*3.1.1 Culture-dependent methods*

In the last century, most of the microbiome studies were based on culturebased methods. Almost for the last 300 years, this approach mainly relied on main identification features like colony features, bacterial growth, and selection of some biochemical typing and microscopic investigation of culturable microbes in lab condition. In the 1980s, large numbers of gram-negative bacterial species were identified from the fecal samples [10]. Later on, many species have been identified and phylogenetically classified by using fermentation profiling or *in vitro* requirements of bacterial species. It has contributed enormously to the identification of microbial agents and given birth to a new branch, i.e., microbial ecology [11]. The culture-based method is still considered as the gold standard protocol for the identification of new species and provided a deep understanding related to the microbial world. They are a cheap and most credible method of bacterial identification. But they could not be proven completely effective against anaerobic and not amenable bacterial species. It is already given that more than 30% of bacterial species cannot be grown outside from their habitat. Moreover, gut microbiota not only includes the bacteria but also consists of bacteriophages, archaea, fungal species, and single-celled eukaryotes. Hence, we need more wide investigative approaches to cover all the microbial agents involved and contribute to the stable form of gut microbiota.

#### **Figure 1.**

*Summary of techniques used to phylogenetic classification and functional characterization of the human gut microbiome.*

**7**

*3.1.3 Microfluidics assays*

*Genomic Techniques Used to Investigate the Human Gut Microbiota*

The significance of culture-dependent methods cannot be undermined for the identification of microbes from the gut microbial community. Therefore, microbiologists have rediscovered and focused once again to revive culture-based methods by adding many sophisticated instrumentations and suitable growth media. This has allowed growing most of the unculturable bacteria that were earlier thought to be impossible in a lab environment. Hence, it will allow to know more about the functional aspects of gut microbiome that include its composition, microbial gene expression, metabolic pathways and host-bacteria relationships [12]. Actually, diverse types of favorable growing and incubation conditions are required to grow unculturable microbes that are provided by the new culturomics procedures. Currently, more than 50% of bacterial species that were earlier identified by classical 16S rRNA metagenomics could be re-identified with the help of culturomics. Simultaneously, it will also allow isolating hundreds of new bacterial species in the

The culturomics is a multistep protocol that includes sample preparations and their diversification under different growth conditions that promote the growth of fastidious bacteria but, simultaneously, also cease the growth of few microbes. The targeted samples are subjected to further MALDI-TOF mass spectroscopy-based investigations such as a comparison of newly obtained protein spectra in recent protein databases. If the applied method could fail to establish the identification of bacteria, then the sample is processed for NGS-based 16sRNA metagenomic methods. Based on the 16sRNA gene sequencing, various toxicogenomics principles are applied to classified new species in phyla or family. Culturomics is quite an effective growth strategy particularly in microbes that are involved in mechanistic networks or intricate host-microbiome interactions. More recently, many culture techniques, for example, gel microdroplets, microculture, and microbial chips, provide very diverse growth conditions; hence, a large number of unknown microbes are able to grow [14]. Although new methods are quite helpful in the identification of new microbial species, e.g., from gut microbial ecosystem, these are also used to study human vaginal and urinary microbiota. Currently, almost 2671 new species have been identified by using culturomics ranging from commensals to pathogens, for example, 31 new bacterial species that belong to *Synergistetes* or *Deinococcus-Thermus* phyla. But, there are certain demerits like nonavailability of suitable culture media and growth conditions that allow the growth of uncultured bacteria in an artificial environment [15]. Moreover, certain bacteria grow in a highly intrigue environment inside the human gut because several microbes use common metabolites as a food and live in symbiotic and mutual interrelationship inside the gut environment.

Microfluidics systems or cell on-chip offers a specific microenvironment for biochemical reactions. Microfluidics comprises numerous microchannels enshrined on the glass or polymer surface such as polydimethylsiloxane [16]. These channels are linked to each other that are based on principles of mixing, pumping, sorting, or offering biochemical environment; hence it can produce a suitable environment for microbial reactions. Recently, great advances have been made in this area; consequently, high-throughput screening, multiplexing, and automation of biochemical reactions could be achieved [17]. Microfluidics technique is also applied in the studies of gut microbiota; hence, some scientists called it gut-on-chip. With microchips, many uncultured microbes are identified because it provides specific growth environment and nutrition required for these bacterial growths, for example,

*DOI: http://dx.doi.org/10.5772/intechopen.91808*

gut microbial ecosystem in the near future [13].

*3.1.2 Culturomics*

### *3.1.2 Culturomics*

*Human Microbiome*

(3) latest techniques are described (**Figure 1**).

*3.1.1 Culture-dependent methods*

**3.1 Phylogenetic analysis of microbial community**

data related to uncultured microorganisms. This helped us in the identification of new microbe species inside the gastrointestinal tract. But there are many important issues associated with the accurate and proper investigation of a gut ecosystem like sample preparation, storage, and handling from the human as well as animal subjects. In the current chapter, total techniques under three major headings (1) culture-dependent methods, (2) culture-independent genomic technologies, and

In the last century, most of the microbiome studies were based on culturebased methods. Almost for the last 300 years, this approach mainly relied on main identification features like colony features, bacterial growth, and selection of some biochemical typing and microscopic investigation of culturable microbes in lab condition. In the 1980s, large numbers of gram-negative bacterial species were identified from the fecal samples [10]. Later on, many species have been identified and phylogenetically classified by using fermentation profiling or *in vitro* requirements of bacterial species. It has contributed enormously to the identification of microbial agents and given birth to a new branch, i.e., microbial ecology [11]. The culture-based method is still considered as the gold standard protocol for the identification of new species and provided a deep understanding related to the microbial world. They are a cheap and most credible method of bacterial identification. But they could not be proven completely effective against anaerobic and not amenable bacterial species. It is already given that more than 30% of bacterial species cannot be grown outside from their habitat. Moreover, gut microbiota not only includes the bacteria but also consists of bacteriophages, archaea, fungal species, and single-celled eukaryotes. Hence, we need more wide investigative approaches to cover all the microbial agents involved and contribute to the stable form of gut

*Summary of techniques used to phylogenetic classification and functional characterization of the human gut* 

**6**

**Figure 1.**

*microbiome.*

microbiota.

The significance of culture-dependent methods cannot be undermined for the identification of microbes from the gut microbial community. Therefore, microbiologists have rediscovered and focused once again to revive culture-based methods by adding many sophisticated instrumentations and suitable growth media. This has allowed growing most of the unculturable bacteria that were earlier thought to be impossible in a lab environment. Hence, it will allow to know more about the functional aspects of gut microbiome that include its composition, microbial gene expression, metabolic pathways and host-bacteria relationships [12]. Actually, diverse types of favorable growing and incubation conditions are required to grow unculturable microbes that are provided by the new culturomics procedures. Currently, more than 50% of bacterial species that were earlier identified by classical 16S rRNA metagenomics could be re-identified with the help of culturomics. Simultaneously, it will also allow isolating hundreds of new bacterial species in the gut microbial ecosystem in the near future [13].

The culturomics is a multistep protocol that includes sample preparations and their diversification under different growth conditions that promote the growth of fastidious bacteria but, simultaneously, also cease the growth of few microbes. The targeted samples are subjected to further MALDI-TOF mass spectroscopy-based investigations such as a comparison of newly obtained protein spectra in recent protein databases. If the applied method could fail to establish the identification of bacteria, then the sample is processed for NGS-based 16sRNA metagenomic methods. Based on the 16sRNA gene sequencing, various toxicogenomics principles are applied to classified new species in phyla or family. Culturomics is quite an effective growth strategy particularly in microbes that are involved in mechanistic networks or intricate host-microbiome interactions. More recently, many culture techniques, for example, gel microdroplets, microculture, and microbial chips, provide very diverse growth conditions; hence, a large number of unknown microbes are able to grow [14]. Although new methods are quite helpful in the identification of new microbial species, e.g., from gut microbial ecosystem, these are also used to study human vaginal and urinary microbiota. Currently, almost 2671 new species have been identified by using culturomics ranging from commensals to pathogens, for example, 31 new bacterial species that belong to *Synergistetes* or *Deinococcus-Thermus* phyla. But, there are certain demerits like nonavailability of suitable culture media and growth conditions that allow the growth of uncultured bacteria in an artificial environment [15]. Moreover, certain bacteria grow in a highly intrigue environment inside the human gut because several microbes use common metabolites as a food and live in symbiotic and mutual interrelationship inside the gut environment.

#### *3.1.3 Microfluidics assays*

Microfluidics systems or cell on-chip offers a specific microenvironment for biochemical reactions. Microfluidics comprises numerous microchannels enshrined on the glass or polymer surface such as polydimethylsiloxane [16]. These channels are linked to each other that are based on principles of mixing, pumping, sorting, or offering biochemical environment; hence it can produce a suitable environment for microbial reactions. Recently, great advances have been made in this area; consequently, high-throughput screening, multiplexing, and automation of biochemical reactions could be achieved [17]. Microfluidics technique is also applied in the studies of gut microbiota; hence, some scientists called it gut-on-chip. With microchips, many uncultured microbes are identified because it provides specific growth environment and nutrition required for these bacterial growths, for example,

microfluidics-based model (human-microbial cross talk (HuMiX)). The HuMiX provide gastrointestinal-like environment for the co-growth of human epithelial cell and obligate anaerobe *Bacteroides caccae* cells [18]. Recently developed iChip containing multiple microchambers which are further divided into hundreds of miniature multiple cells has been used to grow bacteria. This technique mainly acts by providing a selective supply of nutrients to an inoculated single bacterial cell onchip. Another chip-based method l-tip also acts on the same principles as iChip, but it allows bacterial cell multiplication in a gel and supplies required nutrients which are essential for growth [19]. Microfluidics is the combination of gel-based methods and sophisticated instruments, for example, first we grow a single bacterial cell, then amplify its genome, and, finally, sequence its genome that helps in identifying new species [20]. Recently, TM7, bacterium, and *Sulcia muelleri* could be identified which produced very unique metabolites. By using the same method, 34 various bacterial strains are identified and phylogenetically classified.
