Pedosphere lS(1): 18-24, 2006

ISSN 1002-0160/CN 32-1315/P

@ 2006 Soil Science Society of China
Published by Elsevier Limited and Science Press

A Review of Methods for Studying Microbial Diversity in Soils*'

LIU Bing-Ru', JIA Guo-Mei', CHEN Jian' and WANG Gang1i2,*2

'Key Laboratory of Arid and Grassland Agroecology a t Lanzhou University, Ministry of Education, Lanzhou 730000
(China). E-mail: liubr02@st.lzu.edu.cn
2School of Life Science, Lanzhou University, Lanzhou 730000 (China)
(Received September 14, 2005; revised December 12, 2005)

ABSTRACT
Soil microorganisms play a central role in decomposing organic matter, in determining the release of mineral nutrients,
and in nutrient cycling. Recently, extensive studies have focused on soil microbial diversity. However, understanding the
diversity of this complex microbial community in the soil environment is a challenging task. Thus, it is important to
master and comprehend appropriate methods for studying soil microbial diversity. Concepts of soil microbial diversity
and major methods of study are briefly introduced in this paper. Then, the application of biochemical-based and molecular-
based techniques in this area, and their advantages and disadvantages are evaluated. Based on recent related research,
perspectives for studying microbial diversity in soils are presented.

Key Words: bacteria, diversity, fungi, molecular, soil

INTRODUCTION

Soil microorganisms comprise much of the Earth's biodiversity and have a critical role in biogeochemi-
stry cycles (BGC) as well as ecosystem functioning (Green et al., 2004). Because soil microorganisms
play a central role in decomposing organic matter, in determining the release of mineral nutrients, and
in nutrient cycling, they affect soil nutrient contents, chemical-physical properties, and consequently,
primary productivity (Rutigliano e t aZ., 2004). Moreover many anthropogenic activities, such as urban
development, agriculture, pollution, and the use of pesticides can potentially affect soil microbial diver-
sity (Zhang et aZ., 2004). Therefore, soil microbial diversity is fundamental for sustaining environmental
management and for evaluating soil quality (Anderson, 2003; Schloter et al., 2003; Li e t aZ., 2005).
Understanding the complex diversity of the microbial community in the soil environment has proven
to be a challenging task. This has arisen not only because of methodological limitations, but also of a
lack of taxonomic knowledge. In recent years, extensive studies have focused on soil microbial diversity,
hoping for in-depth understanding of the soil black box (Tiedje e t al., 1999). Thus, it is very important
to use and comprehend appropriate methods of studying soil microbial diversity.

THE SOIL MICROBIAL DIVERSITY CONCEPT

Soil microbial diversity is composed of species diversity and genetic diversity, as well as ecosystem
biodiversity (Solbrig, 1991). Species diversity consists of species richness, the total of species present,
species evenness, and the distribution of species ((dvrebs, 2000). However, the traditional species defi-
nition was based on higher plants and animals and is not readily applied to prokaryotes (Godfray and
Lawton, 2001) or asexual organisms. Microbial diversity has usually been taken as the number of in-
dividuals assigned to different taxa and their distribution among taxa. consequently, microorganisms
have been referred to collectively as functional groups or guilds. Typically, microbial diversity studies
have included the relative diversities of communities across a gradient of stress, disturbance, or other

*lProject supported by the National Key Basic Research and Development Program of China (No. 2002CB111505).
*2Corresponding author. E-mail: wangg@lzu.edu.cn.
METHODS FOR STUDYING SOIL MICROBIAL DIVERSITY 19

biotic or abiotic differences (Hughes et al., 2001).

MEASURING SOIL MICROBIAL DIVERSITY

Because of taxonomic and methodological limitations, studying species and genetic diversity has been
difficult. A fundamental problem with many traditional physiological and biochemical methods has been
their dependency on cultivation of the microorganisms and/or analysis of their phenotypic expression
(e.g., respiration, enzymes, and catabolic potential). However, even when they have demonstrated
metabolic activity many microorganisms cannot be cultivated under laboratory conditions. In addition,
due to low gene expression under the test conditions, using biochemical test kits has led to fairly
common negative results (Torsvik et al., 1998). At present only two approaches are known to overcome
this problem, namely the use of signature lipid biomarkers (SLB), such as phospholipid fatty acids
(PLFA), and nucleic acid technologies (molecular biology). In most of these methods multivariate data
(or fingerprints) were produced and analyzed with principal component analysis (PCA) or canonical
variate analysis (CVA). Thus, soil microbial communities are characterized according to their phenotypic
and genetic diversity, and methods to measure microbial diversity in soils have been categorized into
two groups: biochemical-based techniques and molecular-based techniques (Kirk et aL, 2004).

BIOCHEMICAL-BASED TECHNIQUES

Plate counts

As a traditional and culture-dependent method, the plate count is fast, inexpensive, and can directly
provide information on the active, heterotrophic component of the population (Tabacchioni et al., 2000).
It has been estimated that about 5000 bacterial species have been described (Pace, 1997), but only
approximately 0.1% to 1% of the soil bacterial population can be cultured by standard laboratory
practices (Torsvik et al., 1998). Limitations include growth conditions such as temperature, pH, and
light. Moreover, an estimated 1.5 million species of fungi exist in the world, but unlike bacteria, current
standard laboratory methods cannot be used to culture many fungi (van Elsas et al., 2000).

Community level physiological profiles (CLPP) and sole carbon source utilization (SCSU)patterns

CLPP method is a means of investigating the physiological diversity present in soils. These profiles
reflect how the microbial communities could potentially utilize a range of carbon substrates. Differences
in utilization patterns are interpreted as differences in the major active members of the microbial
community. For instance, in the BIOLOG system, 95 different carbon sources are used to produce a
metabolic profile of microorganisms (Garland and Mills, 1991).
As it is simple, useing an automated measuring apparatus, and yielding a great deal of information
about important functional attributes of microbial communities, the technique has become popular;
nonetheless, the analysis and interpretation of such data are often complicated (Broughton and Gross,
2000). There are also other drawbacks. The BIOLOG systems only assess the metabolic diversity of
culturable bacteria, and soil fungi and slow-growing bacteria have minimal influence on the microbial
metabolic profile. Additionally, the BIOLOG sole C-source test plates contain high concentrations of
carbon sources (Campbell et al., 1997), and TTC (triphenyltetrazolium chloride). Moreover, the plates
are buffered at nearly neutral pH, which is quite different from the pH of some acid or alkaline soils,
leading to some limitations for partial microorganisms that have adapted well to acidic or alkaline
soils. Many of these factors have presented disadvantages when determining soil microbial community
structure.

Fatty acid methyl ester (FAME) and phospholipid fatty acid (PLFA) analyses

Fatty acid methyl ester (FAME) method provides information about the microbial community com-
20 B. R. LIU et al.

position based on groupings of fatty acids (Ibekwe and Kennedy, 1998; Broughton and Gross, 2000).
Fatty acids make up a relatively constant proportion of the cell biomass, and signature fatty acids can
differentiate major taxonomic groups within a community (Kirk et al. 2004). Therefore, a change in
the fatty acid profile would represent a change in the microbial biomass and community structure.
The PLFA technique has been used to elucidate different strategies employed by microorganisms
to adapt to changed environmental conditions under wide ranges of soil types, management practices,
climatic origins, and different perturbations (Zelles, 1999). To simplify evaluating procedures and
improve assessment of soil microbial communities, since only subgroups assumed to be involved in
key processes would be investigated, Zelles (1999) proposed classification of PLFAs into a number of
chemically different subgroups.

MOLECULAR-BASED TECHNIQUES

During the last few years, to study the distribution and activity of microorganisms in the environ-
ment, microbial ecologists have switched more and more to molecular strategies. A number of approaches
have been developed to detect microbes in soils, such as nucleic acid hybridization, fluorescent in situ
hybridization (FISH), DNA cloning and sequencing, polymer chain reaction (PCR)-based methods, etc.

Nucleic acid hybridization and fluorescent in situ hybridization (FISH)

Nucleic acid hybridization using specific probes is an important qualitative tool in molecular bacterial
ecology (Theron and Cloete, 2000). These hybridization techniques can be performed on extracted
DNA and RNA, or in situ hybridization can be conducted at the cellular level. The FISH method
has also been used successfully to study the spatial distribution of bacteria in biofilms (Thurnheer et
al., 2004). However, with respect to sensitivity, some limitations to the standard FISH method that
prevents detection of cells with low ribosome content have been noted. Low physiological activity was
often correlated with low ribosome content per cell, therefore slow-growing or starving cells may not
be detected (Amann et aL, 1995). To overcome this limitation, FISH has adopted a tyramime signal
amplification technique, which allowed the analysis of slow-growing microorganisms (Pernthaler et al.,
2002). Also, a disadvantage of nucleic acid hybridization or FISH is the lack of sensitivity unless
sequences are present in high copy number (Pernthaler et al., 2002).

Guanine plus cytosine ( G + C) content

+
Difference in the guanine plus cytosine (G C) content of DNA can be used to study soil bacterial
diversity (Niisslein and Tiedje, 1999). This procedure is based on the knowledge that microorganisms
differ in their G + C content and that taxonomically related groups only differ between 3% and 5%
(Tiedje et al., 1999). This method provides a coarse level of resolution as different taxonomic groups
+
may share the same mol percentage range of G C. The melting curves provide microbial community
profiles indicative of the overall genetic diversity. Even if this analysis is considered to be low resolution,
it can be used to indicate overall changes in microbial community structure, especially when the diversity
is low. An advantage of this approach includes no PCR bias with all DNA extractions, quantification, or
uncovering rare members in the microbial populations. Thus, some of the less dominant microorganisms
in the community that PCR might not detect without fractionation can be detected and analyzed.
However, it requires large quantities of DNA (Tiedje et al., 1999).

PCR- based techniques

In diversity studies to overcome the limitations of culture-based methods molecular techniques based
on polymer chain reaction (PCR) have been used. This detection protocol can be widely utilized in
ecological and environmental research. DNA extracted directly from the environment can act as a tem-
plate for PCR. PCR targeting the 16s rDNA has been used extensively to study prokaryote diversity
METHODS F O R STUDYING SOIL MICROBIAL DIVERSITY 21

and allows identification of prokaryotes (Pace, 1997), whereas 18s rDNA and internal transcribed spacer
(ITS) regions are increasingly used to study fungal communities. Target DNA (16S, 18S, or ITS) is
amplified using universal or specific primers, and the resulting products are separated in different ways.
Then, the amplified PCR product can be hybridized with primers to provide specific information on the
community (Pace, 1997).
Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE).
DGGE and TGGE are two similar methods for studying microbial diversity. Theoretically, DGGE
can separate DNA with one base-pair difference (Miller et al., 1999). TGGE uses the same principle
as DGGE except the gradient is temperature rather than chemical denaturants. These techniques were
originally developed to detect point mutations in DNA sequences. Advantages of DGGE and TGGE
include being reliable, reproducible, rapid, and somewhat inexpensive; providing concurrent analysis of
multiple samples; and having the ability to follow changes in microbial populations (Muyzer, 1999).
Random amplified polymorphic DNA (RAPD). Randomly amplified polymorphic DNA poly-
merase chain reaction (RAPD-PCR) is a simple and rapid method for identification of useful genetic
markers and determination of organismal genetic diversity at various taxonomic levels (Welsh and Mc-
Clelland, 1990; William et al., 1990). With this technique, PCR amplifies with random primers; the
amplified products are analyzed with electrophoresis stained with ethidum bromide; and the gel images
are analyzed with imaging systems. Then RAPD bands are scored as binary presence or absence char-
acters, to assemble a matrix of RAPD phenotypes. The percentage of polymorphic bands is utilized
to measure genetic diversity. Compared with other molecular markers, advantages of this method are
that it is simpler, fast, and abundant in polymorphic DNA. Nevertheless, the short primers result in
low repetition.
Amplified fragment length polymorphism (AFLP). AFLPs are PCR-based markers for the rapid
screening of genetic diversity. The key feature of AFLP-PCR is its capacity for simultaneous screen-
ing of many different DNA regions distributed randomly throughout the genome. In essence, AFLP
methods allow PCR amplification to detect polymorphisms of genomic restriction fragments. AFLP
markers have proven useful for assessing genetic differences among individuals, populations, and inde-
pendently evolving lineages, such as species (Vos et al., 1995). The main disadvantage of AFLP-PCR is
the difficulty in identifying homologous markers (alleles), rendering this method less useful for studies
that require precise assignment of allelic states, such as heterozygosity analysis. However, because of
the rapidity and ease with which reliable, high-resolution marks can be generated, AFLPs are emerging
as a powerful addition to the molecular toolkit of ecologists and evolutionary biologists (Mueller and
Wolfenbarger, 1999).
Restriction fragment length polymorphism (RFLP) and terminal restriction fragment length poly-
morphism (T-RFLP). RFLP is a culture-independent technique for assessing microbial community
diversity using a DNA sequencer. To obtain useful results, one must ensure digestion completeness and
the reproducibility of the RFLP banding pattern. In general, PCR-amplified rDNA is digested with
base pairs cutting restriction enzyme. Different lengths are detected using agarose or non-denaturing
polyacrylamide gel electrophoresis (PCGE) in the case of communities analysis (Tiedje et al., 1999).
T-RFLP follows the same principle as RFLP except that one PCR primer is labeled with a fluorescent
dye. T-RFLP fingerprints are often used to track spatial and temporal changes in microbial diversity
(Moeseneder et al., 1999; Urakawa et al., 2000).
Automated ribosomal intergenic spacer analysis (ARISA) and ribosomal intergenic spacer analysis
(RISA). Automated ribosomal intergenic spacer analysis (ARISA), a commonly used DNA-based
community fingerprinting method (0vrebs, 2000) , is a high-resolution, highly reproducible technique for
detecting differences among complex fungal communities (Ranjard et al., 2001). RISA is based on the
length polymorphism of the ribosomal intergenic spacer region between the 16s and 23s rRNA genes
(Borneman and Triplett, 1997). The non-coding ribosomal internal spacer region is variable in both
size and nucleotide sequence even within closely related strains, and the method has been successfully
22 B. R.LIU et al.

used to characterize, classify, and type strains, as well as to fingerprint simple communities and mixed
populations (Nagpal et al., 1998; Ranjard et al., 2000). Ribosomal intergenic spacer analysis (RISA)
exploits variability in the length of the internal transcribed spacer regions of rRNA genes to sort samples
rapidly into operational taxonomic units (OTUs). Members of different species may share the same ITS
fragment size (Ranjard et al., 2001). Although ARISA assays a different taxonomic resolution than
species level, it is a consistent measure of community composition. Consequently, differences between
two OTU assemblages directly reflect changes in species composition (Green et al., 2004).
Single-strand conformational polymorphism (SSCP). SSCP, based on separation of PCR-amplified
rRNA and rDNA molecules, has been used successfully to analyze the structure and dynamics of micro-
bial communities (Schwieger and Tebbe, 1998). The method is based on the differential intra-molecular
folding of single-stranded DNA that is itself dependent upon DNA sequence variations. Thus, DNA se-
condary structure alters the electrophoresis mobility of the single-stranded PCR amplifications enabling
them to be resolved. SSCP has been used to differentiate between pure cultures of soil microorganisms
and to distinguish community fingerprints of uncultivated rhizospheric microbial communities from dif-
ferent plants (Schwieger and Tebbe, 1998). SSCP analysis should, in principle, be easier to carry out
than DGGE or TGGE, as no primers with GC-clamp or specific apparatus for gradient gels are required.
A limitation of the method, in addition to potential PCR bias, however, is that a single bacterial species
may yield several bands due to the presence of several operons or more than one conformation of the
single-stranded PCR amplifications.
Another approach to identify community members is to apply specific enrichments to enhance the
growth of the microorganism of interest. This strategy is particularly useful in studies of functional
groups or guilds (Lynch et al., 2004).

FUTURE PERSPECTIVES

The current ability to study and understand soil microbial diversity is wrought with taxonomic and
methodological limitations. So, soil microbiologists have attempted to ameliorate molecular methods.
More recently, bioinformatics and microarray technology have been used in life sciences (McLachlan et
al., 2004; Mount, 2004); and these new methods could be valuable in soil microbial diversity studies.
Although molecular methods have the advantage of obtaining information about uncultivable organi-
sms, they also have limitations that cannot be ignored. Therefore, it is difficult to state whether one
technique of studying soil microbial diversity is better than another. In order to obtain the broadest
picture and the most information, the best way to study soil microbial diversity would be to use a
variety of tests with different endpoints and degrees of resolution.
One of the primary challenges in modern microbial ecology is effectively and accurately assessing total
microbial diversity, particularly with regard to detection of uncultivable and fastidious microbial species
and those present in low abundance. Because of the soil’s innate heterogeneity and spatial distribution
of the microorganisms, very little is known about spatial and temporal variability of microorganisms
in soil. So using a combination of geostatistical analyses to describe spatial distribution of subsurface
microorganisms together with power analyses to assess the required sample size should reduce variability
in sampling and provide a more representative sampling regime.
The present knowledge level of microbial diversity and function in the soil, the link between structural
diversity and function of below- and above-ground ecosystems, as well as methods to study plant-
microbesoil interactions are relatively limited. In order to understand the complexity of interacting
biological, chemical, and physical factors, botanists, microbiologists, pedologists, and ecologists should
cooperate to further the cause of science (Dobrovol’skaya et al., 2001).

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