Culture-based methods in microbial ecology: limitations
As culture-dependent assessment favors only fraction of microbial community, these methods failed to provide infonnation on whole bacterial community present in an environmental sample. The “great plate count anomaly” by Staley and Konopka (1985) clearly proved that the diversity of bacteria that is observed under microscope and grown in petri dishes shows vast disparity. The major problem with culture- depeudeut method arises from the fact that artificial medium in any laboratory supports the growth of only a fraction of total organisms. The reason for divided growth may be sunnised by the fact that: (i) laboratory conditions failing to support ecological niches that are encountered in complex environment (ii) as replication rate varies for bacterial species, the culturable fraction deforms during growth because fast growing species outcompete the slow growing ones (iii) growth requirements such as nutrients and their concentrations, optimum pH, osmotic conditions and temperanire of all microbial species are unknown and unpredictable (iv) interdependency of bacteria on then host or each other, viz., co-culturing and endosymbiotic relationship cannot be provided in the laboratory conditions.
The diversity of bacteria with its unique and expanded biosynthetic pathway leading to differentiated growth conditions and parameters restricted researchers to identify wiiole bacterial community present in a particular environment. With increase in knowiedge and understanding of bacterial ecology, various methodological improvements were adapted. These developments include the simulation of environmental parameters at laboratory conditions to increase the chance of cultivation. However, at the end, only 1% of the total microbial community is cultivable in any environment.
Culture-independent/molecular methods for microbial diversity analyses
Culture-dependent epoch gives a huge collection of bacteria and provides information on genetics, physiology and evolution of grown bacteria. However, huge disparity between cultured and actual diversity has increased the importance of culture-independent methods. In addition to this, culturing takes several days for analysis. The utilization of advanced techniques and molecular approaches (Fig. 3) publicized that the bacterial diversity is far superior than predicted until now. The diversity through culture-independent analysis involves PCR based methods (DGGE, T-RFLP. ARISA, ARDRA and SSCP) and non-PCR based methods (Fluorescence In Situ Hybridization (FISH) and DNA microarray). In general, these techniques include isolation of metageuome (genome of total microbiota) directly from the environmental source, amplification of molecular marker and finally the sequencing of nucleic acid that determines the bacterial diversity. The principal target for assessmg the bacterial diversity has been 16S rRNA gene. Other functional genes such as one coding RNA polymerase В (rpo В), methane mono-oxygenase (mmo A), nitrogenase (nif H), nitrite reductase (nir S/nir K) and ammonium mono-oxygenase (ото A) are effectively employed as markers to delineate bacterial diversity (Nocker et al. 2007).
PCR based techniques
The advent of polymerase chain reaction (PCR) by Mullis et al. (1986) led to the rapid and efficient improvement in molecular studies. Recombinant DNA technology and genetic engineering has PCR and cloning as their core processes. PCR targeting molecular markers such as prokaryotic 16S rDNA, eukaryotic 18S rDNA and internal transcribed spacer (ITS) region have been extensively studied for identification and prediction of phylogenetic relationship and therefore to explore the yet uncultured bacterial community. The major culture-independent techniques that rely on PCR are clone library, DGGE, T-RFLP. ARDRA. ARISA and SSCP.
The most suitable method to analyze bacterial diversity is to amplify the 16S rRNA gene from an environmental sample and then to determine the sequence of individual gene (DeSantis et al. 2007). Obtained nucleotide sequences are compared with known sequences in public database and allocated to most relative taxonomic group.
First step in this method involves amplification of nearly full-length 16S rRNA gene from metagenomic DNA of an environmental sample. Amplified and purified DNA fragments are cloned into ТА cloning vector (e.g., pGEM-T Easy), followed by transforming Escherichia coli DH5a competent cells. A known volume of transformed cells is spread plated on agar plate containing appropriate antibiotics. The insert from positive colonies is re-amplified and sequenced by standard Sanger’s dideoxy method.
In diversity assessment, the major limitation of a typical clone library of 16S rDNA is analysis and sequencing of individual clones. A typical clone library of 16S rDNA contains nearly 1,000 clones that constitute only small portion of the overall diversity of an environmental sample. Previous smdy by Dunbar et al. (2002) has shown that an environmental sample, viz., soil necessitates the analysis of more than 40,000 clones to document 50% of total richness.
Denaturing or temperature-gradient gel electrophoresis (DGGE/TGGE)
DGGE is a molecular fingerprinting technique employed for determination of complex microbial diversity. This technique distinguishes short fragment of DNA based on their melting characteristic and applied to ascertain sequence variations in number of genes from various organisms. It relies on the fact that during electrophoresis, single stranded DNA migrates slower than double stranded similitude due to elevated interaction of exposed nucleotide to the gel matrix in the single stranded molecule. The procedure includes the electrophoretic migration of bacterial 16S rDNA fragments in polyaciylamide gel containing a linear increasing gradient of chemical denaturants (usually urea and formamide). As double stranded DNA molecule passes through the gradient of denaturants, at a particular denaturant concentration, a transition from helical to partially melted molecules occur (DNA molecules start to denature) and thereby then migration is retarded due to newly acquired branched structure (Fig. 4a). The differences in melting behavior cause the DNA fragments to stop migr ating at different position in the denaturing gel and thereby effective separation. The denaturation depends on %GC content and therefore in this technique. DNA fragments that are identical in length but different in sequences can be separated.
DNA molecules containing low GC content may totally separate into single stranded (complete denaturation) and may not form any detectable band. Therefore, in a later modified method (Sheffield et al. 1989), to increase the detection sensitivity of single-base variation by DGGE, a 40-base-pair G+C-rich sequence (designated as GC-clamp) was incorporated by PCR onto the 5'-end of one of the primers. This results in partially melted structure in which GC-clamp prevents the complete denaturation (Fig. 4b).
This technique became very popular and has been successfully applied for microbial ecological studies such as profiling community complexity (Muyzer et al. 1993, Viszwapriya et al. 2015), to compare DNA extraction methods (Ariefdjohan et al. 2010, Dilhari et al. 2017), to study seasonal variation (Alonso-Saez et al. 2007, Oberbeckmann et al. 2014), bio fouling diversity (Ivnitsky et al. 2007, Belgini et al. 2018, Rajeev et al. 2019) and to assess the impacts of anthropogenic activities on bacterioplaukton population (Jeffries et al. 2016).
One of the major limitations of this method is the separation of relatively short (500 bp) DNA fragments and therefore it is most commonly applied to analyze one or two of nine hyper-variable regions (Vj-V9) of 16S rRNA gene, unlike other fingerprinting techniques that target the analysis of fiill-length 16S rRNA gene. PCR primers targeting the hypervariable region (V3, V3-V4, V6, V6-Vg and Vj-V3) are
Fig. 4. Illustration of denaturing gradient gel electrophoresis: (a) Parallel DGGE gel showing increasing gradient (top-bottom) of denaturant (urea and fonnamide). Heterogeneous population of amplified hypervariable-16S rDNA fragments electrophoresed parallel to the direction of electrophoresis. Once the DNA molecule reaches at particular concentration of denaturant in the gel, molecules start to denature and their migration halt. High content of GC needs higher concentration of denaturant to achieve denaturation. (b) Generally, molecules containing lower' GC content may not form any band due to complete denaturation; to prevent it, a GC-rich (typically 40 base pair- long) DNA is added to the 5'-end of PCR amplicons. (c) DGGE employed to characterize and compare the planktonic (lane 2) and biofilm-forming (lane 3) bacterial diversity on artificial surfaces at the south coast of India (Rajeev et al. 2019). Metagenomic DNA extracted from both communities were used as templates for PCR amplification of hypervariable Y3 region (-180 bp) of 16S rDNA and were applied onto 11% polyacrylamide gel. Each gel contained a linear gradient of denaturants urea and fonnamide (100% denaturant concentration corresponds to 7 M urea [w/v] and 40% [v/v] deionised fonnamide). Electrophoresis was performed at constant voltage (100 V) and temperature (60°C) for 15 h in IX TAE buffer using INGENYPHORU system (Tire Netherlands). After electrophoresis, the gel was stained for 45 min in ethidium bromide (0.5 pg ml"1), rinsed for 30 min in IX TAE buffer and DGGE profile was visualized using gel documentation system.
used to amplify the intervening fragment(s) of 16S rDNA from uncharacterized bacterial community. The heterogeneous mixture of PCR amplified genes (mixed PCR products) is then separated by parallel DGGE. This allows the identification, comparison and relative abundance of dominant taxa present in any environmental sample (Fig. 4c).
In DGGE, each band represents a single microbial species (phylotype) and its intensity corresponds to the abundance. However, methodological limitations of DGGE should be noted. For example, DGGE fingerprinting relies on PCR amplification of 16S rDNA, which itself can be present in multiple copies with variation in sequences (Nubel et al. 1996) and therefore represent multiple bands even from single bacterial species. Similarly, co-migration of bands containing different DNA sequences but similar melting behavior can underestimate the community composition (Casamayor et al. 2000). Temperature gradient gel electrophoresis (TGGE) is another variant and relies on the same principle of DGGE except that a temperature gradient is applied to separate the DNA molecules rather than chemical denaturant.
Most of the published reports have targeted the hyper-variable V3 region (Muyzer et al. 1993, Viszwapriya et al. 2015) of the 16S rRNA gene, as it is a relatively short fragment containing higher nucleotide diversity. The major advantages of these techniques are then affordability for ordinary laboratories, fast and the relative easy interpretation of the results. Moreover, individual bands of interest can be excised from the gel and representative phylotype can be identified through sequencing.