single genomic source) or heterogeneous microarrays (probes from different genomic sources) [68]. In principle, DNA microarray technique is based on hybridization of the target DNA molecules (single cell genomes or community genomes) to the array probes detected by measuring change in fluorescence signals (probe or target DNA can be tagged with several fluorescent dyes such as Cy3 or Cy5). The fluorescence signals from each of the probe-target hybridization spots are designated for quantification using mean signal intensity of each signal spot relative to its local background by signal-to-noise ratio (SNR) and measured using commercially available image analysis softwares. DNA Microarrays are amenable for rapid, sensitive and quantitative as well as simultaneous monitoring of several microbial populations within complex ecosystems. Based on the probes utilized in the fabrication of an array or depending on their applications, microbial ecological microarrays can be classified into several different types [68]. Of these different types of ecological microarrays; phylogenetic oligonucleotide arrays (POA), functional gene arrays (FGA) and whole-genome arrays (WGA) are most frequently employed in bioremediation studies. Phylogenetic oligonucleotide arrays (POA) or PhyloChips are constructed using short stretches of known oligonucleotide sequences based on rRNA genes from different microbial phyla and are amongst the most commonly used microarrays to decipher microbial community structures in environmental samples. Due to huge numbers of rRNA gene sequences available from public databases (such as RDP-II, ARB, NCBI, EMBL and DDBJ) along with easy in silico accessibility of online rRNA-targeted probe design and probe match tools, designing probes for a POA experiment becomes very convenient and user friendly. However, shorter stretches of oligonucleotide probes are less effective in resolving species-level phylogeny for some bacterial lineages [68]. Loy et al. 2005 [69] used a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes for diversity analysis of the betaproteobacterial order ‘‘Rhodocyclales” in activated sludge samples from an industrial wastewater treatment plant. RHC-PhyloChip was successful in detection of uncultured Zoogloea, Ferri bacterium/Dechloromonas and Sterolibacterium related bacterial lineages from the activated sludge samples that play an important role in waste water bioremediation process. An advanced version of PhyloChips is the Isotope Array, which is based on incorporation of radioactivity into rRNA of microbes incubated with radioactively labeled substrates [70]. Isotope array approach of hybridization of the radioactive rRNA of growing cells to PhyloChips enables microbial community diversity and activity to be assessed simultaneously using fluorescence and radioactivity detection modules.

8. Ribosomal Intergenic Spacer Analysis (RISA)

The RISA method makes use of the length and sequence heterogeneities that are present in the intergenic spacer (IGS) region between the small (SSU) and the large subunit (LSU) rRNA genes in the rRNA operon. It is a PCR-based technique that amplifies the region between the 16S and 23S rRNA genes. The IGS region, depending on the species, has both sequence and length (50–1500 bp) variability [71] and this unique feature facilitates taxonomic identification of organisms [72]. RISA has been used to distinguish between different strains and closely related species of Staphylococcus [73, 74], Bacillus [75, 76], Vibrio [77, 78], and other medically important microorganisms. In environmental studies, RISA has been used to detect microbial populations involved in the degradation of PAH at low temperature under aerobic and nitrate-reducing enriched soil conditions [79]. RISA has also been used to define microbial diversity and community composition in freshwater environments [80]. RISA is a very rapid and simple fingerprinting method but its application in microbial community analysis from contaminated sources is limited partly due to the limited database for ribosomal intergenic spacer sequences is not as large or as comprehensive as the 16S sequence database. Kostka et al. [81] used ARISA to determine the diversity of hydrocarbon degrading bacteria and bacterial community response in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico. Their findings indicated that oil contamination from the Deepwater Horizon had a profound impact on the abundance and community composition of autochthonous bacteria in the beach sands. Also members of the γ-proteobacteria (Alcanivorax, Marinobacter) and α-proteobacteria (Rhodobacteraceae) were identified as the key players in crude oil degradation.

9. Nucleic Acids Based Stable Isotope Probing (SIP)

Nucleic acids-based stable isotope probing (SIP) is a novel approach which directly links the microbial community structure with its function without the need for cultivation of individual micro-organisms. In principle, SIP technique consists of providing heavy isotope-labeled substrates (e.g. 13C-labeled substrates) to microbial communities and separation of the total cellular pool of nucleic acids within these microbial communities by isopycnic density gradient ultra-centrifugation [82]. The total extracted nucleic acids will form two different centrifugal zones, one with 13C-labeled (high buoyant density) and the other with C12-containing nucleic acid fragments (low buoyant density). Functional microbial communities that utilized the heavy isotope-labeled substrates can be identified from the resultant 13C-labeled nucleic acid fragments using molecular techniques discussed above. Natural C12-containing nucleic acid fragments are usually used as negative control in the SIP experiments to differentiate between active (13C-labeled) and inactive (C12-containing) microbial populations. SIP technique has been applied using wide variety of xenobiotic compounds to delineate the active microbial populations that utilize these compounds as substrates in cellular metabolism. Singleton et al. [83] supplied 13C-napthalene, 13C-salicylate and 13C-phenanthrene into PAH contaminated soils of a bioreactor to identify enriched bacterial degraders of PAHs within the bioreactor community DNA. In this DNA-SIP experiment, analysis of distinct 16S rDNA-based taxa within 13C-labeled community nucleic acids using DGGE fingerprinting technique identified Pseudomonas sp., Ralstonia sp. as degraders of naphthalene and salicylate and Acidovorax sp. as degraders of phenanthrene hydrocarbons. The SIP-approach in complementation with molecular fingerprinting or sequencing method can identify potential degraders of xenobiotics; however, the actual biodegradative pathways cannot be identified using this technique.

10. Molecular Biosensors / Bioreporters

In bioremediation of PAHs, in situ aerobic biodegradation of PAHs has been shown to be one of the most effective remediation strategies for detoxification of PAHs contaminated sites. In this sense, better understanding of the ecological behaviour of indigenous PAHs-degrading microorganisms and potential inoculants strains is required to optimise their potential use in bioremediation processes. However, it is impossible nowadays to reveal the whole veridical information of the ecological behaviour of microorganisms present in PAHs-contaminated environment. One of the feasible approaches to date is to mark a target strain before mixing with other microorganisms and then detect its activities to discover the role of this microorganism. With the increase of such microorganisms, theoretically, it is possible to elucidate the relationship between each microorganism in degrading of PAHs. Nevertheless, up to now, such research was preferable rare.

Environmental biosensors have made significant advancement towards monitoring of pollutants at contaminated sites as they have the unique ability to measure the interaction of specific compounds (pollutants) with biological systems through highly sensitive bio-recognition processes (signals). A biosensor is composed of a biosensing component interfaced with a transducing element that produces a measurable signal. Biosensing systems cover a wide array of integrated devices that utilize enzymes, antibodies, and sections of organs or tissues depending upon their applications [84]. A molecular biosensor has a recombinant plasmid as the biological component. It has a specific promoter, whose expression is sensitive to a target molecule and uses the reporter system to generate the signal. The promoters can be turned on or off with specific molecules, hence they provide the required specificity in signal generation. The generation of signals is directly proportional to the expression of the promoter. A biomarker, or marker gene, is defined as a DNA sequence, introduced into an organism, which provides a distinct genotype or phenotype to facilitate monitoring in a given environment [85]. Bundy et al. [86] showed use of single species biosensor responses to monitor recovery of oil polluted soil. Majority of bioreporters used in the study of microbial ecology are genetically engineered organisms in which responsive promoters are fused with suitable reporter genes, including lacZ coding for bgalactosidase, lux genes for the luciferase system, gfp for green fluorescent protein (GFP) and inaZ for ice nucleation protein [87]. The gene of green fluorescent protein (gfp) from the jellyfish Aequoria victoria, possess several advantages over other visual marker genes. The introduction of gfp into microorganisms is a useful tool with which to study microbial distribution, gene expression and protein interactions. The combination of transposon tagging with fluorescent reporter genes like gfp may lead to the development of internal markers for in situ detection of PAHs-degrading mycobacteria. Bastos et al. [88] chromosomally labeled an Alcaligenes faecalis strain isolated from an Amazonian soil sample and after PCR and Southern blot analyses confirmed that the gfp gene was integrated into the chromosome and the addition of the gfp marker did not affect phenol degradation ability compared with the wild-type. Environmental mycobacteria are considered promising for the biological clean-up of PAHs-contaminated environment for their peculiar physiological and structural properties. However, the molecular study of PAHs-degrading mycobacteria is hampered by the lack of adequate tools and methods. Wattiau et al. [89] employed suitable vectors carrying the pAL5000-based plasmids containing the green fluorescent protein (gfp) gene to investigate PAHs-degrading mycobacteria. Although they demonstrate the suitability of the pAL5000 replicon for the development of recombinant DNAbased studies in PAHs-degrading Mycobacterium spp., difficulties were encountered during their study in terms of electroporation efficiency and stability of plasmid constructs. Based on the above mentioned researches, Dandie et al. modified the transformation systems, especially transposon-based mycobacterial vectors to enhance electroporation efficiency. In their studies, the vectors pEM32 and pEM42 were employed to be electroporated into Mycobacterium sp. strain [90]. The electroporation condition was not satisfying similar to the results obtained by Wattiau et al. which demonstrated standard methods were inadequate for electroporation of environmental mycobacterial isolates and a systematic investigation of electroporation conditions and growth/preparation of electrocompetent cells is required to fully optimise transformation conditions for this strain [89].

11. Metagenomic Libraries and Pyrosequencing

Metagenomic libraries are constructed by direct cloning of DNA fragments extracted from an environmental sample in a suitable vector (e.g. plasmid, phage, fosmid, cosmid or bacterial artificial chromosomes BAC), which is then transformed into a suitable host strain. Cloned DNA fragments are then analyzed using either sequence based or function-based screening procedures. Many studies have reported construction and screening of metagenomic libraries to identify genes involved in bioremediation. Martin et al. [91] constructed metagenomic libraries to decipher ecological and metabolic functions of microbial communities involved in enhanced biological phosphate removal (EPBR) systems. This remarkable metagenomic study addressed the 30 year-old mystery of phosphate removal in EPBR systems by determining the complete genome and the associated phosphate accumulation genes within an uncultured, yet dominant poly-phosphate accumulating micro-organism (POA) known as Candidatus Accumulibacter phosphatis. In a similar study, Suenaga et al. [92] constructed fosmid libraries from metagenomic DNA fragements recovered from sludge samples. The resulting library was screened for extradiol dioxygenases (EDOs) using catechol as a substrate, which yielded 91 EDO-positive clones. Sequencing of the inserts within these positive clones depicted new EDO gene subfamilies involved in degradation of aromatic compounds. Metagenomics has now become a widespread approach to discover novel biocatalysts or gene products involved in biodegradation of anthropogenic compounds. However, the scope of constructing metagenomic libraries from environments having lower microbial abundance is very limited. These limitations have been over-come by application of whole community genome amplification (WCGA) based on principle of multiple displacement amplification (MDA), improving the accessibility and efficacy of metagenomic gene discoveries from lowbiomass environments. In this technique, all the metagenomic DNA is evenly amplified which ensures the representativeness of community microbial genomes [93]. Initial WGA (whole genome amplification) reactions utilized PCR-based techniques such as degenerate oligonucleotide primed PCR and primer extension PCR. However, these were limited by non-specific artifacts of amplification, strong bias and short amplification products. Majority of researchers now utilize the multiple displacement amplification (MDA) for whole genome amplification, this procedure involves an isothermal (300C) strand displacement synthesis in which the highly processive phi29 DNA polymerase repeatedly extends random primers on the template as it concomitantly displaces previously synthesized copies [94]. MDA has been applied in many metagenomic studies, for example Gonzalez et al. [93] used MDA as a pre-PCR enrichment step to alleviate the problem of co-extraction of humic acids and exopolysaccharides associated with low concentration of biomass in samples from cave, meadow soil and wastewater treatment systems. In this study, MDA from a defined mixture of pure-culture DNA accurately reflected the microbial community composition using denaturing gradient gel electrophoresis fingerprints. A yet another innovative technical breakthrough in the field of metagenomics is the massively parallel pyrosequencing also known as metagenomic pyrosequencing. Many pyrosequencing based chemistries and instruments are now commercially available, such as the Genome Sequencers from Roche/454 Life Sciences [GS-20 or GS-FLX;], the 1G Analyzer from Illumina/Solexa and the SOLiD System from Applied Biosystems (solid.appliedbiosystems.com). 454 Life Sciences has scaled up this technology enabling massively parallel sequencing of more than 300,000 sequences at once. In addition to the massive parallelization, the 454 technology does not require cloning from metagenomic DNA, thus eliminating many of the problems that are associated with this step of metagenomics. The only limitation of pyrosequencing is the short read lengths of approximately 250–400 bp that provide poor phylogenetic information as compared to full length 16S rRNA gene sequences (~1500 bp). However, these limitations can be over-come by using error-correcting barcoded primers [94] or by accurate taxonomic assignments of 16S rRNA sequence reads obtained from massively parallel pyrosequencers [96]. Hamady et al. [95] constructed error-correcting DNA barcodes that allow massively parallel pyrosequencing in a single run; this approach was successful in characterization of 16S rRNA gene sequences representing microbial communities in 286 environmental samples.

12. Transcriptomics and Metatranscriptomics in Bioremediation

Transcriptomic or metatranscriptomics tools are used to gain functional in-sights into the activities of environmental microbial communities by studying their mRNA transcriptional profiles [97]. Transcriptomic analyses entail the following basic steps; (1) extraction and enrichment of the total mRNA, (2) cDNA synthesis, (3) followed by either microarray hybridization of cDNA or sequencing of the complete cDNA transcriptome. Jennings et al. [98] performed transcriptomics analysis on a cisdichloroethene (cDCE)-assimilating Polaromonas sp. JS666 strain in order to identify the genes upregulated by cDCE using DNA microarrays. In this study, whole-genome expression arrays for Polaromonas sp. strain JS666 were synthesized and hybridized with the cDNA isolated from the strain JS666 during its growth on cDCE. The genes upregulated by cDCE were determined to be antioxidant proteins, ABC transporters and sodium/solute symporters. Moreover, it was hypothesized that a major degradation pathway involving carbon-chloride cleavage and a minor degradation pathway involving monooxygenase-catalyzed epoxidation were responsible for biodegradation of cDCE by Polaromonas sp. JS666 strain. In a similar study, Holmes et al. [99] deciphered the transcriptome of Geobacter uraniireducens strain growing in uranium-contaminated subsurface sediments. In this study, a whole-genome microarray analysis comparing sediment-grown G. uraniireducens with cells grown in defined culture medium indicated that there were 1084 genes that had higher transcript levels during growth in uranium contaminated sediments. It was observed that thirty four c-type cytochrome genes involved in Fe(III) and U(VI) reduction were upregulated in the sediment grown cells of G. uraniireducens. The above mentioned studies demonstrate that it is feasible to monitor gene expression of micro-organisms growing in presence of anthropogenic contaminants. Recent advances in direct extraction of mRNA from archaeal, bacterial and eukaryotic microbial cells have enabled researchers to obtain the gene expression profile of the entire microbial community also known as ‘‘metatranscriptome”. Extraction of this metatranscriptomes coupled with pyrosequencing or construction of cDNA microarrays provides a useful tool to monitor transcriptional activities of entire microbial communities (Urich et al., 2008). Urich et al. [100] employed a metatranscriptomic approach to simultaneously obtain information on both structure and function of soil microbial communities. In this study, the total community RNA was extracted and randomly reverse transcribed into cDNA, this resultant cDNA was subjected to pyrosequencing in order to produce 193, 219 rRNA-tags with valid taxonomic information, together with 21, 133 mRNA-tags exhibiting functional capabilities of the soil microbial communities. Transcriptomics approaches discussed above have a wide-applicability in linking structure and function of environmental microbial communities by performing a single experiment.

13. Pahbase, a Freely Available Functional Database of Polycyclic Aromatic Hydrocarbons (Pahs) Degrading Bacteria

Microbial population is a highly diverse and a ubiquitous group among the living world. One of the novel features of the microbes relates to their versatility in utilizing a large numbers of natural and manmade compounds. This property proves highly valuable in bioremediation for the complete destruction and removal of pollutants [101]. Contamination of soils and sediments by Polycyclic Aromatic Hydrocarbons (PAHs) is widespread, which raises enormous environmental concerns. It has been observed that PAH degradation in soil is dominated by bacterial strains belonging to a very limited number of taxonomic groups such as Sphingomonads, Burkholderia, Pseudomonas, Bacillus, Micrococcus and Mycobacterium [102-109][102] Members of these genera are specialized in the degradation of aromatic chemicals [110, 111]. As such, bioremediation may provide relatively low-cost and less intensified technology with high public acceptance. Bioinformatics based analysis and prediction is playing a pivotal role in understanding and capturing the in-depth knowledge of biological molecules particularly with reference to proteomics and genomics. Although with this advancement, there have been only limited efforts on the collection of all relevant information for a specific field of interest. With this realization, present study focuses on the wide spread data and information related to the occurrence and potential of PAH degrading bacteria. The information and detailed account on these bacteria are quite limited and scattered in scientific journals. Therefore, details from the research papers were extracted, analyzed and presented in form of a precise informative database: PAHbase reflecting the diversity and functional analysis of PAHs degrading bacteria.

PAHbase is a freely available functional database of Polycyclic Aromatic Hydrocarbons (PAHs) degrading bacteria. The database consists of relevant information obtained from scientific literature and databases. The database provides a comprehensible representation of PAH degrading bacteria with reference to its occurrence, phylogeny, and stress adaptation, potential to withstand extreme conditions, biodegradative ability, metabolic pathways and genetic basis of the degradation. The narrow search and limit options of the constructed database provide comparable information from the relevant PAH degrading candidates. The user friendly approach of PHP front end facilitates to add sequences of reported entries leading to scientific information for the specific purpose. The functional PAH database available freely on internet under URL: www.pahbase.in.

14. Conclusions

As mentioned above, PAHs as one kind of the most serious marine pollutants now has become a worldwide environmental problem and posed harmful threaten to humanbeings. Studies about PAHs biodegradation by microbes is a most focused branch in marine environment research field and research emphasis has been changed from finding PAHs-degrading microorganisms into metabolic pathways of microbes, genetic regulation and construction of high efficiency engineering microorganisms. Techniques introduced in this article has become powerful tool in biology and should be applied more intensively in studying of PAHs biodegradation and therefore provide prospective achievements in future. At the same time, as the rapid development of science, new molecular techniques such as meta-genomics sequencing (meta sequencing), DNA stable-isotope probing (DNA-SIP), function- driven screening method were also introduced into microbial ecological research and manifested powerful prospect. However, the microflora in PAHs-contaminated environment is pretty complex and therefore it is difficult to discover the nature of the biodegradation process by using solely one kind of methods. In this sense, only the integrated utilization of molecular techniques and other approaches could the nature of PAHs biodegradation be discovered.

15. Future Prospects

Developing efficient and consistent bioremediation strategies requires in depth understanding of the parameters governing the community structures and metabolic functioning of innate microbial communities. In this review, we described the recent advances in molecular and -omics technologies that have become an integral tool to manage and monitor bioremediation processes. Emergence of specialized techniques for monitoring the microbial genome, transcriptome, proteome, metabolome and fluxome in this ‘‘integrative- omics” era have paved way towards development and successful execution of efficient bioremediation strategies. However, the interpretation and management of the massive data generated by these ‘‘-omics” approaches still requires development of efficient statistical algorithms and bioinformatics tools. The progress made by recent molecular and ‘‘omics” explorations in microbial biodegradation and/or biotransformation have an enormous impact on our efforts to sustain industrialized societies with a cleaner environment.

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