Monitoring the efficacy of bioremediation.

The general acceptance of bioremediation technology as an environmentally sound and economic treatment for hazardous waste requires the demonstration of its efficacy, reliability and predictability, as well as its advantages over conventional treatments. An effective monitoring design includes protocols for treatment-specific, representative sampling, control and monitoring: these should take into account abiotic and biotic pollutant fate processes in all relevant process compartments. A number of well-established and novel chemical and molecular biological monitoring techniques and parameters are available. Logical and balanced combinations of both chemical and biological monitoring parameters should be used to demonstrate complete degradation and detoxification of a hazardous waste as well as the biological nature of the process. At each process-scale level, a set of general criteria should be used for systematic evaluation of the overall efficacy of bioremediation.

Heat shock gene expression in continuous cultures of Escherichia coli.

Temperature inducible systems for the controlled expression of recombinant genes are finding increasing industrial applications. These involve either short or long term exposure of the process culture to superoptimum temperatures. It is well known that bacteria respond to a sudden increase in their environmental temperature with an immediate transient increase in the synthesis rates of specific heat shock proteins. The use of continuous flow processes for the production of recombinant proteins would allow higher productivity and smaller scale bioreactors. However, the induction patterns of heat shock proteins in continuous culture after defined heat shocks are not well defined despite a large amount of information which is now available concerning heat shock protein induction in batch cultures. An overview of this information is presented to enable a better understanding of the response in continuous cultures. The latter was investigated by monitoring the transient expression of a representative heat shock gene, htpG, in E. coli in continuous culture. The relative magnitude of the response was found to be both temperature and exposure time dependent, but growth rate independent. Changing medium composition resulted in both different steady and transient state expression levels.

Recovery of exponentially growing cultures of Klebsiella pneumoniae NCIB 418 after heat shocks.

Exponentially growing cultures of Klebsiella pneumoniae were subjected to heat shocks in the superoptimal and supermaximal temperature ranges for growth on glucose in a defined mineral salts medium. Transitory changes in the specific growth rate constant during recovery were evident. The response was heat shock temperature and exposure time dependent. Cell viability determinations, based on colony counts, indicated complete recovery from heat treatments at superoptimal temperatures. In contrast, at supermaximal temperatures, discrepancies in colony counts on different agars were observed. The kinetic response of the specific growth rate constant after a heat shock at supermaximal temperatures is explained by segregation within the bacterial population.

Utility of phenomenological models for describing temperature dependence of bacterial growth.

We compared three unstructured mathematical models, the master reaction, the square root, and the damage/repair models, for describing the relationship between temperature and the specific growth rates of bacteria. The models were evaluated on the basis of several criteria: applicability, ease of use, simple interpretation of model parameters, problem-free determination of model parameters, statistical evaluation of goodness of fit (chi 2 test), and biological relevance. Best-fit parameters for the master reaction model could be obtained by using two consecutive nonlinear least-square fits. The damage/repair model proved to be unsuited for the data sets considered and was judged markedly overparameterized. The square root model allowed nonproblematical parameter estimation by a nonlinear least-square procedure and, together with the master reaction model, was able to describe the temperature dependence of the specific growth rates of Klebsiella pneumoniae NCIB 418, Escherichia coli NC3, Bacillus sp. strain NCIB 12522, and the thermotolerant coccobacillus strain NA17. The square root and master reaction models were judged to be equally valid and superior to the damage/repair model, even though the square root model is devoid of a conceptual basis.

Some effects of growth conditions on steady state and heat shock induced htpG gene expression in continuous cultures of Escherichia coli.

Most of the data concerning heat shock gene expression reported in the literature are derived from batch culture experiments under substrate and nutrient sufficient conditions. Here, the effects of dilution rate and medium composition on the steady state and heat shock induced htpG gene expression have been investigated in continuous cultures of Escherichia coli, using a chromosomal htpG-lacZ gene fusion. During steady state growth temperature dependent patterns of the relative htpG expression were found to be largely similar, irrespective of the growth condition. However, nitrogen-limited growth resulted in a markedly reduced specific steady state htpG expression as compared to growth under carbon limitation or in complex medium, correlating qualitatively with the total cellular protein content. During heat shock, tight temperature controlled expression was evident. While the relative heat shock induced expression was largely identical at various dilution rates in a given growth medium, significantly different response patterns were observed in the three growth media at any given dilution rate. From these results a clearly temperature regulated htpG expression during both, steady and transient state growth in continuous culture is evident, which is further significantly affected by the growth condition used.

Specific and quantitative assessment of naphthalene and salicylate bioavailability by using a bioluminescent catabolic reporter bacterium.

A bioassay was developed and standardized for the rapid, specific, and quantitative assessment of naphthalene and salicylate bioavailability by use of bioluminescence monitoring of catabolic gene expression. The bioluminescent reporter strain Pseudomonas fluorescens HK44, which carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism, was used. The physiological state of the reporter cultures as well as the intrinsic regulatory properties of the naphthalene degradation operon must be taken into account to obtain a high specificity at low target substrate concentrations. Experiments have shown that the use of exponentially growing reporter cultures has advantages over the use of carbon-starved, resting cultures. In aqueous solutions for both substrates, naphthalene and salicylate, linear relationships between initial substrate concentration and bioluminescence response were found over concentration ranges of 1 to 2 orders of magnitude. Naphthalene could be detected at a concentration of 45 ppb. Studies conducted under defined conditions with extracts and slurries of experimentally contaminated sterile soils and identical uncontaminated soil controls demonstrated that this method can be used for specific and quantitative estimations of target pollutant presence and bioavailability in soil extracts and for specific and qualitative estimations of napthalene in soil slurries.

Kinetics and response of a Pseudomonas fuorescens HK44 biosensor.

The reporter bacterium, Pseudomonas fluorescens HK44 (HK44), was characterized in an immobilized state to investigate utility for deployment as a remote sensor in the subsurface. A packed-bed reactor with alginate-immobilized HK44 simulated hydrodynamic conditions such as might be found in a subsurface environment. The reporter bacterium, HK44, harbors a reporter plasmid, pUTK21, which contains a transcriptional fusion between the nahG gene in the lower pathway of the catabolic plasmic NAH7 and a luxCDABE gene cassette. The upper nah pathway and the lux pathway in pUTK21 are induced by salicylate. The lux enzymes catalyze the light reaction. HK44 demonstrated a quantitative relationship between salicylate concentration and degradation. Light intensity mimicked salicylate concentration, whereas degradation was first order in biomass and first order in salicylate concentration, with a degradation constant of 2.23 x 10(-2) dm(3) g(-1) min(-1).

Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium.

An optical whole-cell biosensor based on a genetically engineered bioluminescent catabolic reporter bacterium was developed for continuous on-line monitoring of naphthalene and salicylate bioavailability and microbial catabolic activity potential in waste streams. The bioluminescent reporter bacterium, Pseudomonas fluorescens HK44, carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism. Exposure to either compound resulted in inducible bioluminescence. The reporter culture was immobilized onto the surface of an optical light guide by using strontium alginate. This biosensor probe was then inserted into a measurement cell which simultaneously received the waste stream solution and a maintenance medium. Exposure under defined conditions to both naphthalene and salicylate resulted in a rapid increase in bioluminescence. The magnitude of the response and the response time were concentration dependent. Good reproducibility of the response was observed during repetitive perturbations with either naphthalene or salicylate. Exposure to other compounds, such as glucose and complex nutrient medium or toluene, resulted in either minor bioluminescence increases after significantly longer response times compared with naphthalene or no response, respectively. The environmental utility of the biosensor was tested by using real pollutant mixtures. A specific bioluminescence response was obtained after exposure to either an aqueous solution saturated with JP-4 jet fuel or an aqueous leachate from a manufactured-gas plant soil, since naphthalene was present in both pollutant mixtures.