**2. Extremophilic environments and associated microbial diversity**

The physicochemical characteristics of extremophilic environments observed on the planet Earth are quite diverse and they are often studied with regards to temperature, pH, salt concentration, nutrient availability etc. Some of the typical extremophilic environments widely studied include thermophilic environments, psychrophilic environments, halophilic environments, acidophilic environments, subterranean habitats, and hyper-arid environments [2, 8, 32]. Representative niches for each of these environments have been scanned with both cultivationdependent and cultivation-independent approaches [19, 20, 24, 33]. A brief description of some of the representative extremophilic environments and the associated microbial diversity is presented below.

#### **2.1 Thermophilic environments**

Studies pertaining to thermophilic environments initiated in the 1970s and 1980s with the isolation of several novel hyperthermophiles. Subsequent studies led to the discovery of deep-sea hydrothermal vents and consequent addition of isolation of a wide range of hyper thermophilic microorganisms belonging to the 'archaeal' domain of the life [34]. During the 1990s, with the advent of culture-independent characterization of microbial diversity using 16S rRNA gene pool sequencing, the thermophilic environments e.g. hydrothermal vents were analyzed [35–37]. These studies could define the composition and diversity of the microbial communities present within the representative thermophilic environments and characterized the prokaryotic phylotypes amongst diverse thermophilic environments representing the temperature gradients from 60o C to 120o C [35–37]. However, the understanding of the functions associated with microbial diversity and the intra-species, interspecies interaction remained poorly defined.

A few of the culture-independent studies on thermophilic environments, which analyzed the sequence of the entire metagenomic DNA pool rather than just the phylogenetic marker gene, identified dominance of sulfur- recycling genes amongst the dominant phylotypes within the sulfur-rich deep-sea vents [35]. Similarly, the

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

These unique capabilities enable them not only survive but also thrive in extremes of physicochemical parameters [3–6]. The idea that microorganisms might survive in such extreme environments and the term 'extremophile' was first proposed in the 1970s by Robert MacElroy. Conventionally, extremophilic microorganisms have been defined by their ability to grow optimally under environments characterized by extreme temperature, pH, pressure, and salinity etc. [7, 8]. It is argued that survival and growth under extreme environments require stabilization of cellular components and enzymes so that their optimal functionality is maintained. Therefore, extremophilic microorganisms are proposed to be one of the greatest reservoirs of the wide spectrum of exclusive enzymes and metabolites with significant biotechnological applications [9–15]. In addition, the extremophilic microorganisms are now also being regarded to have the pivotal role in maintaining the balance of global biogeochemical cycles [16–18]. With this understanding, there has been a continued increase in the scientific interest in isolation and characterization of extremophilic microorganisms. The same is clearly reflected by the fact that many new extremophilic microorganisms have been isolated and cultured in laboratories all over the world during the past 2–3 decades [19, 20]. Still, much of the physiological and phylogenetic diversity of extremophilic microorganisms remains rather unexplored. Given the ability of extremophilic microorganisms to thrive in the extreme environments; their taxonomic, genetic and metabolic characterization is widely regarded as an indispensable step towards harnessing their true potential. The progress in this line of scientific endeavor has remained hampered due to the vast majority of microbial biodiversity within extremophilic environments comprising of the lineages that are recalcitrant to traditional culturing techniques based isolation and purification approaches [21]. In absence of purified cultures of extremophilic microorganisms, the access to their genetic and metabolic diversity has remained obscure as only until recently, cultivability was the single most important prerequisite for having access to the genetic complement of individual organisms. This limitation has been circumvented to a great extent with the implementation of culture-independent approach (i.e. metagenomics). The 'state of the art metagenomics technologies,' allow not only to develop a theoretical and mechanistic understanding of the possible role of extremophilic microorganisms in biogeochemical cycles but also assess the genetic & metabolic potentials (e.g. discover novel enzymes and proteins for industrial applications) of the uncultured extremophilic microbial population [22–25]. Having mentioned that, it is also pertinent to remark that even with the implementation of improved cultivation methodologies and metagenomics characterization, the understanding of the 'black box of extremophilic microbial diversity' has improved only marginally over the period of last 2 decades. The optimal exploitation of their potential still remains elusive. This situation could be attributed to the following reasons: (i) despite the everimproving cultivation methodologies, most of the extremophilic microorganisms are not yet amenable to laboratory culturing which use traditional reductionist culturing approaches; (ii) the microbial biomass densities within extremophilic environments are often too less to yield enough DNA for carrying out effective culture-independent analyses (e.g. metagenomics, metatranscriptomics, and recombinant cloning of a gene of interest); and (iii) inability to annotate novel genetic complements during post-sequencing analyses of metagenomic due to lack

**64**

of reference sequences in the nucleotide databases [24].

This situation demands continued improvement of technical methodologies towards assessing and harnessing the genetic and metabolic diversity of extremophilic microorganisms from even the minute quantities of retrievable metagenomic DNA. Some of the developments in this aspect have focused on improving the recovery of metagenomic DNA from extremophilic environments [26]. Yet another

prevalence of hydrogen oxidation genes was observed in hydrogen-rich deep-sea hyperthermophilic vents [38–40]. A few other studies have identified the critical genetic signatures (e.g. genes for alternative mechanisms of nitrogen utilization) of the microbial communities surviving within the thermophilic environments. Some of the recent culture-independent studies on samples collected from thermophilic environments have indicated for the occurrence of the significantly higher diversity of CRISPR compared to the metagenomes of the mesophilic microbial diversity [41–43].

Even with increasing frequency of reports showing the identification of novel genetic and metabolic mechanisms prevalent in thermophilic environments; the comprehensive understanding about key genetic elements which determine the composition as well the function of the microbial diversity within the thermophilic environments is only poorly understood. It is not yet established how physicochemical factors contribute to shaping up the composition and structure of the microbial diversity of any thermophilic environment. The scenario is expected to improve only through the inclusion of physicochemical information along with full community metagenome data.

### **2.2 Psychrophilic environments**

The psychrophilic environments are characterized by extremely low temperatures. Just like the thermophilic environments, they also represent one of the most thoroughly investigated extreme environments [21, 44]. It is noteworthy that unlike the thermophilic environments, the microbial diversity within psychrophilic environments consists of both eubacteria and archaea [45]. The biodiversity and adaptive strategies of psychrophilic microorganisms have been extensively studied. Results from some of the representative metagenomic studies on the psychrophilic environment have shown microbial community diversity and complexity to be significantly higher than other environments [45, 46]. The most note-worthy studies on psychrophilic environments have been carried out on samples from Antarctic continent, which harbors sub-glacial ice habitat. These studies have reported the dominance of 'chemoautotrophs' that are capable of tapping reduced iron and reduced sulfur compounds as the source of energy [47]. Other studies with psychrophilic environments have recognized the presence of 'chemolithotrophic' bacterial and archaeal communities [45, 47]. These share a close phylogenetic relationship with microorganisms able to use reduced nitrogen, and iron compounds as the source of energy. With regards to the psychrophilic environments, it is generally accepted that 'availability of organic metabolizable carbon' is the single most dominant factor determining the microbial activity, diversity, and dynamics.

#### **2.3 Acidophilic environments**

Acidophilic environments have emerged as 'extremophilic environments of choice' for studies on mechanisms and genetic elements determining the survival of life under extreme environments. A number of studies had reported attempts for isolation of microorganisms from acidophilic environments. Culture-independent studies with respect to acidophilic environments were first carreid out with a natural acidophilic biofilm sample [48]. Subsequent studies in this regard were carried out on samples collected from an Acid Mine Drainage located at different parts of the world [49–52]. The data obtained with these samples showed the microbial community structure to have a poor diversity with presence of only chemoautotrophic consortia largely comprising members of genera *Leptospirillum* and *Ferroplasm* [48]. The genetic signatures observed within the Acid Mine Drainage

**67**

**metabolites**

*Harnessing the Genetic Diversity and Metabolic Potential of Extremophilic Microorganisms…*

metagenomes indicated for molecular mechanisms for acidophilic survival through implmentation of unique carbon metabolic pathways for Carbon metabolism, Nitrogen fixation and iron oxidation [53]. The community composition of Acid Mine Drainage samples were found to have significant contrast to the naturally occuring acidophilic biofilms that has *Acidithiobacillus*, *Acidimicrobium* and *Ferrimicrobium* as the dominant genera present within the community [49, 52, 54]. The other noticeably dominant microbial extremophilic taxa in acid mine drainages

Like other extremophilic environments, the microbial community structure and

diversity of the halophilic environments has also been subject of great scientific curiosity. Several culture dependent and culture independent studies have been carried in past 2–3 decades for the assessment of the microbial diversity thriving within the halophilic environments [57–59]. The research findings from some of the most important studies have been thoroughly reviewed. Studies pertaining to halophilic microorganisms have got greatly benefitted with the implementation of cultivation independent approaches for microbial diversity analyses. Metagenomic analyses of the samples collected from multiple hypersaline systems (e.g. Tyrell Lake, Crystallizer Ponds) have indicated presence of high phylotypic diversity with the dominance of halophilic archaeon in particular [60–63]. The whole DNA pool metagenome sequencing of halophilic samples followed by *de novo* assembly and annotation resulted in discovery of a dominant novel uncultivated archaeal class viz., Nanohaloarchaea [60]. This study also revealed occurrence of a unique combination of amino acids which increase the structural flexibility and osmo-resistance of the protein elements. Another characteristic feature of the genetic resources associated with microbial diversity within halophilic environment was discovered in an independent study and it was observed to be the prevalence of Halo-resistance mechanisms orchestrated through synthesis of solutes (such as glycine, betaine, ectoine and trehalose etc.) that are compatible with high salt concentrations [64].

**3. Extremophilic microorganisms: invaluable source of novel** 

many helpful functions, such as enabling defense mechanisms etc. [66–69],

It is suggested that extremophile enzymes would be more suitable and stable for use in industrial biotechnology applications than those obtained from mesophilic microbial species [9, 21, 70]. Also, the unusual secondary metabolites isolated from extremophilic microorganisms are steadily being characterized as drug molecules with unique potential and applications. One of the recently published studies

Microorganisms surviving in the extreme environments are being looked up to as they could help treat a wide spectrum of human illnesses, from ovarian cancer, migraine, high blood pressure, ovarian cancer and lung cancer to Alzheimer's disease. This doctrine has emerged out of the understanding that extremophilic environments present very hostile conditions that impose serious threat to survival of any organism exposed to them [8, 65]. However, extremophilic microorganisms which thrive under such hostile environment must be doing it by synthesizing unusual, but potentially very useful, secondary metabolites. Probably, the best studied molecules produced by extremophilic microorganisms are (i) biocatalytic proteins that are often referred as extremozymes; and (ii) secondary metabolites that are not directly required growth of the microorganism, yet they often perform

*DOI: http://dx.doi.org/10.5772/intechopen.82639*

**2.4 Halophilic environments**

was Ferroplasm and Thermoplasmatales archaea [55, 56].

*Harnessing the Genetic Diversity and Metabolic Potential of Extremophilic Microorganisms… DOI: http://dx.doi.org/10.5772/intechopen.82639*

metagenomes indicated for molecular mechanisms for acidophilic survival through implmentation of unique carbon metabolic pathways for Carbon metabolism, Nitrogen fixation and iron oxidation [53]. The community composition of Acid Mine Drainage samples were found to have significant contrast to the naturally occuring acidophilic biofilms that has *Acidithiobacillus*, *Acidimicrobium* and *Ferrimicrobium* as the dominant genera present within the community [49, 52, 54]. The other noticeably dominant microbial extremophilic taxa in acid mine drainages was Ferroplasm and Thermoplasmatales archaea [55, 56].

#### **2.4 Halophilic environments**

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

prevalence of hydrogen oxidation genes was observed in hydrogen-rich deep-sea hyperthermophilic vents [38–40]. A few other studies have identified the critical genetic signatures (e.g. genes for alternative mechanisms of nitrogen utilization) of the microbial communities surviving within the thermophilic environments. Some of the recent culture-independent studies on samples collected from thermophilic environments have indicated for the occurrence of the significantly higher diversity of CRISPR compared to the metagenomes of the mesophilic microbial

Even with increasing frequency of reports showing the identification of novel genetic and metabolic mechanisms prevalent in thermophilic environments; the comprehensive understanding about key genetic elements which determine the composition as well the function of the microbial diversity within the thermophilic environments is only poorly understood. It is not yet established how physicochemical factors contribute to shaping up the composition and structure of the microbial diversity of any thermophilic environment. The scenario is expected to improve only through the inclusion of physicochemical information along with full

The psychrophilic environments are characterized by extremely low temperatures. Just like the thermophilic environments, they also represent one of the most thoroughly investigated extreme environments [21, 44]. It is noteworthy that unlike the thermophilic environments, the microbial diversity within psychrophilic environments consists of both eubacteria and archaea [45]. The biodiversity and adaptive strategies of psychrophilic microorganisms have been extensively studied. Results from some of the representative metagenomic studies on the psychrophilic environment have shown microbial community diversity and complexity to be significantly higher than other environments [45, 46]. The most note-worthy studies on psychrophilic environments have been carried out on samples from Antarctic continent, which harbors sub-glacial ice habitat. These studies have reported the dominance of 'chemoautotrophs' that are capable of tapping reduced iron and reduced sulfur compounds as the source of energy [47]. Other studies with psychrophilic environments have recognized the presence of 'chemolithotrophic' bacterial and archaeal communities [45, 47]. These share a close phylogenetic relationship with microorganisms able to use reduced nitrogen, and iron compounds as the source of energy. With regards to the psychrophilic environments, it is generally accepted that 'availability of organic metabolizable carbon' is the single most dominant factor determining the microbial activity, diversity, and dynamics.

Acidophilic environments have emerged as 'extremophilic environments of choice' for studies on mechanisms and genetic elements determining the survival of life under extreme environments. A number of studies had reported attempts for isolation of microorganisms from acidophilic environments. Culture-independent studies with respect to acidophilic environments were first carreid out with a natural acidophilic biofilm sample [48]. Subsequent studies in this regard were carried out on samples collected from an Acid Mine Drainage located at different parts of the world [49–52]. The data obtained with these samples showed the microbial community structure to have a poor diversity with presence of only chemoautotrophic consortia largely comprising members of genera *Leptospirillum* and *Ferroplasm* [48]. The genetic signatures observed within the Acid Mine Drainage

**66**

diversity [41–43].

community metagenome data.

**2.2 Psychrophilic environments**

**2.3 Acidophilic environments**

Like other extremophilic environments, the microbial community structure and diversity of the halophilic environments has also been subject of great scientific curiosity. Several culture dependent and culture independent studies have been carried in past 2–3 decades for the assessment of the microbial diversity thriving within the halophilic environments [57–59]. The research findings from some of the most important studies have been thoroughly reviewed. Studies pertaining to halophilic microorganisms have got greatly benefitted with the implementation of cultivation independent approaches for microbial diversity analyses. Metagenomic analyses of the samples collected from multiple hypersaline systems (e.g. Tyrell Lake, Crystallizer Ponds) have indicated presence of high phylotypic diversity with the dominance of halophilic archaeon in particular [60–63]. The whole DNA pool metagenome sequencing of halophilic samples followed by *de novo* assembly and annotation resulted in discovery of a dominant novel uncultivated archaeal class viz., Nanohaloarchaea [60]. This study also revealed occurrence of a unique combination of amino acids which increase the structural flexibility and osmo-resistance of the protein elements. Another characteristic feature of the genetic resources associated with microbial diversity within halophilic environment was discovered in an independent study and it was observed to be the prevalence of Halo-resistance mechanisms orchestrated through synthesis of solutes (such as glycine, betaine, ectoine and trehalose etc.) that are compatible with high salt concentrations [64].
