For Use in Modern Biotechnology

There is great interest in the characteristics of procaryotes isolated from the outflow mixing regions above deep hydrothermal vents that release water at 250 to 350°C. This is because these procaryotes can grow at temperatures as high as 113°C. The problems in growing these microorganisms, often archaea, are formidable. For example, to grow some of them, it will be necessary to use special cultur-ing chambers and other specialized equipment to maintain water in the liquid state at these high temperatures.

Such microorganisms, termed hyperthermophiles, with optimum growth temperatures of 80°C or above (see p. 126), confront unique challenges in nutrient acquisition, metabolism, nucleic acid replication, and growth. Many of these are anaerobes that depend on elemental sul-

fur as an oxidant and reduce it to sulfide. Enzyme stability is critical. Some DNA polymerases are inherently stable at 140°C, whereas many other enzymes are stabilized in vivo with unique thermoprotectants. When these enzymes are separated from their protectant, they lose their unique thermostability.

These enzymes may have important applications in methane production, metal leaching and recovery, and for use in immobilized enzyme systems. In addition, the possibility of selective stereochemical modification of compounds normally not in solution at lower temperatures may provide new routes for directed chemical syntheses. This is an exciting and expanding area of the modern biological sciences to which microbi-ologists can make significant contributions.

Observations of microbial growth at temperatures approaching 113°C in thermal vent areas, or of hyperther-mophiles, (Box 28.2; see also Box 6.1) indicate that this area will continue to be a fertile field for investigation. For some successful microorganisms, an extreme environment may not be "extreme" but required and even, perhaps, ideal. Thermophilic microorganisms (pp. 126; 463)

1. What are the main factors that lead to the creation of extreme environments?

2. Why are molecular techniques possibly changing our view of these environments?

3. What is unique about Ferroplasma acidarmanus?

28.5 Methods in Microbial Ecology

A wide variety of techniques can be used to evaluate the presence, types, and activities of microorganisms as populations, communities, and parts of ecosystems (table 28.8). Measurements made by these techniques can span a range of time scales and physical dimensions. In marine, freshwater, sewage, and plant root environments, as examples, responses can be measured in seconds and minutes. For deep marine and soil organic matter changes, a time scale of years, decades, or even centuries may be required. The physical scale used in a study may range from a single bacterium and its microenvironment to a lake, ocean, or an entire plant-soil system.

As noted at the beginning of this chapter, a fundamental problem in studying microorganisms in nature is the inability to culture and characterize most organisms that can be observed. This long-standing problem is now being approached by the use of molecular techniques, by which nonculturable microorganisms can be characterized and compared with known genomic sequences (see chapter 19).

Microbial community diversity can be assessed by several approaches, including molecular phylogeny based on analyses of small subunit (SSU) ribosomal RNA (see pp. 433-35). Small amounts of DNA also can be recovered from environmental samples or individual cells and "amplified" by use of the polymerase chain reaction (PCR). The polymerase chain reaction (pp. 326-27)

As noted in table 28.8, some of these techniques are limited in terms of the types of samples that can be analyzed. This may be due to low microbial populations (marine and some freshwater samples) or high concentrations of interfering organic matter or particulates in samples. In contrast, the newer molecular procedures, such as direct DNA extraction, DNA amplification fingerprinting (DAF), 16S and 18S RNA-based phylogenetic analysis, PCR, and DNA probe and hybridization techniques, are applicable to a wider variety of samples.

Recently gel array microchips containing mixtures of probes, called "genosensors," or microarrays, have been developed (see pp. 354; 1018). These allow the detection of small subunit riboso-mal RNA from mixed populations. In addition, probes can detect specific groups of microorganisms such as the iron- and manganese-oxidizing sheathed bacteria by the use of 16S rRNA-based probes.

Many newer and more sensitive procedures are now available, including the use of radioactive substrates and sophisticated techniques to measure the viability and activity of individual microorganisms. Hybridization techniques can be used to "probe" colonies or single cells to determine if they contain specific DNA or RNA sequences. The technique of whole-cell hybridization has progressed to the point that "subcluster-specific" probes have been developed. These allow the simultaneous detection of different mi-crobial types in the same preparation (see figure 29.9, p. 643).

In most studies employing these molecular approaches to analyze complex microbial communities, nucleic acids have been extracted from the sample, followed typically by cloning and further genomic and phylogenetic analyses. The specific source of the nucleic acids that are being studied is not known. Because of

Table 28.8 Methods Used to Study Microorganisms in Different Environmentsa

Environment

Characteristic Evaluated

Technique Employed or Property Measured

Marine

Freshwater

Sewage

Soil

Food

Nutrients

Chemical analysis (e.g., C, N, P) COD (chemical oxygen demand) BOD (biochemical oxygen demand)

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Microbial biomass

Photosynthetic pigments

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