1. The Bio-oxidation Pattern As a Tool to Study Biodegradation with On-line Measurement Jongtai Jung (Professor/Ph. D) (Professor/Ph. D) Major of Environmental Engineering College of Urban Science, University of Incheon

3. – Target compound – Microorganism – Reactor type – Parameters to be measured – Analysis method – The way to enhance reaction etc. Factors to be considered (in biodegradation study)

4. Determination of biodegradability of an organic toxic compound can be a lengthy procedure due to cumbersome analytical steps for the organic compound. The research work which takes long time can be shortened to answer the most basic question about response of the bioculture when challenged with an unknown compound. Determination of biodegradability

5. Biochemical oxygen demand(BOD) is a measurement of amount of dissolved oxygen required to oxidize an organic chemical by microorganisms Among the techniques for respirometeric and direct oxygen uptake measurements, the ones most often used are the manometric and direct oxygen uptake measurements using galvanic cells oxygen probes or polarographic electrodes. The polarographic technique which uses a galvanic cell oxygen probe is better and simpler technique which when used properly can provide quick measurement of oxygen uptake rates. For aerobic process, determination of biodegradability of an organic toxic compound can be done by on-line monitoring of dissolved oxygen(DO) and pH level Oxygen Uptake and biodegradability

6. To demonstrate the effectiveness of monitoring DO, pH and monitoring the bio-oxidation pattern of an immobilized cell bioreactor. This bio-oxidation pattern will show that whether the unknown compound can be biodegraded or not, and how long it will take for the unknown compound to be completely biodegraded Purpose of this work

7. Experimental Set-up(1) 1) Recirculation flow-type bioreactor, - Reactor size : 6.4 cm in diameter 20 cm in length.. 2) Reservoir - Reservoir size : 11.4 cm in diameter 25.4 cm in length 3) Total reaction volume - 2 liters including the reservoir.

8. Experimental Set-up(2) 1) Culture medium - 100 ppm MgCl 2, - 0.5 ppm FeCl 3 - 10 ppm MgSO 4, - 10 ppm K 2 PO 4 2)Oxygen supply - Air (1.5 liter/min for EG, TEG) - H 2 O 2 (1% v/v for MMA, Styrene) 3)Recirculation flow rate : 325ml/min. 4)Chosen Substrate - MMA(Methyl methacylate), Styrene - Ethylene Glycol(EG), Tetraethylene Glycol(TEG)

9. Activated sludge(Mixed microbial population) from Waste water treatment plant 100 g alginate-immobilized activated sludge How to immobilize - Distilled water - Concentrated sludge(50 mg dry biomass/ g of pallet) - 0.5% sodium chloride - 1% sodium alginate - 0.1 mol/liter CaCl 2 - Distilled water and Conc. Pellets in a ratio 5:2 mixed with NaCl and Sodium Alginate in a blender - The homogeneous cell suspension was then extruded using a syringe pump into CaCl 2 solution to obtain the immobilized bacterial beads Microorganism

10. 1) Time based control - Oxygen is supplied periodically by a timer - No matter what concentration is, it’s control is the supplying air as an oxygen source for certain period of time and then turning it off for another period of time in consistant bases. - The ups and downs in the DO profile are due to intermittent supply of air which was turned on and off by a timer. Experiments to be performed(1)

11. 1) Time based control - Since the reactor was operated in batch mode, the oxygen consumption and requirement changed along with substrate concentration. - Consequently the DO level is never constant, but always varied. It decreased slowly, and passed through a minimum before rising back to the original level. - This typical pattern may be observed. Experiments to be performed(2)

12. 2) Set point control - The DO levels are maintained using a set point control logic with a minimum error bound. - No matter how much time is consumed, set point control is the supplying H 2 O 2 used as an oxygen source till a certain concentration which is already set and automatically turning it off Experiments to be performed(3)

13. 2) Set point control - The controller has capabilities to perform A/D(analog to digital) conversion, do real time graphing of input variables, and to do data logging. - The microprocessor based controller was obtained from Omega Engineering, This was necessary to determine the consumptions for substrate by keeping the DO level essentially constant. Experiments to be performed(4)

14. 1)Dissolved Oxygen concentration : - Clark-type dissolved oxygen probe - Chart recorder. 2) MMA, Styrene, EG, TEG Concentration : - Perkin Elmer 8500 Gas Chromatograph, - Detector : FID 3)pH : - pH probe (Orion Cat. No. 91-04) - pH meter (Corning Model 250, NY) Analytical Methods

15. Fig 1. Experimental set-up of the bioreactor

16. Results & Discussions on Time Based Control(1) - Fig.2 shows DO profile and methyl methacylate(MMA) concentration profile in an experiment to study MMA biodegradation. -The key is an unique pattern(initiation, acceleration, completion) of the DO curve which forms upon injection of a substrate

17. Fig 2. Oxygen Conc. pattern with Time based Control on MMA biodegradation, AB: normal, B: injection, BC : initiation, CD: acceleration, DE: completion

18. - Section AB shows the baseline DO consumption rates in absence of any organic compound. - Supply of DO by air was controlled on a time basis. - In this case the DO level dropped initially on injection of MMA at point B and remained in a consultant range from 2.5 to 4ppm(CD, acceleration) until concentration of MMA reached 5ppm before rising up to normal level from 6.7 to 7.7ppm(DE, completion). Results & Discussions on Time Based Control(2)

19. - The disappearance of MMA coincides with rising in DO levels. - In this experiment pH was also monitored and it showed same trends as that shown by DO profile including the initiation, acceleration and completion of the reaction(Fig.3). - The fall in pH value is due to CO 2 which is a byproduct of biodegradation reaction while aerating the medium the pH would rise because CO 2 was being displaced. Results & Discussions on Time Based Control(3)

20. Fig 3. pH pattern with Time Based Control on MMA biodegradation AB: normal, B: injection, BC: initiation, CD: acceleration, DE: completion

21. - At point B in Fig.3, 100ppm MMA was injected in the reservoir, which initiated a sharp increase in the product of CO 2 (BC). - For the next about 7 hours, reaction rate acceleration as seen by the decrease in MMA concentration as well as a decrease in the overall pH level. - After 7 hours, the pH level started rising back to the baseline level, and the MMA concentration was reduced to less than 1ppm. - Section DE indicates completion of the reaction. The disappearance of MMA coincides with rise in pH. Results & Discussions on Time based Control (4)

22. - Fig.4 shows biodegradation of styrene(50ppm) and the associated DO and pH profiles. - It should be noted here that since styrene is highly volatile. Air was not used as a source of DO. Instead hydrogen peroxide was used - DO level were maintained by a set point control method using a commercially available data acquisition and control package. - The set point in this case was set to 7.6ppm DO with 0.01ppm error bound. - But the variation in DO levels from the set point resulted from error bound and system lag phase until oxygen senor sensed in chamber the pumped H 2 O 2, mixed in the reservoir. Results & Discussions on Set Point Control(1)

23. Fig 4. Oxygen conc. pattern with Set Point Control injected styrene and hydrogen peroxide as substrate and oxygen source, AB: normal, b: injection, BC: initiation, CD: acceleration, DE: completion.

24. - As seen in the Fig.4, the variation in DO levels from the set point is more when the system is operating without the organic substrate. This is because when H 2 O 2 is injected (When DO goes below 7.6ppm) the overall DO concentration attains higher levels since there is no significant consumption of DO in the absence of any organic substrate. - When styrene was injected at point B, the slowly DO variation from the set point decreases because as oxygen was being generated at the same time it was also consumed due to the presence of styrene. The section CD indicates acceleration of the reaction. Results & Discussions on Set Point Control (1)

25. - Later when the styrene is essentially degraded the variation in DO level from set point relations to the original values. - The time of disappearance of styrene and rise in DO level coincide and indicates that the reaction is complete without formation of intermediates. Results & Discussions on Set Point Control (2)

26. - The change in pH in this case is different from that seen in MMA experiments. - Here the pH begins to fall and does not rise up to its original value at the end of the experiment. This happens because any CO 2 that is generated does not leave the system. - At point B, 50ppm styrene was injected in the reservoir, which initiated a sharp increase in the product of CO 2 (BC). Results & Discussions on Set Point Control (3)

27. - For the next about 7 hours, reaction rate accelerated as seen by the decrease in the overall pH level as well as the decrease in MMA concentration. - After 7 hours, the pH level does not rise back to the baseline level, even though the MMA concentration was reduced to less than 1ppm. - Section DE indicates completion of the reaction. It is shown that the slope on decreasing pH level is different on each stages. - There the pH reaches a constant of the reaction. Results & Discussions on Set Point Control (4)

28. - Fig.5 shows profiles of DO, pH and ethylene glycol during experiments to study degradation of ethylene glycol. - As before the DO profile shows trends of initiation, acceleration, and completion. But this time the rise in DO doesn't coincide with the disappearance of ethylene glycol. - Ethylene glycol disappears in 160 min. before DO levels rise back to normal. - This trend indicates a different degradation mechanism where intermediates are formed. - In this case ethylene glycol was being converted to formaldehyde and as a result DO levels remained low until the formaldehyde was also degraded. - The formation of formaldehyde was confirmed by GC analysis. Pattern with partial oxidation(1)

29. Fig 5. Oxygen conc. and pH pattern with Time Based Control on ethylene glycol biodegradation, AB: normal, B: injection, BC: initiation, CD: acceleration, DC: completion.

30. GC peak when the only defined medium was injected

31. GC peak when the only defined medium with ethylene glycol was injected without biomass.

32. GC peak at 10 minutes after injection of ethylene glycol

33. GC peak at 25 minutes after injection of ethylene glycol

34. GC peak at 40 minutes after injection of ethylene glycol

35. GC peak at 150 minutes after injection of ethylene glycol

36. GC peak at 300 minutes after injection of ethylene glycol

37. GC peak after biodegradation is over

38. - Fig.6 provides solution to our original objective which was to develop an on-line method to follow biodegradation experiments without analysis of the organic substrate. - In the final experiment with tetraethylene glycol(100ppm) it was demonstrated how this can be done. - The profiles of DO and pH follow identical patterns as seen in earlier case. The regions of reaction initiation acceleration and completion can be clearly seen. - Tetraethylene glycol was degraded and its rate of degradation was computed without its analysis. - In this experiment whole reaction time 660 minutes means the time while TEG itself and all intermediates which can be biodegraded are degraded. Pattern with partial oxidation(2)

39. Fig 6. Oxygen concentration and pH pattern with Time Based Control on TEG biodegradation, AB: normal, B: injection, BC: initiation, CD: acceleration, DC: completion

40. 1)The bio-oxidation pattern on biodegradation of organic compound in real time which has initiation and acceleration completion stages through monitoring DO and pH using on-line measurement with time based and set point control technique. 2)From these profiles the degradation rates of the organic compounds in question can be estimated easily. 3)It is shown that this pattern can be applied on biodegradation with partial oxidation too. Conclusions (1)

41. 4) It is a tool that not only gives a good estimate of the oxygen demand for a given organic substrate but also it takes in less time than that required in BOD. 5) This tool can be used as an easy and inexpensive technique to control and monitor biological wastewater treatment processes. Conclusions (2)

42. 5) It can overcome the disadvantages of conventional oxygen uptake measurement with their low resolution in time and provides a method mainly for the use under dynamic process conditions. Conclusions (3)

43. Thank you very much for listening