ECO29-UVA: Application of chemostat systems to include adaptation of microbial communities in persistency testing (CHEMADAPT)

Description

Persistence in the environment is a key property in assessing the environmental risks of organic chemicals. Currently, persistence is most often determined using screening tests such as the OECD ready biodegradability tests and sometimes with simulation tests designed to model environmental conditions. Screening tests such as OECD 301 are designed to identify compounds that are mineralised rapidly and are not suited to determining degradation rates under environmentally realistic conditions. This is because the conditions in these tests are very different to those in natural systems, with for example high concentrations of test chemicals and nutrients and relatively short exposure times. The short exposure times (typically 28 days) do not allow for adaptation of microbial communities to a new chemical, while in the natural environment microorganisms tend to adapt to pollutants upon long term exposure, allowing for the development of efficient and fast degradation over time

Adaptation to chemicals can occur at several levels (Van der Meer, 2006). The microbial communities may shift in species composition and the abundances of these species, favouring the growth of degrading species. Within a degrading population, the growth of one strain over another might be favoured. At the level of the individual cell, genetic adaptations may occur due to mutations, horizontal transfer of DNA and recombination events. Adaptation to chemicals can also occur at the biochemical level, influenced by the environmental conditions under which microbial populations are growing. For example, it is possible that the growth under carbon-limited conditions is more likely to lead to adaptation than conditions where microorganism are exposed to a wide variety of potential organic substrates (e.g. Marozava et al., 2014a; 2014b). Therefore, in order to take account of the effect of adaptation on persistence, approaches should be used in which environmentally realistic scenarios are used and in which the various possibilities for adaptation can be determined, and preferably be quantified. Therefore, ideally, the conditions in the experimental system used should be strictly defined in order to investigate their influence on adaptation. Batch systems such as the ready biodegradability tests are unsuitable for this purpose since conditions are constantly changing as nutrients and substrates are consumed within a limited period of time, while biomass and products accumulate. Continuous culture systems such as chemostats and retentostats offer excellent opportunities to allow microorganisms to adapt to new chemicals under defined and environmentally relevant conditions for, in theory, infinite periods of time.

Continuous culturing systems are bioreactors in which  microorganisms can be grown in a physiological steady state under controlled and constant environmental parameters such as  dissolved oxygen concentration, nutrient and chemical concentrations, pH and cell density (Bull 2010). In chemostats fresh medium enters the bioreactor and spent medium and biomass continuously leave the bioreactor. In retentostats, spent medium also leaves the bioreactor but the biomass is retained by the use of a biomass filter, preventing the loss of potentially degrading bacteria. Both systems are very suitable to study environmental processes in the laboratory under growth conditions that well mimic natural in situ conditions: low growth rates and low substrate concentrations (Lin et al. 2009). Chemostats are also very suitable for studying adaptive evolution of a strain and its functional basis (Gresham and Hong, in press) As a consequence, these systems are well suited to studying how environmental parameters affect adaptation to and degradation of organic chemicals. Previously, we have used chemostat and retentostats cultures of surface water and soil microorganism to study biodegradation of pesticides at environmentally relevant concentrations (Schrap et al. 2000, Sprenger et al. 2003; Ralebitso-Senior et al. 2003). Degradation of dicloran and parathion in continuous cultures of microbial communities from agricultural areas took place without requiring adaptation but degradation rates depended on the presence of selective substrates. We propose to use a similar approach to study the adaptation of microbial populations to persistent chemicals, how this is affected by the presence of organic substrates and nutrient conditions and the use of adapted microbiota in ready biodegradability tests. The contrasting features of chemostats and retentostats are expected to help evaluate how microbial communities adapt to chemical pollution and how the adaptation process relates to microbial (functional) diversity. In chemostats the biomass and thus microbial communities are continuously refreshed and the most adapted microbes will dominate while in retentostats microorganisms, and thus also the different stadia of adaptation, are maintained at high diversity in the reactor.

These systems will be used to address the following hypothesis and research questions: long-term exposure leads to biodegradation of chemicals that  are initially persistent. To what extent is this influenced by environmental conditions (e.g. chemical concentration, nutrient levels, complexity of the microbial community)? At what level does the community adapt? Does species composition change? What is the role of population changes in biodegradation of new chemicals? Does selection of competent bacteria initially present in very low numbers play a dominant role? Which molecular events contribute to adaptation?

The anticipated results of this project will contribute to a better understanding of the role of adaptation in determining the rates of biodegradation of chemicals and to the development of guidelines for persistency tests in which the role of adaptation is accounted for.