Analysis of routine biodegradation tests

Humankind has produced over 70.000 chemicals that are all sooner or later released into the environment. In the sixties resistant pesticides and persistent detergents caused environmental pollution. Later the first tests on biodegradability were conceived and the Environmental Protection Agency formed the Office of Pollution Prevention and Toxics (OPPT) in 1977. Nowadays, new chemicals are tested for toxicity and biodegradability before they are admitted to the consumer market. These tests are carried out world-wide according to the guidelines established by the Organization for Economic Co-operation and Development, the European Union, the International Organization for Standardization, and the Environmental Protection Agency.

Task forces on biodegradation tests of, for example, the Society of Toxicity and Environmental Chemistry (SETAC) and of the industry recognize shortcomings in the protocols and in the interpretation of standardized biodegradation tests, in particular with the analysis of the test results. Three areas have been selected to work on in this project:

These topics have been selected because "standard" models fail to capture these important aspects of microbial degradation.

Flocculated growth

Microbes in activated sludge tanks mostly occur in flocs rather than in cell suspensions.

A microbial floc starts to grow with a diameter equal to the maximum thickness of the living layer, develops a dead kernel (indicated in black) and disintegrates. The biomass in the living layer is redistributed to new flocs.
Flocculation results in a limited supply of substrate to the bacteria inside the flocs, which reduces the biodegradation rate of organic compounds by several orders of magnitude. A simple two-parameter extension of growth models for cell suspensions was developed to account for the ensuing reduction of the degradation rate. The additional parameters represent floc size at division and diffusion length. The biomass of small flocs initially increases exponentially at a rate equal to that of cell suspensions. After this first phase, the growth rate gradually decreases and finally the radius becomes a linear function of time. At this time flocs are large and have a kernel of dead biomass. This kernel arises when the substrate concentration decreases below the threshold level at which cells are just able to pay their maintenance costs. We deduced an explicit approximative expression for the interdivision time of flocs, and thereby for the growth of flocculated microbial biomass at constant substrate concentrations. The model reveals that the effect of stirring on degradation rates occurs through a reduction of the floc size at division. The results can be applied in realistic biodegradation quantifications in activated sludge tanks as long as substrate concentrations change slowly.

Co-metabolism

The availability of multiple carbon/energy sources often enhances the biodegradation of recalcitrant compounds. We classified and modelled different modes of multiple substrate utilization in a systematic way, using the concept Synthesizing Unit (generalized enzyme). According to this concept, substrates can be substitutable or complementary; their uptake (or processing) can be sequential or parallel. We show how the different modes of multiple substrate interaction can be described by a single general model. From the general model, we derive simple expressions for co-metabolism of non structurally analogous substrates. Both the general and the co-metabolism model have the advantage that they can be used in combination with any microbial growth model.

Result of model fit against data from Schukat et al 1983 on the degradation of 3-chloroaniline (3CA) by Rhodococcus with glucose as the primary substrate. Apart from a background disappearence rate, 3CA is only degraded in the presence of glucose, which is well-captured by the model. All 14 curves have been fitted simulteneously.

The application of co-metabolism model to experimental data shows that the general model constitutes a useful framework for modeling aspects of multiple substrate utilization.

Adaptation

In their natural environment microorganisms encounter changes in feeding conditions, involving either nutrient concentrations or nutrient types. They have to adapt to the new conditions in order to survive. We modelled the slow microbial adaptation in response to changes in the availability of substrates. The model is based on reciprocal (or mutual) inhibition of expression of both the substrate-specific carriers and the associated assimilatory machinery. The inhibition kinetics is derived from the kinetics of Synthesizing Units. The model accounts for interaction among carriers by diffusion limitation. The number of required adaptation parameters is one less than the number of substrates that is involved. An interesting property of the adaptation model is that the presence of a single limiting resource results in a constant maximum specific substrate consumption rate for fully adapted microorganisms. Because the maximum specific consumption rate is not a function of substrate concentration, for growth on one substrate, the Monod and Pirt models for instance are still valid. Other adaptation models known to us do not fulfil this property. The simplest version of our model describes adaptation during diauxic growth, using only one preference parameter and one initial condition. The applicability of the model is exemplified by fitting it to published data from diauxic growth experiments.

Model fit of growth of Pseudomonas oxalaticus (circles) on oxalate (triangles) while adapting to acetate (blocks) on data by Dijkhuizen et al 1980. The typical diauxic growth is well-captured by the adaptation model. All three curves have been fitted simultaneously.

This and other applications show the realism of the model.

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