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Treatment of paper factory effluent using a phenol degrading Alcaligenes sp. under free and immobilized conditions
Bioresource Technology 98 (2007) 714–716
Short Communication
Treatment of paper factory effluent using a phenol degrading Alcaligenes sp. under free and immobilized conditions Indu C. Nair, K. Jayachandran *, Shankar Shashidhar School of Biosciences, M.G. University, Kottayam 686 560, India Received 8 October 2005; received in revised form 13 February 2006; accepted 26 February 2006 Available online 8 June 2006
Abstract Treatment of the paper factory effluent was done with free and immobilized cells of a phenol degrading Alcaligenes sp. d2. The free cells could bring a maximum of 99% reduction in phenol and 40% reduction in chemical oxygen demand (COD) after 32 and 20 h of treatment, respectively. In the case of immobilized cells, a maximum of 99% phenol reduction and 70% COD reduction was attained after 20 h of treatment under batch process. In the continuous mode of operation using packed bed reactor, the strain was able to give 99% phenol removal and 92% COD reduction in 8 h of residence time The optimum flow rate was 2.5 ml/h and the half life period was 76 h. Even after the complete removal of phenol, the strain could further enhance reduction in chemical oxygen demand, which clearly indicated that in the paper factory effluent, this strain could also oxidize organic matter other than phenol. 2006 Elsevier Ltd. All rights reserved. Keywords: Effluent treatment; Phenol degradation; Immobilized Alcaligenes sp.; Packed bed reactor
1. Introduction Phenol and chlorophenols are toxic compounds, often found in industrial effluents such as from pulp and paper, timber, plastics and synthetic polymer, pharmaceutical, pesticide, oil and petrochemical industries (Torres et al., 1998). These phenolic compounds have various degrees of toxicity. Many mesophilic microorganisms including Pseudomonas sp. Streptomyces sp. Acinetobacter sp. and Alcaligenes sp. have been reported to degrade phenol (Indu and Shashidhar, 2004). These microbial cells can be potential tools in the biological treatment of phenolic effluents. There are different kinds of reactor system, which could be used for the treatment of phenolic wastewater using microorganisms, e.g., fluidized bed reactor (Gonzalez et al., 2001), moving bed biofilm reactor (Hosseini and Borghei, 2005) and rotating perforated tube biofilm reactor (Kargi and Eker, 2005). In fluidized bed reactor, Pseudo*
Corresponding author. E-mail address: jayan_chk@rediffmail.com (K. Jayachandran).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.02.034
monas putida immobilized in alginate beads were used. Hosseini and Borghei (2005) used a ratio of phenolic COD to total COD in the range from 0.2 to 1.0. In the case of rotating perforated tube biofilm reactor, an activated sludge supplemented with P. putida was used for the treatment of dichlorophenol containing wastewater. The aim of this work was to use free and immobilized cells of phenol degrading Alcaligenes sp. d2 for the treatment of phenolic paper factory effluent. The liquid waste from the paper industry is generated from the two different sections, one from pulp making process and other from paper making process. The resultant wastewater contains many organic components like lignin, cellulosic materials, sugars and phenols as well as certain inorganic salts (Naik, 1989). 2. Methods 2.1. Sample Raw effluent from Hindustan Newsprint Factory, Kottayam was used for the present study. The effluent
I.C. Nair et al. / Bioresource Technology 98 (2007) 714–716
was collected from the final settling tank before subjecting it to any type of treatment. It was analyzed for COD, biological oxygen demand (BOD) and total suspended solids as per the standard methods (APHA, 1995). Phenol concentrations were determined by a modified spectrophotometric technique (Mordoco et al., 1999). 2.2. Microorganism The microorganism used in the present study was Alcaligenes sp. d2 (Indu and Shashidhar, 2004; Jayachandran et al., 1994). The cells were grown in mineral salt phenol medium (MSPM) with following composition (g/l): (NH4)2SO4, 1; KH2PO4, 1; MgSO4 Æ 7H2O, 0.5; CaCl2, 1 mg/l and phenol 60 mM. 2.3. Preparation of immobilized cells Alcaligenes sp. d2, grown in mineral salt phenol medium (MSPM) for 24 h were harvested by centrifugation at 10,000 rpm for 30 min. These cells were suspended in physiological saline (0.85% NaCl) at a cell concentration of 1 OD and were mixed with 4% sodium alginate in the ratio 1:2. The mixture was added drop-wise to a 0.2 M CaCl2 solution to get alginate entrapped cells (Jayachandran et al., 1994). 2.4. Growth curve of free and immobilized cells MSPM was inoculated with 1% inoculum of 1 OD concentration in the case of free cells and 100 beads/100 ml in the case of immobilized cells. The quantification of the growth was done by serial dilution of the sample followed by viable counts at regular intervals of 4 h. In the case of alginate-immobilized cells, to recover the bacterium for viable counts, known amount of the beads were immersed in phosphate buffer (1 M, pH 7), and dissolved by vigorous shaking (Muyima and Cloete, 1995).
715
3. Results and discussion The biodegradation of industrial wastewaters can be improved if the microorganism is previously adapted to the respective toxic chemical in the effluent (Zilli et al., 1993). The strain Alcaligenes sp. used in the present study was reported earlier as a potent phenol removing bacteria in mineral salt medium (Indu and Shashidhar, 2004) and could show better result in the treatment of phenolic effluent. The phenolic paper factory effluent used in the present study contained (mg/l) COD 1100, BOD 425, total suspended solids 1620, total heterotrophic bacterial population of 48 · 102, pH 9 and a phenol concentration of 100 mM. A comparison of the growth curve of the Alcaligenes sp. in MSPM and phenolic effluent revealed that the logarithmic phase of the strain in MSPM was from 24 to 32 h (maximum cell yield as 107 in 32 h) and in effluent it was from 8 to 36 h (maximum cell yield as 109 in 36 h) (results not shown). Enhanced growth was a possible indication of better activity of the strain in the effluent. The high growth rate of the strain and the diauxic nature of growth curve in the phenolic effluent showed that (2003). Growth curve of the immobilized cells also revealed the same fact that effluent offered a better growth condition with an earlier logarithmic phase and enhanced growth yield (maximum cell yield of 1010 cells/12 h in MSPM and 1011 cells/22 h in effluent). In the mineral salt phenol medium, 99% phenol reduction was resulted in 32 h with free cells while the same percentage of phenol reduction was achieved in 20 h with immobilized cells (Fig. 1). Immobilized cells were reported to be more stable for the treatment of wastewater (Godjevargava et al., 2003). Immobilized cells of Alcaligenes sp. resulted in a better performance than the free cells in batch process in the removal of phenol COD (Fig. 2) (99% phenol removal in 20 and 32 h with immobilized cells and free cells, respectively). COD reduction was 70% and 85% in 20 and 38 h and 40% and 65% in 20 and 40 h, respectively. However, in both the cases, further increase in COD reduction was
2.5. Batch and continuous treatments 120 Percentage of phenol degradation
Both MSPM and phenolic effluent were taken as 300 ml aliquots in 1-litre flasks and were inoculated with the 18 h old culture at 5% inoculum level in free cell treatment and 300 immobilized beads in immobilized cell treatment (Jayachandran et al., 1994). These flasks were incubated at 28 ± 2 C on a shaker at 150 rpm. Samples were withdrawn at regular intervals of 4 h for phenol and COD estimations. Continuous treatment of the phenolic effluent was performed with a packed bed reactor in a glass column (30 cm length and 6 cm diameter). Immobilized cells in beads (1050) were packed to a height of 25 cm in the glass column carefully without trapping any air bubble and then the reactor was fed with effluent at varying flow rates of 2.5, 5.0, 7.5 and 10 ml/h. Samples were withdrawn and analysed as above.
100 80 free cells
60
immobilized cells
40 20 0 0
20
40
60
Incubation period (hours)
Fig. 1. Phenol degradation in mineral salt phenol medium by free and immobilized cells of Alcaligenes sp. in batch process.
I.C. Nair et al. / Bioresource Technology 98 (2007) 714–716
Percentage reduction in phenol / percentage reduction in COD
716
120 phenol degradation by free cells phenol degradation by immobilized cells cod reduction by free cells
100 80 60 40
cod reduction by immobilized cells
20 0 0
20
40
60
Acknowledgements
Incubation period (hours)
percentage of COD reduction
Fig. 2. Treatment of phenolic effluent with free and immobilized cells of Alcaligenes sp. in batch process.
Percentage of phenol reduction /
remaining ones. Their resistance can be ensured by the degradation activity of the strain used towards most of the waste products present in the wastewater (Godjevargava et al., 2003). The results of the preset study strongly indicated that immobilized Alcaligenes sp. could be effectively used for the safe disposal of the phenolic paper factory effluent, which, however, would need developing system for large-scale treatment.
The authors are thankful to School of Biosciences, M.G. University, Kottayam for providing the facilities for carrying out the above research work.
120
References
100 80 phenol reduction in
60
effluent
40
COD reduction in effluent
20 0 0
5 10 Flow rate (ml/h)
15
Fig. 3. Phenol degradation and COD reduction in effluent by immobilized cells of Alcaligenes sp. by continuous treatment in a packed bed reactor.
obtained even after 99% phenol removal. This showed that the organism was equally capable of utilizing other carbon sources after the complete utilization of phenol. In the continuous treatment of the effluent at different flow rates, 99% removal of phenol and 92% reduction in COD were attained at 2.5 ml/h flow rate (residence time 8 h; Fig. 3). However, on increasing the flow rate from 2.5 to 10 ml/h, both the phenol and COD reduction rate progressively got reduced to 6% and 28%, respectively. The increased flow rate resulted in reduced residence time, hence, showed poor performance On evaluating the performance of the packed bed reactor continuously for 10 days, 76 h was observed as the half-life period of the reactor, which was same for COD reduction and phenol removal. Continuous treatment of the effluent in packed bed reactor gave maximum reduction in phenol content (99%) and COD (92%) in 8 h. The strains used for decontamination of phenolic wastewater should not only be highly active to one of the contaminants but they should also be resistant enough to the
APHA, 1995. Standard methods for the examination of water and wastewater, 19th Ed. APHA, Washington, DC, pp. 5–16. Godjevargava, T., Ivanova, D., Alexieva, Z., Dimova, N., 2003. Biodegradation of toxic organic components from industrial phenol producing wastewater by free and immobilized Trichospora cutaneum R 57. Process Biochemistry 38, 915–920. Gonzalez, G., Herrera, G., Garcia, M.T., Pena, M., 2001. Biodegradation of phenolic industrial wastewater in a fluidized bed reactor with immobilized cells of Pseudomonas putida. Bioresource Technology 80, 137–142. Hosseini, S.H., Borghei, S.M., 2005. The treatment of phenolic wastewater using a moving bed biofilm reactor. Process Biochemistry 39, 1177– 1181. Indu, C. Nair, Shashidhar, S., 2004. Microbial degradation of phenol by a species of Alcaligenes isolated from a tropical soil. Soil Science 3 (4), 47–51. Jayachandran, K., Suresh, P.V., Chandrasekaran, M., 1994. A novel Acinetobacter sp. for treating highly acidic rubber latex centrifugation effluent. Biotechnology Letters 16 (6), 649–654. Kargi, F., Eker, S., 2005. Removal of 2,4,-dichlorophenol and toxicity from synthetic wastewater in a rotating perforated tube biofilm reactor. Process Biochemistry 40, 2105–2111. Mordoco, Angela, Kuek, Clem, Jenkim, Roger, 1999. Continuous degradation of phenol at low concentration using immobilized Pseudomonas putida. Enzyme and Microbial Technology 25, 530–536. Muyima, Y.O., Cloete, T.E., 1995. Growth and uptake of immobilized Acinetobacter cells suspended in activated sludge mixed liquor. Water Research 29, 2461–2466. Naik, B.N., 1989. In: Trivedy, R.K. (Ed.), Pollution Management in Industries. Environmental Publications, Karad, India, pp. 14–20. Torres, L.G., Sanchez-de-la-Vega, A., Beltran, Norma A., Jimenez, B.E., 1998. Production and characterization of a calcium alginate biocatalyst for removal of phenol and chloramphenicol from wastewater. Process Biochemistry 33, 625–634. Zilli, M., Converti, A., delBorghi, M., Ferriolo, G., 1993. Phenol removal from waste gases with a biological filter by Pseudomonas putida. Biotechnology and Bioengineering 41, 693–699.
Short Communication
Treatment of paper factory effluent using a phenol degrading Alcaligenes sp. under free and immobilized conditions Indu C. Nair, K. Jayachandran *, Shankar Shashidhar School of Biosciences, M.G. University, Kottayam 686 560, India Received 8 October 2005; received in revised form 13 February 2006; accepted 26 February 2006 Available online 8 June 2006
Abstract Treatment of the paper factory effluent was done with free and immobilized cells of a phenol degrading Alcaligenes sp. d2. The free cells could bring a maximum of 99% reduction in phenol and 40% reduction in chemical oxygen demand (COD) after 32 and 20 h of treatment, respectively. In the case of immobilized cells, a maximum of 99% phenol reduction and 70% COD reduction was attained after 20 h of treatment under batch process. In the continuous mode of operation using packed bed reactor, the strain was able to give 99% phenol removal and 92% COD reduction in 8 h of residence time The optimum flow rate was 2.5 ml/h and the half life period was 76 h. Even after the complete removal of phenol, the strain could further enhance reduction in chemical oxygen demand, which clearly indicated that in the paper factory effluent, this strain could also oxidize organic matter other than phenol. 2006 Elsevier Ltd. All rights reserved. Keywords: Effluent treatment; Phenol degradation; Immobilized Alcaligenes sp.; Packed bed reactor
1. Introduction Phenol and chlorophenols are toxic compounds, often found in industrial effluents such as from pulp and paper, timber, plastics and synthetic polymer, pharmaceutical, pesticide, oil and petrochemical industries (Torres et al., 1998). These phenolic compounds have various degrees of toxicity. Many mesophilic microorganisms including Pseudomonas sp. Streptomyces sp. Acinetobacter sp. and Alcaligenes sp. have been reported to degrade phenol (Indu and Shashidhar, 2004). These microbial cells can be potential tools in the biological treatment of phenolic effluents. There are different kinds of reactor system, which could be used for the treatment of phenolic wastewater using microorganisms, e.g., fluidized bed reactor (Gonzalez et al., 2001), moving bed biofilm reactor (Hosseini and Borghei, 2005) and rotating perforated tube biofilm reactor (Kargi and Eker, 2005). In fluidized bed reactor, Pseudo*
Corresponding author. E-mail address: jayan_chk@rediffmail.com (K. Jayachandran).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.02.034
monas putida immobilized in alginate beads were used. Hosseini and Borghei (2005) used a ratio of phenolic COD to total COD in the range from 0.2 to 1.0. In the case of rotating perforated tube biofilm reactor, an activated sludge supplemented with P. putida was used for the treatment of dichlorophenol containing wastewater. The aim of this work was to use free and immobilized cells of phenol degrading Alcaligenes sp. d2 for the treatment of phenolic paper factory effluent. The liquid waste from the paper industry is generated from the two different sections, one from pulp making process and other from paper making process. The resultant wastewater contains many organic components like lignin, cellulosic materials, sugars and phenols as well as certain inorganic salts (Naik, 1989). 2. Methods 2.1. Sample Raw effluent from Hindustan Newsprint Factory, Kottayam was used for the present study. The effluent
I.C. Nair et al. / Bioresource Technology 98 (2007) 714–716
was collected from the final settling tank before subjecting it to any type of treatment. It was analyzed for COD, biological oxygen demand (BOD) and total suspended solids as per the standard methods (APHA, 1995). Phenol concentrations were determined by a modified spectrophotometric technique (Mordoco et al., 1999). 2.2. Microorganism The microorganism used in the present study was Alcaligenes sp. d2 (Indu and Shashidhar, 2004; Jayachandran et al., 1994). The cells were grown in mineral salt phenol medium (MSPM) with following composition (g/l): (NH4)2SO4, 1; KH2PO4, 1; MgSO4 Æ 7H2O, 0.5; CaCl2, 1 mg/l and phenol 60 mM. 2.3. Preparation of immobilized cells Alcaligenes sp. d2, grown in mineral salt phenol medium (MSPM) for 24 h were harvested by centrifugation at 10,000 rpm for 30 min. These cells were suspended in physiological saline (0.85% NaCl) at a cell concentration of 1 OD and were mixed with 4% sodium alginate in the ratio 1:2. The mixture was added drop-wise to a 0.2 M CaCl2 solution to get alginate entrapped cells (Jayachandran et al., 1994). 2.4. Growth curve of free and immobilized cells MSPM was inoculated with 1% inoculum of 1 OD concentration in the case of free cells and 100 beads/100 ml in the case of immobilized cells. The quantification of the growth was done by serial dilution of the sample followed by viable counts at regular intervals of 4 h. In the case of alginate-immobilized cells, to recover the bacterium for viable counts, known amount of the beads were immersed in phosphate buffer (1 M, pH 7), and dissolved by vigorous shaking (Muyima and Cloete, 1995).
715
3. Results and discussion The biodegradation of industrial wastewaters can be improved if the microorganism is previously adapted to the respective toxic chemical in the effluent (Zilli et al., 1993). The strain Alcaligenes sp. used in the present study was reported earlier as a potent phenol removing bacteria in mineral salt medium (Indu and Shashidhar, 2004) and could show better result in the treatment of phenolic effluent. The phenolic paper factory effluent used in the present study contained (mg/l) COD 1100, BOD 425, total suspended solids 1620, total heterotrophic bacterial population of 48 · 102, pH 9 and a phenol concentration of 100 mM. A comparison of the growth curve of the Alcaligenes sp. in MSPM and phenolic effluent revealed that the logarithmic phase of the strain in MSPM was from 24 to 32 h (maximum cell yield as 107 in 32 h) and in effluent it was from 8 to 36 h (maximum cell yield as 109 in 36 h) (results not shown). Enhanced growth was a possible indication of better activity of the strain in the effluent. The high growth rate of the strain and the diauxic nature of growth curve in the phenolic effluent showed that (2003). Growth curve of the immobilized cells also revealed the same fact that effluent offered a better growth condition with an earlier logarithmic phase and enhanced growth yield (maximum cell yield of 1010 cells/12 h in MSPM and 1011 cells/22 h in effluent). In the mineral salt phenol medium, 99% phenol reduction was resulted in 32 h with free cells while the same percentage of phenol reduction was achieved in 20 h with immobilized cells (Fig. 1). Immobilized cells were reported to be more stable for the treatment of wastewater (Godjevargava et al., 2003). Immobilized cells of Alcaligenes sp. resulted in a better performance than the free cells in batch process in the removal of phenol COD (Fig. 2) (99% phenol removal in 20 and 32 h with immobilized cells and free cells, respectively). COD reduction was 70% and 85% in 20 and 38 h and 40% and 65% in 20 and 40 h, respectively. However, in both the cases, further increase in COD reduction was
2.5. Batch and continuous treatments 120 Percentage of phenol degradation
Both MSPM and phenolic effluent were taken as 300 ml aliquots in 1-litre flasks and were inoculated with the 18 h old culture at 5% inoculum level in free cell treatment and 300 immobilized beads in immobilized cell treatment (Jayachandran et al., 1994). These flasks were incubated at 28 ± 2 C on a shaker at 150 rpm. Samples were withdrawn at regular intervals of 4 h for phenol and COD estimations. Continuous treatment of the phenolic effluent was performed with a packed bed reactor in a glass column (30 cm length and 6 cm diameter). Immobilized cells in beads (1050) were packed to a height of 25 cm in the glass column carefully without trapping any air bubble and then the reactor was fed with effluent at varying flow rates of 2.5, 5.0, 7.5 and 10 ml/h. Samples were withdrawn and analysed as above.
100 80 free cells
60
immobilized cells
40 20 0 0
20
40
60
Incubation period (hours)
Fig. 1. Phenol degradation in mineral salt phenol medium by free and immobilized cells of Alcaligenes sp. in batch process.
I.C. Nair et al. / Bioresource Technology 98 (2007) 714–716
Percentage reduction in phenol / percentage reduction in COD
716
120 phenol degradation by free cells phenol degradation by immobilized cells cod reduction by free cells
100 80 60 40
cod reduction by immobilized cells
20 0 0
20
40
60
Acknowledgements
Incubation period (hours)
percentage of COD reduction
Fig. 2. Treatment of phenolic effluent with free and immobilized cells of Alcaligenes sp. in batch process.
Percentage of phenol reduction /
remaining ones. Their resistance can be ensured by the degradation activity of the strain used towards most of the waste products present in the wastewater (Godjevargava et al., 2003). The results of the preset study strongly indicated that immobilized Alcaligenes sp. could be effectively used for the safe disposal of the phenolic paper factory effluent, which, however, would need developing system for large-scale treatment.
The authors are thankful to School of Biosciences, M.G. University, Kottayam for providing the facilities for carrying out the above research work.
120
References
100 80 phenol reduction in
60
effluent
40
COD reduction in effluent
20 0 0
5 10 Flow rate (ml/h)
15
Fig. 3. Phenol degradation and COD reduction in effluent by immobilized cells of Alcaligenes sp. by continuous treatment in a packed bed reactor.
obtained even after 99% phenol removal. This showed that the organism was equally capable of utilizing other carbon sources after the complete utilization of phenol. In the continuous treatment of the effluent at different flow rates, 99% removal of phenol and 92% reduction in COD were attained at 2.5 ml/h flow rate (residence time 8 h; Fig. 3). However, on increasing the flow rate from 2.5 to 10 ml/h, both the phenol and COD reduction rate progressively got reduced to 6% and 28%, respectively. The increased flow rate resulted in reduced residence time, hence, showed poor performance On evaluating the performance of the packed bed reactor continuously for 10 days, 76 h was observed as the half-life period of the reactor, which was same for COD reduction and phenol removal. Continuous treatment of the effluent in packed bed reactor gave maximum reduction in phenol content (99%) and COD (92%) in 8 h. The strains used for decontamination of phenolic wastewater should not only be highly active to one of the contaminants but they should also be resistant enough to the
APHA, 1995. Standard methods for the examination of water and wastewater, 19th Ed. APHA, Washington, DC, pp. 5–16. Godjevargava, T., Ivanova, D., Alexieva, Z., Dimova, N., 2003. Biodegradation of toxic organic components from industrial phenol producing wastewater by free and immobilized Trichospora cutaneum R 57. Process Biochemistry 38, 915–920. Gonzalez, G., Herrera, G., Garcia, M.T., Pena, M., 2001. Biodegradation of phenolic industrial wastewater in a fluidized bed reactor with immobilized cells of Pseudomonas putida. Bioresource Technology 80, 137–142. Hosseini, S.H., Borghei, S.M., 2005. The treatment of phenolic wastewater using a moving bed biofilm reactor. Process Biochemistry 39, 1177– 1181. Indu, C. Nair, Shashidhar, S., 2004. Microbial degradation of phenol by a species of Alcaligenes isolated from a tropical soil. Soil Science 3 (4), 47–51. Jayachandran, K., Suresh, P.V., Chandrasekaran, M., 1994. A novel Acinetobacter sp. for treating highly acidic rubber latex centrifugation effluent. Biotechnology Letters 16 (6), 649–654. Kargi, F., Eker, S., 2005. Removal of 2,4,-dichlorophenol and toxicity from synthetic wastewater in a rotating perforated tube biofilm reactor. Process Biochemistry 40, 2105–2111. Mordoco, Angela, Kuek, Clem, Jenkim, Roger, 1999. Continuous degradation of phenol at low concentration using immobilized Pseudomonas putida. Enzyme and Microbial Technology 25, 530–536. Muyima, Y.O., Cloete, T.E., 1995. Growth and uptake of immobilized Acinetobacter cells suspended in activated sludge mixed liquor. Water Research 29, 2461–2466. Naik, B.N., 1989. In: Trivedy, R.K. (Ed.), Pollution Management in Industries. Environmental Publications, Karad, India, pp. 14–20. Torres, L.G., Sanchez-de-la-Vega, A., Beltran, Norma A., Jimenez, B.E., 1998. Production and characterization of a calcium alginate biocatalyst for removal of phenol and chloramphenicol from wastewater. Process Biochemistry 33, 625–634. Zilli, M., Converti, A., delBorghi, M., Ferriolo, G., 1993. Phenol removal from waste gases with a biological filter by Pseudomonas putida. Biotechnology and Bioengineering 41, 693–699.