Lea Ellegaard-Jensen:
Fungal degradation of pesticides - construction of microbial consortia for bioremediation

Date: 15-11-2012    Supervisor: Søren Rosendahl

Pesticides are used worldwide on agricultural land as well as in urban areas. This use has often led to contamination of the environment with serious effects on our natural resources. Frequent pesticide use and spills have led to deterioration of soil quality and pesticide leaching has resulted in groundwater contamination. New technologies are therefore needed for cleaning up contaminated soil and water resources. This PhD was part of the project entitled Microbial Remediation of Contaminated Soil and Water Resources (MIRESOWA) where the overall aim is to develop new technologies for bioremediation of pesticide contaminated soil and water. The objectives of this PhD were to investigate fungal degradation of pesticides and following to construct microbial consortia for bioremediation.

In Manuscript I the fungal degradation of the phenylurea herbicide diuron was studied. Isolates of soil fungi of the genus Mortierella were tested for their ability to degrade diuron. The fungi were incubated in liquid culture with diuron on an orbital shaker at 8oC. The results showed that three of the five strains tested could degrade diuron, and molecular analysis revealed that these three strains constituted a closely related phylogenetic group, while the two non-degraders were located more distantly on the phylogenetic tree. In addition, it was examined whether the fungi utilized diuron as a carbon or nitrogen source. Here the most efficient diuron degrading strain Mortierella sp. LEJ701 was applied in liquid cultures with different levels of carbon and nitrogen. Degradation of diuron was fastest in carbon and nitrogen rich media while degradation was very restricted at low nutrient levels, making it unlikely that Mortierella utilize diuron as carbon or nitrogen source. The degradation kinetics of these experiments showed that diuron degradation was followed by formation of the metabolites 1-(3,4-dichlorophenyl)-3-methylurea, 1-(3,4-dichlorophenyl)urea and an hitherto unknown metabolite. This metabolite was subsequently biosynthesised, purified and analyzed by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). On the basis of these analyses the metabolite was suggested to be 1-(3,4-dichlorophenyl)-3-methylideneurea. This study especially brings new insights into the phylogenetic link between fungal diuron degraders, but also to the fungal degradation pathway of diuron.

The next two manuscripts dealt with constructing fungal-bacterial consortia and examining whether their degradation would be superior to that of the single strains in unsaturated systems. In Manuscript II a consortium was created for degradation of the pesticide metabolite 2,6-dichlorobenzamide (BAM). A consortium with Mortierella sp. LEJ702 and the BAM-degrading Aminobacter sp. MSH1 as well as the single strains were introduced into small sand columns. The sand was spiked with [ring-U-14C]-BAM and mineralization could thus be determined from the amount of evolved 14CO2. In addition, the effect of moisture content was examined by adding water corresponding to 0, 1.7, 5 or 10% of water holding capacity (WHC) to the sand. A faster mineralization of BAM was obtained by the consortium compared to Aminobacter sp. MSH1 alone, especially at the lower moisture contents. These results were supported by chemical analyses of 14C residues extracted from the sand. Additionally, it was investigated whether bacterial transport was enhanced in the presence of Mortierella. This was done by extracting DNA from the top layer of the sand followed by quantitative real-time polymerase chain reaction (qPCR) analysis. Results demonstrated that the number of Aminobacter transported to the top sand was greatly enhanced in the presence of Mortierella, suggesting that the fungal hyphae act as transport vectors for the bacteria in the sand. Finally, the distribution of 14C-BAM in the sand was studied in novel setup. It was found that the presence of Mortierella slightly enhanced BAM distribution. From this work it is evident that the fungal-bacterial consortium is capable of enhancing BAM-degradation in unsaturated systems, and may therefore be a promising application for soil bioremediation.

In Manuscript III two- and three-member consortia were constructed with bacterial and fungal diuron degraders. The purpose of this was to create a consortium which was superior for diuron degradation in unsaturated system and to investigate the interactions between the microorganisms in this consortium. The synergy leading to a more efficient degradation could either be a result of co-operative catabolism or physical interactions between the organisms improving growth and/or distribution of fungi and bacteria. The bacterial strains applied were Sphingomonas sp. SRS2, Variovorax sp. SRS16 and Arthrobacter globiformis D47 and the fungal strains were Mortierella sp. LEJ702 and Mortierella sp. LEJ703. In the experimental setup a layer of sterile glass beads was added between the organisms and the sand column above; simulating air-filled gaps in soil. [Ring-U-14C]-diuron was mixed into the sand to a concentration of 100 μg diuron kg-1. Degradation was measured as the amount of 14C-diuron mineralized and as 14C residues in the sand at experimental termination. Mineralization results established the three-member consortium LEJ702/SRS16/D47 as the most efficient transforming 32% of the diuron to 14CO2, while the single strains or other consortia mineralized no more than 10%. Furthermore, analyses of 14C residues in the sand showed that production of diuron metabolites by this consortium was minimal. The interactions between the organisms in the consortia were examined by phospholipid fatty acid analysis (PLFA) and 16S rDNA PCR using strain-specific primers. The molecular results suggested that the presence of Mortierella sp. LEJ702 enhanced distribution of SRS16 and D47 in the sand. Only fungal phospholipid fatty acids could be quantified. From those, however, it was apparent that the fungal growth was severely inhibited in the presence of A. globiformis D47. On the other hand, this effect was somewhat alleviated in the three-member consortium LEJ702/SRS16/D47. This study is the first to show that a three-member consortium of both fungal and bacterial degraders can indeed increase pesticide degradation.