B1ODEGRAUASI KLOROLIGNIN OLEH PSEUDOMONAS PUTIDA ATCC 45491DAN PHANEROCHAETE CHRYSOSPORIUM BKMF 1767

• Main Author: Pinkan Aditiawati
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/5539
• Institution: Institut Teknologi Bandung

<b> Abstract : </b><p align=\"justify\">Wastewater from the bleaching process in the pulp and paper industry contains a chlorinated organic compound known as chlorolignin. Chlorolignin has many damaging effects on the environment since low molecular weight of chlorolignin is toxic in nature, mutagenic, and carcinogenic, while high molecular weight chlorolignin is difficult to degrade. Physical and chemical treatments of wastewater have been conducted, but the results are not satisfactory due to the high cost required. Conventional biological treatment using full scale activated sludge and anaerobic or aerobic lagoon system are capable of removing only 19 - 66% of the chlorolignin and more toxic compounds are also produced.<br /> <br /> The research was conducted on the chlorolignin degradation by a microbiological method in which single cultures of Pseudomonas putida and Phanerochaete chrysosporium were used, after which both cultures were used consecutively. It is known that P. chrysosporium has been used to degrade lignin in the process of the color removal of Bleach Plant Effluent, nevertheless this study will attempt to increase ligninolytic activity of the microorganisms by optimalization of environmental conditions that influence the microorganisms. On the other hand, study upon P. putida is conducted in order to discover the dehalogenation and degradation capability towards chlorolignin as a substrate - as studies concerning this matter have not been done. The microorganisms had potentials to be used in the waste treatment due to their wide substrate specificity. The use of a mixed culture in a consecutive manner is expected to increase the degradation rate of chlorolignin and furthermore would result in a higher percentage of chlorolignin removal without the production of the toxic intermediate compounds.<br /> <br /> The research was performed in several stages using synthetic chlorolignin. Initially, the study was done on the degradation of chlorolignin by P. chrysosporium, then by P. putida, and in the next step a consecutive application of P. putida and P chrysosporium was conducted. The degradation of chlorolignin was done using a stirred liquid medium in a 100 mL erlenmeyer flask andpgraded further using a 500 mL flask. Optimized parameters were the initial pH, the amount of inoculum, agitation, glucose concentration as a cosubstrate, and the chlorolignin concentration.<br /> <br /> The degradation of chlorolignin by P. chrysosporium, P. putida and the consecutive mixed cultures were observed through the change in the chlorolignin content, the concentration of Cl- ion released, and the color of the medium at certain time intervals. The consecutive degradation of chlorolignin by P. putida and P. chrysosporium was also observed through the change in the molecular weight distribution using reversed-phase HPLC, the product of the degradation process by HPLC using C18 column, and the determination of the intermediate compounds as well as their molecular weights by GCMS. Toxicity was evaluated by biological assay using Staphylococcus aureus.<br /> <br /> Experimental results show that delignification process of 0.3 g/L chlorolignin by P. chrysosporium in a medium having a high C and N ratio, where glucose was used as cosubstrate and source of C while ammonium tartrate as a N source, occurred in two steps. In the first step, low molecular weight chlorolignin penetrated the cell membrane while the high molecular weight chlorolignin was adsorbed by the fast growing mycelia. The dry weight of P. chrysosporium observed increased 14 times within 48 hours. The second step was induction of ligninolitic activity at the iodophase of growth, when the C and N ratio was low. The delignification of chlorolignin reached 52.9%, while dechlorination was 34.4% in 12 days and the pH changed from 4.5 to 3.3. The whole study was conducted at 37°C, pH 4.5, with an inoculum concentration of 10%, and agitation at 150 rpm.<br /> <br /> The delignification of 0.3 g/L chlorolignin by P. chrysosporium in a medium with low C and N ratio was caused by the ligninolytic activity induced on the first day and absorbtion process, while the growth rate was inhibited. The dry weight increased only 1.5 times while ligninolysis reached 68%, and dechlorination reached 57.5% in 5 days. The biological assay indicated that the ligninolyticproduct was toxic. Increase of chlorolignin from 0.3 g/L to 0.6 g/L in a medium with low C and N concentration were able to inhibit the rate of ligninolysis and dechlorination of chlorolignin by P. chrysosporium. The result of ligninolysis was only 68,32%; dechlorination was 68,32% in 8 days, at 37°C, initial pH of 4,5, inoculum concentration of 10%, and agitation was 150 rpm for the initial 2 days and later decreased to 100 rpm.<br /> <br /> P. chrysosporium growth rate has been proved to be higher in growth media (MB2) containing a high C/N ratio with chlorolignin, compared to ligninolytic media with lower C/N ratio and containing chlorolignin, while in media with lower C/N ratio showed higher rates of delignification and dechlorination compared to media with high C/N ratio. Ligninolysis and dechlorination of 0.3 g/L chlorolignin by P. putida was carried out in a medium with high C and N ratio. The cell density of P. putida increased to 19 times of the initial value during 2 days of the ligninolysis process. Lygninolysis by P. putida were able to reduce chlorolignin to 47.8% and dechlorination 31.2% in 7 days. The increase of chlorolignin from 0.3 g/L to 0.6 g/L at the high C and N concentration medium were unable to inhibit the dechlorination of chlorolignin but were able to diminish the ligninolysis rate. Dechlorination of chlorolignin reached 60% while the ligninolysis was only 44.2% in 7 days at 30°C, initial pH 7, 10% inoculum and agitation at 150 rpm. Results show that P. putida has the ability to degrade and dehalogenate chlorolignin.<br /> <br /> The consecutive ligninolysis and dechlorination by P. putida during 2 days and followed by P. chrysosporium yielded a better result, where ligninolysis reached 96.3% and dechlorinization reached 66.4% in 8 days, at optimal environmental condition for P. putida and P. chrysosporium with no intermediate toxic compounds produced. Studies conducted on the chlorolignin degradation using a mixed culture showed an improved result compared to degradation using single culture of P. putida or P. chrysosporiumA sample of the degradation product was further analyzed using infrared spectrophotometer to detect the C-Cl binding during the degradation. After 10 days of degradation, the C-Cl binding was still observed. Reverse-phase HPLC indicated a change in the molecular weight distribution of chlorolignin. A large concentration of high molecular weight chlorolignin was found in the initial sample, but after degradation for 7 days the concentration of high molecular weight chlorolignin was reduced, while low molecular weight chlorolignin i. e. guaiacol (MW 124) showed the highest concentration.<br /> <br /> HPLC results using ODS C18 column also showed the composition and kind of low molecular weight chlorolignin fragments. These fragments were able to disappear and reappear as degradation products of low and high molecular weight chlorolignin. The results of the HPLC studies by gradient as well as isocratic elution showed that guaiacol (MW 124) and veratryl alcohol (MW 168) were almost always produced as products of the mixed culture of P. chrysosporium and P. putida.<br /> <br /> GCMS studies indicated the changes of the compound composition of samples from 0, 2, 6, and 10 days degradation process. There were 5 different kinds of compounds found prior to degradation. The sample of the second day showed that the degradation product contained 6 different low molecular weight chlorolignins, some were newly formed compounds which resulted from the degradation process. On the sixth day, the degradation products showed the presence of 7 chlorolignin compounds, which 2 of them were different compared to the compounds found after 2 days of the degradation process. A sample obtained after 10 days of the degradation process indicated a mixture of 5 compounds, where 2 of them were different from those of 6 days degradation products. Compound composition change within sample collected 2, 6 and 10 days were caused by chlorolignin degradation to compounds with lower molecular weight - which finally were mineralized to CO2 and H2O. The molecular weight of the compounds formed