Methods for examining biodegradative microbial populations

4.3.1.1 Sampling methods and sample handling

Samples for analysis should be representative and homogenized. All sam- ples should be collected into sterile containers (e.g. Whirl-Pak bags, Nasco) with clean implements and swabbed with 70% ethanol (w/v). Samples should be stored

at low temperature (e.g. 4◦C) in the dark. If they are not analyzed immediately

the samples should be stored frozen at−20◦C (assuming that the soil freezes

regularly), until analysis.

4.3.1.2 Enumeration of hydrocarbon degraders

Methods for enumeration of hydrocarbon degraders have been reviewed by Rosenberg (1992). Typically, most probable number (MPN) techniques are employed (Braddock and McCarthy 1996; Aislabie et al. 1998). The MPN method counts viable hydrocarbon-degrading organisms and is a statistical method based on probability theory. A suspension of cells in buffer is serially diluted ten-fold in buffer to reach a point of extinction. The dilutions are used to inoculate mul- tiple tubes containing growth media, typically Bushnell Haas minimal media, and sterile hydrocarbon(s) are added to each tube. It is important that the hydro- carbon(s) provided as growth substrate be representative of site contamination. For example, if remediating a site contaminated with jet fuel, then jet fuel should be used as growth substrate rather than a long chain alkane such as hexadecane. It is crucial to include sufficient controls, both positive (containing known hydrocarbon-degrading microbes) and negative (lacking viable microbes). To determine the ‘‘end points” for MPN tubes, a number of parameters may be used either singly or in combination, including turbidity due to cell growth and disruption of hydrocarbon films (Brown and Braddock 1990). Sometimes a redox dye is used to indicate substrate oxidation (Johnsen et al. 2002), or

the growth substrate is spiked with an appropriate14_{C-labeled compound and}

the endpoint is determined by trapping 14_CO

2 (Atlas 1979). Incubation times

and temperatures used should reflect the environment. Commonly, tempera-

tures ranging from 4–20◦C are used to enumerate hydrocarbon degraders in

cold soils, with incubation times from 2–8 weeks. Incubation times should be longer than those commonly used for enumerations at moderate temperatures. The pattern of positive and negative growth results is then used to estimate the concentration of hydrocarbon-degrading microbes in the original sample (the MPN of hydrocarbon-degrading microbes) by comparing the observed pat- tern of results with a table of the statistical probabilities of obtaining those results.

Liquid from MPN tubes exhibiting growth can be plated onto solid media for the isolation of hydrocarbon bacteria for subsequent investigations.

4.3.1.3 Detection of hydrocarbon degradation genes in soil

Molecular tools used for detection of hydrocarbon degradation genes in cold soils have been described by Whyte et al. (1999a; 2002a) and Margesin et al. (2003). DNA is extracted from the soil and the genes of interest are amplified using PCR. Catabolic genes targeted include those that encode alkane hydroxy- lase (alkB), catechol 2,3-dioxygenase (C23DO) and naphthalene dioxygenase (ndoB). The principle of PCR is the generation of copies of two strands of DNA by a repeated sequence of denaturation of double-stranded DNA, synthesis of com- plementary strands, followed by the next round of denaturation and synthesis. Complementary oligonucleotides (primers) are required for synthesis, designed from knowledge of the sequence flanking or within the genes of interest. Since each newly synthesized complementary strand can serve as a template for the next round of PCR, the amount of DNA synthesized increases exponentially, and from a few starting DNA molecules, 20 cycles can generate over a million copies. The PCR products are visualized by agarose gel electrophoresis. To ver- ify amplification of the correct PCR fragment, they are analyzed by Southern blotting and hybridization with DNA probes specific for hydrocarbon degra- dation genes or by sequencing. The PCR detection limits for these analyses are generally 100–1000 colony forming units per g of soil (A. K. Bej, personal communication).

To enumerate gene copy number, quantitative PCR methods such as com- petitive and real-time PCR assay are now being developed for investigations of hydrocarbon degrading bacteria (Mesarch et al. 2000; Laurie and Lloyd-Jones 2000). Competitive PCR (cPCR) includes a competitive sequence that serves as an internal control in each PCR reaction. In contrast, real-time PCR uses either a fluorogenic probe to detect a specific PCR product, or a DNA binding dye that detects double-stranded DNA, and measures fluorescence emitted continuously during the amplification reaction. cPCR requires post PCR analyses whereas real- time PCR does not.

The main advantage of a molecular approach is that it is rapid. Information on the genetic potential of the indigenous microbial community to degrade the contaminant(s) of concern can be obtained within days rather than weeks. However, a major limitation is dependence on existing catabolic gene sequences (for design of probes and primers) that are probably only a small fraction of the extant sequences in nature. Hence, inability to detect known degradative genes in a sample may indicate that the known genes are present in numbers

below detection limits or alternatively that other ‘‘unknown” catabolic genes are involved in degradation at the site under investigation.

4.4 Future research

Bioremediation technology is underpinned by fundamental knowledge of pure microbial cultures, typically bacteria, able to degrade contaminants of concern. Further work that will enhance application of bioremediation to cold soils includes the development of specific microbial isolation methods that could result in the cultivation of new hydrocarbon degraders, and subsequent iden- tification of hydrocarbon degradation genes as yet undescribed. Deposition of these new degradative gene sequences in public databases such as GenBank will lead to the design of additional gene probes for culture-independent in situ study. Further elucidation of cold/stress adaptations (desiccation; freeze-thaw; cold shock proteins) in combination with growth on or exposure to hydro- carbons could reveal metabolic strategies unique to cold-adapted hydrocarbon degraders.

Some activities that are currently known only from laboratory experiments must be demonstrated in cold soils. For example, the role of heterotrophic nitro- gen fixers in cold soil hydrocarbon degradation and nutrient balance remains to be quantified in situ. The importance of hydrocarbon co-metabolism by microbial consortia may be more important in cold soils than in temperate soils, but this possibility has not yet been tested. Neither hydrocarbon degradation at sub-zero temperatures nor cold temperature anaerobic hydrocarbon degradation has been examined extensively in situ.

Recent advances include the development of DNA microarrays to describe the composition and function of microbial communities (Denef et al. 2003). This method will eventually enable simultaneous monitoring of multiple members of a community. Gene chip technology is already being developed for detect- ing hydrocarbon catabolism genes in contaminated environments (L. Whyte and C. W. Greer, personal communication). In the near future it should be possible to detect and evaluate, within hours, the known genetic potential for hydrocarbon degradation residing in a natural population. This technology will be a pow- erful adjunct to the traditional culture-based methods and laboratory studies described above. It would be constructive to perform bioremediation trials incor- porating molecular techniques to promote and test new methods of determin- ing community composition and diversity in hydrocarbon-impacted cold soils. The succession of phylotypes during hydrocarbon degradation would add to our understanding of bioremediation processes in cold soils.

Temperature effects on biodegradation