**5. Foot prints of proteomics**

microarrays and their applicability for bacterial functional genomics (Denef et al. 2003). Optimal parameters were found to be 50-C6- amino-modified 70 mers printed on CMT-GAPS II substrates at a 40 *m*M concentration combined with the use of tyramide signal amplification labelling. Based on most of the known genes and pathways involved in biodegradation and metal resistance, a comprehensive 50-mer-based oligonucleotide microarray was developed for effective monitoring of biodegrading populations (Rhee et al. 2004). This type of DNA microarray was effectively used to analyze naphthaleneamended enrichment, and soil microcosms demonstrated that microflora changed differen‐ tially depending on the incubation conditions (Cho and Tiedje. 2002 ).A global gene expression analysis revealed the co-regulation of several thusfar- unknown genes during the degradation of alkylbenzenes (Kuhner et al. 2005). Besides this, DNA microarrays have been used to determine bacterial species, in quantitative applications of stress gene analysis of microbial genomes and in genome-wide transcriptional profiles (Muffler et al. 2002.,

**Figure 1.** Work flow of gene array analysis. Diagrammatic representation of DNA microarray data analysis and relative

limitations under each category of data analysis during data mining.

Greene and Voordouw. 2003).

378 Applied Bioremediation - Active and Passive Approaches

The terms 'proteomics' and 'proteome' were introduced in 1995 (Wasinger et al. 1995), which is a key postgenomic feature that emerged from the growth of large and complex genome sequencing datasets. Proteomic analysis is particularly vital because the observed phenotype is a direct result of the action of the proteins rather than the genome sequence.Traditionally, this technology is based on highly efficient methods of separation using two-dimensional polyacrylamide gel electrophoresis (2-DE) and modern tools of bioinformatics in conjunction with mass spectrometry (MS) (Hochstrasser. 1995). However, 2-DE has been considered to be a limited approach for very basic and hydrophobic membrane proteins in compartmental proteomics. In bioremediation, the proteome of the membrane proteins is of high interest, specifically in PAH biodegradation, where many alterations in any site specific bacterium affects cell-surface proteins and receptors (Sikkema et al. 1995). The improvements in 2-DE for use in compartmental proteomics have been made by introducing an alternative approach for multidimensional protein identification technology (MudPIT) (Paoletti et al. 2004). MS has revolutionized the environmental proteomics towards the analysis of small molecules to peptides and proteins that has pushed up the sensitivity in protein identification by several orders of magnitude followed by minimizing the process from many hours to a few minutes (Aebersold and Mann. 2003). The advancement in MS techniques coupled with database searching have played a crucial role in proteomics for protein identification.

Matrixassociated laser desorption/ionization time-of-flight MS (MALDI-TOF-MS) is the most commonly used MS approach to identifying proteins of interest excised from 2-DE gels, by generation of peptidemass fingerprinting (Aebersold and Mann. 2003., Aitken and Learmonth. 2002., Landry et al. 2000). Surface-enhanced laser-desorption-ionization MS (SELDI-TOF-MS) is the combination of direct sample fractions on a chip integrated with MALDI-TOF-MS analysis (Merchant and Weinberger. 2000., Seibert et al. 2005). A variety of differentially expressed signature proteins were analysed using SELDITOF- MS in blue mussels (Mytilus edulis) exposed to PAHs and heavy metals (Knigge et al. 2004). The liquid chromatography MS (LC-MS) technique has begun to open a new analytical window for direct detection and identification of potential contaminants in water (Joo and Kim. 2005). In addition, the metab‐ olites and degradation products have been taken into account to assess the fate of organic contaminants such as pesticides, surfactants, algal and cynobacterial toxins, disinfection byproducts or pharmaceuticals in the environment and during water treatment processes (Joo and Kim. 2005).

### **6. Interaction of interactomics**

Genome-wide mRNA profiling is unable to provide any information about the activity, arrangement, or final destination of the gene products, the proteins. Various proteomic approaches, on the other hand, can successfully provide the straight answers. It is very rare that any protein molecule acts as a unique pillar during the physiological response in biore‐ mediation process of any contaminant when cellular proteins and various other related cellular expressions are on crest (Muffler et al. 2002., Kuhner et al. 2005., Eyers et al. 2004., Segura et al. 2005). In general, cellular life is organized through a complex protein interaction network, with many proteins taking part in multicomponent protein aggregation. The detection of these aggregated proteins, i.e. 'interactomics', is usually based upon affinity tag/pull down/MS/MS approaches at a proteome level (Lee and Lee. 2004., Coulombe et al. 2004., Gingras et al. 2005). Studies on protein–protein interaction and supermolecular complex formation repre‐ sent one of the main directions of functional proteomics and/or second generation proteomics.

**Microorganism Relevance to bioremediation Web site for genome**

Reductive dechlorination of chlorinated solvents to ethylene. The 16S rRNA gene sequence of D. ethanogenes is closely related to sequences that are enriched in subsurface environments in which chlorinated solvents are being degraded

Anaerobic oxidation of aromatic hydrocarbons and reductive precipitation of uranium. 16S rRNA gene sequences closely related to known *Geobacter* species predominate during anaerobic in situ bioremediation of aromatic hydrocarbons and

Main organism for elucidating pathways of anaerobic metabolism of aromatic compounds, and

aerobically degrading a wide variety of organic contaminants. Excellent organism for genetic engineering of bioremediation capabilities.

Representative of ubiquitous genus of perchloratereducing microorganisms and capable of the anaerobic oxidation of benzene coupled to nitrate

Reductive dechlorination of chlorinated solvents and phenols. *Desulfitobacterium* species are widespread in a variety of environments.

chromium. An actual role in contaminated environments is yet to be demonstrated.

reduce U(vi) to U(iv) in culture, but *Shewanella* species have not been shown to be important in metal reduction in any sedimentary environments.

Highly resistant to radiation and so might be genetically engineered for bioremediation of highly

regulation of this metabolism.

*Pseudomonas putida* Metabolically versatile microorganism capable of

uranium.

reduction.

*Desulfovibrio vulgaris* Shown to reductively precipitate uranium and

*Shewanella oneidensis* A closely related *Shewanella* species was found to

radioactive environments.

**Table 1.** Genomes of microorganisms pertinent to bioremediation.

*Dehalococcoides ethanogenes*

Geobacter sulfurreducens Geobacter metallireducens

*Rhodopseudomonas*

*Dechloromonas aromatica*

*Desulfitobacterium*

*hafniense*

*Deinococcus radiodurans*

*palustris*

**documentation**

http://www.tigr.org

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http://www.jgi.doe.gov http://www.tigr.org

http://www.jgi.doe.gov

http://www.tigr.org

http://www.jgi.doe.gov

http://www.jgi.doe.gov

http://www.tigr.org

http://www.tigr.org

http://www.tigr.org

The growing demands of genomics and proteomics for the analysis of gene and protein function from a global bioremediation perspective are enhancing the need for microarraybased assays enormously. In the past, protein microarray technology has been successfully implicated for the identification, quantification and functional analysis of protein in basic and applied proteome research (Labaer and Ramachandran. 2005). Other than the DNA chip, a large variety of protein-microarraybased approaches have already been verified that this technology is capable of filling the gap between transcriptomics and proteomics (Liu and Zhu. 2005). However, in bioremediation, microarray-based protein–protein interaction studies still need to make progress to understand the chemotaxis phenomenon of any site specific bacterium towards the environmental contaminant.

#### **7. Revolution of genomics**

A drastic innovation in the study of pure cultures has been brought by the application of genomics to bioremediation. (Nierman & Nelson, 2002). Next generation genome sequencing techniques play a vital role in advancing the understanding of physiological and genomic features of microorganisms relevant to bioremediation. Complete, or nearly complete, genome sequences are now available for several organisms that are important in bioremediation (Table. 1). The notions of researches have been changed after the application of bioremediation to the advanced sciences like genomics which gave different answers. For example, molecular analyses have indicated that *Geobacter* species are important in the bioremediation of organic and metal contaminants in subsurface environments. The sequencing of several genomes of microorganisms of the genus *Geobacter,* as well as closely related organisms, has significantly altered the concept of how *Geobacter* species function in contaminated subsurface environ‐ ments. For instance, before the sequencing of the *Geobacter* genomes, *Geobacter* species were thought to be non-motile, but genes encoding flagella were subsequently discovered in the *Geobacter* genomes (Childers et al. 2002) Further investigations revealed that *Geobacter metal‐ lireducens* specifically produces flagella only when the organism is growing on insoluble Fe(ra) or Mn(IV) oxides. Genes for chemotaxis were also evident in the *Geobacter* genomes, and experimental investigations have revealed that *G. metallireducens* has a novel chemotaxis to Fe(II), which could help guide it to Fe(III) oxides under anaerobic conditions. Pili genes are present and are also specifically expressed during growth on insoluble oxides (Childers et al.


**Table 1.** Genomes of microorganisms pertinent to bioremediation.

mediation process of any contaminant when cellular proteins and various other related cellular expressions are on crest (Muffler et al. 2002., Kuhner et al. 2005., Eyers et al. 2004., Segura et al. 2005). In general, cellular life is organized through a complex protein interaction network, with many proteins taking part in multicomponent protein aggregation. The detection of these aggregated proteins, i.e. 'interactomics', is usually based upon affinity tag/pull down/MS/MS approaches at a proteome level (Lee and Lee. 2004., Coulombe et al. 2004., Gingras et al. 2005). Studies on protein–protein interaction and supermolecular complex formation repre‐ sent one of the main directions of functional proteomics and/or second generation proteomics.

The growing demands of genomics and proteomics for the analysis of gene and protein function from a global bioremediation perspective are enhancing the need for microarraybased assays enormously. In the past, protein microarray technology has been successfully implicated for the identification, quantification and functional analysis of protein in basic and applied proteome research (Labaer and Ramachandran. 2005). Other than the DNA chip, a large variety of protein-microarraybased approaches have already been verified that this technology is capable of filling the gap between transcriptomics and proteomics (Liu and Zhu. 2005). However, in bioremediation, microarray-based protein–protein interaction studies still need to make progress to understand the chemotaxis phenomenon of any site specific

A drastic innovation in the study of pure cultures has been brought by the application of genomics to bioremediation. (Nierman & Nelson, 2002). Next generation genome sequencing techniques play a vital role in advancing the understanding of physiological and genomic features of microorganisms relevant to bioremediation. Complete, or nearly complete, genome sequences are now available for several organisms that are important in bioremediation (Table. 1). The notions of researches have been changed after the application of bioremediation to the advanced sciences like genomics which gave different answers. For example, molecular analyses have indicated that *Geobacter* species are important in the bioremediation of organic and metal contaminants in subsurface environments. The sequencing of several genomes of microorganisms of the genus *Geobacter,* as well as closely related organisms, has significantly altered the concept of how *Geobacter* species function in contaminated subsurface environ‐ ments. For instance, before the sequencing of the *Geobacter* genomes, *Geobacter* species were thought to be non-motile, but genes encoding flagella were subsequently discovered in the *Geobacter* genomes (Childers et al. 2002) Further investigations revealed that *Geobacter metal‐ lireducens* specifically produces flagella only when the organism is growing on insoluble Fe(ra) or Mn(IV) oxides. Genes for chemotaxis were also evident in the *Geobacter* genomes, and experimental investigations have revealed that *G. metallireducens* has a novel chemotaxis to Fe(II), which could help guide it to Fe(III) oxides under anaerobic conditions. Pili genes are present and are also specifically expressed during growth on insoluble oxides (Childers et al.

bacterium towards the environmental contaminant.

**7. Revolution of genomics**

380 Applied Bioremediation - Active and Passive Approaches

2002). Genetic studies have indicated that the role of the pili is to aid in attachment to Fe(III) oxides, as well as facilitating movement along sediment particles in search of Fe(III).

This energy-efficient mechanism for locating and reducing Fe(ra) oxides in *Geobacter* species contrasts with the strategies for Fe(III) reduction in other well-studied organisms, such as *Shewanella* and *Geothrix* species. These other organisms release Fe(III) Chelators, which solubilize Fe(m) from Fe(m) oxides (Nevin and Lovley. 2002), and electron shuttling com‐ pounds, which accept electrons from the cell surface and then reduce Fe(m) oxides (Newman and Kolter. 2000., Nevin and Lovley. 2002).These strategies make it possible for *Shewanella* and *Geothrix* species to reduce Fe(III) without directly contacting the Fe(m) oxide. However, the synthesis of chelators and electron shuttles requires a significant amount of energy, and the lower metabolic energy requirements of the *Geobacter* approach is the probable explanation for the fact that *Geobacter* species consistently outcompete other Fe(III)-reducing microorgan‐ isms in several subsurface environments (Nevin and Lovley. 2002).Understanding this, and numerous other previously unsuspected physiological characteristics of *Geobacter* species, is important in guiding the manipulation of conditions in subsurface environments to optimize the ability of *Geobacter* species to remove organic and metal contaminants from polluted groundwater.

The study of the physiology of other microorganisms with bioremediation potential, the genomes of which have been sequenced, is now accelerating in a similar manner. With the completed genome sequences, it is possible using whole-genome DNA microarrays to analyse the expression of all the genes in each genome under various environmental conditions. Using pro-teomic techniques, it is possible to identify which proteins are expressed (Nierman & Nelson, 2002).Such genome-wide expression analysis provides important data for identifying regulatory circuits in these organisms (Baldi and Hatfield. 2002).This is significant as the mechanisms that control the regulation of the catabolic and respiratory genes that are the most important in bioremediation are largely unknown. As genetic systems for these environmen‐ tally significant organisms become available, it is possible to elucidate the function of the many genes of previously unknown function and to decipher bioremediation pathways. For example, the availability of the Geobacter genomes and a genetic system for these organisms is leading to the elucidation of which of the more than 100 c-type cytochromes that are apparent in the genome are important in electron transfer to metals (Lloyd et al. 2003., Leang et al. 2003)*.*

Treatability study is a process, in which samples of the contaminated environment are incubated in the laboratory and the rates of contaminant degradation or immobilization are documented (Rogers and McClure. 2003). Giving little insight into the microorganisms that are responsible for the bioremediation, such studies provide an estimate of the potential metabolic activity of the microbial community. When bioremediation processes are researched in more detail, attempts are generally made to isolate the organisms responsible(Rogers. et al. 2003).The isolation and characterization of pure cultures has been, and will continue to be, crucial for the development and interpretation of molecular analyses in microbial ecology (Fig. 1).The recovery of isolates that are representative of the microorganisms responsible for the bioremediation process can be invaluable because, as outlined below, studying these isolates provides the opportunity to investigate not only their biodegradation reactions, but also other

**Figure 2.** Evolution of increasingly sophisticated studies of pure cultures and their application to the study of microbi‐

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al communities

2002). Genetic studies have indicated that the role of the pili is to aid in attachment to Fe(III)

This energy-efficient mechanism for locating and reducing Fe(ra) oxides in *Geobacter* species contrasts with the strategies for Fe(III) reduction in other well-studied organisms, such as *Shewanella* and *Geothrix* species. These other organisms release Fe(III) Chelators, which solubilize Fe(m) from Fe(m) oxides (Nevin and Lovley. 2002), and electron shuttling com‐ pounds, which accept electrons from the cell surface and then reduce Fe(m) oxides (Newman and Kolter. 2000., Nevin and Lovley. 2002).These strategies make it possible for *Shewanella* and *Geothrix* species to reduce Fe(III) without directly contacting the Fe(m) oxide. However, the synthesis of chelators and electron shuttles requires a significant amount of energy, and the lower metabolic energy requirements of the *Geobacter* approach is the probable explanation for the fact that *Geobacter* species consistently outcompete other Fe(III)-reducing microorgan‐ isms in several subsurface environments (Nevin and Lovley. 2002).Understanding this, and numerous other previously unsuspected physiological characteristics of *Geobacter* species, is important in guiding the manipulation of conditions in subsurface environments to optimize the ability of *Geobacter* species to remove organic and metal contaminants from polluted

The study of the physiology of other microorganisms with bioremediation potential, the genomes of which have been sequenced, is now accelerating in a similar manner. With the completed genome sequences, it is possible using whole-genome DNA microarrays to analyse the expression of all the genes in each genome under various environmental conditions. Using pro-teomic techniques, it is possible to identify which proteins are expressed (Nierman & Nelson, 2002).Such genome-wide expression analysis provides important data for identifying regulatory circuits in these organisms (Baldi and Hatfield. 2002).This is significant as the mechanisms that control the regulation of the catabolic and respiratory genes that are the most important in bioremediation are largely unknown. As genetic systems for these environmen‐ tally significant organisms become available, it is possible to elucidate the function of the many genes of previously unknown function and to decipher bioremediation pathways. For example, the availability of the Geobacter genomes and a genetic system for these organisms is leading to the elucidation of which of the more than 100 c-type cytochromes that are apparent in the genome are important in electron transfer to metals (Lloyd et al. 2003., Leang et al. 2003)*.*

Treatability study is a process, in which samples of the contaminated environment are incubated in the laboratory and the rates of contaminant degradation or immobilization are documented (Rogers and McClure. 2003). Giving little insight into the microorganisms that are responsible for the bioremediation, such studies provide an estimate of the potential metabolic activity of the microbial community. When bioremediation processes are researched in more detail, attempts are generally made to isolate the organisms responsible(Rogers. et al. 2003).The isolation and characterization of pure cultures has been, and will continue to be, crucial for the development and interpretation of molecular analyses in microbial ecology (Fig. 1).The recovery of isolates that are representative of the microorganisms responsible for the bioremediation process can be invaluable because, as outlined below, studying these isolates provides the opportunity to investigate not only their biodegradation reactions, but also other

oxides, as well as facilitating movement along sediment particles in search of Fe(III).

groundwater.

382 Applied Bioremediation - Active and Passive Approaches

**Figure 2.** Evolution of increasingly sophisticated studies of pure cultures and their application to the study of microbi‐ al communities

aspects of their physiology that are likely to control their growth and activity in contaminated environments. However, before the application of molecular techniques to bioremediation, it was uncertain whether the isolated organisms were important in bioremediation *in situ*,or whether they were 'weeds' that grew rapidly in the laboratory but were not the primary organisms responsible for the reaction of interest in the environment.

than 99% similarity to the MTBE-degrading organism, strain PM-1,which is available in pure

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The primary limitation of the 16S rRNA technique is that knowledge of the phylogeny of the organisms associated with bioremediation does not necessarily predict important aspects of their physiology (Pace. 1997., Achenbach and Coates. 2000). For example, microorganisms with 16S rRNA sequences closely related to the TCE-degrader D. ethanogenes can differ in the chlorinated compounds that they can degrade (He et al. 2003., Bunge et al. 2003), and predicting which of these compounds an uncultured organism will degrade might not be apparent from analysis of its 16S rRNA sequence alone (Hendrickson et al. 2002). Predicting physiology from phylogeny is even more difficult if there are no closely related organisms available in pure

Based on an overall analysis of transcriptomics and proteomics, the comprehensive analysis of wholegenome sequencing is especially helpful to understand bioremediation-relevant microorganisms whose physiology has not yet been studied in detail. Global gene expression using DNA microarray technology, very much depends on the degree of coverage of the cellular mRNA and cellular proteins, whereas the coverage of the whole genome represents all the genes of an organism by definition. Cellular mRNA levels do not display as wide a dynamic range as the encoded proteins (Gygi et al. 1999). Thus, whole genome arrays are believed to provide a much more comprehensive overview of the actual gene expression

According to global gene expression studies, both transcriptomics and proteomics support the view that the DNA array technologies record changes in gene expression more completely than the proteomics (Muffler et al. 2002., Kuhner et al. 2005., Eymann et al. 2002). Therefore, genomics data is deemed necessary to complement the proteomics approach (Hegde et al. 2003). However, proteomics would retain its central position in functional transcriptomics and/ or genomics. The protein molecules, but not the mRNAs, are the key players in an on-site microbial mineralization reaction; the later are one of the highly unstable transmitters on the path from the genes to the ribosome, but each protein molecule represents the end product of gene expression (Kuhner et al. 2005). Complete protein profiling provides not only information on the individual organism, but also information on the fate and destination of protein molecules inside and outside the cell that can only be discovered via a joint transcriptomics,

MetaRouter is a system for maintaining heterogeneous information related to Biodegradation in a framework that allows its administration and mining (application of methods for extract‐

**9. Comparative analysis of Omics in bioremediation**

culture (Hristova et al. 2003).

pattern than proteomic studies.

proteomics and interactomics approach (Figure 3).

**10. Bioinformatics in bioremediation**

culture.

### **8. The 16S rRNA approach**

A significant advance in the field of microbial ecology was the finding that the sequences of highly conserved genes that are found in all microorganisms, most notably the 16S rRNA genes, could provide a phylogenetic characterization of the microorganisms that comprise microbial communities (Pace et al. 1986., Amann et al. 1995).This was a boon to the field of bioremediation because it meant that by analysing 16S rRNA sequences in contaminated environments, it was possible to determine definitively the phylogenetic placement of the microorganisms that are associated with bioremediation processes (Rogers and McClure. 2003., Watanabe and Baker. 2000)

One of the surprises from the application of the 16S rRNA approach to bioremediation has been the finding that, in some instances, microorganisms that predominate during bioreme‐ diation are closely related to organisms that can be cultured from subsurface environments (Lovley. 2001).This contrasts with the general dilemma in environmental microbiology that is, it can be difficult to recover the most environmentally relevant organisms in culture (Amann et al. 1995). For example, in polluted aquifers, in which microorganisms were oxidizing contaminants with the reduction of Fe (m) oxides, there was a significant enrichment in microorganisms with 16S rRNA sequences that were closely related to those of previously cultured Geobacter species (Rooney-varga et al. 1999., Snoeyenbos-West et al. 2000., Roling et al. 2001).Coupled with the fact that Geobacter species in pure culture are capable of oxidizing organic contaminants with the reduction of Fe(III) oxide (Lovley et al. 1989),this indicated that Geobacter species are important in contaminant degradation in situ. Geobacter species can also remove uranium from contaminated water by reducing soluble U(vi) to insoluble U(iv) (Lovley et al. 1991). 16S rRNA sequence analysis showed that, when acetate was added to uranium-contaminated groundwater to promote micro-bial reduction of U(vi), the number of Geobacter species increased by several orders of magnitude, accounting for as much as 85% of the microbial community in the groundwater (Anderson et al. In Press, Holmes et al. 2002) In aquifers in which the indigenous microbial community was degrading the solvent trichlor‐ oethene (TCE), 16S rRNA sequences that are ~99% identical to the 16S rRNA sequence of a pure culture of the TCE-degrader Dehalococcoides ethanogenes, were detected (Fennell et al. 2001., Richardson et al. 2002., Hendrickson et al. 2002).Marine sediments with high rates of anaerobic naphthalene degradation were found to be specifically enriched in microorganisms with 16S rRNA sequences closely related to NaphS2, an anaerobic naphthalene degrader that is available in pure culture (Hayes and Lovley. 2002). There was a close correspondence between the potential for aerobic degradation of the fuel oxygenate methyl tert-butyl ether (MTBE) in groundwater and the number of organisms with 16S rRNA sequences that had more than 99% similarity to the MTBE-degrading organism, strain PM-1,which is available in pure culture (Hristova et al. 2003).

The primary limitation of the 16S rRNA technique is that knowledge of the phylogeny of the organisms associated with bioremediation does not necessarily predict important aspects of their physiology (Pace. 1997., Achenbach and Coates. 2000). For example, microorganisms with 16S rRNA sequences closely related to the TCE-degrader D. ethanogenes can differ in the chlorinated compounds that they can degrade (He et al. 2003., Bunge et al. 2003), and predicting which of these compounds an uncultured organism will degrade might not be apparent from analysis of its 16S rRNA sequence alone (Hendrickson et al. 2002). Predicting physiology from phylogeny is even more difficult if there are no closely related organisms available in pure culture.
