Effect of maize rhizosphere on degradation of 3-chlorobenzoate by Pseudomonas B13 or Alcaligenes L6.*


For years polychlorinated benzoates (PCBs) have been used in agriculture and industrial use, and therefore a lot of PCBs were introduced in nature. PCBs can be degraded partly by the indigenous microflora into even more toxic intermediates. These intermediates, monochlorinated benzoates like 3-chlorobenzoate (3CBA), appeared to be accessible to further degradation by other bacteria. The isolated Pseudomonas B13 is one of those bacteria. Clean up of contaminated sites can be done by classical methods or by the still developing method of in situ bioremediation, which means the use of bacteria introduced to soil. This thesis has been carried out to obtain information on the bioremediation of 3CBA by Pseudomonas B13.
The presence of a rhizosphere may have a positive effect on the degradation activity by the bacteria as it is a nutrient rich site in soil, the root system offers a surface easy to adsorb to, can induce enzyme production and it's a buffered zone, of which all contribute to an increased microbial activity, called the "rhizosphere effect". To detect a possible rhizosphere effect in bioremediation of 3CBA the following steps are executed. First an extended literature research has been carried out on the topics of the different fully aerobic degradation pathways of 3CBA, the location of the genes involved and the soil characteristics. Then pre experiments were done to obtain the most optimal conditions for the final experiment, of which the following parameters has been tested: the disappearance of 3CBA (HPLC), the quantitative analysis of the introduced bacteria (M( plates) and the physiological status of the inoculant (TGGE).
Seven different degradation pathways in the degradation of 3CBA can be divided (see overview). 4CC, 3CC and catechol can be cleaved by a meta- or ortho-ring fission (by intradiol oxygenases). Genes encoding the enzymes for the different pathways are mainly located on plasmids like pJP4, pBR60, pWR1, pAC25 and pAC27. However sometimes the genes are located on translocatable elements or on the chromosome. Genes encoding the enzymes of the relatively evolutionary ancient ortho-cleavage pathway can sometimes be found on the chromosome and often the initial benzoate dioxygenase is located on the chromosome.
The pre-experiment with the Kujenbuch chambers showed that the optimal 3CBA concentration for incubation was 0.40 µmol/g soil. The mesh size of the nylon net had to be less than, or equal to, 120 µm to prevent root penetration. The optimal moisture content of the soil was 15%. A more stable set up for the water supply had to be found. The incubation temperature appeared to be best at 26°C in the climate room. The next pre experiment was done to compare the wild type strain Pseudomonas B13 with its constructs #3 and #6, which contained the chromosomally inserted markers spectinomycin and Xgluc. #6 Was most equal to the wild type strain B13 regarding to 3CBA analysis. However the constructs died when 3CBA wasn't present anymore, indicating the inserted sequence interfere with catabolic genes, as the wild type strain was still culturable on the M9 plates. The incubation period of the final experiment was set on 12 days. Results made clear that inoculation of the soil was far more effective in bioremediation of 3CBA than the presence of maize rhizosphere. A decrease in 3CBA content of respectively a 5-fold and an additional =< 25% in presence of the maize rhizosphere has been observed. Though within the time of the experiment almost all 3CBA has been disappeared. Quantitative analysis of the inoculant didn't show larger cell numbers in presence of the maize rhizosphere. Further, there might be the possibility that the inoculant show plant growth promoting activity. Interfering parameters influencing 3CBA degradation positively were the temperature, the indigenous microflora and possibly the moisture content. The set up of the system can use some further improvements as well as the extraction method for 3CBA.

For more information about the theoretical background and the results + conclusions, feel free to contact me and I'll send you the digital version of my report.

* This 6 months thesis was carried out in 1997 at the Department of Microbiology, section Molecular Ecology at the Wageningen Agricultural University, the Netherlands.