Chapter 2: LITERATURE REVIEW
2.4 TECHNIQUES FOR ESTIMATING SOIL MICROBIAL DIVERSITY
2.4.1 Non-molecular techniques
2.4.1.4 Substrate utilization
Analysis of community functioning based on substrate utilization (metabolic) profiling, measures biodiversity of communities rather than of species (Pankhurst, 1997). Substrate utilization patterns provide reliable ‘fingerprints’ of microbial populations and may indicate metabolic potential or microbial community function (Kennedy and Gewin, 1997; Buyer et al., 2001). A simple approach to studying microbial functional diversity is based on the number of C substrates utilized by the community, the two most commonly used methods being the BIOLOG® microplate identification system (BIOLOG Inc., Hayward, California, USA) and substrate induced respiration (SIR) (Pankhurst, 1997).
2.4.1.4.1 The BIOLOG method
Garland and Mills (1991) published a method for the functional characterization of heterotrophic microbial communities. This was based on sole-carbon source utilization as a community-level physiological profiling (CLPP) approach, using
commercially available BIOLOG GN 96-well microtitre plates. The BIOLOG system was originally devised to identify pure bacterial cultures, mainly Gram-negative (GN) species of clinical importance. The 95 different substrates in the GN plates were selected from a set of 500 carbon sources, for their ability to distinguish clinically important species from among 6000 bacterial strains (Konopka et al., 1998; Hartmann et al., 2007). To identify Gram-positive bacteria, BIOLOG GP plates have been developed. Sixty two substrates are common to both GN and GP plates, with a further 33 substrates unique to each (Konopka et al., 1998). In addition, wells in the GN and GP plates contain tetrazolium violet dye. This is reduced to purple-coloured insoluble formazan when oxidation of the C compounds by microbial metabolic activity occurs, thus enabling catabolic diversity to be assessed (De Fede et al., 2001; Kirk et al., 2004). The production of formazan is directly related to the reducing equivalents formed by the inoculated community, and subsequently, by the growing cells within a well (Smalla et al., 1998).
Although the system was designed to monitor respiratory activity rather than growth, bacteria can grow in the microplate wells. If the inocula are diluted, colour does not develop until the cell concentration per well has reached 108 cells ml-1. Therefore, at an inoculum density of < 108 cells ml-1, colour development will be a sigmoidal function of time, in which: (i) the initial lag phase is a period when the inoculum grows to a population of 108 cells ml-1; (ii) the linear phase is a period of microbial metabolic activity on a specific substrate; and (iii) the plateau represents the formazan yield produced by particular microbes from a specific substrate. All phases are therefore, affected by the microbes’ physiology, with the initial lag phase being highly dependent on the inoculum cell density (Konopka et al., 1998).
CLPP analysis usually requires extraction of microbial cells from environmental samples (Smalla et al., 1998). However, as researchers found that non-culturable cells from mixed communities also responded to substrate supply, the method was adapted to characterize the functional potential of these communities. Numerous studies have shown that habitat-specific and reproducible patterns of carbon utilization are produced by such microbial communities (Hartmann et al., 2007).
Prokaryote community analysis and ecological studies are now possible using BIOLOG EcoPlates™, which were created specifically for this purpose. Each microplate contains 31 of the most useful carbon sources, together with tetrazolium dye, for soil community analysis, replicated three times (Appendix D, Table D1) (Liu et al., 2007a, b; Zhang et al., 2008). The typical procedure when using EcoPlates involves inoculating environmental samples directly into the microplate wells, either as aqueous samples, or suspensions of soil, sludge or sediment. The microplates are then incubated and plate readings taken at defined time intervals, using a microplate reader, to give optical density (OD) values. CLPPs are then assessed for pattern development (similarity), the rate of colour change in each well, and the richness of well response (diversity) (BIOLOG, 2008).
When using CLPP, soil samples present the greatest challenge as microbes bind to soil particles (Konopka et al., 1998). To overcome this problem, vigorous shaking or homogenisation of soils suspended in either sterile distilled water, phosphate buffer or saline, is used to release bacteria from particles (Calbrix et al., 2005; Chen et al., 2007; Liu et al., 2007a). The suspension is then allowed to settle or, in some protocols, centrifuged, as the presence of soil particles in the inoculum causes inaccurate OD readings (Calbrix et al., 2005). The supernatant is retained and usually diluted before use (Govaerts et al., 2007; Liu et al., 2007a, b).
As fungi cannot be detected by the GN and GP plates, because they do not reduce tetrazolium violet and the dye is toxic to many fungal species, fungus-specific microplates (namely, SF-N and SF-P MicroPlates™) have been developed. The SF-N plates (Appendix E, Table E1) are identical to the GN microplates and the SF-P plates to the GP microplates, except that they lack the tetrazolium redox dye (BIOLOG, 2008). Testing fungi in SF-N and SF-P microplates is very simple and uses a protocol similar to that for testing bacteria and yeast. Modifications include the use of special culture media to promote sporulation, preparation of lower density inocula in a gel- forming colloid rather than in water or saline, and inoculation of 100 µl per well instead of 150 µl. Plates are incubated at a low temperature (~26˚C) for several days and growth is measured by an increase in turbidimetry at OD650. BIOLOG FF microplates and a FF database have also been developed for fungal analyses (BIOLOG, 2008).
The advantages of CLPP assays include their ease of use for replicated large-scale studies. The microplates provide a fast, simple and accurate method for studying microorganisms that is an alternative, or is complementary, to traditional macroscopic and microscopic methods. Also, CLPP is more sensitive than PLFA analysis for monitoring microbial community and ecological changes. CLPP provides broad coverage of a variety of microbial samples, both bacterial and fungal, and has many different applications. For example, it can distinguish spatial and temporal changes in microbial communities: it has been used to detect population changes in soil, water, wastewater, activated sludge, compost and industrial waste: and it also has clinical applications (BIOLOG, 2008).
Limitations of CLPP are that culturable microbes are required, the system is sensitive to inoculum density, and favours fast-growing microorganisms (Buyer et al., 2001;
De Fede et al., 2001; Kirk et al., 2004). Culture conditions in BIOLOG microplates are thought to have a harmful effect on a large proportion of the inoculated bacteria, as microbial diversity decreases continuously during the first week of incubation. In addition, the relative proportion of species in a microbial community may change during microplate incubation, and a long incubation time induces cell lysis and the formation of extracellular storage polymers (Calbrix et al., 2005). Thus CLPP analysis does not necessarily reflect the functional potential of the dominant community members but is biased towards those populations that grow best under the assay conditions (Ros et al., 2008).
2.4.1.4.2 Substrate induced respiration (SIR)
To avoid some of the drawbacks of the BIOLOG method, Degens and Harris (1997) developed substrate induced respiration (SIR), an in situ approach that does not require prior extraction and cultivation of soil microorganisms. Short-term SIR responses to 36 organic substrates added directly to soil are measured by assessing CO2 efflux from soil by gas chromatography, thus revealing catabolic diversity of active bacterial and fungal communities.