FORMULATION OF BACTERIAL CONSORTIA AND STUDYING THEIR SYNERGISTIC EFFECT ON TREATMENT OF EFFLUENT

Transcription

1 CHAPTER 5 FORMULATION OF BACTERIAL CONSORTIA AND STUDYING THEIR SYNERGISTIC EFFECT ON TREATMENT OF EFFLUENT 5.1. Introduction Based on the biodegradability, the industrial pollutants have been classified as low, moderate and highly biodegradable. This is primarily due to the microorganisms used for the biodegradation of different constituents present in individual industries. Pulp and paper industrial wastewater is complex in nature. Since the inception of waste water treatment plants, activated sludge process is being used for the biodegradation/ treatment of wastewater. Inspite of great endeavours exercised for the engineering component of the waste water treatment plants the biological part has been totally neglected. However, the biological component is one of the most important component because the biological entities play major role in biodegradation of the chemical constituents present in the wastewater. It is quite possible to alter the biodegradability of pulp and paper industry effluent if the microbial diversity is explored for specific biodegradation. In the previous objective the microorganisms were isolated based on their biodegradability of different dyes and substrates. But, it is difficult for single microorganisms to degrade the complex pulp and paper wastewater. Therefore, in the present study different permutation combinations (consortia were formulated) were made in order to treat the pulp and paper wastewater holistically. Microbial consortium was used to treat wastewater because it has long been known that they are much more efficient at degrading complex organic wastes than single isolate. In a microbial consortium one might find any number of organisms with different metabolic capabilities. This could include organisms that are proteolytic (are able to degrade proteins and amino acids); organisms that are saccharolytic (able to degrade various sugars); organisms that are lipolytic (able to digest lipids or fats); and organisms that are cellulytic/lignotytic (able to degrade cellulose/lignin or plant matter). These different metabolic capabilities allow the consortium to work together in degrading a variety of complex waste streams. 130

2 5.2. Material and Methods Chemicals and Reagents All chemical used in this study were of analytical grade. Nutrient broth, agar powder, Tween 80 and glycerol were obtained from Hi-media. The routine chemicals were procured from S.D. fine, Qualigens and Merck India limited. Distilled water was used throughout the study Equipments The equipment used during the present study includes laminar air flow (Kartos International), electronic balance (Sartorious), ph meter (Lab India), autoclave (Yorco), incubator shaker (New-Brunswick-Innova 4300), centrifuge (Sorvall-RC 5B Plus), spectrophotometer (Pharmaspec UV 1700, Shimadzu), digital burette (Eppendorf), COD digestor (Spectralab), vortex mixer and magnetic stirrer (Matrix) Glassware s / Plastic Wares Storage bottles, Petri dishes were procured from Tarson. Measuring cylinders, beakers, Erlenmeyer flasks, were procured from Borosil Treatment of Effluent by using Single Isolate In previous objective, six bacteria were isolated form three different soil samples. The list of bacteria screened on the basis of their substrate degradation capability is shown in Table 5.1. Table 5.1: List of bacteria selected on the basis of degradation of lignocellulose S.No. Laboratory Name for the Isolate Isolates selected on the basis of degradation 1 PNP 1 Lignin 2 PNP 3 Lignin 3 PNP 5 Cellulose 4 PNP 6 Cellulose 5 PNP 8 Brich wood xylan 6 PNP 9 Brich wood xylan 131

3 Inoculum Preparation The inoculums were prepared by inoculating one loopful of individual bacterial isolates, separately in 50 ml of sterilized nutrient broth having 0.01% Tween-80. Inoculated broths were incubated in an orbital shaker at 35ºC for 16 hrs so as to obtain actively growing mother cultures. These mother cultures (100µl) were used for sub-culturing 100 ml NB and incubated at 35ºC under shaking conditions at 200 rpm for a period of hrs. The obtained cultures were harvested by centrifugation at 4ºC and 7000 rpm followed by washing twice with sodium phosphate buffer (ph ). The supernatant of all the cultures were discarded and pellets were stored for the further experiments Treatment Bacterial pellets were inoculated in effluent in the ratio of 1:10 i.e., 10% inoculum was added in 100 ml of effluent in 500 ml conical flask. In each flask the nitrogen and phosphorous (in the form of urea and di ammonium phosphate) was added in the ratio of 100:5:1 (BOD: N: P). In order to check the activity of the single isolate under same conditions, a blank containing only wastewater (without any inoculum) was paced. All the flasks were incubated in an orbital shaker at 35 C and 200 rpm for 48 hrs (in order to acclimatize the bacteria). After 48 hrs, flasks were removed and kept at static condition for sludge settling. After settling approximately 90% of the treated effluent was replaced with the untreated effluent in the flask with the addition of urea and di ammonium phosphate as the source of nitrogen and phosphorous and all the flasks were again kept in shaker at 35 C and 200 rpm for 24 hrs. After 24 hrs the treatment cycle was repeated in same manner i.e., flasks were kept undisturbed in static position, 90% of the treated effluent was replaced with 90% of untreated effluent with the addition of nitrogen and phosphorous (figure 5.1). The collected treated effluent from all the flasks was analyzed for COD and colour reduction (procedures as mentioned in chapter 3). 132

4 Figure 5.1: Experimental representation for the treatment of pulp and paper wastewater using single bacterial isolate Formulation of Consortia From the above experiment it was clear that single isolate was not able to degrade the pulp and paper industrial effluent. The nature of pulp and paper industry effluent is quite complex as it contains a number of organic components, for example, lignin, tannic acid, resin, cellulose, and hemicellulose which are difficult to be degraded by a single bacterial isolate. So, there was need for the formulation of effective microbial consortium which can biodegrade the pollutants in effluent effectively. Formulation of consortia was done by using six bacteria isolated from three different samples. Each consortium consists of three bacteria. Formulation of consortia was done by following permutation combination in which repetition was not allowed and order of the bacteria does not matter. Twenty consortia were formulated by using the following formula. List of bacterial consortia is given in table 5.2. n! / (n-r)! (r)! Where, n (total number of bacteria) = 6 and r (bacteria in each consortia) = 3 133

5 Table 5.2: List of bacterial consortia and their respective bacteria S.No. Laboratory name for the consortia Number of isolates 1 Consortia 1 PNP 1, PNP 3, PNP 5 2 Consortia 2 PNP 6, PNP 3, PNP 1 3 Consortia 3 PNP 8, PNP 1, PNP 3 4 Consortia 4 PNP 3, PNP 1, PNP 9 5 Consortia 5 PNP 1, PNP 5, PNP 6 6 Consortia 6 PNP 1, PNP 5, PNP 8 7 Consortia 7 PNP 5, PNP 9PNP 1 8 Consortia 8 PNP 1, PNP 6, PNP 8 9 Consortia 9 PNP 3, PNP 6, PNP 8 10 Consortia 10 PNP 1, PNP 8, PNP 9 11 Consortia 11 PNP 3, PNP 5, PNP 6, 12 Consortia 12 PNP 5, PNP 8, PNP 3 13 Consortia 13 PNP 3, PNP 5, PNP 9 14 Consortia 14 PNP 1, PNP 6, PNP 9 15 Consortia 15 PNP 3, PNP 9, PNP 6 16 Consortia 16 PNP 9, PNP 3, PNP 8 17 Consortia 17 PNP 6, PNP 8, PNP 5, 18 Consortia 18 PNP 6, PNP 5, PNP 9 19 Consortia 19 PNP 8, PNP 5, PNP 9 20 Consortia 20 PNP 6, PNP 8, PNP Treatment by using Formulated Bacterial Consortia Inoculum Preparation The inoculum was prepared by inoculating one loopful of individual bacterial isolates, separately in 50 ml of sterilized nutrient broth having 0.01% Tween-80. Inoculated broths were incubated in an orbital shaker at 35ºC, 200 rpm for 16 hrs to obtain actively growing mother cultures. These mother cultures were used for sub-culturing and hence 100µl of the mother culture was inoculated into 100 ml of fresh NB. The inoculated flasks were incubated at 35ºC under shaking conditions at 200 rpm for a period of hrs The culture was harvested by centrifugation at 4ºC and 7000 rpm followed by washing twice with sodium phosphate buffer (ph ). Supernatant was discarded and the pellets were stored for the further experiments. 134

6 Treatment At the time of experiment, different pellets were re-suspended according to the 20 consortia designed. The pellets were inoculated in effluent in the ratio of 1:10 i.e., 10% inoculum was added in 100 ml of effluent. In each flask the nitrogen and phosphorous was added in the ratio of (BOD: N: P) 100:5:1., in order to check the activity of the bacterial consortia, blank containing only wastewater with no inoculums was placed in parallel with the experiment. All the flasks were incubated in the orbital shaker at 35 C and 200 rpm for 48 hrs (in order to acclimatize the bacteria). After 48 hrs the flasks were removed and kept undisturbed in static position to settle the sludge. After settling approximately 90% of the treated effluent was replaced with the untreated effluent in the flask with the addition of nitrogen and phosphorous. The treated effluent was discarded. All the flasks were again kept in shaker at 35 C and 200 rpm for 24 hrs. After 24 hrs the cycle was repeated in similar manner i.e., flasks were kept undisturbed in static position, 90% of the treated effluent was replaced with 90% of untreated effluent with the addition of nitrogen and phosphorous (figure 5.2). The collected treated effluent from all the flasks was analyzed for COD and colour reduction. The results were statistically analyzed by using software Minitab 16. Figure 5.2: Experimental representation for the treatment of pulp and paper wastewater using bacterial consortia 135

7 Treatment by using Selected Consortia From above experiment consortia 9 and 14 were selected for further studies. The experiment was repeated three times in order to check the repeatability among the results. Experiment was repeated in the similar manner as mentioned in section To Study the Growth Pattern of Bacterial Consortium One loopful of bacterial culture of selected consortium 9 (PNP 3, PNP6 and PNP 8) was inoculated in 25 ml of nutrient broth separately to get active mother culture. The inoculated flasks were incubated at 35 C and 200 rpm for 6-8hrs. Similar concentration (~2.8 x 10 5 CFU/ml) of each mother culture i.e., 1ml of each isolate was inoculated in 100 ml of nutrient broth collectively. The flask was incubated at 35 C and 200 rpm for 24 hrs. After every 4 hrs 1 ml of sample was withdrawn, serially diluted, then spreaded on nutrient agar and incubated to calculate CFU/ml in order to check the growth of each isolated in the presence of other isolate. This experiment was carried out in triplicates Results Treatment of Effluent by using Single Isolates The selected single bacterial isolates were screened for their capability to degrade the pulp and paper industrial effluent. Two wastewater parameters COD and colour were analyzed by placing the experiment Estimation of Chemical Oxygen Demand by using Single Isolates The percentage reduction in COD values of the effluent by using single isolates is depicted in table 5.3. The COD of untreated wastewater was estimated to be around 730 mg/l. For each isolates three treatment cycles were placed and samples were collected in between and analyzed. Results of isolate PNP 1 showed that after 24 hrs the achieved reduction was up to 28.22% with the COD value of 524 mg/l. On completing the 2 nd cycle the percentage reduction was 29.45% with the COD value of 515 mg/l. After last cycle of 24 hrs the final reduction by using isolate PNP 1 was 136

8 31.51% with the reduce value of COD 500 mg/l. In the similar manner the results were observed for isolate PNP 3 for three cycles. After first cycle the achieved reduction was 29.45% with the COD value 515 mg/l, observation showed that after completing second cycle the reduction attain was 32.88% with the reduced COD value 490 mg/l. Finally after third cycle the observed reduction was 30.14% with the COD value 510 mg/l. While observing the result for isolate PNP 5 the reduction for three cycles was 39.04% after 1 st cycle, 40% after 2 nd cycle, after 3 rd cycle. The COD values for the three cycles were 445 mg/l 438 mg/l and 432 mg/l respectively. In case of isolate PNP 6 the reduction after first cycle was up to 40.41% with the reduced COD value 435 mg/l. After second cycle the achieved reduction was 42.19% with the COD value 422 mg/l. Final stage cycle showed the reduction value of 41.78% with slight increase in COD value i.e., 425 mg/l. COD was estimated after placing the experiment with isolate PNP 8 and it was observed that after the completion of cycle 1 the achieved reduction was up to 35.34% with the COD value of 485 mg/l. The sample was collected after 2 nd cycle the observed value for COD was 472 mg/l with the achieved reduction 35.34%. After final cycle the reduction attains was 36.30% with the COD value 465 mg/l. The results of last isolate i.e., PNP 9 showed the reduction values of 23.29% after first cycle, 26.85% after second cycle and finally 28.77% after third cycle. The reduced COD values for the three cycles were 560 mg/l, 534 mg/ and 520 mg/l respectively. In order to check whether the biodegradation of the pulp and paper wastewater is due to the inoculated isolates, a blank containing only wastewater is placed along with the experiment. The results revealed that the achieved reduction after first cycle was 20.0% with the COD value of 584 mg/l. After completing the second cycle the reduction was up to 21.23% with the COD value 575 mg/l. Finally the sample from third cycle was analyzed and it was observed that the reduction was up to 21.92% with the reduced COD valued 570 mg/l. 137

9 Table 5.3: Reduction of COD after different cycles of 90% replacement of effluent sample by using single isolate COD of the effluent without treatment 730 mg/l (Control) Isolate 1 Cycles 2 3 Mean Percentage reduction w.r.t control / cycle Mean Blank PNP PNP PNP PNP PNP PNP The samples collected from three cycles were subjected to COD analysis followed by colour estimation by using standard method (APHA 1998). The mean value of three cycles for each isolates shown in figure 5.3. The results were compared with control i.e., untreated wastewater. Final reduction achieved by using isolate PNP 1 was 29.73% with the mean COD value of three cycles of 513 mg/l. PNP 3 gave the final reduction of 30.82% with the reduced COD value of 505 mg/l. While comparing the results of other isolates PNP 5, PNP 6, PNP 8 and PNP 9 it was observed that the achieved percentage reduction was 39.95%, 41.46%, 35.07% and 26.30% respectively with the COD value of mg/l, mg/l, mg/l and mg/l respectively. Blank was placed in order to check the activity of single isolate because it is the fact, that wastewater itself contains some microflora, sometimes it is possible that by getting favorable conditions they get activated and degrade the sample. The achieved COD value for blank was mg/l. when the percentage reduction was calculated it was about 21.05%. The results showed that the maximum reduction was up to %. Out of which blank value was 21.05% which shows that it is not possible for single isolate to degrade the complex pulp and paper wastewater. 138

10 Note: the bars were plotted on the basis of mean value of the three cycles mentioned in the table above. Figure 5.3: Reduction in COD of effluent by using single bacterial isolates Estimation of Colour by using Single Isolates The percentage reduction in the colour of wastewater by using single isolates is depicted in table 5.4. Estimated colour for the untreated wastewater was 820 PCU. For each isolates three cycles were placed and samples were collected in between for analysis. In order to check whether the isolates were working or it s just the incubation conditions responsible for the biodegradation of the pulp and paper wastewater. Blank was placed which was without any inoculum. The results revealed that the achieved reduction after 1 st cycle was 4.88% with the colour value 780 PCU. After completing the 2 nd cycle the reduction was up to 6.10% with the colour value 770 PCU. Finally the sample from third cycle was analyzed and it was observed that the reduction was up to 6.71% with the reduced colour valued 765 PCU. The results for isolate PNP 1 showed that after 24 hrs, achieved reduction was up to 13.41% with the colour value 710 PCU. On completing second cycle the reduction value was 17.07% with the colour value of 680 PCU. After last cycle of 24 hrs, the final reduction by using isolate PNP 1 was 17.68% with the reduce value of colour 675 PCU. In the similar manner the results were observed for isolate PNP 3 for three cycles. After cycle 1, the achieved reduction was 17.07% with the colour value 680 PCU, observation showed that after completing 139

11 second cycle the reduction attain was 17.68% with the reduced colour value 675 PCU. Finally after 3 rd cycle the observed reduction was 19.02% with the colour value 664 PCU. While observing the result for isolate PNP 5 the reduction for three cycles was 7.32% after 1 st cycle, 8.54% after 2 nd cycle and 10% after 3 rd cycle. The colour values for three cycles were 760 PCU, 750 PCU and 738 PCU respectively. In case of isolate PNP 6 reduction after 1 st cycle was up to 10.98% with the reduced colour value of 730 PCU. After 2 nd cycle the achieved reduction was with the colour value 722 PCU. Final stage cycle showed the reduction value of 12.68% with slight increase in colour value i.e., 716 PCU. The colour of treated effluent was estimated after placing the experiment with isolate PNP 8 and it was observed that after the completion of cycle 1 the achieved reduction was up to % with the colour value of 725 PCU. The sample was collected after 2 nd cycle the observed value for colour was 718 PCU with the achieved reduction of 12.44%. After final cycle the reduction attains was 13.41% with the colour value of 710 PCU. The results of last isolate i.e., PNP 9 showed the reduction values of 8.54% after 1 st cycle, 10.98% after 2 nd cycle and finally 9.51% after 3 rd cycle. The reduced colour values for the three cycles were 750 PCU, 730 PCU and 742 PCU respectively. Table 5.4: Reduction of colour after different cycles of 90% replacement of effluent sample by using single isolate Colour of the effluent without treatment 820 PCU (Control) Isolate 1 Cycles 2 3 Mean Percentage reduction w.r.t control / cycle Mean Blank PNP PNP PNP PNP PNP PNP

12 The colour of treated effluent was estimated after each cycle and finally the mean was calculated. Figure 5.4 shows the mean values of colour reduced by using single isolates and the values above the bars show achieved percentage reduction. Final reduction achieved by using isolate PNP 1 was 16.06% with the mean colour value of three cycles of PCU. The isolate PNP 3 gave the final reduction of 17.93% with the reduced colour value of PCU. The isolate PNP 5 showed the percentage reduction up to 8.62% with the colour value of PCU. While comparing the results of other isolates PNP 6, PNP 8 and PNP 9 it was observed that the achieved percentage reduction was 11.87%, 12.48% and 9.67% respectively with the colour value of PCU, PCU and PCU respectively. The achieved colour value for blank was PCU and when the percentage reduction was calculated it was about 5.89%. Note: The bars were plotted on the basis of mean value of the three cycles mentioned in the table above. Figure 5.4: Reduction in colour of effluent by using single bacterial isolates Treatment of Effluent by using Formulated Consortia The nature of pulp and paper industry effluent is quite complex as it contains a number of organic components, for example, lignin, tannic acid, resin, cellulose, and hemicellulose which are difficult to be degraded by a single bacterial isolate. So, bacterial consortia were used for effective treatment of effluent in minimum time period. 141

13 Estimation of COD by using Formulated Consortia The experiments were performed by using 20 formulated consortia and results were depicted in table 5.5. Treated effluent was analyzed for COD reduction. Control value of the COD of (untreated effluent) was 730 mg/l. The percentage degradation was calculated by comparing the obtained value with respect to control value. For each consortium three cycles were placed and samples were collected in between for analysis. In order to check whether the consortia were working or it s just the incubation conditions responsible for the biodegradation of the pulp and paper wastewater. Blank was placed which was without any inoculum. Results for consortium 1 showed that after 24 hrs the achieved reduction was up to 44.52% with the COD value 405 mg/l. On completing the 2 nd cycle the reduction value was 47.95% with the COD value 380 mg/l. After last cycle of 24 hrs the final reduction by using consortium 1 was 45.75% with the COD value 396 mg/l. In the similar manner the results were observed for consortium 2 for three cycles. After cycle 1 the achieved reduction was 43.84% with the COD value 410 mg/l. It was observed that after completing cycle 2 the reduction attains was 45.89% with the reduced COD value 395 mg/l. Finally after 3 rd cycle the observed reduction was 47.95% with the COD value 380 mg/l. While observing the result for consortium 3 the reduction for three cycles was 40.41% after 1 st cycle, 42.19% after 2 nd cycle and 43.15% after 3 rd cycle. The COD values for the three cycles were 435 mg/l, 422 mg/l and 415 mg/l respectively. In case of consortium 4 the reduction after 1 st cycle was up to 38.90% with the reduced COD value 446 mg/l. After 2 nd cycle the achieved reduction was 40.82% with the COD value 432 mg/l. Final stage cycle showed the reduction value of 42.74% with slight decreased in COD value i.e., 418 mg/l. COD was estimated after placing the experiment with consortium 5 and it was observed that after the completion of cycle 1 the achieved reduction was up to 40.82% with the COD value 432 mg/l. The sample was collected after 2 nd cycle and observed value for COD was 425 mg/l with the achieved reduction 41.78%. After final cycle the reduction attains was 43.01% with the COD value of 416 mg/l. The results of consortium 6 showed the reduction values of 44.38% after 1 st cycle, 46.58% after 2 nd cycle and finally 47.95% after 3 rd cycle. The reduced COD values for the three cycles were 406 mg/l, 142

14 390 mg/ and 380 mg/l respectively. Results for consortium 7 showed that after 24 hrs the achieved reduction was up to 47.26% with the COD value of 385 mg/l. On completing the 2 nd cycle the reduction value was 46.03% with the COD value of 394 mg/l. After last cycle of 24 hrs the final reduction by using consortium 7 was 46.85% with the COD value 388 mg/l. In the similar manner the results were observed for consortium 8 for three cycles. After cycle 1 the achieved reduction was 47.67% with the COD value 382 mg/l, observation showed that after completing cycle 2 the reduction attain was 49.32% with the reduced COD value 370 mg/l. Finally after 3 rd cycle the observed reduction was 50.0% with the COD value 365 mg/l. While observing the result for consortium 9 the reduction for three cycles was 47.26% after 1 st cycle, 50.68% after 2 nd cycle, % after 3 rd cycle. The COD values for the three cycles were 385 mg/l, 360 mg/l and 345 mg/l respectively. In case of consortium 10 the reduction after 1 st cycle was up to 42.74% with the reduced COD value 418 mg/l. After 2 nd cycle the achieved reduction was 44.38% with the COD value of 406 mg/l. Final stage cycle showed the reduction value of 46.58% with slight increase in COD value i.e., 390 mg/l. COD was estimated after placing the experiment with consortium 11 and it was observed that after the completion of cycle 1 the achieved reduction was up to 41.37% with the COD value 428mg/l. The sample was collected after 2 nd cycle the observed value for COD was 418 mg/l with the achieved reduction 42.74%. After final cycle the reduction attains was 41.78% with the COD value 425 mg/l. The results of consortium 12 showed the reduction values of 46.30% after 1 st cycle, 47.26% after 2 nd cycle and finally 48.49% after 3 rd cycle. The reduced COD values for the three cycles were 392 mg/l, 385 mg/ and 376 mg/l respectively. Results for consortium 13 showed that after 24hr the achieved reduction was up to 47.95% with the COD value 380 mg/l. On completing the 2 nd cycle the reduction value was 49.04% with the COD value 372 mg/l. After last cycle of 24 hrs the final reduction by using consortium 13 was 47.04% with the reduce value COD 384 mg/l. In the similar manner the results were observed for consortium 14 for three cycles. After cycle 1 the achieved reduction was 48.63% with the COD value 375 mg/l. Observation showed that after completing cycle 2 the reduction attain was 50.68% with the reduced COD value 143

15 360 mg/l. Finally after 3 rd cycle the observed reduction was 51.37% with the COD value 355 mg/l. While observing the result for consortium 15 the reduction for three cycles was 46.58% after 1 st cycle, 47.67% after 2 nd cycle and 48.63% after 3 rd cycle. The reduced COD values for the three cycles were 390 mg/l, 382 mg/l and 375 mg/l respectively. In case of consortium 16 the reduction after 1 st cycle was up to 28.08% with the reduced COD value 525 mg/l. After 2 nd cycle the achieved reduction was 29.32% with the COD value 516 mg/l. Final stage cycle showed the percentage reduction 30.82% with COD value 505 mg/l. COD was estimated after placing the experiment with consortium 17 and it was observed that after the completion of cycle 1 the achieved reduction was up to 39.73% with the COD value 440 mg/l. The sample was collected after 2 nd cycle the observed value for COD was 430 mg/l with the achieved reduction of 41.10%. After final cycle the reduction attains was 41.78% with the COD value 425 mg/l. The results of consortium 18 showed the reduction values of 41.78% after 1 st cycle, 42.19% after 2 nd cycle and finally 41.78% after 3 rd cycle. The reduced COD values for the three cycles were 425 mg/l, 422 mg/l and 425 mg/l respectively. COD was estimated after placing the experiment with consortium 19 and it was observed that after the completion of cycle 1 the achieved reduction was up to 40.41% with the COD value 435 mg/l. The sample was collected after 2 nd cycle the observed value for COD was 430 mg/l with the achieved reduction of 41.10%. After final cycle the reduction attain was 43.01% with the COD value 416 mg/l. The results of last consortium 20 showed the percentage reduction 38.08% after 1 st cycle, 36.99% after 2 nd cycle and finally 35.62% after 3 rd cycle. The reduced COD values for the three cycles were 452 mg/l, 460 mg/l and 470 mg/l respectively. In order to check whether the isolates were working or it s just the incubation conditions are responsible for the biodegradation of the pulp and paper wastewater. Blank was placed which was without any inoculum. The results revealed that the achieved reduction after 1 st cycle was 20.00% with the COD value of 584 mg/l. After completing the 2 nd cycle the reduction was up to 21.23% with the COD value of 575 mg/l. Finally the sample from third cycle was analyzed and it was observed that the reduction was up to 21.92% with the reduced COD valued 570 mg/l. 144

16 Table 5.5: Reduction in COD after different cycles of 90% replacement of effluent by using bacterial consortia COD of the effluent without treatment 730 mg/l (Control) Consortia No. 1 Cycles 2 3 Mean Percentage reduction w.r.t control / cycle Mean Blank The samples collected from three cycles were subjected to COD analysis using standard method. The figure 5.5 showed the mean value of three cycles for each consortium. The results were compared with control i.e., untreated wastewater. Final reduction achieved by using consortium 1 was 42.7% with 418 mg/l as the mean COD value of three 145

17 cycles. Consortium 2 showed the final reduction 43.2% with the reduced COD value 414 mg/l. While comparing the results of other isolates consortium 3, 4, 5 and 6 it was observed that the achieved percentage reduction was 41.92%, 40.82%, 41.87% and 42.3% respectively with the COD value of mg/l, mg/l, mg/l and mg/l respectively. The achieved reduction after three cycles in case of consortia 7, 8, 9, and 10 was mg/l, mg/l, mg/l and 429 mg/l respectively. The achieved percentage reduction for these consortia was 46.71%, 40.00%, 50.23% and 41.2% respectively. The consortium 11 showed the final percentage reduction up to 41.96% with mg/l as the reduction achieved in COD value. The results of consortium 12 showed that the reduced COD value after three cycles was mg/l with the percentage reduction 47.35%. Reduction in COD achieved by using consortium 13 was 48.13% with mg/l as the mean COD value of three cycles. Note: The bars were plotted on the basis of mean value of the three cycles mentioned in the table above. Figure 5.5: Reduction in COD of effluent by using bacterial consortia The achieved reduction by using consortium 14 was 50.23% with the reduced COD value o mg/l. While comparing the results of other consortium 15, 16, 17 and 18 it was observed that the achieved percentage reduction was 47.63%, 36.7%, 40.87% and 41.92% respectively with the COD value mg/l, mg/l, mg/l and mg/l respectively. The consortium 19 showed the percentage reduction up to 146

18 41.51% with the reduced COD value of mg/l. The estimated value of last consortium 20 showed that the achieved value for COD was with the percentage reduction of 36.89%. Blank was placed in order to check the activity of single isolate because it is the fact, that wastewater itself contains some microflora, sometimes it is possible that by getting favorable conditions they get activated and degrade the sample. The achieved COD value for blank was mg/l when the percentage reduction was calculated it was about 21.05% Estimation of Colour by using Formulated Consortia The control value of the colour for the untreated effluent was 820 PCU. The percentage degradation was calculated by comparing the obtained value with control. Results for consortium 1 showed that after 24 hrs the achieved reduction was up to 33.54% with the colour value 545 PCU. On completing the second cycle the reduction was 34.15% with the colour value 540 PCU. After last cycle of 24 hrs the final reduction by consortium 1 was 32.32% with the colour value 555 PCU. In the similar manner the results were obtained by using consortium 2 for three cycles. After cycle 1 the achieved reduction was 33.90% with the colour value 542 PCU. Observation showed that after completing cycle 2 the reduction attains was 35.37% with the reduced colour value of 530 PCU. Finally after 3 rd cycle the observed reduction was 36.34% with the colour value 522 PCU. While observing the result of consortium 3, the reduction for three cycles was 32.68% after 1 st cycle, 33.54% after 2 nd cycle and 34.15% after 3 rd cycle. The colour values for the three cycles were 552 PCU, 545 PCU and 540 PCU respectively. In case of consortium 4 the percentage reduction after first cycle was up to 33.17% with the reduced colour value 548 PCU. After 2 nd cycle the achieved reduction was 34.15% with the colour value 540 PCU. Third cycle showed 34.88% reduction in colour value it t was 534 PCU. The colour of treated effluent was estimated after placing the experiment with consortium 5 and it was observed that it was reduced up to 23.17% after completion of first cycle with the colour value 630 PCU. The sample was collected after 2 nd cycle and it was observed 25.0% reduction with colour value 615 PCU. After third cycle the achieved reduction was 26.83% with the colour value 600 PCU. The results of consortium 6 showed the reduction values 31.10% after 1 st cycle, 32.93% 147

19 after 2 nd cycle and finally 32.68% after 3 rd cycle. The reduced colour values for the three cycles were 565 PCU, 550 PCU and 552 PCU respectively. Consortium 7 showed that after first cycle of 24h the achieved reduction was up to 32.93% with the colour value 550 PCU. On completing the 2 nd cycle the reduction value was 33.54% with the colour value 545 PCU. After last cycle of 24 hrs the final reduction by using consortium 7 was 35.37% with the colour value 530 PCU. In the similar manner the results were observed for consortium 8 for three cycles. After first cycle the achieved reduction was 36.34% with the colour value 522 PCU. Observation showed that after completing second cycle the reduction attain was 37.80% with the reduced colour value 510 PCU. Finally after third cycle the observed reduction was 37.20% with the colour value 515 PCU. While observing the result for consortium 9 the reduction for three cycles was, 37.20% after 1 st cycle, 37.80% after 2 nd cycle and 37.20% after 3 rd cycle. The colour values for the three cycles were 515 PCU, 510 PCU and 500 PCU respectively. In case of consortium 10, the reduction after 1 st cycle was up to 31.71% with the reduced colour value 560 PCU. After 2 nd cycle the achieved reduction was 32.68% with the colour value of 552 PCU. The final cycle showed the reduction value of 34.15% with slight decreased in colour value i.e., 540 PCU. The colour of- treated effluent was estimated after placing the experiment with consortium 11 and it was observed that after the completion of cycle 1 the achieved reduction was up to 25.61% with the colour value 610 PCU. Sample was collected after 2 nd cycle, observed value of colour was 690 PCU with the achieved reduction of 15.85%. After final cycle reduction was 27.44% with colour value 595 PCU. The results of consortium 12 showed the reduction values of 34.15% after 1 st cycle, 35.37% after 2 nd cycle and finally 34.15% after 3 rd cycle. The reduced colour values for the three cycles were 540 PCU, 530 PCU and 540 PCU respectively. The results of consortium 13 showed that after first treatment cycle of 24 h the achieved reduction was up to 33.54% with the colour value of 545 PCU. On completing the 2 nd cycle the reduction value was 35.37% with the colour value of 530 PCU. After last cycle of 24 hrs the final reduction by using consortium 13 was 34.15% with the reduce colour value 540 PCU. In the similar manner the results were observed for consortium 14 for three cycles. After cycle 1 the achieved reduction was 35.61% with the colour value 528 PCU. Observation showed that after completing cycle 2 the 148

20 reduction attains was 37.20% with the reduced colour value 515 PCU. Finally after 3 rd cycle the observed reduction was 30.02% with the colour value of 500 PCU. While observing the result for consortium 15 the reduction for three cycles was 34.76% after 1 st cycle, 36.59% after 2 nd cycle and 35.98% after 3 rd cycle. The reduced colour values for the three cycles were 535 PCU, 520 PCU and 525 PCU respectively. In case of consortium 16 the reduction after 1 st cycle was up to 30.24% with the reduced colour value 572 PCU. After 2 nd cycle the achieved reduction was 31.10% with the colour value of 565 PCU. The final cycle showed the reduction value of 31.95% with reduced in colour value i.e., 558 PCU. The colour was estimated after placing the experiment with consortium 17 and it was observed that after the completion of cycle 1 the achieved reduction was up to % with the colour value of 710 PCU. The sample was collected after 2 nd cycle the observed value for colour was 695 PCU with the achieved reduction of 15.24%. After final cycle the reduction attains was 14.63% with the colour value of 700 PCU. The results of consortium 18 showed the reduction values of after 1 st cycle, 13.41% after 2 nd cycle and finally 14.02% after 3 rd cycle. The reduced colour values for the three cycles were 725 PCU, 710 PCU and 705 PCU respectively. Colour was estimated after placing the experiment with consortium 19 and it was observed that after the completion of cycle 1 the achieved reduction was up to 13.41% with the colour value of 710 PCU. The sample was collected after 2 nd cycle the observed value for colour was 700 PCU with the achieved reduction of 14.63%. After final cycle the achieved reduction was 15.24% with the colour value of 695 PCU. The results of last consortium 20 showed the reduction values of 12.80% after 1 st cycle, 14.63% after 2 nd cycle and finally 13.17% after 3 rd cycle. The reduced colour values for the three cycles were 715 PCU, 700 PCU and 712 PCU respectively. In order to check whether the isolates were working or it s just the incubation conditions are responsible for the biodegradation of the pulp and paper wastewater. Blank was placed which was without any inoculum. The results revealed that the achieved reduction after 1 st cycle was 4.88% with the colour value of 780 PCU. After completing the 2 nd cycle the reduction was up to 6.10% with the colour value of 770 PCU. Finally the sample from third cycle was analyzed and it was observed that the reduction was up to 6.71% with the reduced colour valued of 765 PCU (Table 5.6). 149

21 Table 5.6: Reduction of colour after different cycles of 90% replacement of effluent sample by using bacterial consortia Colour of the effluent without treatment 820 PCU (Control) Consortia No. 1 Cycles 2 3 Mean Percentage reduction w.r.t control / cycle Mean Blank The samples collected from three cycles were subjected to colour analysis using standard method. The figure 5.6 showed the mean value of three cycles for each consortium. The results were compared with control i.e., untreated wastewater. Final reduction achieved by using consortium 1 was 31.3% with the mean colour value of three cycles of PCU. Consortium 2 gave the final reduction of 30.9% with the 150

22 reduced colour value of PCU. While comparing the results of other isolates consortium 3, 4, 5 and 6 it was observed that the achieved percentage reduction was 33.46%, 34.07%, 25.00% and 32.24% respectively with the colour value of PCU, PCU, PCU and PCU respectively. The achieved reduction after three cycles in case of consortia 7, 8, 9, and 10 was PCU, PCU, PCU and PCU respectively. The achieved percentage reduction for theses consortia were 31%, 30.2%, 38.01% and 32.85% respectively. The consortium 11 showed the final percentage reduction up to 22.97% with the achieved reduction in colour value was PCU. The results of consortium 12 showed that the reduced colour value after three cycles was PCU with the percentage reduction of 30.6%. Note: The bars were plotted on the basis of mean value of the three cycles mentioned in the table above. Figure 5.6: Reduction in colour of effluent by using bacterial consortia The final reduction achieved by using consortium 13 was 31.0% with the mean colour value of three cycles of PCU. The achieved reduction by using consortium 14 was 37.28% with the reduced colour value of PCU. While comparing the results of other isolates consortium 15, 16, 17 and 18 it was observed that the achieved percentage reduction was 31.2%, 31.10%, 14.43% and 13.01% respectively with the colour value of PCU, mg/l, PCU and PCU respectively. The consortium 19 showed the percentage reduction up to 14.43% with the reduced 151

23 colour value of PCU. The estimated value of last consortium 20 showed that the achieved value for colour was 709 with the percentage reduction of 13.54%. Blank was placed in order to check the activity of single isolate because it is the fact, that wastewater itself contains some microflora, sometimes it is possible that by getting favorable conditions they get activated and degrade the sample. The achieved colour value for blank was PCU. When the percentage reduction was calculated it was about 5.89% Statistical Analysis for the Selection of Consortia Analysis of COD Reduction A one-way analysis of variance (ANOVA) was used to tests the hypothesis whether the means of COD reduction by using 20 bacterial consortia equal or not. The null hypothesis for the test is that all means are same. The alternative hypothesis is that one or more population means differ from the others. It was found that p-value for the COD reduction ANOVA was α level for the test is 0.05, it was concluded that there are significant differences in mean COD reduction of effluent by different bacterial consortia. S, R and adjusted R are measures of how well the model fits the data. These values can help to select the model with the best fit. S is measured in the units of the response variable and represents the standard distance data values fall from the fitted values. For a given study, the better the model predicts the response, the lower S is R (R-Sq) describes the amount of variation in the observed response values. Adjusted R is a modified R that has been adjusted for the number of terms in the model. For the percent COD reduction data, S is 2.092, R is 83.84%, and adjusted R equals 76.17% (Table 5.7). Table 5.7: One-way ANOVA: Percentage COD reduction versus bacterial consortia 1 to Consortia. 20 Source DF SS MS F P Factor Error Total S = R-Sq = 83.84% R-Sq(adj) =

24 Table 5.8: Individual 95% CIs for mean based on pooled standard deviation N. The number of observations included for each level of the factor. Mean. The mean of the observations for each level. StDev. The sample standard deviations for each level. Pooled StDev. The pooled standard deviation is an estimate of the common standard deviation for all levels. The results of COD reduction the pulp and paper industry effluent by bacterial consortia analysis indicate that consortium 9 has the highest mean COD reduction (50.2 mg/l) and consortium 16 has the lowest (36.7). The standard deviations for the different consortia are given in the table; the pooled standard deviation is (Table 5.8). Confidence intervals 95% was used for each level of the factor. When the p-value in the analysis of variance table indicates there is a difference among the factor level means, table of individual confidence intervals was used to explore the differences. Each asterisk represents a sample mean. Each set of parentheses encloses a 95% confidence interval for 153

25 the mean of a population. If the intervals for two means do not overlap, it suggests that the population means are different. Tukey s method was further used to compares the means for each pair of factor levels. Results are presented as a grouping table and a set of simultaneous confidence intervals for the difference between pairs of means. The grouping information table was to display, in a summarized format, groups of factor level means that are not significantly different. If a level mean is not in a group, then its mean is significantly different from that group. Table 5.9: Grouping information using Tukey s method N Mean Grouping Cons A Cons A B Cons A B C Cons A B C D Cons A B C D E Cons A B C D E Cons A B C D E F Cons Cons Cons Cons Cons Cons Cons Cons Cons Cons Cons Cons Cons B C D E F G C D E F G H D E F G H D E F G H D E F G H D E F G H E F G H E F G H E F G H F G H F G H G H N The sample size for each factor level. Mean least squares means sorted in descending order. H 154

26 Grouping contains columns of letters that group the factor levels. Levels that share a letter are not significantly different. Conversely, if they do not share a letter, the level means are significantly different. The grouping information for the COD reduction data shows that group A contains consortia 9, 14, 8, 13, 15, 12 and 7. While group B contains consortia 14, 8, 13, 15, 12, 7 and 2. Group C contains consortia 8, 13, 15, 12, 7, 2 and 1. Group D contains consortia 13, 15, 12, 7, 2, 1, 6, 11, 18 and 3. Group E contains consortia 15, 12, 7, 2, 1, 6, 11, 18, 3, 19, 10 and 5. Group F contains consortia 7, 2, 1, 6, 11, 18, 3, 19, 10, 5, 17, and 4. Group G contains consortia 2, 1, 6, 11, 18, 3, 19, 10, 5, 14, 4 and 20. Finally group H contains consortia 1, 6, 11, 18, 3, 19, 10, 5, 17, 4, 20 and 16 (Table 5.9) Analysis of Colour Reduction A one-way analysis of variance (ANOVA) was used to tests the hypothesis whether the means of colour reduction by using 20 bacterial consortia equal or not. The null hypothesis for the test is that all means are same. The alternative hypothesis is that one or more population means differ from the others. It was found that p-value for the colour reduction ANOVA was α level for the test is 0.05, it was concluded that there are significant differences in mean colour reduction of effluent by different bacterial consortia. For the percent colour reduction data, S is 2.021, R is 95.60%, and adjusted R equals 93.51% (Table 5.10). Table 5.10: One-way ANOVA: Percentage colour reduction versus bacterial consortia 1 to Consortia. 20 Source DF SS MS F P Factor Error Total S = R-Sq = 95.60% R-Sq(adj) = 93.51% 155

27 Table 5.11: Individual 95% CIs for mean based on pooled standard deviation The results of colour reduction the pulp and paper industry effluent by bacterial consortia analysis indicate that Consortium 9 has the highest mean colour reduction (38.0 PCU) and consortium 18 has the lowest (13 PCU). The standard deviations for the different consortia are given in the table; the pooled standard deviation is Confidence intervals 95% was used for each level of the factor. Tukey s method again was used to compares the means for each pair of factor levels (Table 5.11). 156

28 Table 5.12: Grouping information using Tukey s method N Mean Grouping Cons A Cons A B Cons A B C Cons A B C Cons A B C Cons A B C Cons Cons Cons Cons Cons Cons Cons Cons Cons Cons B C B C D B C D C D C D C D C D C D D E E Cons Cons Cons Cons F F F F N The sample size for each factor level. Mean least squares means sorted in descending order. The grouping information for the colour reduction data shows that group A contains consortia 9, 14, 4, 3, 10 and 6. While group B contains consortia 14, 4, 3, 10, 6, 1, 15 and 16. Group C contains consortia 4, 3, 10, 6, 1, 15, 16, 13, 7, 2, 12, and 8. Group D contains consortia 15, 16, 13, 7, 2, 12, 8, and 5. Group E contains consortia 5 and 11. Finally group F contains consortia 17, and 8 (Table 5.12). It was observed that in both of the analysis COD and color reduction, group A have the highest mean of percentage reduction (table 5.9 and 5.12). In table 5.9 group A contains 157

29 consortia 9, 14, 8, 13, 15, 12 and 7, while in the case of table 5.12 Group A contains consortia 9, 14, 4, 3, 10 and 6. Consortia 9 and 14 are common and showed maximum COD and colour reduction of pulp and paper industry effluent. Screening of bacterial consortia was done on the basis of COD and colour reduction of effluent. On the basis of these results consortia 9 and 14 were selected for further study Screening of Selected Consortia Estimation of Chemical Oxygen Demand The experiment was repeated thrice in order to screen the effective consortia. As depicted in figure 5.7 consortium 9 was able to reduce the COD up to 52.9% and in case of consortium 14 it was up to 49.0%. Whereas the in the blank, COD reduction was up to 18.8%. Figure 5.7: Reduction in COD of effluent by using screened bacterial consortia (n=3) Estimation of Colour by using Selected Consortia The samples collected after the experiments, were analyzed for colour reduction. It was observed from that consortium 9 was able to reduce colour up to 41.0%, which is more than the reduction value i.e. 38.0% achieved by using consortium 14 In the blank the observed reduction was up to 6.1% only (figure 5.8). 158

30 Figure 5.8: Reduction in colour of effluent by using screened bacterial consortia (n=3) After the comparative analysis between the two consortia (9 and 14), consortium 9 was selected for further studies based on the above mentioned results To Study the Growth Pattern of Bacterial Consortium The consortium consisting of three bacteria, grown collectively in the sample. Question arises that whether the growth of on bacteria will suppress the other or vise a versa. To show the growth pattern of the bacteria collectively in a flask and experiment was designed. After every 4 hours sample was collected and CFU/ml was estimated and it was clear from the calculated values that none of the bacteria suppressed the growth of the other. Results revealed that the total CFU/ml at 0 hr was 8.1 x 10 3 while calculating the CFU/ml for each individual bacterium it was around 2.8 x 10 3 for PNP 3, 2.7 x 10 3 for PNP 6 and 2.6 x 10 3 for PNP 8. After 4 hrs the sample was collected the total CFU/ml was 5.7 x 104 and the calculated value for the individual isolate was 2.1 x 10 4 for PNP 3, 1.9 x 10 4 for PNP 6 and 1.7 x 10 4 for PNP 8. After 8 hrs of incubation the sample was again withdrawn and CFU/ml was calculated for individual isolate. It was observed that for PNP 3 the CFU/ml was 1.4 x 10 5, for PNP 6 it was 1.6 x 10 5 and for PNP 8 it was 1.3 x In the similar fashion the sample was collected and analyzed after 12 hrs of incubation the total CFU/ml was 7.5 x The CFU/ml calculated for individual isolate i.e., PNP 3, PNP 6 and PNP 8 was 2.7 x 10 5, 2.5 x 10 5 and 2.3 x