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Record number 27708
Title Simulation of subsurface biotransformation
Author(s) Bosma, T.N.P.
Source Agricultural University. Promotor(en): A.J.B. Zehnder; G. Schraa. - S.l. : Bosma - ISBN 9789054852216 - 136
Department(s) Microbiology
Publication type Dissertation, internally prepared
Publication year 1994
Keyword(s) microbiële afbraak - geologie - biologie - bodembacteriën - organische verbindingen - organische scheikunde - microbial degradation - geology - biology - soil bacteria - organic compounds - organic chemistry
Categories Microbiology (General)

Hydrophobic organic contaminants like DDT, Polychlorobiphenyls (PCB's) and polyaromatic hydrocarbons (PAH's), have been detected all over the world. They tend to accumulate in the atmosphere and in the soil as a result of their physical and chemical properties. Breakdown mainly proceeds by (photo)chemical reactions in the atmosphere and via microbial transformation in the soil. Microbial transformation can be viewed as part of the ecological process of decomposition, that is, the remineralization of organic material by biota. This Chapter discusses the ecological significance of biotransformation and the dependence of biotransformation rates on enviromnental conditions, and suggests ways to improve the effectiveness of biological soil remediation techniques.

Contaminant cycling in ecosystems
Chemicals are released into the environment by human activities. Normally, they enter the abiotic part of the ecosystem which may be viewed as a contaminant pool (Fig. S. 1). Biota take up contaminants directly from the abiotic environment e.g. via leaves or the skin, or ingest them by feeding on a lower trophic level. Organisms have systems at their disposal to excrete or detoxify contaminants. Excretion brings contaminants back to the contaminant pool, while detoxification results in a decontamination as indicated in Fig. S. 1.

Plants and animals are not always able to detoxify or excrete contaminants after uptake. The inability of organisms to handle xenobiotic compounds may have several causes. One example is the absence of appropriate enzymes to transform the compounds, another the accumulation in (animal) fat tissue before excretion or enzymatic transformation has taken place. Contaminants accumulate in the food chain when organisms are not able to detoxify or excrete them. Accumulation is indicated by the use of different grey shades in Fig. S. 1.

The population of "decomposers" (Fig. S. 1) is specialized in the uptake and conversion of all kinds of dead organic material, like for instance dead animals and plant debris. Decomposers are crucial for the functioning of ecosystems because they recycle nutrients to the nutrient pool. Contaminants which are accumulated in the tissue of organisms are recycled to the contaminant pool simultaneously. Some bacteria and fungi are able to detoxify and mineralize man-made organic compounds like chlorinated benzenes and polyaromatic hydrocarbons. Thus, they prevent their accumulation in the environment. These microorganisms may therefore be viewed as the "decontaminators" of ecosystems (Fig. S. 1), Many micro-organisms live in soil and ground water where hazardous compounds may accumulate. Microbial transformation is the only mechanism leading to the effective detoxification of such compounds. Therefore, it is of interest to know under which environmental conditions biotransformation is inhibited or stimulated. The potential of micro- organisms to transform contaminants under various environmental conditions is discussed in the following together with the factors governing exposure of micro-organisms to contaminants in soil and ground water.

Potential of micro-organisms to transform organic contaminants
The capacity of micro-organisms to detoxify anthropogenic chemicals under similar enviromnental conditions is variable among various habitats. This may be related to previous exposure of the micro-organisms to the compound under consideration. An adapted microflora capable of converting and mineralizing new compounds may evolve after a long exposure time. The microflora in a not pre- exposed environment may not be able to detoxify the same compound. Dichloropropene and 2,4-D (dichlorophenoxy acetic acid) are examples of pesticides that micro-organisms "learned" to transform. Degradation of these compounds in the field can be so rapid nowadays that their effectiveness as pesticide is strongly reduced. As a result, farmers have to apply considerably larger amounts of these pesticides than was necessary in the early times of their use.

Many non-chlorinated organic compounds can be mineralized by aerobic bacteria. Well documented examples are simple aromatic compounds like benzene, toluene, and xylenes. ,More complicated aromatic structures like PAH's are also susceptible to aerobic degradation. Heavily chlorinated compounds are not readily degraded under aerobic conditions. However, anaerobic bacteria have a great potential to dehalogenate all kinds of such chemicals. Dehalogenation changes the environmental impact of the parent compounds considerably. Partly dechlorinated compounds are often more toxic and more mobile than the original compounds. The carcinogenic compound vinylchloride for example, may arise from the anaerobic dechlorination of tetra- and trichloroethene (PER and TRI). The anoxic transformation products are often biodegradable under aerobic conditions. The increased mobility of the more toxic products allows them to travel to aerobic environments where they
can be mineralized. Thus, the anaerobic process of dehalogenation may be an important mechanism to initialize the complete mineralization of heavily chlorinated contaminants in the subsurface environment.

Most of the information regarding the potential of micro-organisms to degrade organic contaminants is obtained from laboratory studies at 20°C. Studies carried out at temperatures down to 4°C, reveal only a slight temperature dependency of aerobic biotransformation rates. Anaerobic dehalogenation rates are reduced and intermediary dehalogenation products accumulate at lower temperatures. It seems that activities of aerobic micro-organisms involved in these processes are less dependent on temperature than those of anaerobes. Therefore, the aerobic removal rates of non- or partly halogenated compounds may be similar in summer and in winter in natural systems, while heavily halogenated compounds will tend to persist more in winter because of the reduced activity of anaerobes.

A microscopic view of soil pollution and micro-organisms
A picture of a versatile microbial community that is able to transform and mineralize a variety of hazardous organic compounds arises from the previous section. Nevertheless, biodegradable organic contaminants can persist in soil for decades. The microbial transformation rate of an organic compound is strongly affected by the potential uptake rate which is influenced by the transport rate to individual micro-organisms, The very slow in situ biotransformation is probably caused by the properties of the soil matrix surrounding the micro-organisms which reduces the transport rate. The microscopic spatial distribution of contaminants and micro-organisms will affect biotransformation rates in soil. This paragraph discusses how a spatial separation between micro- organisms and contaminants may develop in case of pollutions from point and non-point sources.

Soil is polluted from point sources like for instance accidental spills and landfills (local pollution), or from non-point sources like atmospheric deposition and application of pesticides (diffuse pollution). The general characteristic of a local pollution is the presence of high contaminant concentrations in a small volume of soil (Fig. S.2A, upper part). Diffuse pollution is characterized by low contaminant concentrations over a wide area (Fig. S.2B, upper part). The lower part of Fig. S.2 schematically shows the local distribution of micro-organisms and contaminants in both situations. Bacteria are normally present inside soil aggregates. Low concentrations of contaminants flow around these aggregates in the case of diffuse pollution (Fig. S.2B). Local pollution initially contaminates pores around soil aggregates. The easily accessible part in wide pores may be biotransformed rapidly until nutrients become exhausted. This leads to a rapid growth of bacteria in the wide pores. Pollutants which are not biotransformed initially will diffuse into the aggregates. Thus, a situation arises with relatively high numbers of bacteria surrounding contaminated aggregates (Fig. S.2A). Degradation activity is drastically reduced as a result of spatial separation. A similar situation may arise when spots containing pure contaminant exist, where no biological activity is possible anymore. Hence, micro-organisms and contaminants are spatially separated both in the case of local and diffuse pollution. Biotransformation can only take place after diffusion of contaminant through the soil matrix to the micro-organisms.

Computer calculations based on the concept presented in Fig. S.2 show that intra-aggregate processes of sorption and diffusion are of primary importance in determining the kinetics of biotransformation in soil. Effective diffusion rates in soil aggregates can be up to 1-10 orders of magnitude smaller than in water, depending on the characteristics of the soil matrix. As a consequence, biotransformation rates in different soils are subject to the same variation. When the diffusivity in soil aggregates is small, a steep concentration gradient is needed to maintain a flux of nutrients and contaminants that is sufficient to sustain microbial activity. As soon as the contaminant concentration drops below the value that is needed to maintain the gradient, biotransformation will stop. This threshold concentration is inversely proportional to the effective diffusion rate of contaminant. So, residual concentrations after biotransformation are expected to differ by many orders of magnitude, just like the biotransformation rates do.

Optimization of bioremediation techniques to relieve limitation of biotransformation
Limitation of biotransformation is not only the result of slow diffusion rates in soil, but may also be due to physiological or thermodynamic factors, to the presence of undissolved pollutants, or to the coupling of pollutant to soil organic matter via covalent bonds (bound residue formation). All these factors may result in reduced biotransformation rates. A strong association between the pollutants and the soil matrix especially develops at sites which have been polluted for years or decades already, Bound residue formation and extremely slow diffusion into small, highly tortuous pores have both been proposed as causes for this strong association. As a result, bioremediation is particularly difficult for these so-called "aged" pollutants (Fig. S.2A,

Dissolution rates of undissolved pollutants may be enhanced by dispersing the pure component through the soil, and by the addition of surfactants that increase maximum dissolution rates. These methods have been shown to increase biotransformation rates in practice. However, surfactants do not dissolve bound residues which are covalently bound to organic matter. In addition, they do not increase diffusion rates in small, highly tortuous pores, The existence of bound residues and the extremely slow diffusion rates are causes for high residual concentrations that remain after bioremediation. Therefore, surfactants are not expected to decrease these residual concentrations.

The possible application of procedures that enhance bound residue formation as means of bioremediation is disputed. It can be argued that pollutants which are present as bound residues are not hazardous anymore, having lost their specific chemical characteristics. Thus, they have also lost their biological activity. However, pollutants not only bind to the humus fraction of the soil, but also to dissolved or colloidal fractions of soil organic matter. This may lead to a mobilization of pollutants instead of the intended immobilization. In addition, dioxinlike products are formed when pollutants with phenolic or carboxylic groups bind to each other. The use of applications involving enhancement of bound residue formation requires that the possible hazards are better understood and that ways are provided to prevent them.

Considerable residual concentrations will always remain after bioremediation of "aged" pollutions, due to strong sorption and incorporation in organic matter, unless special measures are taken to mobilize the pollutants. In an ex situ scheme, the soil may be pulverized to increase biotransformation rates and decrease residual concentrations. It is imaginable to remove the mobile fraction of pollutant in a relatively short time via a biological treatment during in situ remediation. The residual immobilized fraction which is trapped inside soil aggregates is biologically and chemically inactive. It should be sufficient to monitor and control pollutants that are slowly desorbing from the soil aggregates in an "after-care" phase. An approach may be to monitor the concentration level in the macropores continuously and to stimulate biotransformation by the addition of nutrients as soon as some critical level is reached. An alternative would be to apply a slow pump-and-treat method continuously, The after-care phase can be stopped as soon as total pollution concentrations in the soil are below acceptable limits.

New pollutions have to be treated biologically as soon as possible to achieve optimal results because long contact times between pollutants and soil have a negative effect on the expected result of bioremediation. A possible strategy is to include biological soil treatment in human activities which almost inherently lead to soil pollution with organic chemicals. Thus, the establishment of a strong association between contaminants and soil can be prevented. This strategy has shown to be effective at tank stations where leaking of benzine or diesel is unavoidable.

Introduction of specialized bacteria is used as a strategy to enhance the biotransformation of compounds that are not degraded by the indigenous microflora. The success of the addition of micro- organisms depends on their ability to reach the contamination, to survive, and to carry out the desired reaction. A better understanding and control of the transport of bacteria in soil and ground water will help to optimize techniques for bioremediation which employ introduced bacteria. Surface characteristics of bacteria and soil particles together with the ionic strength of the flowing water control the transport of bacteria under saturated flow conditions. The adhesion of bacteria to soil particles is positively correlated with the hydrophobicity of bacteria and the ionic strength of the flowing water. Hence, the ionic strength of the water in which bacteria are introduced can be used to control microbial transport and attachment. If a low ionic strength is used, bacteria may travel long distances and disperse around the point where they are introduced. On the other hand, a high ionic strength will generally stimulate the attachment of bacteria to the solid phase and may prevent bacteria from moving away from the polluted site.

Concluding remarks
Microbial transformation is required to achieve detoxification of hydrophobic organic contaminants that accumulate in soil. Micro-organisms can therefore be viewed as a subpopulation of the decomposers with a special function, namely detoxification of the environment. The effectiveness of microbial transformation can severely be reduced by the relative immobility of organic compounds in the soil matrix where micro-organisms live. Limitations resulting from slow diffusion can only be removed effectively during ex situ remediation, e.g. by pulverizing the contaminated soil. During a biological in situ treatment the bulk of contamination can be removed rapidly. The treatment should be followed by an aftercare period in which the possible leaking of the residual amount is monitored to be able to take measures if necessary.

From an ecological stand-point, it can be argued that production and release rates of toxicants have to be smaller than in situ biotransformation rates to keep environmental pollution within acceptable limits. Treatment as close to the source as possible during the manufacturing and use of chemicals will be an important strategy to reach such a goal. The use of pesticides should be regulated such that the amount applied in a growth. season is completely transformed in situ in the same season.

Discussions with Clay L. Montague were very helpful in the conception of the Synopsis.

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