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PubMed1995

Construction of a rhizosphere pseudomonad with potential to degrade polychlorinated biphenyls and detection of bph gene expression in the rhizosphere.

The genetically engineered transposon TnPCB, contains genes (bph) encoding the biphenyl degradative pathway. TnPCB was stably inserted into the chromosome of two different rhizosphere pseudomonads. One genetically modified strain, Pseudomonas fluorescens F113pcb, was characterized in detail and found to be unaltered in important parameters such as growth rate and production of secondary metabolites. The expression of the heterologous bph genes in F113pcb was confirmed by the ability of the genetically modified microorganism to utilize biphenyl as a sole carbon source. The introduced trait remained stable in laboratory experiments, and no bph-negative isolates were found after extensive subculture in nonselective media. The bph trait was also stable in nonselective rhizosphere microcosms. Rhizosphere competence of the modified F113pcb was assessed in colonization experiments in nonsterile soil microcosms on sugar beet seedling roots. F113pcb was able to colonize as efficiently as a marked wild-type strain, and no decrease in competitiveness was observed. In situ expression of the bph genes in F113pcb was found when F113pcb bearing a bph'lacZ reporter fusion was inoculated onto sugar beet seeds. This indicates that the bph gene products may also be present under in situ conditions. These experiments demonstrated that rhizosphere-adapted microbes can be genetically manipulated to metabolize novel compounds without affecting their ecological competence. Expression of the introduced genes can be detected in the rhizosphere, indicating considerable potential for the manipulation of the rhizosphere as a self-sustaining biofilm for the bioremediation of pollutants in soil. Rhizosphere bacteria such as fluorescent Pseudomonas spp. are ecologically adapted to colonize and compete in the rhizosphere environment. Expanding the metabolic functions of such pseudomonads to degrade pollutants may prove to be a useful strategy for bioremediation.

Bacterial Proteins↗
PubMed1998

Enterococcus faecalis gene transfer under natural conditions in municipal sewage water treatment plants.

The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria). The transfer rate between different strains of E. faecalis under natural conditions was, compared to that under laboratory conditions, at least 10(5)-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn916. In no case was transfer from E. faecalis to another bacterial species detected. By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for the sex pheromone plasmids between different strains of E. faecalis in the municipal sewage water treatment plant of the city of Regensburg ranged from 10(5) to 10(8) events per 4 h, indicating that gene transfer should take place under natural conditions.

Enterococcus faecalis↗
PubMed1994

Biotransformations of Aroclor 1242 in Hudson River test tube microcosms.

A microcosm system to physically model the fate of Aroclor 1242 in Hudson River sediment was developed. In the dark at 22 to 25 degrees C with no amendments (nutrients, organisms, or mixing) and with overlying water being the only source of oxygen, the microcosms developed visibly distinct aerobic and anaerobic compartments in 2 to 4 weeks. Extensive polychlorinated biphenyl (PCB) biodegradation was observed in 140 days. Autoclaved controls were unchanged throughout the experiments. In the surface sediments of these microcosms, the PCBs were biologically altered by both aerobic biodegrading and reductive dechlorinating microorganisms, decreasing the total concentration from 64.8 to 18.0 micromol/kg of sediment in 1140 days. This is the first laboratory demonstration of meta dechlorination plus aerobic biodegradation in stationary sediments. In contrast, the primary mechanism of microbiological attack on PCBs in aerobic subsurface sediments was reductive dechlorination. The concentration of PCBs remained constant at 64.8 micromol/kg of sediment, but the average number of chlorines per biphenyl decreased from 3.11 to 1.84 in 140 days. The selectivities of microorganisms in these sediments were characterized by meta and para dechlorination. Our results provide persuasive evidence that naturally occurring microorganisms in the Hudson River have the potential to attack the PCBs from Aroclor 1242 releases both aerobically and anaerobically at rapid rates. These unamended microcosms represent a unique method for determining the fate of released PCBs in river sediments.

Aerobiosis↗
PubMed2006

Nutrient amendments in soil DNA stable isotope probing experiments reduce the observed methanotroph diversity.

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in non-amended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.

Carbon Isotopes↗
PubMed2004

Effects of Bacillus subtilis O9 biosurfactant on the bioremediation of crude oil-polluted soils.

The application of a surfactant from Bacillus subtilis O9 (Bs) on the bioremediation of soils polluted with crude oil was assayed in soil microcosms under laboratory conditions. Three concentrations of biosurfactant were assayed (1.9, 19.5, and 39 mg kg(-1) soil). Microcosms without biosurfactant were prepared as controls. During the experiment, the crude oil-degrading bacterial population, the aliphatic and aromatic hydrocarbons were monitored in each microcosm. The results indicated that applying Bs did not negatively affect the hydrocarbon-degrading microbial population Concentrations of 19 and 19.5mg (Bs) per kilogram of soil stimulated the growth of the population involved in the crude oil degradation, and accelerated the biodegradation of the aliphatic hydrocarbons. However, none of the assayed Bs concentrations stimulated aromatic hydrocarbon degradation.

Bacillus subtilis↗
PubMed2004

How do grazers affect periphyton heterogeneity in streams?

The effects of grazing by stream invertebrates on algal biomass and spatial heterogeneity were tested experimentally in flow-through microcosms with natural substrates (rocks). One experiment tested the effects of fixed densities of three species of grazers (the caddisfly Allomyia sp. and two mayflies, Epeorus deceptivus and Baetis bicaudatus) on periphyton. Baetis was tested with and without chemical cues from fish predators, which reduced grazer foraging activity to levels similar to the less mobile mayfly (Epeorus). Mean algal biomass (chlorophyll a; chl a) was reduced in grazer treatments compared to ungrazed controls, but there were no differences among grazer treatments. Algal heterogeneity (Morisita index) increased with grazer mobility, with the highest heterogeneity occurring in the Baetis-no fish treatment (most mobile grazer) and the lowest in the caddisfly treatment (most sedentary grazer). A second experiment used a three factorial design, and tested whether initial resource distribution (homogeneous vs. heterogeneous), Baetis density (high vs. low) and fish odor (present vs. absent) affected grazer impact on algal resources. Abundances of Baetis and chl a on individual rocks were recorded to explore the mechanisms responsible for the observed distributions of algae. Initial resource heterogeneity was maintained despite being subjected to grazing. Mean chl a was highest in controls, as in experiment I, and effects of Baetis on algal biomass increased with grazer density. There were no fish effects on algal biomass and no effects of grazer density or fish on algal heterogeneity. At the scale of individual rocks Baetis was unselective when food was homogeneously distributed, but chose high-food rocks when it was heterogeneously distributed. Results of these mechanistic experiments showed that Baetis can track resources at the scale of single rocks; and at moderate densities mobile grazers could potentially maintain periphyton distributions observed in natural streams.

Animals↗
PubMed1999

Bacterial Stimulation in Mixed Cultures of Bacteria and Organic Carbon from River and Lake Waters.

Abstract Interactions between natural bacterial assemblages and dissolved organic carbon (DOC) were investigated in two complementary batch experiments. In the first, a positive relationship was found between the proportion of electron transport system (ETS) active bacteria and the diversity of DOC in microcosms enriched with an increasing number of organic substrates. In a second experiment, bacterial and nutrient dynamics were measured in microcosms with natural bacterial populations and organic matter from rivers and lakes of different trophic levels. The interactions between the bacterial assemblages and DOC from different sources was investigated using source systems (rivers or lakes) and blended (different proportions of river and lake water) batch cultures. In each experiment, the number of total and ETS-active bacteria, the fluorescein diacetate (FDA)-hydrolytic activity, and the total (DOC), biodegradable (BDOC) and refractory (RDOC) dissolved organic carbon were measured four times during 5 days. The results suggested that the temperature, more than trophic level, controlled planktonic bacterial production. Furthermore, bacterial activity was stimulated in micrososms where river and lake waters were mixed. For the oligotrophic microcosms, this observation can be explained by a greater diversity of the organic nutrients ("qualitative" stimulation of bacteria), whereas for the meso-eutrophic microcosms, the production of new pools of dissolved organic carbon (both biodegradable and total) could account for the observed "quantitative" stimulation of the bacteria. These experiments suggest that the mixing of bacteria and organic matter from two different systems can give rise to novel nutrient and bacterial dynamics that are likely similar to those that occur in river-lake ecotones.http://link.springer-ny.com/link/service/journals/00248/bibs/38n3p285.html</hea

Journal Article↗
PubMed2003

Enhancement of aerobic microbial degradation of polychlorinated biphenyl in soil microcosms.

This article reports the results of various biodegradation experiments on polychlorinated biphenyl (PCB)-contaminated sandy soil employing a mixed culture of acclimatized bacteria. Following the optimization of different variables without chemical pretreatment, the elimination rate achieved of Aroclor 1242 in slurry-phase reactors was 61% after four months of treatment, with the presence of biphenyl as cosubstrate being the most important factor affecting PCB biodegradation. The biodegradation occurred as a first-order process, and it proved most effective in respect to dichlorinated biphenyls (100% removal), followed by trichlorinated (92%) and tetrachlorinated biphenyls (24%). The results also showed that the degradability of PCBs in soil may be enhanced by an advanced oxidation pretreatment (Fenton reaction), producing almost 100% elimination of PCBs at the end of the integrated chemical-biological process and 72% mineralization of the intermediates generated during the chemical pretreatment.

Aerobiosis↗
PubMed2006

On the limits of toxicant-induced tolerance testing: cotolerance and response variation of antibiotic effects.

Pollution-induced community tolerance (PICT) as an ecotoxicological test system has been claimed to detect pollutant effects highly specifically and sensitively. However, the specificity might be limited by the occurrence of cotolerance. Another limitation of the application of any ecotoxicological test system lies in variation of the measured responses. We tested the variation and the occurrence of cotolerance experimentally, using antibiotics as toxicants, soil microcosms as microbial communities, and tolerance determination in Biolog plates as PICT detection test. Bacteria have been discussed as being prone to multiple tolerances due to the possible accumulation of multiple resistance genes on mobile genetic elements. However, in our experiments, cotolerance occurred only between antibiotics of the same group (oxytetracycline and tetracycline), as expected from their identical mode of action. Cotolerance between oxytetracycline and tylosin in soil microcosms exposed to oxytetracycline was low, as was cotolerance to oxytetracycline in tylosin-exposed microcosms. We conclude that tolerance development to antibiotics in soils reflects the actual selection pressure rather than a general pattern of multiple resistances. Concerning variation, the PICT effect of tetracycline was well reproducible in two consecutive years. The response variation linked to PICT experiments in controlled microcosms was comparable to that of ecotoxicological test systems of equivalent complexity. In conclusion, our results support an application of the PICT methodology as an effective means to study the soil ecotoxicology of antibiotics.

Adsorption↗
PubMed2003

Survival and alteration of the plasmid-containing microorganism Escherichia coli Z905/pPHL7 introduced into manmade closed aquatic microcosms.

It has been demonstrated that the transgenic microorganism Escherichia coli Z905/pPHL7 (AprLux+) can exist for a long time at an elevated concentration of mineral salts. The microorganism was introduced into microcosms with sterile brackish water (salinity variable from 21 to 22 g l-1) taken from Lake Shira (Khakasia, Russia). The survival of the microorganism was estimated both by measuring the growth of the colonies on solid nutrient media and by the bioluminescence exhibited by the transgenic strain in samples from the microcosms and in the enrichment culture with the added selective factor-ampicillin (50 micrograms/ml). In the enrichment culture, the bioluminescent signal was registered through the 160-day experiment. It has been shown that in the closed microcosms with brackish water the E. coli strain becomes heterogeneous in its ampicillin resistance. The populations of the transgenic strain were mainly represented by isolates able to persist in the medium containing 50 micrograms/ml, but there were also the cells (about 10%) with the threshold of ampicillin resistance not more than 0.05 micrograms/ml. Thus, it was shown that in the microcosms with brackish water and in the absence of the selective factor the transgenic strain survives and retails the recombinant plasmid.

Ampicillin↗
PubMed1992

Survival of Bacillus licheniformis in Seawater Model Ecosystems.

The fate of Bacillus licheniformis DSM 13 was monitored after introduction into laboratory microcosms and mesocosms established in the Knebel Vig estuary, Denmark. The model organism was detected by a combination of immunofluorescence microscopy and nonselective plating followed by colony blotting. This allowed simultaneous quantification of intact cells and culturable cells. B. licheniformis DSM 13 adapted poorly to the conditions in filtered (0.2-mum-pore-size filter) seawater. Results from additional microcosm studies using natural seawater demonstrated that protozoan grazing also was important in regulating the population of the introduced model organism. In experiments using mesocosms, B. licheniformis DSM 13 also showed a rapid die-off. The introduction of the organism led to increased nutrient levels and to increased growth of both autotrophic and heterotrophic components of the plankton community compared with those of control enclosures. Thereby, a more intensive predation impact on the bacterioplankton community was induced. The combination of microcosm and mesocosm experiments provides a scenario in which the influence of single biotic and abiotic factors on survival of introduced organisms can be tested and in which the effect of the introduction on ecosystem structure and function can be evaluated. This test concept might prove useful in risk assessment of genetically modified microorganisms.

Journal Article↗
PubMed2001

Monitoring biodegradation of methyl tert-butyl ether (MTBE) using compound-specific carbon isotope analysis.

Methyl tert-butyl ether (MTBE), the most common gasoline oxygenate, is frequently detected in surface water and groundwater. The aim of this study was to evaluate the potential of compound-specific isotope analysis to assess in situ biodegradation of MTBE in groundwater. For that purpose, the effect of relevant physical and biological processes on carbon isotope ratios of MTBE was evaluated in laboratory studies. Carbon isotope fractionation during organic phase/gas-phase partitioning (0.50 +/- 0.15@1000), aqueous phase/gas-phase partitioning (0.17 +/- 0.05@1000), and organic phase/aqueous-phase partitioning (0.18 +/- 0.24@1000) was small in comparison to carbon isotope fractionation measured during biodegradation of MTBE in microcosms based on aquifer sediments of the Borden site. In experiments with MTBE as the only substrate and a cometabolic experiment with 3-methypentane as primary substrate, MTBE became enriched in 13C by 5.1 to 6.9@1000 after 95 to 97% degradation. For both experiments, similar isotopic enrichment factors were obtained (-1.52 +/- 0.06 to -1.97 +/- 0.05@1000). Biodegradation of TBA, which accumulated transiently in the cometabolic microcosms, was also accompanied by carbon isotope fractionation, with an isotopic enrichment factor of -4.21 +/- 0.07@1000. This study suggests that carbon isotope analysis is a potential tool to trace in situ biodegradation of MTBE and TBA and thus to better understand the fate of these contaminants in the environment.

Biodegradation, Environmental↗
PubMed2002

Waste ecocompatibility in storage and reuse scenarios: global methodology and detailed presentation of the impact study on the recipient environments.

In 1995, the ADEME launched a research program called "Waste Ecocompatibility" in order to define a reliable methodology for measuring the impact of waste in storage or reuse scenarios. The French concept of "Ecocompatibility" is defined as the situation where the pollutant flux from waste disposed of or used in specified conditions is compatible with the environmental acceptance of the receiving environments. The chief feature of this definition is to integrate the evaluation of the three following terms: pollutants emission from the waste, transport of the pollutants from the waste to the receptor cells and the environmental acceptance of the receiving environments. The "Waste Ecocompatibility" program consisted of a literature survey and an experimental part. The literature study aimed to determine factors and waste characteristics to be considered for a reliable ecocompatility assessment, to provide an overview of the available tools for measuring those factors and characteristics and to propose a first approach of the methodology. In the framework of the experimental program, this approach was then applied to three theoretical scenarios to validate the laboratory tools (comparative study of laboratory and field results) and to calibrate the global methodology. This paper deals with the results of the experimental program concerning the impact study on receiving environments: impact on plants and microorganisms living in soil, impacts on soil fauna and aquatic fauna. In other papers we intend to present the operational methodology for the assessment of waste ecocompatibility. It includes bio-assays at laboratory scale (microcosms), pilot scale (mesocosms) and in situ experiments (experimental prairie). To limit the use of in situ experiments other research works are necessary to validate bio-assays at laboratory or pilot scale.

Animals↗
PubMed1994

An evaluation of four types of freshwater model ecosystem for assessing the hazard of pesticides.

Experimental results are reported on four types of freshwater model ecosystem after administration of a single dose of chlorpyrifos. The fate, and primary and secondary effects of chlorpyrifos were compared between the model ecosystems, and were evaluated in the light of the predictive value of the current ecotoxicological hazard assessment procedure for pesticides. 'Slootbox', a fate model used in the ecotoxicological risk assessment of pesticides in the Netherlands, overestimated chlorpyrifos concentrations. The primary effects of chlorpyrifos can be predicted accurately on the basis of single species laboratory toxicity data. The population effects observed in the microecosystems, microcosms, and mesocosms were consistent between all experiments and with the single species tests. Community metabolism, as a functional endpoint, was less sensitive than the structural parameters measured. Secondary effects, both for structural and functional endpoints, varied between the micro- and mesocosm experiments. At the present 'state of the art' in ecotoxicology, no a priori prediction of secondary effects in natural ecosystems will generally be possible. 0.1 times the lowest acute L(E)C50 for chlorpyrifos, as measured in the current ecotoxicological hazard assessment procedure, matched the NOECmesocosm for a single dose of chlorpyrifos. Recovery of populations affected by insecticide stress was found to depend on factors such as life cycle characteristics and ecological infrastructure, in addition to the toxicant concentration. The onset of (potential) recovery is likely to start at an approximate concentration of the EC10(48 h).

Animals↗
PubMed1999

Metabolite formation and toxicity measurements in evaluating bioremediation of a jet-fuel-contaminated aquifer.

The metabolic capabilities of subsurface, jet-fuel-contaminated, aquifer microbial communities were characterized using an ecological approach to biotreatment assessment. A multifaceted experimental design was used that incorporated quantification of metabolite formation and toxicity screening along with the typical microbial activity measurements and pollutant degradation measurements used for assessing bioremediation potential. For select experiments, dissolved oxygen levels and pH in microcosm systems were also monitored. Results suggest that a sizable, metabolically active microbial community exists in both contaminated and uncontaminated areas of the study site. Time course metabolism analyses indicated that the microbial communities were capable of degrading all three test compounds (amino acids, decane, and toluene) without any apparent adaptation period. Measurements of mineralization, cellular uptake, and metabolite formation indicated that metabolite formation was the predominant fate of the target pollutants in the microcosms. The results of toxicity screening time courses indicated that under oxygen-limiting conditions, the potential for the accumulation of toxic, acidic metabolites that could adversely affect the rates and extent of bioremediation existed. The experimental results indicate that the microbial communities at the site possess the metabolic potential for in situ biodegradation of the jet fuel. Care must be taken in the design of an in situ biodegradation treatment system (for this site and perhaps other petroleum-contaminated aquifers) to avoid the development of microaerophilic or oxygen-depleted zones, which could result in possible self-poisoning owing to acidic metabolite accumulation.

Aviation↗
PubMed1994

Contribution of the Global Regulator Gene gacA to Persistence and Dissemination of Pseudomonas fluorescens Biocontrol Strain CHA0 Introduced into Soil Microcosms.

Structural and regulatory genes involved in the synthesis of antimicrobial metabolites are essential for the biocontrol activity of fluorescent pseudomonads and, in principle, amenable to genetic engineering for strain improvement. An eventual large-scale release of such bacteria raises the question of whether such genes also contribute to the persistence and dissemination of the bacteria in soil ecosystems. Pseudomonas fluorescens wild-type strain CHA0 protects plants against a variety of fungal diseases and produces several antimicrobial metabolites. The regulatory gene gacA globally controls antibiotic production and is crucial for disease suppression in CHA0. This gene also regulates the production of extracellular protease and phospholipase. The contribution of gacA to survival and vertical translocation of CHA0 in soil microcosms of increasing complexity was studied in coinoculation experiments with the wild type and a gacA mutant which lacks antibiotics and some exoenzymes. Both strains were marked with spontaneous resistance to rifampin. In a closed system with sterile soil, strain CHA0 and the gacA mutant multiplied for several weeks, whereas these strains declined exponentially in nonsterile soil of different Swiss origins. The gacA mutant was less persistent in nonrhizosphere raw soil than was the wild type, but no competitive disadvantage when colonizing the rhizosphere and roots of wheat was found in the particular soil type and during the period studied. Vertical translocation was assessed after strains had been applied to undisturbed, long (60-cm) or short (20-cm) soil columns, both planted with wheat. A smaller number of cells of the gacA mutant than of the wild type were detected in the percolated water and in different depths of the soil column. Single-strain inoculation gave similar results in all microcosms tested. We conclude that mutation in a single regulatory gene involved in antibiotic and exoenzyme synthesis can affect the survival of P. fluorescens more profoundly in unplanted soil than in the rhizosphere.

Journal Article↗
PubMed2006

Use of microcosms to determine the survival of the fish pathogen Tenacibaculum maritimum in seawater.

The survival of the fish pathogen Tenacibaculum maritimum in different seawater microcosms was investigated during 160 days. The persistence of culturable cells was greater in sterile than in natural seawater. Standard plate counts showed that T. maritimum survived in sterile seawater for more than 5 months at concentration around 10(3) cfu ml(-1). However, T. maritimum proved to be very labile in non-sterile seawater, rendering culturable cells no longer than 5 days. These results were confirmed when DNA-based methods were applied. Regardless of the microcosms used, epifluorescence microscopy counts remained at about 10(6) cells ml(-1) throughout the experiment, even though we can not distinguish T. maritimum in the case of non-sterile microcosms. Resuscitation assays with addition of fresh medium to non-sterile microcosms did not favour the recovery of T. maritimum on solid media. Although morphological changes from filamentous to spheres were observed after 3 days in the non-sterile microcosms, in the case of the sterile microcosms this change was observed at the sixth day. The biochemical, physiological, serological and genetic characteristics were unaffected in the sterile microcosms. The overall results contribute to a better understanding of the behaviour of T. maritimum in natural seawater and suggest that the aquatic bacterial population play an important role in the survival of this fish pathogen.

Animals↗
PubMedDate not supplied

Direct wet surface imaging of an anaerobic biofilm by environmental scanning electron microscopy: application to landfill clay liner barriers.

To contain domestic waste and its associated pollution within a landfill, engineered mineral (clay) barriers are used and are designed to have a permeability of 1 x 10(-9) m/s (Westlake 1995). The rate of permeability of various porous media has shown to be influenced by the clogging of flow paths (media pores) due to biofilm formation (Charckalis and Marshall 1990, Cunningham et al. 1991). The term biofilm is given to describe the colonies of surface adherent microorganisms (Donlan et al. 1994). In this study, permeability experiments were built and modified to act as microcosms to investigate the influence of biofilm formation on the permeability of clay barriers. Traditional scanning electron microscopy methods disrupt or destroy the biofilm and previous anaerobic studies have involved building closed cells (such as miniature continuous culture chambers) that utilise light microscopes (Robin Jones et al. 1997). This paper examines the application of the environmental scanning electron microscope (ESEM) to the direct examination of the clay interface and biofilm formation in situ within the microcosm.

Bacteria, Anaerobic↗