Microbial Diversity in the Genomic Era

Japan

Preface

Surajit Das and Hirak Ranjan Dash

Microbial world is considered to be the largest unexplored reservoir of biodiversity on earth. Microbial systematics and phylogeny are the foundations and guiding light to understand different aspects of microbiology. Owing to the practical significance of identification, diversity, and species designation of microorganisms, microbial genomics offer novel insights for intraspecies variation and development of a potential tool for microbial diversity studies. In this regard, microbial genomes are considered to be highly dynamic and diverse due to the occurrence of horizontal gene transfer (HGT) which adds complexity in terms of understanding the bacterial genome as well as their evolution. Genomics has completely revolutionized every aspects of microbiology. The pioneer work of Carl Woese et al. of comparative analysis of small subunit of rRNA revolutionized the understanding of the species diversity and evolutionary relatedness. However, a uniform gold standard methodology for species designation and study of microbial phylogeny is still lacking.

Over the years, microbiologists have witnessed a paradigm shift in microbial diversity research owing to the development of genetic analyses and genomics studies of microorganisms. The data generated by application of genomics to microbiology should be dealt with multidisciplinary approaches by integrating microbiology with bioinformatics, statistics and mathematical approaches. Advancements in genomic research has led to the discovery of novel diagnostics, discovery of new bioactive compounds, application in bioremediation of toxic elements, and many other discoveries. However, with the increasing knowledge, we realize that we are just scrabbling the surface of the microbial world. Enhanced genetic information is challenging our existing knowledge of bacterial species designation, typing systems as well as the microbial taxonomy.

First, bacterial genome was sequenced in 1995 in Haemophilus influenza. Afterward, significant advancements have been taken place in the field of genomics and sequencing technologies for the assessment of microbial taxonomy and phylogeny. The technological progress has led the advanced techniques to completely dominate the conventional techniques for microbial diversity analysis. Thus, the aim of this book is to discuss about where we stand in the genomics era with respect to the microbial diversity, species designation, and typing. There are many techniques used to determine the genetic relatedness of microorganisms, and this book aims to discuss the techniques used for microbial taxonomy and phylogeny with their applications and respective pros and cons. Though much advanced techniques are available in genomics era for the identification of any unknown bacterium, still a lot of undiscovered microorganisms exist in the world. Thus, the assessment of microbial taxonomy and biosystematic techniques discovered and practiced in the current genomics era with suitable recommendations is the prime focus of this book.

The chapters of this book have been written by the authors with vast research and academic experience in the field of microbial diversity, molecular biology, genomics, and evolution. The whole book has been divided into six sections. Section I introduces the microbial diversity and describes various principles to study diversity in different ecosystems. Section II describes various molecular tools employed to study microbial diversity such as multiplex PCR, omics approach, next generation sequencing, and polyphasic taxonomy. Extremophiles play an important role in maintaining the biogeochemical cycles in the environment and are a huge source for biotechnological applications. Section III describes different genomic tools to study the extremophilic microbial diversity and exploitation of psychrophiles, thermophiles, acidophilic, and alkaliphilic microbes. Huge diversity of microbes imparts varied functions in the environment, and Section IV of the book describes the functional microbial diversity and their study using advanced genomics tools. Section V describes various genomics tools and techniques to study the diversity of microbes associated with infectious diseases and to deduce various preventive measures to promote significant medical and public health. Finally, the limitations and opportunities of the currently available genomics techniques and future directions for microbial diversity studies have been discussed in Section VI.

We have tried with our best possible efforts to accommodate the currently available knowledge, content, research advancements in the field of microbial diversity to an extent suitable for the students, budding and seasoned researchers, medical professionals, policy makers, and practitioners in the field of environmental and medical microbiology. Hope, this book will serve as a key point of reference to various stakeholders involved in microbial diversity study and research.

Section I

Overview of Microbial Diversity

Outline

Chapter 1 Methods of Assessment of Microbial Diversity in Natural Environments

Chapter 2 Metagenomic Achievements in Microbial Diversity Determination in Croplands: A Review

Chapter 3 Genomic Diversity and Evolution of Rhizobia

Chapter 4 Microbial Biodiversity Study of a Brackish Water Ecosystem in Eastern India: The Chilika Lake

Chapter 5 Microbial Diversity and Community Analysis of the Sundarbans Mangrove, a World Heritage Site

Chapter 1

Methods of Assessment of Microbial Diversity in Natural Environments

Anwesha Ghosh and Punyasloke Bhadury,    Integrative Taxonomy and Microbial Ecology Research Group, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, West Bengal, India

Abstract

Understanding structure and function of microbial communities within an environment is a key to understanding processes and fluxes which ultimately have consequences from food security to global climate. In the modern era of microbial ecology, molecular tools offer ways to assess structure and diversity of microbial communities. Moreover, molecular tools can also aid in developing strategies for culture-based approaches to explore microbes. More recently, advent of next-generation sequencing (NGS) has revolutionized our understanding of microbial communities across a range of environments. This chapter discusses some of the tools used in assessing microbial diversity with examples particularly from aquatic bacterioplankton communities. A deeper insight into aspects including applicability of NGS has been also highlighted in this chapter with relevant examples.

Keywords

Microbial diversity; cultured approaches; next-generation sequencing; data processing; third generation sequencing

1.1 Introduction

Microbes are ubiquitous across all environments, mediate major ecosystem level processes, and also represent a large unexplored reservoir of biodiversity (Whitman et al., 1998). In natural environments, microbial communities mediate cycling of major elements and can effectively degrade pollutants (Falkowski et al., 2008; Fuchs et al., 2011). Given the functional importance of microbial communities in natural environments and their potential for application in various areas of biotechnology, therefore it is very important to gain an understanding of their community structure.

Until the late 1970s, microbial communities from natural environments were characterized primarily on culture-based approaches. The basic approach to culture methodology involves enrichment and isolation of pure cultures in defined growth media followed by physiological and functional characterizations. In this approach, several modifications have been undertaken with respect to specific growth curve including oxygen concentration gradient (Emerson et al., 2005). Moreover, culture-based approaches can lead to enrichment bias due to rapid growth of fast growing microbes, and therefore members with slow metabolism that make up microbial communities within a natural environment cannot be accounted for in cultures. Post 1970s, it became evident that less than 1% of the total number of microbes can be cultured under laboratory conditions (Hugenholtz, 2002). Therefore, culture-dependent approach may not represent natural microbial communities, and thus their functional roles in natural environments may get underrepresented (Rappe and Giovannoni, 2003).

In the early 1990s, a series of seminal work on microbial ecology led to the foundation and advancement of molecular microbial ecology along with development of molecular tools. The fundamental work undertaken by Norman Pace and his group established the application of molecular markers (e.g., ribosomal RNAs) for phylogenetic characterization of microbes (Lane et al., 1985; Olsen et al., 1986). Similarly, the invention of polymerase chain reaction (PCR) by Karry Mullis and DNA sequencing by Frederick Sanger et al. revolutionized the field of biological sciences. As a result of these findings along with other protocols such as extraction of total nucleic acids across various natural environments (Tsai and Olson, 1991; Griffiths et al., 2000), there has been development of culture-independent tools to study the diversity and dynamics of microbial communities in a much greater resolution.

As a result, molecular tools can help toward addressing fundamental questions pertaining to microbial communities in natural environments such as (1) what type of microbes are present in a natural environment and how these can vary temporally and spatially? (2) What are the functions of microbes in a natural environment? (3) How environmental parameters can shape the structure and function of microbial communities in a natural environment? To address some of these fundamental questions, methods that utilize constituents of a microbial cell such as the nucleic acids or fatty acids can provide vital information on diversity of microbial communities within a natural environment including functional perspectives.

In comparison to culture-dependent processes, methods which integrate molecular tools can provide better resolution of in situ composition, diversity, and metabolic information of microbial communities across natural environments. In this chapter, an overview has been provided on some of the molecular tools that be used to study microbial diversity in natural environments with examples particularly from marine environment. A general overview on the culture-based approaches has been also discussed. Most importantly, this chapter also provides in-depth information on the NGS-based approaches to study microbial communities.

1.2 Analysis of Microbial Communities Based on Cultured Approaches

The analysis of microbial communities from natural environments has progressed by focusing commonly on process level information. Traditionally, these have involved determination of biomass and respiration rates and expanded to studies on enzyme activities (Hill et al., 2000). The most widely used method relies on the use of variety of culture media. Bacteriologists are well accustomed to the rich medium called Luria–Bertani broth which allows fast growth of a vast number of bacterial species. Design and use of culture media such as Luria–Bertani has provided insights into the commonly known bacterial physiological states including log phase, exponential phase, stationary phase, and death phase. This has further allowed for the calculation of bacterial doubling time and average cell mass. In a well comprehended article by Sezonov et al. (2007), the authors have shown Escherichia coli K-12 strain MG1655 isolated originally from the environment attain steady state at Optical density (OD) (600 nm) of 0.3. Additionally, the authors showed growth of E. coli K-12 ceased at OD600=7. Removal of the cells by centrifugation and reinoculation following addition of glucose shows carbon to be the limiting metabolite in Luria–Bertani media. Such studies have also helped clarify ideas of bacterial steady state. Denbigh (1951) defined the steady state as a time-invariant condition of a system that is open to its environment.

The number of colonies that grow on petri plates was far outweighed by the number of cells that were observed microscopically (Amann, 1911), thereby introducing the concept of unculturable bacteria. This highlighted the lack of critical information regarding bacterial physiology which posed a great challenge in isolating novel bacteria from various environments, particularly from extreme environments (Stewart, 2012). This challenge was overcome to a large extent by the introduction of growing bacteria in their natural environment. The technique of isolation of bacterial cells into sterilized natural water or other media is being frequently used (Schut et al., 1993; Button et al., 1993).

A methodology commonly applied involves preparing dilution cultures by dilution of the original population to near extinction (Button et al., 1993). It is considered that this allows for the growth of oligotrophic bacterial communities compared to traditional culture methods such as agar plates which promote the growth of nutrient-tolerant bacteria. Use of natural seawater has also been employed to establish high-throughput culturing techniques (Connon and Giovannoni, 2002). The authors recorded a range of 0.4%–14.3% culturability from 2484 extinction wells. This technique has helped toward identifying previously uncultured members of SAR11, OM43, SAR92, and OM60/OM241 bacteria phyla across marine environments. Stewart (2012) has extensively discussed some of these methodologies essentially shedding light on alternate ways to study physiology of these unculturable bacteria. The use of diffusion chambers allowed for mimicking the natural settings of microorganisms (Kaeberlein et al., 2002). The authors were able to recover 40 ± 13% of the cells inoculated from intertidal marine sediment. Additionally, they reported two new isolates MSC1 and MSC2. The closest known relatives of MSC1 and MSC2 were Lewinella persica (Order: Sphingobacteriales) and Arcobacter nitrofigilis (Order: Campylobacterales), respectively.

These alternate techniques can help identify members of bacterioplankton from freshwater and marine environments that do not usually grow on culture plates. The most widely accepted reason for the the great plate count anomaly has been attributed to the inability of dormant bacterioplankton cells to adapt to culture plates (Connon and Giovannoni, 2002). Additionally, many of the oligotrophic bacteria generally found in seawater are adapted to fastidious conditionally including those of very diluted nutrient conditions (Button et al., 1993; Connon and Giovannoni, 2002). The high nutrient media used in agar plates often tend to inhibit the isolation of such oligotrophic bacteria by traditional plating techniques. But these techniques are relatively laborious and result in identification of limited number of novel bacteria after prolonged effort encompassing long time periods. Modifications in culture-based methods have increased the isolation of nearly 14.3% of marine bacteria from 0.01% to 0.1% usually found on agar plates (Kogure et al., 1979). In order to gain greater insights into the bacterioplankton communities, the focus shifted to using culture-independent molecular techniques which helped define nearly 92 named phyla within the bacteria domain known as of today (Hug et al., 2016).

1.3 Culture-Independent Techniques

Culture-independent techniques using molecular tools are used to determine different types of microbes such as bacteria that constitute overall microbial communities within an environment. A number of different techniques including clone libraries, terminal restriction fragment length polymorphism (T-RFLP), denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), and NGS [Roche 454 sequencing, Illumina MiSeq and Illumina HiSeq, Pacific Biosciences (PacBio)] are used to elucidate diversity and composition of microbial communities from natural samples. Culture-independent or molecular approaches generally proceed by direct amplification and sequencing of the 16S rDNA molecule. The use of 16S rDNA in community level analysis is made possible due to its ubiquitous presence across all groups of known bacteria. Ever since the proposal by Woese and Fox, the 16S rDNA molecule has been used as molecular clock to track phylogenetic relationships between different groups of bacteria. This molecule is now considered the gold standard in microbial ecology studies (Case et al., 2007). However, one of the primary concerns with the use of 16S rDNA is the variable copy number within genomes of different bacteria. This has led researchers to suggest the use of single copy number genes for microbial community analysis. Case et al. (2007) showed that use of rpoB (beta subunit of bacterial RNA polymerase) could yield phylogenetic resolution comparable to that of the 16S rDNA. In fact at lower taxonomic levels such as species and subspecies, rpoB appears to provide higher phylogenetic resolution than the 16S rDNA molecule. In the following sections, however, the discussion has been focused with respect to 16S rDNA molecule with examples from bacterioplankton communities across environments since this is more widely used as a molecular clock compared to other molecular markers.

1.3.1 Clone Library and Sequencing Approach

The most widely used approach to study microbial communities such as bacterioplankton communities precedes by generation of clone libraries of 16S rDNA (Cottrell and Kirchman, 2000). The ubiquitous presence, availability of primers binding to conserved region, and ease of differentiation between species owing to variable and hypervariable regions make the 16S rDNA a instead molecular marker. Environmental DNA extracted from natural samples is used as a template in PCR to amplify the 16S rDNA which is then cloned, and clone libraries are generated. Partial or full length sequences of these insert containing clones are then obtained using Sanger sequencing approach. Such sequence data that is obtained in the form of chromatograms need careful analyses. Owing to the robust datasets of 16S rDNA that is publicly available today, it is possible to ascertain identity of individual sequences (thereby the clones) to already known groups of bacteria. Availability of easy-to-use tools such as blastn and RDP Classifier (available on the RDP website) makes it possible to affiliate the clones accurately to species level. The sequence information can then be used to undertake molecular phylogenetic studies in order to answer questions pertaining to community composition. Molecular phylogeny based on 16S rDNA sequences has provided insights into diversity and has revealed habitat specificity of bacterioplankton groups (Page et al., 2004). For example, surveys of bacterioplankton communities have revealed that freshwater systems are dominated by β-proteobacteria. Yet, there are numerous examples where the use of clone library and sequencing approach has elucidated the presence of novel bacterial clades, thereby highlighting the uniqueness of bacterioplankton communities across aquatic habitats. A study carried by Larson and Giovannoni (2001) described two novel bacterial clusters, namely, CL120-10 (a cluster affiliated to Verrucomicrobiales) and ACK4 (Actinomycetes) in the ultraoligotrophic Crater Lake, USA. Additionally, the authors showed the presence of different groups of bacterioplankton along a vertical profile of the studied lake. They also found the globally distributed freshwater clade of Polynucleobacter to be absent in Crater Lake during their study period. In another study undertaken by Tang et al. (2015) in Lake Bosten in China also reported similar result. During the studied period, the bacterioplankton community in Lake Bosten was represented by one only sequence of Polynucleobacter. In contrast to freshwater ecosystems, bacterioplankton community compositions from coastal oceans appear to be dominated by α-proteobacteria. Studies conducted in coastal California Pacific Ocean (Cottrell and Kirchman, 2000), Southwestern Atlantic Ocean (Piccini et al., 2006), Blanes Bay (Alonso-Sáez et al., 2007), Chukchi Sea (Malmstrom et al., 2007), and Changjiang estuary (Feng et al., 2009) revealed the dominance of α-proteobacteria across these sites during the study period. Abundance of other bacterioplankton classes such as Gammaproteobacteria has been found in Mooriganga estuary of Sundarbans (Ghosh and Bhadury, 2017). This indicates the fine-scale resolution of the bacterioplankton communities that can be obtained using molecular approach.

Irrespective of the advantage of clone libraries in phylogenetic resolution, the process of generation and analyses using a clone library process is expensive, time-consuming, and laborious (Sanz and Kochling, 2007). Additionally, this process suffers from the obvious biases introduced by PCR. Acinas et al. (2005) highlighted the possible efforts of PCR biases on the overall bacterioplankton community compositions and thus can lead to alteration of microbial diversity across natural environments. The authors observed a decrease in number of unique 16S rDNA sequences (from 76% to 48%) upon reduction in cycles from 35 to 15 + 3. Errors introduced by Taq DNA polymerase also resulted in higher observed diversity in microbial communities. In account of such observations, the authors suggested a decrease in the number of PCR cycles and an additional reconditioning step while performing 16S rDNA PCR. The modified protocol allows for enhanced coverage index, i.e., sequencing fewer number of clones would allow for a clear snapshot of microbial communities. Owing to such difficulties, methods such as DGGE have become more popular to address questions on seasonal changes in microbial communities across a range of environments.

Use of the clone library and sequencing approach proceeds through a PCR step. It has been considered on multiple occasions that the use of PCR often introduces bias. This could lead to differential amplification of only dominant constituents of overall microbial communities. Other factors such as presence of extracellular DNA, inhibition of PCR due to presence of contaminants and formation of artifacts such as chimeric sequences resulting from PCR could be important factors to be considered before determination of microbial communities from different study sites (Wintzingerode et al., 1997). The review article by Wintzingerode et al. (1997) discusses these points extensively and can give a good overview to the readers to address challenges pertaining to PCR.

1.3.2 Terminal Restriction Fragment Length Polymorphism

T-RFLP is a community fingerprinting technique which has been frequently used to study microbial communities across a range of environments including bacterioplankton from marine environments (Stoica, 2009). The 16S rDNA fragment is amplified by using a 5′ fluorescent primer, and the generated amplicons are digested into smaller fragments by using restriction enzymes. This results in terminal restriction fragments of distinct lengths from which the method takes its name (Avaniss-Aghajani et al., 1994). The digested amplicons are separated by polyacrylamide gel electrophoresis or by running it in a capillary gel electrophoresis apparatus equipped with a fluorescence detector. A series of peaks representing fragments of different sizes are obtained that is a proxy for microbial species composition from the study site. Comparison of the number and type of peaks across samples can give an idea of the composition and diversity of microbes across study sites (Liu et al., 1997; Dunbar et al., 2001). T-RFLP analysis has been considered as a rapid, convenient, sensitive, high-throughput, and a highly reproducible method for studying microbial communities (Kitts, 2001). T-RFLP laser detection systems can detect bands and peaks containing as less as <0.1% of the total loaded DNA, thereby being a sensitive fingerprinting method (Hewson and Fuhrman, 2004).

A study in Constanta Bay which is a shallow coastal area of the Black Sea was monitored using T-RFLP. The authors used the restriction enzyme HhaI to digest the PCR amplicons and separated them using an ABI Prism 310 automated capillary sequencer. T-RFLP measurements yielded a total of 74 (operational taxonomic units) OTUs which comprised of both members belonging to bacteria and archaea. A total of 18 bacterial OTUs could be detected across all studied samples, and 16 OTUs were shared at a temporal scale. This method has been found to be highly reproducible and allows for a clear difference between bacterial and archaeal diversity from across samples. Questions on ocean acidification have also been addressed using T-RFLP. Zhang et al. (2013) studied the response of pCO2 gradient on bacterioplankton community structure. Use of this method allowed for analysis and comparison of 159 samples from across 19 sampling points. The T-RFLP patterns were generated by digesting with RsaI. The report suggested that increase in pCO2 levels in the ocean may not directly affect marine bacterial community, and this could have implications for overall functioning of the studied ecosystem. This indicates the ease of this method and its efficiency in detecting board changes in bacterioplankton community structure.

Apart from the known possibilities of underestimation of diversity by T-RFLP (Liu et al., 1997), biases may also arise during sample collection, DNA extraction steps, and PCR (Osborn et al., 2000; Urakawa et al., 2001; Denaro et al., 2005). Biases may also arise due to the choice of restriction enzymes used in T-RFLP analysis. Zhang et al. (2008) used five restriction enzymes (AluI, HaeIII, MspI, Sau3AI, and TaqI) to assess biases associated with the choice of use of digestion enzymes. The study was performed on surface and bottom water samples collected from Victoria Harbor in Hong Kong. The authors found highly variable species richness and diversity indices even though the bacterial community structure remained consistent by the use of different digestive enzymes. Additionally, it has been also argued that T-RFLP provides low phylogenetic resolution as it relies upon a few sequence heterogeneity (Hewson and Fuhrman, 2004). The choice of enzyme and other biases hence may become critical when detecting microbial communities and estimating their diversity within an environment using T-RFLP.

1.3.3 Denaturing Gradient Gel Electrophoresis

The process of DGGE, first described by Muyzer et al. (1993), is a widely used method to analyze genetic diversity of microbial communities. Electrophoresis of PCR-amplified 16S rDNA fragments is performed in polyacrylamide gels prepared with a linearly increasing gradient of denaturants. DGGE is based on the melting of DNA molecules. Once the DNA molecule reaches specific melting temperature, the transition from helical to partially melted state ceases its movement through the gels. As DNA melting temperatures are sequence dependent, sequence variants of the same sized DNA molecule halts are different positions on the polyacrylamide gel (Lerman, 1986). The review article by Lerman (1986) provides an extensive idea on the concepts of DNA melting based on sequence specificity.

One of the primary factors that might affect the results of DGGE is the choice of primers. Sánchez et al. (2007) have used five different primer sets to study the bacterioplankton community in Blanes Bay, Spain, which represents an oligotrophic coastal ecosystem. The authors have shown the representation of bacterioplankton communities by amplification of 16S rDNA by the use of different primer sets. Additionally, it is difficult to detect rare bacterioplankton groups (>0.5%–1% of the total bacterial community) (Kan et al., 2006). Nevertheless, the DGGE technique has proved to be a useful tool in studying bacterioplankton populations from a variety of aquatic habitats including lakes and rivers (Øvreås et al., 1997; Casamayor et al., 2000) to polar regions (Crump et al., 2003).

DGGE has been used in numerous aquatic habitats to study bacterioplankton communities and understand their response to specific environmental parameters. Kan et al. (2006) monitored interannual changes in three stations along Chesapeake Bay, USA over 2 years using DGGE of amplified 16S rDNA. The authors found significant changes in bacterioplankton community structure on a seasonal scale, but repeatable annual patterns over the studied years could be observed. DGGE banding patterns showed higher diversity (higher number of unique bacterial groups) in spring compared to that of summer and fall. The authors could identify common bands across interannual samples indicating that common bacterial groups existing during the same season across years. Similarly, the appearance of unique bands in the polyacrylamide gel indicated the presence of new bacterial groups in the ecosystem during that particular sampling time. A similar study conducted by Kan et al. (2006) highlights the use of DGGE in discerning difference in bacterioplankton communities on a monthly scale in Baltimore Inner Harbor, USA. This site is connected to Chesapeake Bay by Patapsco River, which is the fifth largest tributary of the Chesapeake Bay. Compared to the previous study where the authors found Gammaproteobacteria to be absent during winter in Chesapeake Bay, the present study found Gammaproteobacteria to be present only in winter and early spring. Hence, it can be seen that even when two of these studied sites are only ~22.5 km apart, the bacterioplankton communities are largely variable, and these differences can be resolved using techniques such as DGGE.

1.3.4 Fluorescence In Situ Hybridization

FISH is a method to determine bacterial community composition using 16S rDNA probes thereby eliminating the PCR step (DeLong et al., 1989). This method is based on binding of oligodeoxynucleotide hybridization probes complementary to rDNA sequences. Such probes are tagged with fluorescent dyes that are used for the detection and phylogenetic characterizations of microorganisms under the microscope. The first report by DeLong et al. (1989) hypothesized that the high abundance of cellular ribosomes in actively growing bacteria (10⁴–10⁵ per cell) could allow for direct observation under the fluorescence microscope. The rDNA binding probes are phylogenetic group specific in nature. The authors tested this technique on a mixture of Bacillus megaterium (an eubacterium under Bacillales) and Saccharomyces cerevisiae (an eukaryote) and could observe both cell types by phase contrast microscopy (refer to DeLong et al., 1989 for further details on microscopic observations).

This technique has since been used to study microbial community composition across a range of environments. By coupling with clone library approach, FISH has been used to address questions on broad biogeographic patterns of microbial communities including bacterioplankton communities. Cottrell and Kirchman (2000) used the coastal California bacterioplankton communities to investigate the dominance of α-proteobacteria and Cytophaga–Flavobacteria in marine ecosystems. Direct microscopic analysis using FISH showed a high abundance of Cytophaga–Flavobacteria, whereas clone libraries indicated a dominance of α-proteobacteria. The authors suggested that choice of primers and the lack of amplification by general primers could explain the difference in observed results. This indicates that it may be important to use different uncultured approaches in cohesion to study patterns and diversity of microbial communities across environments, but labor, time, and cost could also become important factors while undertaking a multifaceted approach.

The use of fluorescence probes is often restricted by weak signal produced which cannot be detected (Amann et al., 1990; Lee et al., 1993). This is largely due to slow growth of bacteria in natural environments which tends to result in low rDNA (Lee and Kemp, 1994). There have been attempts to circumvent such problems. For example, in a report by Ouverney and Fuhrman (1997), the authors used chloramphenicol, a broad-spectrum antibiotic, to boost fluorescence signal intensity of problems binding to marine bacteria. They reported an increase in fluorescence upon such treatment in samples collected from three different sites along the Southern California coast. A review by Bouvier and del Giorgio (2003) has extensively discussed the factors that influence detection of bacterial cells using this technique. The authors have discussed the use and effectiveness of EUB338 probe, which is one of the most widely used FISH probes to study bacterial communities from aquatic habitats.

1.4 Next-Generation Sequencing (NGS) or High-Throughput Sequencing (HTS)

With the advent of NGS, the limitations of the cultivation or commonly used fingerprinting techniques could be circumvented (Shan et al., 2015). The rapidly decreasing cost of NGS has largely promoted the use of this sequencing technique in studying diversity and functions of microbial communities across a range of environments. The commonly used sequencing approach involves sequencing of only one molecular marker (for e.g., 16S rDNA) in which case the process is called metabarcoding. Sequencing of multiple molecular markers for elucidation of microbial communities is generally referred to as metagenomics. Use of molecular markers to understand functional aspects is commonly referred to as metatanscriptomics.

1.4.1 Pyrosequencing

For most of the molecular techniques discussed over, a constant concern remained the high cost and low throughput. Given the high cell abundance of bacteria in aquatic habitats (10⁸–10⁹ cells/L) (Ducklow and Carlson, 1992; Zeng et al., 2013a,b), there was need for HTS methods to gain insights into the rarer members within microbial communities in order to understand their functional significance. Sequencing techniques such as pyrosequencing on the Roche 454 GS FLX platform far exceeded the capacity of Sanger sequencing approaches by several orders of magnitude (Tedersoo et al., 2010). Pyrosequencing technology detects the release of pyrophosphate during nucleotide incorporate. Further details on preparation and processing of DNA samples for pyrosequencing can be obtained extensively in the literature (Liu et al., 2012).

One of the primary points to consider in processing of NGS data is quality control. For example, Zeng et al. (2013a,b) investigated bacterioplankton community structure from Chukchi Borderland and Kongsfjorden in the Arctic Ocean. The authors used sequence length cutoff to be ≥150 bp including primer sequence. Additionally, after removal of ambiguous bases, all sequences were matched with the primer sequence and one of the barcode sequences. The minimum sequence identity percentage used in this study was at least 75% match to a previously determined 16S rRNA gene sequence. This lead to rejection of nearly 18% of total generated data. The study found a total of 9 phyla from Kongsfjorden and 11–19 phyla from Chukchi Borderland. Apart from the dominant groups of Proteobacteria, Actinobacteria, and Bacteroidetes, a number of rare bacterial phyla including Candidate division OD1, OP1, and SR11 were found in the study site. Additionally, it was possible to differentiate between the abundance of these rare taxa between different studied stations. This provided a good opportunity to analyze rare members of the bacterioplankton communities.

Similarly, in another study undertaken by Andersson et al. (2010) in the Baltic Sea, bacterioplankton communities were elucidated by sequencing the V6 region of the bacterial 16S rDNA using a mix of five versions of 967 F and four versions of 1046R primers. The data was analyzed after removal of the forward and reverse primer sequences and removal of low-quality sequences. Additionally, after binning into OTUs, the single-read OTUs were filtered out to ensure removal of sequences resulting from sequencing errors. The study showed the widespread dominance of Actinobacteria, Bacteroidetes, Cyanobacteria, Verrucomicrobia, and Proteobacteria in the study site during the studied period. In another extensive study by Fortunato et al. (2012), bacterioplankton community composition was analyzed from 300 water samples. The bacterioplankton communities showed distinct spatial variability and could be grouped into riverine, estuarine, plume, epipelagic, mesopelagic, shelf bottom, and slope bottom. Spatial variability in the observed bacterioplankton communities overwhelmed the differences observed as a result of seasonal variability.

The Roche 454 systems produce a read length of 100–150 bp and can generate approximately 2000,000+ reads per run providing around 20 Mb data. Further improvements in technologies allowed for generation of close to 700 bp, but the high cost and relatively high error rate continues to be a problem in the use of this technology (Liu et al., 2012).

1.4.2 Illumina HiSeq and MiSeq Sequencing

At present, the most widely used sequencing methods for elucidating microbial diversity and community level information involves Illumina sequencing on HiSeq or MiSeq platforms. One of the primary advantages offered by Illumina over pyrosequencing involves generation of longer read lengths. The present technologies allows for generation of a forward R1 and a reverse R2 file which can be stitched together into a contig to former a longer stretch. This essentially provides better resolution of microbial communities owing to longer sized sequences. Illumina sequencing technologies have been widely used in analyzing some very critical questions such as those involving effects of pCO2 on bacterioplankton community structure. A study carried out as part of the EPOCA 2010 Arctic campaign reported the effect of increased pCO2 on Gammaproteobacteria. Alongside, 15 rare taxa were found to be significantly affected by increased pCO2 (Roy et al., 2013). As mentioned previously, use of fingerprinting techniques such as T-RFLP could not detect any significant changes in bacterioplankton community structure under elevated levels of pCO2. But a sequencing technique such as Illumina HiSeq and MiSeq could detect such fine scale differences in abundance of rare taxa owing to greater sequencing depths. Rare bacterioplankton taxa have also been detected in estuaries within mangrove ecosystems using Illumina sequencing approaches (Ghosh and Bhadury, 2017).

Other critical questions such as those involving the effects of increased nutrients owing to anthropogenic inputs on river systems have also been addressed using Illumina sequencing technologies. The Yarlung Tsangpo River in China has experienced an increase in nutrient levels owing to urbanization and tourism. This was further aggravated by building of dams on the river which could essentially alter the movement of water and nutrients along the course of the river. A study undertaken by Wang et al. (2017) did not find any significant difference in bacterioplankton communities in the upstream and downstream of the dam. Soluble reactive phosphorus, ammonium, and turbidity appeared to be the primary environmental parameters driving bacterioplankton community structure in the river which was dominated by members of Firmicutes, Bacteroidetes, and Proteobacteria. Again, owing to deeper insights provided by Illumina HiSeq sequencing, it appeared that increased dissolved nutrients which is a proxy for anthropogenic effects largely influenced bacterioplankton communities, but this may not be a direct reflection of processes such as dam building (Wang et al., 2017). Shifts in bacterioplankton resulting from land-use changes and upstream nutrient inputs have also been assessed in Upper Mississippi River, USA. Nearly 33%–49% of the bacterioplankton communities were represented by bacteria which are typical signatures of anthropogenic impacts (Unno et al., 2013). As observed in Yarlung Tsangpo River, anthropogenic impacts did appear to influence bacterioplankton community structure at the downstream stations. This shows the effectiveness of this process detecting changes in community structure from study sites globally. Further applications of NGS such as those in water quality assessment have been discussed extensively in the review by Tan et al. (2015) and can be referred to for a better understanding the suitability and biases in NGS techniques.

1.4.3 Next-Generation Sequencing Data Processing

An extensive literature and guidelines exist which can help in understanding methods involved in processing NGS data. Processing of NGS data through proper steps can ultimately help unravel microbial diversity. The huge amount of information generated by these sequencing technologies may seem difficult to process particularly to beginners and to people who may not have access to dedicated computing systems required for processing of such datasets. Though as part of this chapter, the aim is not to discuss any data processing software in particular, but tools such as Mothur (Schloss et al., 2009) and QIIME (Caporaso et al., 2010) are known to be user friendly for processing of raw data generated by NGS approach. A stepwise pattern of data processing has been thoroughly discussed in a minireview written by Ju and Zhang (2015).

As mentioned previously, one of the primary concerns with NGS data is quality control. Proper filtering of raw data, removal of ambiguous bases, determining the desired read length of sequences, and trimming of primer and adaptor sequences are absolutely essential steps before downstream analysis. All of the above data filtering steps can be conveniently undertaken in Mothur and QIIME though there does exists other software capable of running these processes. This clean data can be further run through chimera check programs such as ChimeraSlayer to remove chimeric sequences. Presence of chimeric sequences could lead to erroneous output. Most importantly, chimera sequences could be classified as novel bacteria which could lead to incorrect interpretation of microbial community structure and associated diversity. Once the chimera sequences are removed, sequences are aligned using any of the available alignment methods. This initial alignment could help in correct grouping and determination of OTUs. Often the presence of gaps or missing bases could lead identical sequences to be binned into separate OTUs. An initial alignment could help circumvent such errors in binning sequences into OTUs. The cutoff at which OTUs are to be determined is based on the scientific question being addressed which is critical in the case of microbial ecology. Usually, most microbial ecology studies keep the cutoff at 97% identity between sequences but nature of the environment can be also important while determining the cutoff. Representative sequences from each OTU are subsequently selected for taxonomic assignment. This reduces the computation need to process large number of sequences for taxonomic assignment. The available 16S rDNA databases such as RDP-II, SILVA, and Greengenes are all widely used for taxonomic assignment of sequences. This information essentially helps identify each member of microbial community represent the studied environment. The taxonomic level, i.e., phyla, class, order, family, genus, and species level affiliation is possible owing to the robust 16S rDNA databases. However, the use of taxonomic level depends on the scientific question being addressed as part of a study.

One of the primary points that have been discussed extensively when dealing with next-generation sequence data is data normalization. This is particularly important when questions being addressed involve comparison of microbial communities across sites. Sequencing depth can vary significantly between samples. Uneven sequencing depth or number of sequences can affect diversity estimates of biological samples (Ju and Zhang, 2015). The process of normalization essentially helps to equalize the number of sequences across sites. This essentially aids in the comparison of microbial communities between different sites and helps determine diversity. Relative abundances and random sampling (i.e., rarefaction) are commonly used for the process of data normalization (Goodrich et al., 2014).

A more technical point that is often discussed in literature is the use of different primer sets in amplification of the 16S rDNA which is then used to elucidate structure of microbial communities from a particular environment. Additionally, there is focused discussion to understand the implications of use of different stretches, i.e., different hypervariable regions of the 16S rDNA. It is important to understand whether different stretches of the 16S rDNA could impact the overall microbial community structure observed from a study site. Mock communities have been used to study the effect of using different primer sets in elucidating microbial communities using different NGS platforms. For example, comparisons between the observed bacterioplankton communities detected using 454 pyrosequencing and Illumina MiSeq have also been undertaken and discussed extensively in published literature (Tremblay et al., 2015; Clooney et al., 2016).

1.4.4 Third-Generation Sequencers

One of the primary concerns of the presently used sequencing technologies involves the PCR bias. With the advent of third-generation sequencing technologies such as PacBio and Nanopore essentially take care of this issue (Liu et al., 2012). The guiding principle of such sequencing techniques is single-molecule real-time sequencing which uses a modified enzyme and is capable of directly conserving the enzymatic reaction in real time. DNA polymerase that is bound to DNA template is attached to a 50-nm-wide well-called zero-mode waveguides. As the DNA polymerase synthesizes the noncoding DNA strand, a γ-phosphate fluorescently labeled nucleotide is added. Energy penetrates the short distance of the well and excites the fluorophores in the vicinity of the polymerase. As each base is incorporated, a distinctive pulse of fluorescence appears and is detected real-time (Quail et al., 2012). This sequencing technology by PacBio makes it possible to sequence the full length of the 16S rDNA. This increases the accuracy of taxonomic assignments and further supports the claims of novel bacterial branches found from unique study sites (Singer et al., 2016). A study by Singer et al. (2016) assessed bacterioplankton communities using PacBio shotgun sequences as long as PacBio full length sequences (PhyloTags), thereby allowing the validation of this technique in accessing bacterioplankton communities from environments. Tests on mock communities (assemble of 23 bacterial and 3 archaeal species) indicated a strong overlap between data generated by PacBio and by Illumina MiSeq sequencing. But when observing complex bacterioplankton communities from natural samples such as that from Sakinaw Lake, British Columbia, Canada, there existed disparity between the data obtained from PacBio and Illumina. Short-reads sequences from Illumina platform could not detect all members of the bacterioplankton communities and missed multiple bacterial phyla. Use of PacBio platform may provide a high-throughput yet cost-effective means to generate long reads of the 16S rDNA for studying bacterioplankton communities (Singer et al., 2016). Additionally, there appears to be no bias against Guanine-Cytosine (GC)-rich and poor regions in PhyloTag (Quail et al., 2012) which further helps reduce biases observed in bacterioplankton community structure. Further ways in addressing errors associated with PacBio sequences can be read in an article by Singer et al. (2012). Further comparisons between PacBio and Illumina sequencers can be found in an article by Quail et al. (2012).

Nanopore is another example of third generation sequencing. The Nanopore resembles a biopore with a nanoscale diameter (Song et al., 1996). Nanopore sequencing involves passes a single-stranded DNA thread across α-hemolysin pore which essentially disrupts the voltage across the channel. This forms the building block of the nanopore. During sequencing, a continuous ionic flow is applied and disruption in current is detected by standard electrophysiological techniques. By application of a small voltage difference (~100 mV) across the nanopore membrane separating the two chambers containing aqueous electrolytes, the resulting ionic current through the pore is measured (Branton et al., 2009). The output detects the size difference between all deoxyribonucleoside monophosphate each of which has a characteristic current modulation. The two obvious advantages of Nanopore sequencing is generation of long read lengths of >5 kbp and no involvement of fluorescent tags which are commonly employed in all other sequencing techniques (Liu et al., 2012). Further information on potential and challenges of nanopore sequences can be read in an article by Branton et al. (2009).

1.5 Integrating Culture and Molecular Techniques in Understanding Microbial Community Structure and Diversity

There are numerous examples where both culture and molecular techniques have been used to microbial communities from a range of environments. As an example, the case of culturing of members of SAR11, an important constituent of bacterioplankton communities has been discussed. As previously mentioned, SAR11 or Candidatus pelagibacter ubique belongs to the Alphaproteobacteria class of phylum Proteobacteria. SAR11 is abundant and is ubiquitously distributed in global oceans. After the initial cultivation of SAR11 in 2005 (Rappé et al., 2005), Tripp et al. (2008) reported the requirement of sulfur for growth of members of SAR11. Strains such as HTCC1062 and other SAR11 stains have been demonstrated to grow in natural autoclaved seawater supplemented with ammonium, phosphate, and other organic carbon compounds (Tripp et al., 2008). Genome sequence has indicated the presence of incomplete set of genes that could be involved in assimilatory sulfate reduction. Genome study also indicated the presence of multiple transporters for 3-dimethylsulphoniopropionate (DMSP) and methionine. The authors demonstrated incorporation of ³⁵S and ³⁵S[DMSP] in the cells of SAR11. Approximately 50% of uptaken sulfur was incorporated in the cells which resulted in increase in cell number of SAR11. This study conclusively showed that Ca. P. ubique relies exclusively on other plankton for reduced sulfur compounds required for its growth. Moreover, the study also provides a good example of how molecular techniques can aid in our understanding to design culture media that could be used to grow novel bacteria under laboratory conditions. Such approaches are particularly important in elucidating microbial communities across various types of environments.

1.6 Conclusion

The emergence of molecular tools has revolutionized the field of microbial ecology and also helped develop the field of cultured approaches. Nowadays, NGS technologies represent the next technological leap toward massive and faster characterization of microbial communities. In particular, questions pertaining to their diversity including functional diversity can be effectively addressed across a range of environments. Addressing some of these key questions is critical toward understand the functional significance of microbial communities and their role in ecosystem, in particular with respect to biogeochemical cycling. However, appropriate interpretation and management of the massive data generated specifically by the NGS approaches still requires development of efficient statistical algorithms and bioinformatics tools. In other words, rapid advances in computation and modeling are of immediate necessity in order for microbial ecologists to fully exploit the potential of high-throughput technologies and interpretation of microbial diversity.

References

1. Acinas SG, Sarma-Rupavtarm R, Klepac V, Polz MF. PCR-induced sequence artifacts and bias: insights from comparison of two 16S rRNA clone libraries constructed from the same sample. Appl Environ Microbiol. 2005;71(12):8966–8969.

2. Alonso-Sáez L, Balagué V, Sà EL, et al. Seasonality in bacterial diversity in north-western Mediterranean coastal waters: assessment through clone libraries, fingerprinting and FISH. FEMS Microbiol Ecol. 2007;60:98–112.

3. Amann J. Die direkte Zählung der Wasserbakterien mittels des Ultramikroskops. Centralbl Bakteriol II Abt. 1911;29:381–384.

4. Amann R, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol. 1990;56:1919–1925.

5. Andersson AF, Riemann L, Bertilsson S. Pyrosequencing reveals contrasting seasonal dynamics of taxa within Baltic Sea bacterioplankton communities. ISME J. 2010;4:171–181.

6. Avaniss-Aghajani E, Jones K, Chapman D, Brunk C. A molecular technique for identification of bacteria using small subunit ribosomal RNA sequences. Biotechniques. 1994;17(1):144–149.

7. Bouvier T, del Giorgio PA. Factors influencing the detection of bacterial cells using fluorescence in situ hybridization (FISH): a quantitative review of published reports. FEMS Microbiol Ecol. 2003;44(1):3–15.

8. Branton D, Deamer DW, Marziali A, et al. The potential and challenges of nanopore sequencing. Nat Biotechnol. 2009;26(10):1146–1153 https://doi.org/10.1038/nbt.1495.

9. Button DK, Schut F, Quang P, Martin R, Robertson BR. Viability and isolation of marine bacteria by dilution culture: theory, procedures, and initial results. Appl Environ Microbiol. 1993;59(3):881–891.

10. Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S. Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Appl Environ Microbiol. 2007;73(1):278–288.

11. Caporaso JG, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010; https://doi.org/10.1038/nmeth.f.303.

12. Casamayor EO, Schafer H, Baneras L, Pedrós-Alió C, Muyzer G. Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis. Appl Environ Microbiol. 2000;66:499–508.

13. Clooney AG, Fouhy F, Sleator D, et al. Comparing apples and oranges? Next generation sequencing and its impact on microbiome analysis. PLoS ONE 2016; https://doi.org/10.1371/journal.pone.0148028.

14. Cottrell MT, Kirchman DL. Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization. Appl Environ Microbiol. 2000;66(12):5116–5122 https://doi.org/10.1128/AEM.66.12.5516-5122.2000.

15. Connon SA, Giovannoni SJ. High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates. Appl Environ Microbiol. 2002;68(8):3878–3885.

16. Crump BC, Kling GW, Bahr M, Hobbie JE. Bacterioplankton community shifts in an arctic lake correlate with seasonal changes in organic matter sources. Appl Environ Microbiol. 2003;69(4):2253–2268.

17. DeLong EF, Wickham G, Pace N. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989;243:1360–1363.

18. Denaro R, D’Auria G, Di Marco G, et al. Assessing terminal restriction fragment length polymorphism suitability for the description of bacterial community structure and dynamics in hydrocarbon-polluted marine environments. Environ Microbiol. 2005;66:2943–2950.

19. Denbigh KG. The Thermodynamics of the Steady State. London: Methuen; 1951;1.

20. Ducklow HW, Carlson CA. Oceanic bacterial production. Adv Microb Ecol. 1992;12:113–181.

21. Dunbar J, Ticknor LO, Kuske CR. Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities. Appl Environ Microbiol. 2001;67(1):190–197.

22. Emerson D, Floyd MM. Enrichment and isolation of iron-oxidizing bacteria at neutral pH. Methods Enzymol. 2005;397:112–123.

23. Falkowski PG, Fenchel T, DeLong EF. The microbial engines that drive Earth's biogeochemical cycles. Science. 2008;320(5879):1034–1039 https://doi.org/10.1126/science.1153213.

24. Feng BW, Li XR, Wang JH, et al. Bacterial diversity of water and sediment in the Changjiang estuary and coastal area of the East China Sea. FEMS Microbiol Ecol. 2009;70(2):80–92.

25. Fortunato CS, Herfort L, Zuber P, Baptista AM, Crump BC. Spatial variability overwhelms seasonal patterns in bacterioplankton communities across a river to ocean gradient. ISME J. 2012;6:554–563.

26. Fuchs G, Boll M, Heider J. Microbial degradation of aromatic compounds- from one strategy to four. Nat Rev Microbiol. 2011;9(11):803–816.

27. Ghosh A, Bhadury P. Insights into bacterioplankton community structure from Sundarbans mangrove ecoregion using Sanger and Illumina MiSeq sequencing approaches: a comparative analysis. Genom Data. 2017;11:39–42.

28. Goodrich JK, Di Rienzi SC, Poole AC, et al. Conducting a microbiome study. Cell. 2014;158(2):250–262.

29. Griffiths RI, Whiteley AS, O'Donnell AG, Bailey MJ. Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl Environ Microbiol. 2000;66(12):5488–5491.

30. Hewson I, Fuhrman JA. Richness and diversity of bacterioplankton species along an estuarine gradient in Moreton Bay, Australia. Appl Environ Microbiol. 2004;70(6):3425–3433.

31. Hill GT, Mitkowski NA, Aldrich-Wolfe L, et al. Methods for assessing the composition and diversity of soil microbial communities. Appl Soil Ecol. 2000;15:25–36.

32. Hug LA, Baker BJ, Anantharaman K, et al. A new view of the tree of life. Nat Microbiol. 2016; https://doi.org/10.1038/nmicrobiol.2016.48 Article number 16048.

33. Hugenholtz P. Exploring prokaryotic diversity in the genomic era. Genome Biol. 2002;3 reviews0003.1-reviews0003.8.

34. Ju F, Zhang T. 16S rRNA gene high-throughput sequencing data mining of microbial diversity and interactions. Appl Microbiol Biotechnol. 2015;99:4119–4129.

35. Kaeberlein T, Lewis K, Epstein SS. Isolating uncultivable microorganisms in pure culture in a simulated natural environment. Science. 2002;296:1127–1128.

36. Kan J, Crump BC, Wang K, Chen F. Bacterioplankton community in Chesapeake Bay: predictable or random assemblages. Limnol Oceanogr. 2006;51(5):2157–2169.

37. Kitts CL. Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Curr Issues Interst Microbiol. 2001;2:17–25.

38. Kogure K, Simidu U, Taga N. A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol. 1979;25:415–420.

39. Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U.S.A. 1985;82(20):6955–6959.

40. Larson GL, Giovannoni SJ. Unusual bacterioplankton community structure in ultra-oligotrophic Crater Lake. Limnol Oceaogr. 2001;46(3):557–572.

41. Lee S, Kemp PF. Single-cell RNA content of natural marine planktonic bacteria measured by hybridization with multiple 16S rRNA-targeted fluorescent probes. Limnol Oceangr. 1994;39:869–879.

42. Lee S-H, Malone C, Kemp PF. Use of multiple 16S rRNA-targeted fluorescent probes to increase signal strength and measure cellular RNA from natural planktonic bacteria. Mar Ecol Prog Ser. 1993;101:193–201.

43. Lerman LS. Electrophoresis of DNA in denaturing gradient gels. In: New York, NY: Plenum; 1986;221–240. Setlow JK, Hollaender A, eds. Genetic Engineering. vol 8.

44. Liu WT, Marsh TL, Cheng H, Forney LJ. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol. 1997;63(11):4516–4522.

45. Liu L, Li Y, Li S, et al. Comparison of next-generation sequencing systems. J Biomed Biotechnol 2012; https://doi.org/10.1155/2012/251364 Article ID251364.

46. Malmstrom RR, Straza TRA, Cottrell MT, Kirchman DL. Diversity, abundance, and biomass production of bacterial groups in the western Arctic Ocean. Aquat Microb Ecol. 2007;47:45–55.

47. Muyzer G, de Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 1993;59(3):695–700.

48. Olsen GJ, Lane DJ, Giovannoni SJ, Pace NR, Stahl DA. Microbial ecology and evolution: a ribosomal RNA approach. Annu Rev Microbiol. 1986;40(1986):337–365.

49. Osborn AM, Moore ERB, Timmis KN. An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ Microbiol. 2000;2:39–50.

50. Ouverney CC, Fuhrman JA. Increase in fluorescence intensity of 16S rRNA in situ hybridization in natural samples treated with chloramphenicol. Appl Environ Microbiol. 1997;63(7):2735–2740.

51. Øvreås L, Forney L, Daae FD, Torsvik V. Distribution of bacterioplankton in meromictic lake Sælenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragment coding for 16S rRNA. Appl Environ Microbiol. 1997;63:3367–3373.

52. Page KA, Connon SA, Giovannoni SJ. Representative freshwater bacterioplankton isolated from Crater Lake, Oregon. Appl Environ Microbiol. 2004;70(11):6542–6550.

53. Piccini C, Conde D, Alonso C, Sommaruga R, Pernthaler J. Blooms of single bacterial species in a coastal lagoon of the Southwestern Atlantic Ocean. Appl Environ Microbiol. 2006;72(10):6560–6568.

54. Quail MA, Smith M, Coupland P, et al. A tale of three next generation sequencing platforms; comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics. 2012;13:341 https://doi.org/10.1186/1471-2164-13-341.

55. Rappé MS, Giovannoni SJ. The uncultured microbial majority. Annu Rev Microbiol. 2003;57:369–394.

56. Rappé MS, Connon SA, Vergin KL, Giovannoni SJ. Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature. 2005;418 https://doi.org/10.1038/nature00917.

57. Roy A-S, Gibbons SM, Schunck H, et al. Ocean acidication shows negligible impacts on high-latitude bacterial community structure in coastal pelagic mesocosms. Biogeosciences. 2013;10:555–566.

C. Comparison of different denaturing gradient gel electrophoresis primer sets for the study of marine bacterioplankton communities. Appl Environ Microbiol. 2007;73(18):5962–5967.

59. Sanz JL, Kochling T. Molecular biology techniques used in wastewater treatment: an overview. Process Biochem. 2007;42:119–133.

60. Schloss PD, Westcott SL, Ryabin T, et al. Introducing mother: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol. 2009;75(23):7537–7541.

61. Schut F, Vries EJ, Gottschal JC, et al. Isolation of typical marine bacteria by dilution culture: growth, maintenance, and characteristics of isolates under laboratory conditions. Appl Environ Microbiol. 1993;59(7):2150–2160.

62. Sezonov G, Joseleau-Petit D, D’Ari R. Escherichia coli physiology in Luria-Bertani Broth. J Bacteriol. 2007;189(23):8746–8749.

63. Singer E, Bushnell B, Coleman-Ferr D, et al. High-resolution phylogenetic microbial community profiling. ISME J. 2016;10:2020–2032.

64. Shan D, Wei G, Li M, et al. Distribution and diversity of bacterioplankton communities in subtrophical seawater around Xiamen Island, China. Microbiol Res. 2015;175:16–23.

65. Song L, Hobaugh MR, Shustak C, Cheley S, Bayley H, Gouaux JE. Structure of staphylococcal α-hemolysin, a heptameric transmembrane pore. Science. 1996;274:1859–1865 https://doi.org/10.1126/science.274.5294.1859.

66. Stewart E. Growing unculturable bacteria. J Bacteriol. 2012;194(16):4151–4160.

67. Stoica E. Application of T-RFLP analysis to the study of the coastal Black Sea bacterioplankton. Rom Biotechnol Lett. 2009;14(5):4710–4719.

68. Tan B, Ng C, Nshimyimana JP, Loh LL, Gin KY-H, Thompson JR. Next-generation sequencing (NGS) for assessment of microbial water quality: current progress, challenges, and future opportunities. Front Microbiol. 2015; https://doi.org/10.3389/fmicb.2015.01027.

69. Tang X, Xie G, Shao K, et al. Bacterial community composition in oligosaline Lake Bosten: low overlap of Betaproteobacteria and Bacteroidetes with freshwater ecosystems. Microbes Environ. 2015;30(2):180–188.

70. Tedersoo L, Nilsson RH, Abarenkov K, et al. 454 pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases. New Phytol. 2010;188(1):291–301.

71. Tripp HJ, Kitner JB, Schwalbach MS, Dacey JWH, Wilhelm LJ, Giovannoni SJ. SAR11 marine bacteria require exogenous reduced sulphur for growth. Nature. 2008; https://doi.org/10.1038/nature06776.

72. Tremblay J, Singh K, Fern A, et al. Primer and platform effects on 16S rRNA tag sequencing. Front Microbiol. 2015; https://doi.org/10.3389/fmicb.2015.00771.

73. Tsai YL, Olson BH. Rapid method for direct extraction of DNA from soil and sediment. Appl Environ Microbiol. 1991;57(4):1070–1074.

74. Unno T, Gould TJ, Jarvis B, Phillips J, Cotner JB, Sadowsky MJ. Application of Illumina next-generation sequencing to characterize the bacterial community of the Upper Mississippi River. J Appl Microbiol. 2013;115:1147–1158.

75. Urakawa H, Yoshida T, Nishimura M, Ohwada K. Characterization of microbial communities in marine surface sediments by terminal-restriction fragment length polymorphism (T-RFLP) analysis and quinine profiling. Mar Ecol Prog Ser. 2001;220:47–57.

76. Wang P, Wang X, Wang C, Miao L, Hou J, Yuan Q. Shifts in bacterioplankton diversity and structure: influence of anthropogenic disturbances along the Yarlung Tsangpo River on the Tibetan Plateau, China. Sci Rep. 2017; https://doi.org/10.1038/s41598-017-12893-4 Article number 12529.

77. Whitman WB, Coleman DC, Wiebe WJ. Prokaryotes: the unseen majority. Proc Natl Acad Sci U.S.A. 1998;95(12):6578–6583.

78. Wintzingerode FV, Göbel UB, Stackebrandt E. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev. 1997;21:213–229.

79. Zhang R, Thiyagarajan V, Qian P-Y. Evaluation of terminal-restriction fragment length polymorphism analysis in contrasting marine environments. FEMS Microbiol Ecol. 2008;65:169–178.

80. Zhang R, Xia X, Lau SCK, Motegi C, Weinbauer MG, Jiao N. Response of bacterioplankton community structure to an artificial gradient of pCO2 in the Arctic Ocean. Biogeosciences. 2013;10:3679–3689.

81. Zeng Y, Yu Y, Li H, He J, Lee SH, Sun K. Phylogenetic diversity of planktonic bacteria in the Chukchi Borderland region in summer. Acta Oceanol Sin. 2013a;32(6):66–74.

82. Zeng Y-X, Zhang F, He J-F, et al. Bacterioplankton community structure in the Arctic waters as revealed by pyrosequencing of 16S rRNA genes. Antonie Van Leeuwenhoek. 2013b;103:1309–1319.

Further Reading

1. Borneman H, Triplett EW. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl Environ Microbiol. 1997;63(7):2647–2653.

2. Zhang Y-X, Yu Y, Qiao ZY, Jun HY, Li HR. Diversity of bacterioplankton in coastal seawaters in Fildes Peninsula, King George Island, Antarctica. Arch Microbiol. 2014;196:137–147.

Chapter 2

Metagenomic Achievements in Microbial Diversity Determination in Croplands

A Review

Agnieszka Wolińska,    Department of Biochemistry and Environmental Chemistry, The John Paul II Catholic University of Lublin, Lublin, Poland

Abstract

The knowledge that biodiversity is an essential factor for maintaining a proper condition of soils is commonly widespread. However, the loss of microbial diversity as an effect of anthropogenic activities remains in the category of worldwide problem. Until recently, our knowledge about the soil biodiversity was limited by laboratory methods based on the microbial cultivability. Fortunately, by metagenomic tools development, our cognizance of unavailable till now information regarding a microbial group called as viable but not cultivable significantly increased. In this paper, an overview of the next-generation sequencing methods, recommended to soil analysis, is presented. Moreover, description of croplands microbiome as an effect of metagenomic analyses is shown, with emphasizing on soil factors and the way of land mode that directly impacted on microbial community. Finally, biological parameters testifying about soil biological degradation phenomenon are discussed and some microorganisms groups are recommended as factors of soil quality.

Keywords

Metagenomic tools; soil biodiversity; croplands; wastelands; microbial indicators

Acknowledgments

The author acknowledges the financial support from the National Science Centre in Poland (DEC-2013/09/D/NZ9/02482). Many thanks are also addressed to Prof. Mieczysław Błaszczyk from Life Science University in Warsaw for his inspiration and wise criticism during paper preparation.

2.1 Introduction

Soil is the most heterogeneous, the most diverse, and the most abundant environment of life organisms that exists in nature (Neelkanta and Sultana, 2013). On microscale (<1 mm³), soil provides numerous microhabitats per gram (Sikorski, 2015). At the same time, soils are the most difficult and the most challenging samples for analyzing (Neelkanta and Sultana, 2013). Despite the fact that Earth hosts >10³⁰ cells, 2.5·10²⁹ is found in the soil environment (Whitman, 2009). Only 1 g of soil is usually inhabited by enormous number of bacterial cells (10¹⁰), whereas species diversity may vary from 4·10³ to 5·10⁴ (Torsvik et al., 1990; Roesch et al., 2007), and microbial