ABSTRACT

This book discusses petroleum spill bioremediation, the use of spectroscopy to identify microbial metabolic pathways, the detoxification of mercury by using recombinant mercury-resistant bacteria, and the use of manganese-oxidizing bacteria for bioremediation.

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Fluorescent antibody techniques have allowed for the direct identification and enumeration of individual bacteria in environmental samples without requiring prior growth in culture media (Bahlool and Schmidt 1980, Cloete and Steyn 1988, Macario et al. 1989). The technique involves the use of specific antibodies raised against surface markers of defined pure cultures that are either labelled directly with fluorescent dye molecules or via a fluorescent secondary antibody. This approach has yielded important insights into the spatial distribution of microorganisms, but it suffers from a number of disadvantages. For example, expression of the antigen may be influenced by environmental factors; false-positive and false-negative results may be obtained due to cross-reactivity or lack of reaction; non-specific binding of antibodies may result in high levels of background fluorescence; and production of specific antibodies requires a pure culture of the organism of interest (Cloete and de Bruyn Various recombinant DNA techniques have subsequently been developed that are independent of cultivation methods (Fig. 1). These techniques provide ways of detecting and quantifying specific phylogenetic groups of microbes on 16S rDNA sequences, and relevant structural genes provide ways of monitoring microbial populations of environmental and industrial systems. In addition to these tools, a number of emerging technologies such as the use of biomarker genes are being increasingly used to monitor with great precision and accuracy the behaviour of microbes in the environment.

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analyses of rRNA sequences can identify so-called signature sequence motifs on various taxonomic levels which are targets for an evolutionary-based identification (Giovannoni et al. 1988, Manz et al. 1992, Amann et al. 1995). In addition, the development of robust DNA cloning techniques and the polymerase chain reaction (PCR) have facilitated higher resolution analyses of more complex communities using 16S rRNA sequence analysis. It has been proposed that the highly variable, selectively neutral, intergenic spacer regions between the 5S and 16S or 16S and 23S rRNA genes might provide better targets (Aakra et al. 1999, Fisher and Triplett 1999). For community structure analysis, however, it seems rather unlikely that other parts of the bacterial genome could fully replace the 16S rRNA gene as target sites due to insufficient sequence information for comparisons.

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The simplest and currently the most widely adopted method to obtain 16S rRNA genes from the environment is through the use of PCR. rRNA genes can be amplified directly from the total community DNA using rRNA-specific primers, and then cloned using standard methods (Giovannoni et al. 1990). By taking advantage of the highly conserved nature of rRNA, universal primers capable of annealing to rRNA genes from all three domains (Archaea, Bacteria, Eukarya) or primers designed to amplify rRNA genes from a particular group of organisms can be used. Following PCR amplification, the amplified products can be cloned. Commercially available kits exploit the fact that PCR-amplified products have an overhanging 3' deoxyadenosine residue at each end when certain DNA polymerases are used. This allows cloning of the product into a sequencing-ready vector containing a complementary deoxy-thymidine overhang, in many cases, without requiring the product to be purified or further modified. Alternatively, the PCR products can be cloned by "filling" overhanging residues followed by blunt-end ligation procedures.

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sequences allows for the design of PCR primer sets that specifically amplify the genes encoding the enzyme from DNA samples extracted from the natural environment (Henckel et al. 1999, Meyer et al. 1999, Mesarch et al. 2000). Although the PCR method is both specific and sensitive, such standard reactions are not quantitative. To obtain quantitative data from PCR-based analyses, statistical methods based on most probable number (MPN) estimations have been used (Wand et al. 1997). In MPN-PCR, DNA extracts are diluted before PCR amplification and limits are set on the number of genes in the sample by reference to known control dilutions. Another way to quantify PCR-amplified products for comparison is to include an internal control in the PCR reaction (Leser et al. 1995, Mesarch et al. 2000). Here, a known amount of target DNA is added to a PCR reaction containing DNA from the mixed microbial population. The known target DNA is complementary to the same primers and thus competes with the target sequences in the extracted DNA sample. By preparing a dilution series of the known and unknown DNA species, it is possible to quantify the amount of product produced from die complementary gene in the extracted DNA. The known DNA target can be generated by cloning the gene of interest or purifying the PCR-amplified product after which a deletion is introduced to give a differently sized PCR product. There exists many variations of the standard PCR technique. The sensitivity and specificity of the PCR may be improved by adopting a nested approach. The initial amplification is carried out with a pair of primers that are not organism-specific, whereafter a second round of amplification is conducted on the product using primers specific for an organism with target sites internal to the first primer pair (el Fantroussi et 1997, Levesque et al. 1997, Rheims and Stackebrandt 1999).

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The easiest way of detecting specific nucleic acid sequences or genes of interest is through direct hybridization of a probe to microbial nucleic acid extracts. Whole-cell DNA or RNA is extracted from the environmental sample and fixed to a positively charged membrane, e.g. nylon or nitrocellulose. Bacterial colonies can also be replica-plated from agar plates to membranes and their nucleic acids exposed in situ following lysis for subsequent hybridization. Probes may be used to detect genes in the bacterial genome (Southern blots) or to detect mRNA or rRNA (Northern blots). For the in situ identification of individual whole cells it is necessary to make the cells permeable to oligonucleotide probes hybridizing with rRNA. These hybridization techniques rely on the specific binding of nucleic acid probes to complementary DNA or RNA (target nucleic acid). The probes are single strands of nucleic acid with the potential of carrying detectable marker molecules highly specifically to complementary target sequences, even if these sequences account for only a small fraction of the target nucleic acid. Either DNA or RNA can serve as a nucleic acid probe, but for a number of reasons (e.g. ease of synthesis and stability), most studies have employed DNA probes (Holben and Hedje 1988). Two general types of probes that have been developed are DNA probes complementary to a single gene or a small region of a gene and DNA probes complementary to genus- or species-specific regions of 16S rRNA for use in whole cell in situ hybridization (FISH).

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and inexpensively produced. During oligonucleotide synthesis a variety of marker or linker sequences can be introduced to the 5' end of the oligonucleotide. The principal conjugate fluorochromes are derivatives of fluorescein or rhodamine, although labels such as digoxigenin and biotin have also been used (Fuhrman et al. 1994, Amann et al. 1995). The design of rRNA-targeted group- or species-specific probes should be based on a good rRNA sequence database and be performed in a computer-assisted way using appropriate software. The organisms encompassed by a probe varies according to the region of the 16S rRNA selected as the hybridization target. Species-specific probes complement the most variable regions, while more general probes target more conserved regions of the molecule. The principal steps involved in the design of probes are: the alignment of rRNA gene sequences; the identification of sequence idiosyncrasies; the synthesis and labelling of complementary nucleic acid probes; and the experimental evaluation and optimization of the probe specificities and assay sensitivities using cultured reference strains (Manz et al. 1992, Devereux et al. 1992).

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A direct correlation between the growth rates of bacterial cells, the average ribosome contents and the probe-conferred fluorescence has been reported (DeLong et al. 1989). This has been used to estimate the growth rates of individual cells in situ (Poulsen et al. 1993, Moller et al. 1995). Often it is also important to obtain information about how the functional components of an ecological system relate to the organization of the system. In communities that have an inherent architecture, such as biofilms and floes, the question of where various organisms are located is of interest. These determinations are difficult to make with conventional epifluorescence microscopy. By coupling in situ hybridization with fluorescently-labelled rRNA-targeted oligonucleotide probes with confocal laser scanning microscopy (Caldwell et al. 1992), it is possible to place the labelled microbes in a three-dimensional reconstruction of the intact microbial community (Moiler et al. 1996, Schramm et al. 1996, Manz et al. 1999, Sekiguchi et al. 1999).

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depending on the particular thermostable DNA polymerase used, the fidelity of the PCR may vary (Speksnijder et al. 2001). Although careful analysis of secondary interactions should identify discrepancies due to misincorporation of nucleotides during PCR, there is a danger that the presence of novel taxa may be assumed as a result of infidelity in DNA replication. Many prokaryotes have several rRNA operons and even though the rRNA coding regions are usually highly similar, they are not necessarily identical. The cloning step in rRNA analysis separates not only the rRNA genes of different organisms but also the different genes. Thus, slightly different gene fragments could originate from one strain and would not indicate the presence of closely related organisms (Farrelly et al. 1995). This is of concern when making conclusions about biodiversity from data obtained with rRNA gene clone libraries.

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slowly growing natural populations. Various approaches have been adopted in order to improve the sensitivity. These have included the use of multiple probes labelled with a single fluor (Lee et al. 1993); or labelled with multiple fluors (Trebesius et al. 1994) and enzyme-linked probes or detection systems that allow signal amplification (Lebaron et al. 1997, Schonhuber et al. 1999). The latter indirect approach not only has the potential for signal amplification, but may also be used in natural samples showing high levels of autofluorescence. Any thorough identification method has to include positive and negative controls. False-positive results may either be caused by cells emitting autofluorescence upon excitation or by nonspecific binding of the probe to nontarget cells. Samples should therefore be checked for autofluorescence before hybridization and a negative control with a fluorescent oligonucleotide not complementary to rRNA has to be applied to check for sequence-independent nonspecific binding. Such non-specific binding may be due to interaction of the dye compound of the probe with hydrophobic cell components. Failures to detect cells containing target sequences (false-negatives) may originate from cells with either low cellular ribosome content or limited permeability of the cell periphery for the fluorescent probe (Manz et al. 1992). With the rapidly expanding database of 16S rRNA sequences, the problem of probe specificity has become more apparent and the design of probes is becoming increasingly difficult. These problems are also applicable to PCR and other oligonucleotide-dependent techniques. The problem of probe specificity may be overcome by using multiple specific oligonucleotide probes targeting different sites on the rRNA molecule and labelled with different fluorochromes. While a single oligonucleotide target sequence may be found in a number of related taxa, the probability that target sites for three designed oligonucleotides are found in a nontarget organism is, however, much reduced.

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without the necessity for cultivation of the cells (Rattray et al.1990). The light output is indicative of a metabolically active population of cells, since luciferase enzymes are dependent on cellular activity reserves, or reducing equivalents for bioluminescence. If the cells are growing, the light output is proportional to the number of cells in the sample. However, after long-term incubation in harsh environments, microbial cells often become starved or stressed and the light production from luciferase enzymes declines as a response to the change in cellular energy status (Duncan et al.1994). Therefore, in situbioluminescence is not a reliable indicator of microbial biomass under starvation conditions. The substrate and energy limitations for the luciferase reporter system may be overcome by adding nutrients to the sample in order to activate the microbial population (Meikle et al.1992, Duncan et al.1994). Alternatively, total protein from the environmental sample may be extracted. Then, the energy source can be added directly to the protein extract in vitroand the luciferase enzyme activity can be correlated to the specific luciferase-tagged microbial biomass in the sample (Moller et al.1995).

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), confocal laser scanning microscopy (Eberl et al. 1997) or enumerated by flow cytometry (Tombolini et al. 1997). A further advantage of GFP, over other biomarkers, is the fact that no other energy source or substrate addition is required, other than oxygen during initial formation of the chromophore. Therefore, the GFP biomarker holds tremendous promise for elucidation of specific bacterial numbers and their behaviour, colonization, distribution, interaction, and movement in situ in a diversity of environmental sample types. Additionally, mutations have been introduced into the GFP gene in order to produce fluorescence signals with altered properties (Heim et al. 1994, Delagrave et al. 1995, Crameri et 1996, Heim and Tsien 1996). For example, one of the mutants (P4) is a

chapter |68 pages

community structure and function, there is a clear trend to combine molecular measures of species composition and the abundance of important microbial groups with measurement of particular processes and environmental parameters. Such studies have the potential to relate community structure to function and activity in complex microbial communities. For example, community structure and function have been analysed through a combination of whole cell in situ hybridization and microsensors. rRNA-based localizations of ammonia- and nitrite-oxidizing bacteria were performed on a nitrifying biofilm following microelectrode measurements for Oj, N20 and N02/N 03' (Schramm et al. 1996, Santegoeds 1998). A good correlation of community structure and function could be demonstrated on a microscopic scale. The distribution of sulfate-reducing and methanogenic bacteria was also determined in a similar manner, with respect to activity (Raskin et al. 1994). Although environmental biotechnology and bioremediation in general could largely benefit from these multidisciplinary analyses of the structure and function of complex microbial communities, the role of classical microbial ecology should not be underestimated. Molecular studies complemented by appropriate culture-based investigations will assist in identifying organisms that are truly representative of those important in nature. It is only by selecting a range of appropriate tools in a complementary fashion that some of the mysteries of microbial ecology can be unlocked and the wealth of novel biodiversity presented by natural microbial communities can be harvested. A.,

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level of the shellfish toxicity is still low, but it increases to a higher level and reaches a maximal level after most of the dinoflagellates disappear from the environment. There is always about a 1 week time lag between the peaks of these parameters (Ogata et al. 1982, Sekiguchi et al. 1989). This phenomenon is difficult to explain if the toxins in the dinoflagellates transfer to the shellfish via the food chain. However, it is hard to evaluate the balance of the toxin amount between dinoflagellates and shellfish in a field survey in which samples of shellfish and plankton are collected periodically at a station set in the field, even though the frequency of the sampling is increased. Thus, the nature of the balance of the toxins be­ tween shellfish and dinoflagellates has not been clarified by data from field surveys. Recently, we examined this problem by feeding dinoflagel­ lates to shellfish. As handling of the shellfish during the experiments was found to affect much the feeding behavior of the shellfish (Sekiguchi et al. 2001a), a single specimen of shellfish was reared in a single tank, and the known amount of the cultured cells of dinoflagellate was fed to the shellfish specimen (Sekiguchi et al. 2001b). These experiments showed that shellfish accumulate toxins by ingesting the toxic dinoflagellate, and they release a part of the toxins to the environmental water even while ingesting the dinoflagellate. When the feeding stopped, the shellfish ex­ creted the toxins continuously. The profile of excreted toxins was similar to that accumulated in the shellfish, that is, shellfish release the toxin components non-selectively. Interestingly, the amount of toxins accumulated in the shellfish was not parallel to that of dinoflagellate cells fed to the shellfish (Fig. 8). At the earlier period of the experiment when shellfish were ingesting the cells, the amount of toxins accumulated in the shellfish was often more than that in the dinoflagellates fed to the shellfish. In the later period when the feeding was stopped, the sum of the toxins in the shellfish and rearing water had decreased to a level which was less than that introduced from the dinoflagellates cells, showing that some amount of toxins disappeared from the experimental system. However, this level recovered to the almost same level as that derived from the fed cells of the dinoflagellate, when they were further reared. These facts indicate that a part of the toxins was transformed to an unknown form which could not be detected by chemi­ cal analysis, such as HPLC. The unknown form of the toxins is gradually transformed to toxins again in the shellfish which can be detected by the chemical analysis. The unexpected high level of toxins accumulated in the shellfish, more than the fed amount of toxins, may show that an unknown form of toxins occurs also in the dinoflagellate fed to the shellfish. Thiol compounds of biological origin, such as GSH, are thought to be involved in the formation of the unknown form of toxins. Proteinous thiols such as that with a cystein residue may be involved in the formation of the

chapter |6 pages

has been used in various fields of the survey. Proteins showing specific affinity to STX, such as sodium channel proteins, are known to possess an important role in neurophysiology. Affinity columns prepared with agar­ ose resin bound with STX could be a useful tool for isolating these proteins more easily and rapidly. Toxification of shellfish by PSP toxins is now a world-wide problem. Monitoring has been conducted in many countries to avoid the marketing of toxic shellfish. For such monitoring, a mouse bioassay (Hollingworth and Wekell 1990) is used in most of the countries as the official method of analysis. The mouse assay for PSP toxins is a simple and reliable method, but it has problems because it is an animal assay, especially because continued use of an animal assay is increasingly difficult to justify on ethical grounds. Various alternative methods have been developed for analysis of PSP toxins, such as HPLC methods (Oshima 1995b, Sullivan et 1985), binding assay (Davio and Fontelo 1984, Deucette et al. 1994) and

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