The Importance of Laboratory Cultivation in Microbial Ecology

Microorganisms and their physiology have historically been studied with pure cultures considered to be representatives of a particular taxonomic or functional group. This approach has proved invaluable because it is possible to select for organisms with a particular metabolism of interest. For example, autotrophic ammonia-oxidizing bacteria, which are typically present at very low abundance in the environment, can be selected in laboratory growth medium containing only ammonia as an energy source, bicarbonate as a carbon source, and inorganic nutrients. This principle can be used to enrich and cultivate organisms from any environment with particular metabolic capabilities (Table 1).

When a microorganism is isolated in pure culture it opens the door to extremely detailed analysis of many aspects of its biology, for example, its response to the environment, its metabolic capabilities right through to determination of the complete sequence of its genome. However, the very strength of the method - selective enrichment - is also its greatest limitation, as it often results in the isolation of bacteria that are not representative of the most important organisms in situ. This has been recognized for many years but it is only with the development of culture-independent methods of microbial community analysis that the organisms genuinely predominant in the environment have been identified. This has led to the refinement of microbial culture techniques to obtain principal players from microbial ecosystems in pure culture. Conventionally, laboratory growth media contain high concentrations of substrates that are generally ^1000 times greater than found in situ. Indeed, many microorganisms are specifically adapted to oligotrophic

Table 1 Enrichment conditions for various microbial metabolisms

Electron acceptora

Electron donor*

Principal carbon

Photosynthesis (light driven) Oxygenic photosynthesis Anoxic photosynthesis

Aerobic or nitrate-reducing respiration Heterotrophs Metal-oxidizing bacteria Ammonia oxidizers Nitrite oxidizers Sulfur oxidizers

Hydrogen-oxidizing bacteria Anaerobic respiration Sulfate-reducing organisms Iron- and manganese-reducing organisms Chlorate-reducing organisms Dehaloginating bacteria

Anaerobic methane-oxidizing consortium Anaerobic ammonia oxidizers Methanogensd

Fermentation

NADP NADP

O2/NO3~ O2/NO3 O2 O2

O2/NO3 O2/NO3

ClO33

Halogenated organic compounds SO43, NO3

NO23 CO2

H2, H2S, S0, S2o33, organic compounds

A wide variety of organic compounds

S23, S0, S2O33, SO33, organosulfur compounds H2

Organic compounds, H2 Organic compounds

Organic compounds Fatty acids, H2

CH4, acetate

H2, CH3OH, acetate, methylated compounds

Complex and simple organic substrate serves as both electron acceptor and electron donor, e.g., starch, organic acids

Inorganic Inorganic

Organic

Inorganic

Inorganic

Inorganic

Inorganic

Inorganic

Organic and inorganic Organic

Organic Organic

Organic

Inorganic

Organic or inorganic Organic b,c source aThe oxidation of electron donors coupled to the reduction of electron acceptor supplies energy for ATP synthesis.

bIn addition to energy generation, organisms also require a source of carbon for growth. Heterotrophs typically utilize carbon from their organic substrate while chemolithoautotrophic and photosynthetic organisms convert inorganic carbon into biomass (autotrophy) using, respectively, the reduction potential of an electron donor or, light-driven reduction of NADP to NADPH. Note many chemolithoautotrophs can also utilize organic carbon (mixotrophy) and many heterotrophic organisms derive a proportion of their carbon from iCO2.

cAll microorganisms have additional individual growth requirements, e.g., N, P, S, and trace elements. They also have temperature, pH, salinity, and pressure optima.

dHydrogenotrophic methanogens reduce CO2 to methane using hydrogen while acetoclastic methanogens disproportionate acetate to methane and CO2.

(low-nutrient) conditions and when placed in media with high substrate concentrations such organisms are rapidly outcompeted by fast-growing opportunists. These obstacles can be overcome by the use of a combination of low-nutrient-growth media and methods to separate individual cells from low abundance, but fast-growing competitors. This can be achieved by procedures as simple as inoculum dilution to remove quantitatively insignificant but rapidly growing microorganisms or microencapsulation procedures that separate individual cells in their own miniature culture free from competition. It is also possible to physically isolate individual cells and inoculate them into a particular growth medium free from competitors. Tools such as flow-activated cell sorting (FACS) using a flow cytometer or micromanipulation with laser tweezers can be used to achieve this objective.

Pelagibacter ubique was unknown until 1990 when it was discovered as a dominant member of the world's ocean planktonic bacterial community. It was identified purely on the basis of 16S rRNA sequences recovered directly from seawater samples and given the designation SAR11 (it was first identified in the Sargasso Sea, hence SAR). Bacteria related to P. ubique are now known to be one of the most numerous bacterial groups in the world with a global population of approximately 1028 individuals. Following the realization of its global abundance in marine environments, the work of isolating the bacterium was initiated. The organism proved to be slow growing and its ecological success is based upon its ability to utilize organic compounds as growth substrates, but only when they are present at very low concentrations. This can in part be attributed to the very small size of the cells (c. 0.01 mm3) which provides an optimal surface area-to-volume ratio for the assimilation of substrates at low concentrations. In addition, P. ubique has one of the smallest genomes of any free-living bacterium (1.3 Mbp compared to about 4.6 Mbp for Escherichia coli). It encodes most of the genes required for an independent existence and very little noncoding DNA. Pelagibacters extremely streamlined genome may well be an important adaptation to live in an environment with very low availability of growth substrates.

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