Review Article

Structuring Microbial Community from the Environment: A review on methods

Rakesh Kumar1*, Deepika Chaudhary1, Anju Kumari2, Khushboo Sihag1 Rashmi1

1Department of Microbiology, CCS Haryana Agricultural University, Hisar India
2 Center for Food Science & Technology, CCS HAU Hisar

J Innov Biol (2014) Volume 2, Issue 1: Pages: 226-233

Abstract: Microorganisms are highly diverse group of organisms and constitutes around 60% biomass on earth. They have both positive and negative impacts on human beings. These microbes are being studied by two methods: culture dependent and culture independent method. Culture dependent techniques cultivate microbes in laboratories on specific media. This technique is used for dominant populations only. Also, all microbes cannot be cultivated as their nutritional requirements are very complex and unknown. Culture independent techniques are mainly based on molecular methods. These methods directly deal with nucleic acid extraction, proteins or lipids from environmental samples. These types of methods are fast, accurate and reliable. These techniques provide specific information regarding present microbial community in a particular area.

Received: 03 January 2015
Accepted: 27 February 2015
Published: 31 March 2015

Corresponding Author:
Kumar R.,
Department of Microbiology, CCS Haryana Agricultural University, Hisar India

Keywords: Microbial community, DGGE, Uncultured microbes, metagenomics, Microarray

Microbial communities are dominant on earth. It constitutes approx 60 % of biomass present on earth, comprises about 4.6 × 1030 prokaryotic cell units (Whitman et al. 1998). Among huge populations of microorganisms only 1 % are culturable by laboratory practices. Our knowledge related to these microbial communities is very limited. Soils have a very diverse range of microorganisms which count bacteria, fungi, algae and actinomycetes. These microbes have so many species which differ in their morphology to functions. Soil microorganisms have an influence on plant nutrition (George et al. 1995; Timonen et al. 1996), plant health (Srivastava et al. 1996; Filion et al. 1999), soil fertility (Yao et al. 2000) and soil structure (Dodd et al. 2000). Microorganisms have capability to produce plant growth promoting substances, i.e. hormones, antibiotics and chelating substances which have great impact on soil health also. The understanding of these microbial populations helps to sustain soil structure, health and soil fertility which ultimately help farming practices to cope up with increased food demand. Soil microbial properties studied are mainly on a functional basis, which include study of biomass, respiration rate and enzyme activities. Community level studies till now gets less attention. Microbial behavior is very complex in community due to the interaction among different species. So there is a cumulative response of all present population of microbes. Thus, the study of microbial population serves as an important and sensitive indicator of soil health. Soil microbiologists’ face challenges to study the types and functions of microorganisms present in soil pool in situ. Soil related problems such as its loss, degradation and contamination are some of the emergencies that mankind must resolve in the third millennium to safeguard the planet and ensure survival of mankind. The microorganism’s effect is so much important that the study regarding this is more sensitive than chemical and physical property changes in environmental conditions. Soil microflora changes are very crucial for property of the soil, which have been already discussed above (Insam 2001).
This review discusses the various methods for determining microbial diversity in the soil. In microbial terminology, diversity is expressed as species richness and species evenness in a given habitat. Culture dependent and culture independent are two types of methods used for studying microbial diversity. In culture dependent method direct plate count and community level physiological profiling (CLPP) are most common. Phospholipid fatty acid analysis (PLFA) also can analyze soil microbial community which is a biochemical based method. Eilers et al. (2000) argued that conventional method is selective and biased towards the growth of specific microorganisms. Molecular techniques are culture independent methods which generate valuable information on microbial diversity and community structure (Nakatsu et al. 2000). The majority of molecular method is based on examination of nucleic acids, either directly or by using the PCR amplified product. Examples of methods those based on direct analysis of nucleic acids are DNA: DNA reassociation kinetics (Torsvik et al. 1990), nucleic acid hybridization (Buckley et al. 1998), FISH (Kirchoff et al. 1997) and microarray (Small et al. 2001; Rhee et al. 2004). Diversity of bacteria most commonly studied by 16S rDNA gene, which occurs in all bacteria and even shows variation among species. A number of techniques are based on PCR based Single strand conformation, specific melting nature or slight differences in sequences. These include restriction fragment length polymorphism (RFLP) (Porteous et al. 1997), single strand conformation polymorphism (SSCP) (Schwieger and Tebbe, 1998), ribosomal intergenic spacer analysis (RISA) (Ranjard et al. 2000) or ARISA (automated RISA) (Cardinale et al. 2004), DGGE (denaturing gradient gel electrophoresis) (Muyzer, 1999), TGGE (temperature gradient gel electrophoresis). These all techniques have their own advantages and disadvantages which will be compiled in this review later (Table 1). Our goal is to place all techniques related to the study of microbial community under the same roof in the prescribed manner.
Conventional / traditional methods for analysis of soil microbial community
Dilution plating on selective media:
Microbial diversity is studied by selective plating (pour and spread plate method) on specific media and taking viable counts respectively. These techniques are culture dependent traditional methods which are used for assessing soil microbial community. This relay on using the variety of culture media for maximum recovery of different microbial population from the same soil sample (Balestra and Misaghi 1997; Mitsui et al. 1997). It has been estimated that only 1 % of microorganism in soil are culturable on available medium (Torsvik et al. 1990; Atlas and Bartha, 1998). These methods are fast, inexpensive and provide information on the active population of soil. But the limitation is that media for all kinds of populations are not available and microbes from adhering surface are difficult to detach i.e. biofilms, soil aggregates. On medium plates fast growing microorganisms show their growth quickly, so slow growing microorganisms may not be detected or show a problem in detection.
Community level physiological profiling (CLPP)
For studying functional diversity there are several methods which measure microbial processes or enzymatic reactions. These methods provide potential information rather than real because these are carried out at optimum conditions (Nannipieri et al. 2003). CLPP is the culture dependent more widely used method (Garland and Millis, 1991; Zak et al. 1994; Konopka et al. 1998). In this method Microplates are used which contain up to 95 different carbon sources. The utilization pattern of carbon sources by communities those present in soil extracts provides potential functional information. The contribution of fungi community to soil functions cannot be determined by using this approach (Nannipieri et al. 2003). To overcome these limitations biolog developed fungal specific plates SFN2 and SFP2, which do not have tetrazolium salt (Classen et al. 2003). Biolog introduced Eco-plates (Choi and Dobbs 1999) containing growth medium, tetrazolium salt and add site specific carbon sources for analysis of samples (Campbell et al. 1997). The soil samples added in these plates, monitored over time for utilization of the substrate ability of community and speed by which utilize carbon sources, is read by Elisa reader. Multivariate analysis is carried out for soil functional diversity assessment. Communities are considered to be functionally similar, if the utilization profile of these carbon sources from one community clusters matches with that from another community. If obtained profile of different communities would be different. CLPP data are used to express the functional diversity of soil (Bending et al. 2002).
This method can be used for studying microbial community diversity at plant rhizosphere (Ellis et al. 1995; Grayston et al. 1998), contaminated sites (Derry et al. 1998; Konopka et al. 1998), arctic soils (Derry et al. 1999) and inoculation of microorganisms (Bej et al. 1991). It has the same drawback as dilution plating. The fast growing community will result in color change and may show false functional diversity.
FAME (Fatty acid methyl ester analysis)
Fatty acids make up a relatively constant proportion of the cell biomass and signature fatty acids also exist which differentiate major taxonomic group in the community. This property is used in this FAME analysis, which provides information on the basis of grouping of fatty acids (Ibekwe and Kennedy, 1999). In the study of fatty acids, if the variation arises means microbial communities also have been different in the particular soil sample. In this method, fatty acids are directly extracted from soil samples, analyzed by using gas chromatography (Ibekwe and Kennedy, 1999). The profile of different samples is compared by using multivariate analysis. Ibekwe and Kennedy (1999) studied microbial communities in the rhizospheric soil samples from field and greenhouse condition by using CLPP and PLFA methodology. Results showed clearly differences in both samples corresponded to biolog plates. Presence or absence of signature fatty acid determines which type of microbes presents in particular community. This method has limitations when we consider total organisms. For example, study of fungal diversity required spores (130-150) but this method cannot study spores. So minor species of fungus are not detected by this method (Graham et al. 1995). Cellular fatty acids also influenced by environment factors like temperature and nutrition that cause problem in profiling. Appropriate signature molecules are not known for all microorganisms, that is also problematic for the accuracy of the method. In general, this method cannot be used to characterize microorganism to the species level.
Molecular methods
Soil microbial diversity cannot be determined only by traditional approaches. There is strong evidence that most of soil bacteria observed under the microscope are viable and active, but unable to form visible colonies on agar plates (Amann et al. 1995). So the diversity of these unculturable microbes is mainly studied by molecular methods which also comprise cultured microorganisms. Molecular techniques are based on extraction, purification and characterization of nucleic acids from soil samples. These provide a more accurate measurement in soil microbial diversity. A number of approaches have been developed for studying microbial diversity, i.e. DNA reassociation, cloning, sequencing, PCR based method including DGGE/TGGE, RISA, ARISA etc.
G + C content
The microorganisms have specific G + C content in their genomes. Taxonomically related groups of microorganism differ in G + C content only by 3-5 % (Tiedjee et al. 1999). This difference in G + C content used to study bacterial diversity of soil communities (Nusslein and Tiedjee, 1999). This method helps to provide a nearby estimation of different taxonomic groups, which may share the same G + C range. This method is not based on PCR, so PCR biases do not affect this. It is a quantitative method which extracts all DNA, including DNA from a rare species. This technique has only drawback that it requires large amounts of DNA and some soil may not have adequate microbial DNA (Tiedjee et al. 1999).
Nusslein and Tiedjee (1999) studied changes in microbial diversity from a vegetative cover of forest land to pasture land in Hawaiian soil. They used three molecular methods, including G + C content, amplified ribosomal DNA restriction analysis (ARDRA) and rDNA sequence analysis. These methods detected a difference in the community which concludes that plants have a strong impact on the microbial community composition. These researchers used three different methods as complementary groups of tests for studying microbial community more thoroughly.
FISH (Fluorescent In situ Hybridization)
Taxon specific oligonucleotide probes are used for hybridization of rRNA either 16S or 23S rRNA in this technique. These probes are fluorescently labeled which can be visualized by confocal laser microscopy. This method detects diversity in natural habitats directly by quantification and identification of microorganism group (Amann et al. 1995; Macnaughton et al. 1996; Kenzaka et al. 1998). Different dyes for fluorescence can be used having different strengths. Multiple probes can be designed which have a role in increasing the strength of fluorescence signal which helps in easy detection (Ludwig et al. 1997). FISH is helpful to study single microorganism within a population and dynamics of whole populations. This technique has a major role in studying biocontrol agents and bioremediation due to its nature of tracking microorganisms which are released into the environments (Kirchoff et al. 1997; Wullings et al. 1998). Despite of so the usefulness of this technique major drawback is observed in poor soil as the dyes may not penetrate the cells due to their small size and thicker walls. Usefulness of the technique is that whole cells are fixed in this technique so problem associated with DNA extraction, PCR amplification and cloning may be avoided (Felske and Akkermans, 1998).
Nucleic acid hybridization
DNA from soil samples is extracted, purified, denatured and then allowed for renaturation. This renaturation depends upon the type of sequences present in extracted DNA. If sequences are similar, then reannealing is very fast. If more complexity present in sequences of genetic material than reannealing is slower. DNA reassociation depends upon the diversity index, which is value of cot1/2. The time which is required for reassociation of half of the DNA is cot ½ values also called half association value (Torsvik et al. 1998). The measurement of this reassociation / hybridization of DNA is also giving information about the genetic complexity of microbial communities. This method is helpful to measure the soil microbial diversity (Torsvik et al. 1990, 1996). Similarities between communities can be studied by this method using hybridization kinetics (Griffiths et al. 1999). The use of specific probes in nucleic acid hybridization detection is an important qualitative and quantitative tool in molecular study of microbial ecology (Clegg et al. 2000, Theron and Cloete, 2000).
DNA microarray technique
In DNA microarray technique, single array contains thousands of DNA sequences; those have high specificity (Cho and Tiedje, 2001). This array can be gene specific or DNA fragments with less than 70 % hybridization of environmental samples (Greene and Voordauw, 2003). Specific gene can be nitrate reductase or nitrogenase can be used which also provide information about functional diversity. This method is also helpful in studying microbial diversity. This technique has advantage that it is not affected by PCR biases. It contains thousands of target gene sequences. However, it detects only most abundant species.
PCR based approaches
PCR is a technique which amplifies only particular sequences of DNA by using universal or specific primers. This specific DNA sequences can be used in different technique for studying molecular microbial diversity. In these methods, DNA was extracted from the samples, purified and 16S, 18S or internal transcribed spacer region is amplified.
DGGE (Denaturing gradient gel electrophoresis)/ TGGE (Temperature gradient gel electrophoresis)
These techniques are basically developed for detection of point mutation in the DNA sequence. Muyzer et al. (1993) expanded the use of DGGE to study microbial diversity. DGGE and TGGE both have similar methods for studying; the only difference is that in DGGE chemical denaturants are used, whether in TGGE temperature is acting as a denaturant. 16S/18S rRNA sequences are amplified by PCR and used as template DNA for seminasted PCR in which forward primer is having 30-40 base pair GC clamp, which ensure that at least part of the DNA remains double stranded. After this, separation is carried out on polyacrylamide gel having a gradient of denaturing chemicals (urea, formamide). Due to denaturation, DNA melts in domains, which are sequence specific, will migrate through gel (Muyzer, 1999). The GC clamp remains double stranded making a y loop which is held within by PAG matrix. The resulting genetic profile / finger prints on gel represent community structure, approximation number of population within amplified community. TGGE method utilizes heat as the main mechanism for unraveling and denaturation of DNA. The number and position of fragments reflect the dominating bacteria in the community. Limitations related to these methods include PCR biases (Wintizingerode et al. 1997), sample handling (Theron and Cloete, 2000) and extraction efficiency of DNA. DNA fragments of different sequences may have similar mobility in polyacrylamide gel. So one band cannot represent one species always (Gelsomino et al. 1999). Advantages of this method are single base pair sequence can also be detected. This type of analysis is useful to study metabolically active microbial populations and rapid screening of microbial communities in different samples (Nakatsu et al. 2000).
SSCP (Single Strand Conformation Polymorphism)
In this technique one primer is phosphorylated at 5’ end, which is digested with lambda exonuclease. These strands are separated by electrophoresis but at low temperature in a polyacrylamide gel. This method is based upon the differential intramolecular folding of SS-DNA, which depends upon variation in DNA sequences. It can be used to study bacterial/ fungal community diversity (Peters et al. 2000; Stach et al. 2001), succession of bacterial communities (Peters et al. 2000), rhizosphere communities (Schmalenberger et al. 2001). This method has limitations, including PCR biases; single bacterial species may yield several bands due to the presence of several operons or more than one conformation of SSPCR amplicon. One of the disadvantages of SSCP is separation of only small DNA fragments ranging from 150 to 400 bp. Moreover, a single-strand DNA sequence can form more than one stable conformation and this fragment can be represented by multiple bands (Rastogi and Sani, 2011; Cetecioglu et al. 2012). Advantages of this technique over DGGE are no requirement of primers with specific GC clamp and specific apparatus for the gradient.
RFLP (Restriction Fragment Length Polymorphism)/ T-RFLP
RFLP is based on the amplification of DNA from the sample. Amplified PCR DNA fragments are digested with restriction enzymes and separated by agarose gel electrophoresis or denaturing acrylamide gel electrophoresis. The bands in the gel reflect the population of all restriction fragments for at least the major members of the community (Massoldeya et al. 1995). In general, this approach has been used most frequently on isolates as part of a clone screening step prior to sequencing (Pace et al. 1996). Recently, the technique has been used to probe community structure (Massoldeya et al. 1995). It is a useful mean to detect changes in the communities, but has a little utility in quantitating diversity in complex communities. An advanced form of RFLP is terminal RFLP. The initial description of this technique was presented by Liu et al. (1997). This technique is based on PCR amplifications of 16S rDNA with specific primers. The primers are labeled with a fluorescent tag at the terminus resulting in labeled PCR- products. The products are cut with several restriction enzymes, one at a time. Since the PCR products are labeled at the terminus, only the terminal fragments of a restriction digest are detected by the sequencer. It has been used for defining microbial communities in soil (Dunbar et al. 2000; Hack et al. 2004).
ARDRA (amplified ribosomal DNA restriction analysis)
ARDRA is a powerful tool for bacterial identification and classification at species level this performs with fluorescent PCR product, restriction enzyme digestion carried out and fragments are separated on automated DNA sequencing gel (Pukall et al. 1998; Sklarz et al. 2011). This is a rapid method for monitoring of microbial communities over time, or comparing biodiversity in response to changing environmental conditions. Sometimes it is not possible to separate the restriction profiles obtained from microbial communities by agarose or polyacrylamide electrophoresis (Rastogi and Sani, 2011). Major limitation of ARDRA is that it provides little or no information about the type of microorganisms, those present in the sample (Gich et al. 2000). This method was used to evaluate the biodiversity of cyanobacteria on stone monuments in the Boboli Gardens in Florence (Tomaselli et al. 2000), in the detection of microorganisms forming a biofilm on the Bayon temple sandstone of Angkor Thom, Cambodia (Lan et al. 2010)
RISA (Ribosomal intergenic spacer analysis)/ automated ribosomal intergenic spacer analysis (ARISA)
RISA is based on length polymorphism of the ribosomal intergenic spacer region between 16S and 23S rRNA genes (Ranjard et al. 2000). This region is varying in strain to strain and code for tRNA, which helps to differentiate between closely related species also (Fisher and Triplett 1999). Polymorphism of this method is detected by silver staining. In ARISA the forward primer is fluorescently labeled and is automatically detected. The disadvantage of RISA is that it requires large quantities of DNA, more time consuming, require silver staining that is somewhat insensitive and the resolution tends to be low (Fisher and Triplett, 1999). Also silver staining is an old technique and very few are using nowadays. The main limitation of ARISA is the large number of peaks in the case of using “universal primers”. In addition, it is very difficult to interpret results for fingerprints obtained from uncultured microorganisms (Popa et al. 2009). ARISA increases the sensitivity of the method and reduces the time, but is still subject to the traditional limitations of PCR (Fisher and Triplett, 1999).

Highly repeated sequence characterization or microsatellite regions
Many organisms, both prokaryotic and eukaryotic, contain highly repetitive short DNA sequences that are 1–10 base pairs long repeated throughout their genomes (Longato and Bonfante, 1997). Depending on the rate of evolution, these sequences may be diagnostic and allow differentiation down to the species or strain level (Zeze et al. 1996). This method, also termed rep-PCR, has been used for identification of bacteria since it provides a genomic fingerprint of chromosome structure, and chromosome structure is considered to be variable between strains (Tiedjee et al. 1999).

Conclusion and future prospective
A number of methods are currently available for studies on soil microbial communities. The use of molecular techniques for investigating microbial diversity in soil communities continues to provide new understanding of the distribution and diversity of organisms in soil habitats. Despite the utility of culture-independent techniques, there remains a general need to cultivate microorganisms from soil habitats to better understand their role in soil processes. Future studies of soil microbial communities must necessarily rely on a combination of both culture-dependent and culture-independent methods and approaches. Only then will we be able to develop a more complete picture of the contribution of specific microbial communities to the overall quality and health of agricultural soils.

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