Berith Elkær Knudsen:
Bacterial Degradation of Pesticides – Construction of Microbial Consortia for Bioremediation

Date: 15-11-2012    Supervisor: Søren Rosendahl

This PhD project was carried out as part of the Microbial Remediation of Contaminated Soil and Water Resources (MIRESOWA) project, funded by the Danish Council for Strategic Research (grant number 2104-08-0012).

The environment is contaminated with various xenobiotic compounds e.g. pesticides. Bioaugmentation i.e. addition of specific degrader organisms, has been suggested as an environmentally friendly and economically competitive strategy for cleaning polluted sites. Several organisms have been isolated, capable of degrading different compounds. However the capacity to degrade the desired compound is just one requirement for successful bioaugmentation. There are several challenges that need to be overcome in order for bioaugmentation to be sufficiently efficient. The purpose of this PhD project was to study the degradative abilities of different bacteria, and, in collaboration with a fellow PhD student, to construct fungal-bacterial consortia in order to potentially stimulate pesticide degradation thereby increasing the chance of successful bioaugmentation. The results of the project are reported in three article manuscripts, included in this thesis.

In manuscript I, the mineralization of 2,6-dichlorobenzamide (BAM) by Aminobacter MSH1 was studied in a sand column set up. We wanted to examine if presence of the fungus Mortierella LEJ702 affected BAM mineralization by Aminobacter MSH1, and furthermore to study the effect of moisture content on the mineralization. The results showed a positive effect of fungal presence, even though the Mortierella fungus is incapable of degrading BAM. At all the moisture contents studied, the mineralization rate was higher in the treatments with the fungal-bacterial consortium, than by the Aminobacter alone. Moreover, at the three lower moisture contents, an overall higher mineralization was obtained with the fungal-bacterial consortium. specific PCR revealed that presence of the Mortierella significantly enhanced the transport of Aminobacter MSH1. These results indicate that using a fungal-bacterial consortium consisting of Mortierella LEJ702 and Aminobacter MSH1 could potentially improve bioremediation of BAM.

An important prerequisite for bioaugmentation is the potential to produce the degrader strain at large quantities within reasonable time. The aim of manuscript II, was to optimize the growth medium for Aminobacter MSH1 and to elucidate optimal growth conditions. Moreover we wanted to examine if it was possible to produce MSH1 at large scale in a bioreactor while keeping the BAM mineralization ability intact. The results of this study showed that optimizing the medium greatly affected growth. An elementary analysis revealed the composition of Aminobacter MSH1, thereby providing information of potentially limiting factors. For instance, the analysis revealed that MSH1 contain approximately 10 times more iron than an average bacterium. After the medium optimization, growth of MSH1 under different pH and temperature conditions was studied both in shake flasks and in bioreactors, in order to establish the optimal growth conditions. The results showed that fastest growth was obtained with pH 7 and at a temperature between 20 and 25ºC. These conditions were used in a scale-up, to study the potential for large scale production. Results revealed that it was possible to produce MSH1 in large amounts within 12-24 hours. Moreover, mineralization experiments showed that the capacity to mineralize BAM was kept intact, and that maximal mineralization rate was not influenced by the state of the cells at the time of harvest (in exponential or stationary phase). This potential to rapidly produce MSH1 while maintaining the mineralization capacity underlines that Aminobacter MSH1 could be a promising strain for bioaugmentation purposes.

Manuscript III deals with diuron degradation by two fungi, Mortierella LEJ702 and Mortierella LEJ703, and three bacteria; Sphingomonas SRS2, Variovorax SRS16 and Arthrobacter globiformis D47. The degradation capacity of each strain individually as well as two- and three-member consortia was studied in a sand column set up. Glass beads were added to the set up to create a dry patch, separating the organisms and the diuron-spiked sand. The results revealed that the three-member consortium consisting of LEJ702, SRS16 and D47 mineralized three times more diuron than all other treatments tested. This shows that even though the two fungi are closely related, the outcome when used in consortia may differ markedly. Phospholipid fatty acid analysis revealed that D47 had an inhibitory effect on the fungal growth; however the effect on LEJ702 was lessened in presence of SRS16. This effect of SRS16 was not seen on LEJ703. These results stress the importance of testing the consortia, as it is impossible to predict the outcome of the created consortia, based on the degradative capacities on the individual strains. However, this study also shows that increased biodegradation of diuron could be obtained by using a fungal-bacterial consortium, thereby indicating a potential for use in bioaugmentation.