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
email: sehrawatrk@gmail.com

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

IntroductionReferences
Introduction
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.

    Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Mol Biol Rev 59:143-169
    Atlas RM, Bartha R (1998) Microbial Ecology: Fundamentals and Applications. Benjamin/Cummings, Redwood City, CA, pp 694.
    Balestra GM, Misaghi IJ (1997) Increasing the efficiency of the plate counting method for estimating bacterial diversity. J Microbiol Meth 30:111-117
    Bej AK, Perlin M, Atlas RM (1991) Effect of introducing genetically engineered microorganisms on soil microbial community diversity. FEMS Microbiol Ecol 86:169-176
    Bending GT, Turner MK, Jones JE (2002) Interactions between crop residues and soil organic matter quality and the functional diversity of soil microbial. Soil Biol Biochem 34:1037-1082
    Buckley DH, Graber JR, Schmidt TM (1998) Phylogenetic analysis of nonthermophilic members of the kingdom Crenarchaeota and their diversity and abundance in soils. Appl Environ Microbiol 64:4333-4339
    Campbell CD, Grayston SJ, Hirst DJ (1997) Use of rhizosphere carbon sources in sole carbon source tests to discriminate soil microbial communities. J Microbiol Meth 30:33-41
    Cardinale M, Brusetti L, Quatrini P, Borin S, Puglia AM, Rizzi A, Zanardini E, Sorlini C, Corselli C, Daffonchio D (2004) Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl Environ Microbiol 70:6147-6156
    Cetecioglu Z, Ince O, Ince B (2012) Gel electrophoresis based genetic fingerprinting techniques on environmental ecology. In Gel electrophoresis – advanced techniques. Magdeldin S, eds, pp 52-66. InTech, Croatia.
    Cheneby D, Philippot L, Hartmann A, Henault C, Germon JC (2000) 16S rDNA analysis for characterization of denitrifying bacteria isolated from three agricultural soils. FEMS Microbiol Ecol 34:121-128
    Cho JC, Tiedje JM (2001) Bacterial species determination from DNA–DNA hybridization by using genome fragments and DNA microarrays. Appl Environ Microbiol 67:3677-3682
    Choi KH, Dobbs FC (1999) Comparison of two kinds of Biolog microplates (GN and ECO) in their ability to distinguish among aquatic microbial communities. J Microbiol Methods 36:203-213
    Classen AT, Boyle SI, Haskins KE, Overby ST, Hart SC (2003) Community-level physiological profiles of bacteria and fungi: plate type and incubation temperature influences on contrasting soils. FEMS Microbiol Ecol 44:319-328
    Clegg CD, Ritz K, Griffiths BS (2000) % G+C profiling and cross hybridisation of microbial DNA reveals great variation in below-ground community structure in UK upland grasslands. Appl Soil Ecol 14:125-134
    Derry AM, Staddon WJ, Kevan PG, Trevors JT (1999) Functional diversity and community structure of micro-organisms in three arctic soils as determined by sole-carbon-source-utilization. Biodivers Conserv 8:205-221
    Derry AM, Staddon WJ, Trevors JT (1998) Functional diversity and community structure of microorganisms in uncontaminated and creosote-contaminated soils as determined by solecarbon-source-utilization. World J Microbiol Biotechnol 14:571-578
    Dodd JC, Boddington CL, Rodriguez A, Gonzalez C, Mansur I (2000) Mycelium of arbuscular mycorrhizal fungi (AMF) from different genera: form, function and detection. Plant Soil 226:131-151
    Dunbar J, Ticknor LO, Kuske CR (2000) Assessment of microbial diversity in fourth southwestern United States soils by 16S Rrna gene terminal restriction fragment analysis. Appl Environ Microbial 66:2943-2950
    Eilers H, Pernthaler J, Glockner FO, Amann R (2000) Culturability and in situ abundance of pelagic bacteria from the North Sea. Appl Environ Microbiol 66:3044-3051
    Ellis RJ, Thompson IP, Bailey MJ (1995) Metabolic profiling as a means of characterizing plant-associated microbial communities. FEMS Microbiol Ecol 16:9-18
    Erdogan E, Sahin F, Namli A (2013) Phospholipid fatty acids analysis-fatty acid methyl ester (PLFA-FAME) changes during bioremediation of crude oil contamination soil. African J Soil Sci 1:1-8
    Everett K, Rees J, Pushparajah I, Janssen B, Luo Z (2010) Advantages and disadvantages of microarrays to study microbial population dynamics- a minireview. NZ Plant Prot 63:1-6
    Felske A, Akkermans ADL (1998) Spatial homogeneity of abundant bacterial 16S rRNA molecules in Grassland soils. Microbial Ecol 36:31-36
    Filion M, St-Arnaud M, Fortin JA (1999) Direct interaction between the arbuscular mycorrhizal fungus Glomus intraradices and different rhizosphere microorganisms. New Phytol 141:525-533
    Fisher MM, Triplett EW (1999) Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl Environ Microbiol 65:4630-4636
    Garland JL, Millis AL (1991) Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization. Appl Environ Microbiol 57:2351-2359
    Gelsomino AC, Keijzer G, Cacco, Van Elsas (1999) Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis. J Microbiol Methods 38:1-15
    George E, Marschner H, Jakobsen I (1995) Role of arbuscular mycorrhizal fungi in uptake of phosphorous and nitrogen from soil. Crit Rev Biotechnol 15:257-270
    Gich FB, Amer E, Figueras JB, Abella CA, Balaguer MD, Poch M (2000) Assessment of microbial community structure changes by amplified ribosomal DNA restriction analysis (ARDRA). Int Microbiol 3:103-106
    Graham JH, Hodge NC, Morton JB (1995) Fatty acid methyl ester profiles for characterization of Glomalean fungi and their endomycorrhizae. Appl Environ Microbiol 61:58-64
    Grayston SJ, Wang S, Campbell CD, Edwards AC (1998) Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol Biochem 30:369-378
    Greene EA, Voordauw G (2003) Analysis of environmental microbial communities by reverse sample genome probing. J Microbiol Methods 53:211-219
    Griffiths BS, Ritz K, Ebblewhite N, Dobson G (1999) Soil microbial community structure: effects of substrate loading rates. Soil Biol Biochem 31:145-153
    Hack E, Zechmeister-Boltenstern S, Bodrossy L, Sessitsch A (2004) Comparison of diversities and compositions of bacterial populations inhabiting natural forest soils. Appl Environ Microbial 70:5057-5065
    He Z, Deng Y, Nostrand JDV, Tu Q, Xu M, Hemme CL , Li X, Wu L, Terry JG, Yin Y, Liebich J, Terry CH, Zhou J (2010) GeoChip 3.0 as a high-throughput tool for analyzing microbial community composition, structure and functional activity. Inter Society for Microbial Ecol 4:1167-1179
    Huang WE, Stoecker K, Griffiths R, Newbold L, Daims H, Whiteley AS, Wagner M (2007) Raman–FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. Environ Microbiol 9:1878-1889
    Ibekwe AM, Kennedy AC (1999) Fatty acid methyl ester (FAME) profiles as a tool to investigate community structure of two agricultural soils. Plant Soil 206:151-161
    Insam H (2001) Development in soil microbiology since mid 1960s. Geoderma 100:389-402
    Kakavas VK, Konstantinos KV, Plageras P (2008) PCR-SSCP: a method for the molecular analysis of genetic diseases. Mol Biotechnol 38:155-63
    Kaur A, Chaudhary A, Raushik R (2005) Phospholipid fatty acid as a bioindicator of environment monitoring and assessment in soil ecosystem. Curr Sci 89:1103-1112
    Kenzaka T, Yamaguchi N, Tani K, Nasu M (1998) rRNA-Targeted fluorescent in situ hybridization analysis of bacterial community structure in river water. Microbiol 144:2085-2093
    Kirchoff G, Schloter M, Assmus B, Hartmann A (1997) Molecular microbial ecology approaches applied to diazotrophs associated with non-legumes. Soil Biol Biochem 29:853-862
    Kirk JL, Beaudette LA, Hart M, Moutoglis P, Klironomos JN, Lee H (2004) Methods of studying soil microbial diversity. J Micro Methods 58:169-188
    Kirk NL, Ward JR, Coffroth MA (2005) Stable Symbiodinium composition in the seafan Gorgonia ventalina during temperature and diseasestress. Biol Bull 209:227-234
    Konopka A, Oliver L, Turco RF (1998) The use of carbon source utilization patterns in environmental and ecological microbiology. Microb Ecol 35:103-115
    Kujur M, Patel AK (2014) PLFA Profiling of soil microbial community structure and diversity in different dry tropical ecosystems of Jharkhand. Int J Curr Microbiol App Sci 3:556-575
    Lan W, Li H, Wang WD, Katayama Y, Gu JD (2010) Microbial community analysis of fresh and old microbial biofilms on Bayon temple sandstone of Angkor Thom, Cambodia. Microb Ecol 60:105-115
    Liu WT, Marsh TL, Cheng H, Forney LJ (1997) Characterisation of microbial diversity by determining terminal restriction length polymorphisms of genes encoding 16S rDNA. App Environ Microbiol 63:4516-4522
    Longato S, Bonfante P (1997) Molecular identification of mycorrhizal fungi by direct amplification of microsatellite regions. Mycol Res 101:425-432
    Ludwig W, Bauer SH, Bauer M, Held I, Kirchhof G, Schulze R, Huber I, Spring S, Hartman A, Schleifer KH (1997) Detection and in situ identification of representatives of a widely distributed new bacterial phylum. FEMS Microbiol Lett 153:181-190
    MacNaughton SJ, Booth T, Embley TM, O’Donnell AG (1996) Physical stabilization and confocal microscopy of bacteria on roots using 16S rRNA targeted fluorescent-labeled oligonucleotide probes. J Microbiol Meth 26:279-285
    Massoldeya AA, Odelson DA, Hickey RF, Tiedje JM (1995) Bacterial community fingerprinting of amplified 16S and 16S–23S ribosomal DNA gene sequences and restriction endonuclease analysis (ARDRA). In: Akkermans, A.D.L., van-Elsas, J.D., de-Bruijn, F.J. (Eds.), Molecular Microbial Ecology Manual. Kluwer Academic Publishing, Boston, pp. 3.3.2 1-3.3.2 8.
    Mitsui H, Gorlach K, Lee HJ, Hattori R, Hattori T (1997) Incubation time and media requirements of culturable bacteria from different phylogenetic groups. J Microbiol Meth 30:103-110
    Morozova D, Leta D, Würdemanna H (2013) Analysis of the microbial community from a saline aquifer prior to CO2 injection in Ketzin using improved Fluorescence in situ Hybridisation method. Energy Procedia 40:276-284
    Moter A, Göbel UB (2000) Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. J Microbiol Methods 41:85-112
    Muyzer G (1999) DGGE/TGGE a method for identifying genes from natural ecosystems. Curr Opin Microbiol 2:317-322
    Muyzer G, de Waal DC, Uitterlinden AG (1993) Pro!ling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-ampli!ed genes coding for 16S rRNA. Appl Environ Microbiol 59:695-700
    Nakatsu CH, Torsvik V, Øvreas L (2000) Soil Community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Sci Soc Am J 64:1382-1388
    Nannipieri P, Ascher J, Ceccherini MT, Landi L, Pietramellara G, Renella G (2003) Microbial diversity and soil functions. Eur J Soil Sci 54:655-670
    Nocker A, Burr M, Camper A (2007) Genotypic microbial community profiling: a critical technical review. Microb Ecol 54:276-289
    Norland KI, Southam G, Tyliszczak T, Hu Y, Karunakaran C, Obst M, Hitchcock AP, Warren LA (2009) Microbial architecture of environmental sulfur processes: a novel syntrophic sulfurmetabolizing consortia. Environ Sci Technol 43:8781-8786
    Nüsslein K, Tiedjee JM (1999) Soil bacterial community shift correlated with change from forest to pasture vegetation in a tropical soil. Appl Environ Microbiol 65:3622-3626
    Okubo A, Sugiyama S (2009) Comparison of molecular fingerprinting methods for analysis of soil microbial community structure. Ecological Research 24:1399-1405
    Pace NR (1996) New perspective on the natural microbial world: molecular microbial ecology. ASM News 62:463-470
    Peters S, Koschinsky S, Schwieger F, Tebbe CC (2000) Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation-polymorphism-based genetic profiles of small-subunit rRNA genes. Appl Environ Microbiol 66:930-936
    Pinar G, Garcia M, Gimento D, Fernandez JL, Ettenauer J, Sterflinger K (2013) Microscopic, chemical and molecular biological investigation of the decayed medieval stained window glasses of two Catalonian churches. Int Biodeter Biodegr 84:388-400
    Popa R, Mashall MJ, Nguyen H, Tebo BM, Brauer S (2009) Limitations and benefits of ARISA intra-genomic diversity fingerprinting. J Microbiol Methods 78:111-118
    Porteous LA, Seidler RJ, Watrud LS (1997) An improved method for purifying DNA from soil for polymerase chain reaction amplification and molecular ecology applications. Mol Ecol 6:787-791
    Pukall R, Brambilla E, Stackebrandt E (1998) Automated fragment length analysis of fluorescently labeled 16S rDNA after digestion with 4-base cutting restriction enzymes. J Microbiol Methods 32:55-63
    Ranjard L, Nazaret S, Gourbiere F, Thioulouse J, Linet P, Richaume A (2000) A soil microscale study to reveal the heterogeneity of Hg (II) impact on indigenous bacteria by quantification of adapted phenotypes and analysis of community DNA fingerprints. FEMS Microbiol Ecol 31:107-115
    Rastogi G, Sani RS (2011) Molecular techniques to asses microbial community structure, function and dynamics in the environment. In Microbes and microbial technology: agricultural and environmental applications. Ahmad I, Ahmad F, Pichtel J, eds, pp 29-57. Springer, New York.
    Rhee SK, Liu X, Wu L, Chong SC (2004) Detection of Genes Involved in Biodegradation and Biotransformation in Microbial Communities by Using 50-Mer Oligonucleotide Microarrays. Appl Environ Microbiol 70:4303-4317
    Schmalenberger A, Schwieger F, Tebbe CC (2001) Effect of primers hybridizing to different evolutionarily conserved regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling. Appl Environ Microbiol 67:3557-3563
    Schwieger F, Tebbe CC (1998) A new approach to utilise PCR-single-strand conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl Environ Microbiol 64:4870-4876
    Simon C, Rolf D (2011) Metagenomic analysis : Past and future trends. Appl Environ Microbiol 77:1153-1161
    Sklarz MY, Angel R, Gillor O, Soares IM (2011) Amplified rDNA restriction analysis (ARDRA) for identification and phylogenetic placement of 16S rDNA clones. In Handbook of Molecular Microbial Ecology I: Metagenomics and Complementary Approaches. de Bruijn FJ, eds, pp 59-60. John Wiley & Sons, Inc., Hoboken, New Jersey.
    Small K, Wieland G, Buchner A, Zock A, Parzy J, Kaiser S, Roskot R, Heuer H, Berg G (2001) Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Appl Environ Microbiol 67:4742-4751
    Srivastava D, Kapoor R, Srivastava SK, Mukerji KG (1996) Vesicular arbuscular mycorrhiza—an overview. In: Mukerji, K.G. (Ed.), Concepts in Mycorrhizal Research. Kluwer Academic Publishing, Netherlands, pp. 1-39.
    Stach JEM, Bathe S, Clapp JP, Burns RG (2001) PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods. FEMS Microbiol Ecol 36:139-151
    Theron J, Cloete TE (2000) Molecular techniques for determining microbial diversity and community structure in natural environments. Crit Rev Microbiol 26:37-57
    Tiedjee JM, Asuming S, Nusslein K, Marsh TL, Flynn SJ (1999) Opening the black box of soil microbial diversity. Appl Soil Ecol 386:1-14
    Timonen S, Finlay RD, Olsson S, Soderstrom B (1996) Dynamics of phosphorous translocation in intact ectomycorrhizal systems: non-destructive monitoring using a B-scanner. FEMS Microbiol Ecol 19:171-180
    Tischer K, Zeder M, Klug R, Pernthaler J, Schattenhofer M, Harms H, Wendeberg A (2012) Fluorescence in situ hybridization (CARD-FISH) of microorganisms in hydrocarbon contaminated aquifer sediment samples. Syst Appl Microbiol 35:526-532
    Tomaselli L, Lamenti G, Bosco M, Tiano P. (2000) Biodiversity of photosynthetic microorganisms dwelling on stone monuments. Int Biodeter Biodegr 46:251-258
    Torsvik V, Daae FL, Sandaa RA, Ovreas L (1998) Review article: novel techniques for analysing microbial diversity in natural and perturbed environments. J Biotechnol 64:53-62
    Torsvik V, Goksoyr J, Daae FL (1990) High diversity in DNA of soil bacteria. Appl Environ Microbiol 56:782-787
    Torsvik V, Sorheim R, Goksoyr J (1996) Total bacterial diversity in soil and sediment communities a review. J Ind Microbiol 17:170-178
    Whitman WB, Coleman DC, Wiebe WJ (1998) Prokaryotes: The unseen majority. Proceedings of the National Academy of Sciences of the United States of America 95:6578-83
    Wintizingerode FV, Gobel UB, Stackebrandt E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21: 213-229
    Wullings BA, van Beuningen AR, Janse JD, Akkermanns ADL (1998) Detection of Ralstonia solanacearum which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes. Appl Environ Microbiol 64:4546-4554
    Yao H, He Z, Wilson MJ, Campbell CD (2000) Microbial biomass and community structure in a sequence of soils with increasing fertility and changing land use. Microb Ecol 40:223-237
    Zak JC, Willig MR, Moorhead DL, Wildman HG (1994) Functional diversity of microbial communities: a quantitative approach. Soil Biol Biochem 26:1101-1108
    Zeze A, Hosny M, Gianinazzi V, Dulieu H (1996) Characterization of a highly repeated DNA sequence (SC1) from the arbuscular mycorrhizal fungus Scutellospora castanea and its detection in planta. Appl Environ Microbiol 62:248-2443