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Aerobic biological treatment of olive mill wastewater by olive pulp bacteria
ARTICLE IN PRESS
International Biodeterioration & Biodegradation 60 (2007) 209–214 www.elsevier.com/locate/ibiod
Aerobic biological treatment of olive mill wastewater by olive pulp bacteria G. Tziotzios, S. Michailakis, D.V. Vayenas Department of Environmental and Natural Resources Management, University of Ioannina, Seferi 2, 30100 Agrinio, Greece Received 7 February 2007; received in revised form 28 February 2007; accepted 2 March 2007 Available online 16 April 2007
Abstract The capability of olive fruit bacteria to remove chemical oxygen demand (COD) and phenolic compounds from olive mill wastewater (OMW) using flasks and packed bed reactors was tested. Batch aerobic experiments were performed in flask reactors, with OMW at different dilutions (20%, 50%, and 100%). The maximum phenolic and dissolved COD removal reached up to 82–90% for the dilutions of 20%, 50%, and 100%, in 11, 23, and 30 days, respectively. Experiments were also conducted in a bench and a pilot-scale packed bed reactor, operated in the laboratory and at an olive mill, respectively. At 28 1C the maximum phenolic and dissolved COD removal, recorded at the bench-scale reactor, reached about 60% and 70%, respectively, for a time period of only 27 h. At the pilot-scale reactor, where the maximum daily temperature varied between 6 and 20 1C, the maximum phenolic and d-COD removal reached about 55% and 70% in 5 and 3 days, respectively. These results indicate an economically feasible method for OMW biodegradation. r 2007 Elsevier Ltd. All rights reserved. Keywords: Olive mill wastewaters; Biodegradation; Phenolic compounds; Packed bed reactors
1. Introduction The olive oil industry is very important in the Mediterranean countries, both in terms of wealth and tradition (Roig et al., 2006). Spain is the main world producer followed by Italy, Greece, Turkey, Syria, and Tunisia. Hence, this area is especially affected by olive mill waste pollution. However, many other countries such as Argentina, Australia, and South Africa are becoming emergent producers since they are promoting intensive olive tree cultivation. The extraction and manufacture of olive oil are carried out in numerous agro-industrial units in the Mediterranean countries, which generate an aqueous phase formed by the water content of the fruit combined with what was used to wash and process the olives: the combination is the socalled olive mill wastewater (OMW) and the annual production is estimated to an amount over 107 m3 (Benitez et al., 1997). Typically, the weight composition of OMW is Corresponding author. Tel.: +30 2641074117; fax: +30 2641074172.
E-mail address: [email protected] (D.V. Vayenas). 0964-8305/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2007.03.003
83–96% water, 3.5–15% organics, and 0.5–2% mineral salts. The organic fraction is composed of sugars (1.0–8.0%), N-compounds (0.5–2.4%), organic acids (0.5–1.5%), fats (0.02–1%) as well as phenols and pectins (1.0–1.5%) (Greco et al., 1999). The maximum biological oxygen demand (BOD) and chemical oxygen demand (COD) reach concentrations of 100 and 220 kg/m3, respectively. As regards to phenols, low-molecular weight compounds (hydroxytyrosol, tyrosol, catechol, methylcatechol, caffeic acid) are usually present in OMW, along with catechol-melaninic polymers. Owing to the content of these effluents, the environmental problems and potential hazards caused by OMW has prompted many countries to limit its discharge and to develop new technologies for reducing the pollutant power, such as the different chemical and biological treatments that have been investigated lately. Moreover, some extensive and detailed reviews have been recently published (Roig et al., 2006; Niaounakis and Halvadakis, 2004; Azbar et al., 2004; Hamdi, 1996). OMW is currently concentrated by evaporation in open pools, but this method is not satisfactory as a black
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foul-smelling sludge, difficult to remove, is produced. Various physicochemical methods have been proposed and tested but the high cost of these methods deteriorated their wide application. Biological methods used for OMW treatment are aerobic-activated sludge and anaerobic digestion. Although aerobic biological treatments succeed higher efficiencies, these processes are limited by the unbearable cost of the continuously provided mechanical aeration. A major limitation of anaerobic digestion of OMWs is inhibition of methanogenic bacteria by simple phenolic compounds and certain organic acids (Hamdi, 1996). The use of filamentous fungi for OMW pretreatment has been shown to reduce OMW toxicity and improve the biodegradability in anaerobic digestion (Assas et al., 2002). Many microorganisms have been tested: the fungi Pleurotus ostreatus, Bacillus pumilus, Panus tigrinus, the yeast Yarrowia Lipolytica, etc. (Fadil et al., 2003; Fountoulakis et al., 2002; Tomati et al., 1991; Ramos-Cormenzana et al., 1996; D’Annibale et al., 2004; Garcı´ a Garcı´ a et al., 2000; Scioli and Vollaro, 1997). The application of fungi on a large scale is limited by the difficulty of achieving sterilised conditions in open-air reactors. The aim of the present work was the utilisation of olive fruit bacteria for the removal of COD and phenolic compounds from OMWs using flask, bench and pilotscale packed bed reactors, under non-sterilised aerobic conditions. 2. Materials and methods 2.1. OMW OMW were obtained from two olive oil factories (three-phase centrifugal) located at Agrinio (Western Greece). Immediately after sampling, the OMWs were maintained at 20 1C throughout the experimentation period. Their composition is shown in Table 1. OMWa refers to the wastewater used for the experimental series in the flask reactors, and OMWb refers to the wastewater used in the bench and pilotscale packed bed reactors. The difference in the composition of the two OMWs is mainly attributed to different water addition amounts during the extraction process.
2.2. Media Olive fruit bacteria were grown in a phenol-based mineral media previously used by Tziotzios et al. (2005), consisting of 1 g K2HPO4, 0.644 g Na2HPO4, 1.3375 g NH4Cl, 0.2 g MgSO4 7H2O, 0.2 g KCl, and Table 1 Composition of OMW Parameter
OMWa
OMWb
Total suspended solids, TSS (g/L) Volatile suspended solids, VSS (g/L) Dissolved phenols (g syringic acid/L) Dissolved chemical oxygen demand, d-COD (g/L) Biological oxygen demand, BOD (g O2/L) pH
59.69 55.65 6.7 50.72 35.79 5.18
41.01 34.03 2.9 15.05 10.64 5.5
0.02 g yeast extract per litre of tap water. Phenol was added as the sole carbon and energy source. The addition of readily grown bacteria into the flask and bench-scale packed bed reactor containing OMW was accompanied by the addition of NH4Cl, K2HPO4, and MgSO4 7H2O, keeping the proportion of dissolved-COD:N:P:Mg ¼ 100:5:1:1 steady. In the case of the pilot-scale packed bed reactor operated nearby an olive mill, the same proportion was kept steady using common agricultural fertilisers.
2.3. Analytical methods Various parameters were measured during the experiments: total suspended solids (TSS), volatile suspended solids (VSS), dissolved (after filtration of OMW) COD, BOD were determined as proposed by Standard Methods (APHA, 1995). Phenol and d-phenolic (with respect to syringic acid) concentrations were determined spectrophotometrically according to the Folin–Ciocalteu method (Waterman and Mole, 1994), using a JASCO V-530 UV/vis spectrophotometer. Dissolved oxygen (D.O.) and pH were measured with a HANNA HI-9143 Microprocessor D.O. meter and a HANNA pH-211 Microprocessor pH meter, respectively.
2.4. pH, temperature Throughout the experiments in the flask and bench-scale packed bed reactors, the temperature was fairly constant at about 28 1C and the pH ranged from 5.570.2 (initial pH) to 6.570.2 (end of the experiment). The maximum daily temperature in the pilot-scale packed bed reactor operated in an olive mill ranged between 6 and 20 1C, and the pH ranged from 5.570.3 to 7.470.3 at the end of the experiment.
2.5. Selection of the bacterial community The original inoculum of bacteria was taken from olive pulp. This source was selected, since olives and OMW contain a significant amount of phenolics (Fountoulakis et al., 2002; Borja et al., 1992). At first, sodium acetate was used as a carbon source. Enrichment and acclimation of phenol-degrading bacteria was achieved in a flask reactor by the gradual replacement of sodium acetate by phenol up to a concentration of 100 ppm. Bacterial samples taken at this point showed, using 16S rRNA sequencing, that the dominant bacterial gena of the mixed culture were Alcaligenes and Acinetobacter. When complete phenol (100 ppm) degradation was recorded, an inoculum was transferred to the reactors, containing OMW and the appropriate nutrient medium.
2.6. Flask, bench and pilot-scale packed bed reactors Erlenmeyer flasks of 1 L were used as flask reactors. Different dilutions of OMW (20%, 50%, and 100%) were tested. The inoculum used in the flask reactors was 50 mL. Aquarium pumps were used to provide aeration. The bench-scale packed bed reactor established in the laboratory consisted of a Plexiglas tube, 160 cm high and with a 9 cm internal diameter. This filter height is typical of a full-scale industrial filter. Since it is the loadings (hydraulic and phenolics) per unit cross-sectional area that matter, no scale-up was necessary. Two different sizes of rippled plastic hollow tubes were tested as the support material of this reactor. This particular support material was chosen, since it is a cheap electrological material and commercially easy to find. The small-sized plastic tubes were of 1.6 cm internal diameter and 3.02 cm in length, with a specific surface area of 500 m2/m3, and filter porosity 0.8. The large-sized plastic tubes were of 2.65 cm internal diameter and 3.95 cm in length, with a specific surface area of 301.9 m2/m3, and filter porosity 0.95. The depth of support media in the filter was 143 cm. Filter backwash due to pore clogging from biomass growth was necessary and was performed at the end of each batch experiment using high water and air upward velocities. The produced sludge was subsequently treated in a composting unit.
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The pilot-scale packed bed reactor operated in the olive mill consisted of a cylindrical polyethylene tank with a conical base, 1.5 m in height and 1.8 m of internal diameter. Along the lower parts of the tank there were 11 ventilation ports, through which air could circulate. Inside the tank, a basket of 1.2 m in height and 1.75 m in diameter was placed. The basket was filled with the support material up to 1 m. The support material of this reactor was rippled plastic hollow tubes (commercial electrical tube), 3.85 cm internal diameter and 4 cm in length, with a specific surface area of 207.8 m2/m3, and porosity 0.93. OMW (500 L) was kept in a nearby tank, and was continuously recirculated in the reactor, from the top, by a rotary distributor.
2.7. Packed bed reactor startup During bench-scale packed bed reactor startup, inoculum (50 mL/L of working volume) was taken from the flask reactor with acclimated phenol bacteria and added to the packed bed reactor with appropriate amounts of nutrient medium, i.e., a concentrated phenol solution mixed with the influent feed to ensure bacterial growth. The initial phenol concentration in the filter was 500 ppm. Sufficient aeration was provided in the base of the packed bed reactor through an air pump. When complete phenol degradation was recorded, the filter was recharged with 500 ppm phenol and nutrients. The inoculum (50 mL/L of working volume) used in the recharges came from the bulk liquid of the filter, just before unloading. The inoculum volume was gradually reduced as bacterial attachment on the support medium was observed. When no further inoculum addition was required, and the filter reached a steady cycle-to-cycle performance after approximately 30 days, the experimental series with OMW took place. At the startup period of the pilot-scale packed bed reactor, inoculum (10 L) was added to the reactor in the beginning of each operating cycle. Inoculum addition was continued until biofilm formation was observed attached to the support material. The reactor reached steady cycle-to-cycle performance at approximately 40 days.
Fig. 1. d-Phenols concentrations in flask reactors with different dilutions of OMWa (20%, 50%, and 100%).
3. Results and discussion 3.1. Flask reactors with OMWa Experiments with OMWa were conducted in 1 L flask reactors (0.5 L working volume) with different OMW dilutions (20%, 50%, and 100%). Even though aquarium pumps were used to provide aeration, the D.O. concentration in the flasks hardly reached 1.5 ppm due to the high dissolved COD content. Three replicates of each dilution were performed and 50 mL inoculum of phenol-degrading bacteria was added to each flask. Duration of the operating cycles was determined by the time that no further reduction was observed. The concentrations of dissolved phenols and dissolved COD at specific time intervals for the three replicates of each dilution are shown in Figs. 1 and 2, respectively. Significant reduction in both d-phenolic and d-COD concentration was recorded. Particularly, for the dilutions of 20%, 50%, and 100%, it took 11, 23, and 30 days, respectively, to record a final 82.62%, 86.01%, and 90.44% reduction of d-phenols concentration. The d-COD reduction, for the same dilutions and time periods, reached 86.14%, 87.42%, and 91.11%, respectively. Such high performance strongly suggests that olive pulp bacteria are capable of degrading the d-phenol and d-COD of OMW.
Fig. 2. d-COD concentrations in flask reactors with different dilutions of OMWa (20%, 50%, and 100%).
3.2. Bench-scale packed bed reactor with OMWb The bench-scale packed bed reactor was initially operated in a draw-fill mode with plain phenol at a concentration of 500 ppm. When it finally reached steady state, the operation mode was switched to draw-fill mode with recirculation, and the experimental series with OMWb took place. The working volume of OMWb was 6 L, held in a bucket under the reactor, and was continuously
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Fig. 3. Schematic drawing of the bench-scale packed bed reactor.
recirculated (Fig. 3). The liquid in the reactor was trickled, so that air could circulate through it, thus the reactor was never flooded. Thus, D.O. concentration was maintained above 4.5 ppm during the experiments. This was precisely the benefit of using such an operating mode in the reactor. Since no external use of oxygen supply was required, the operational cost of the reactor was reduced drastically. Four operating cycles with 6 L of OMWb were performed for the two different sizes of support material tested. At the end of each operating cycle, the filter was backwashed using 5 L of water and high air upward velocity. The d-phenols concentration and d-COD concentration of the four operating cycles for each support media are shown in Figs. 4 and 5, respectively. A comparison between Figs. 1, 2 and Figs. 4, 5 shows clearly the effect of bacteria immobilisation on inert supports. The duration of the operating cycles in the case of the packed bed reactor is reduced to only 27 h, when compared to the duration of several days in the case of the flask reactors with suspended bacteria. The support material provides a large surface for bacterial attachment, thus increasing biomass concentration in the system. Furthermore, the use of larger tubes as support material does not seem to have a particular effect on the reactor’s performance. Excessive biomass growth due to high organic content, as well as the suspended solid concentration of OMW, led to rapid occupation of the extra empty space of the reactor. The d-phenols and d-COD degradation, for the two kinds of support media tested, reached about 60% and 70% for a time period of only 27 h. 3.2.1. BOD of treated and untreated OMWb in bench-scale packed bed reactor BOD measurements of OMWb were performed before and just after the wastewater treatment. Duplicates of each
Fig. 4. d-Phenols concentration in the bench-scale packed bed reactor with OMWb, loaded with different support media.
Fig. 5. d-COD concentration in the bench-scale packed bed reactor with OMWb, loaded with different support media.
sample were undertaken. At the initial BOD measurement, it was added in the BOD test bottle phenol seed from acclimatised phenol bacteria, while a seed control BOD test bottle was also prepared containing only phenol bacteria seed. BOD concentrations of the initial and the posttreatment OMWb were 10636 and 1192 mg O2/L, respectively. This corresponds to an approximately 90% BOD reduction after the treatment of OMWb, indicating that no further biological degradation is possible.
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3.3. Pilot-scale packed bed reactor with OMWb The pilot-scale packed bed reactor was operated at a draw-fill mode with recirculation. The OMWb working volume was 500 L. Also in this case, no external oxygen supply was used. In the startup period, inoculum of readily grown phenol bacteria was added at the beginning of each operating cycle. Inoculum addition was continued until were observed biofilm formations at the support material. Sampling during the draw-fill cycles was performed on a daily basis and not at shorter time intervals, due to the long distance from the laboratory to the olive mill. All samples were taken at the same time of the day, when the maximum temperature of the sample was recorded. During the day, the wastewater temperature in the reactor dropped down to 7–10 1C of the maximum. The d-phenols and d-COD concentrations of the samples are shown in Fig. 6. The sample’s pH and temperature are shown in Fig. 7. It was observed that, for each operating cycle, d-COD removal reached about 70% in 3 days, while d-phenols removal reached about 55% in 5 days. The temperature during these operating cycles ranged between 16 and 20 1C. A sudden temperature drop to 6 1C, on day 37, slowed down the filter’s performance until temperature reached its previous values. This result indicates that packed bed reactor is a useful equipment in OMW biodegradation at controlled temperature conditions. Sludge generation should also be considered to avoid limitation of process applicability. Experimental studies for sludge generation and handle are under way. However, kinetic experiments of olive pulp bacteria in batch cultures using phenol as carbon source (data not shown) revealed
Fig. 7. pH and temperature variation in the pilot-scale packed bed reactor, during draw-fill operating cycles with recirculation.
that about 67% of phenol is converted to biomass. It is anticipated that a similar bacterial yield would be recorded for OMW. Thus, sludge production and handle would be a problem for the proposed process, as well as for all biological methods. By default, attached growth processes produce less sludge than activated sludge processes and this is an advantage of the proposed method. In order to proceed with the produced sludge, a subsequent composting process could be designed. The particular wastewater used in the bench- and pilotscale packed bed reactors had low COD and phenolic content, mainly due to the increased water addition during the extraction process. Dilution of a typical (more concentrated) wastewater is not proposed in any case, since it is against the Greek legislation. It is expected that the packed bed reactors performance would remain almost the same even for more concentrated OMW, since sufficient aeration is the limiting step of the process and the particular operating mode ensures sufficient aeration. However, the produced sludge would be increased significantly, and for this reason sludge composting should be mandatory. 4. Conclusions Based on the experiments conducted in this study, the following conclusions are drawn:
Fig. 6. d-Phenols and d-COD removal (%) in the pilot-scale packed bed reactor, during draw-fill operating cycles with recirculation.
The use of indigenous bacteria populations from olive pulp provides a certain advantage and ensures durability under various operating conditions. Packed bed reactors proved very promising equipment for d-COD and phenolic removal from OMW, providing a support material for biofilm structure development and consequently high biomass concentration.
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The time needed for each operating cycle at the packed reactors is greatly reduced compared to that in the flask reactors. Draw-fill operation with recirculation of the packed bed reactors proved to be a very effective operating mode, since it ensures high d-COD phenolic removal efficiencies (60% phenolic and 70% d-COD removal). Draw-fill operation with recirculation reduces the operational cost of the reactor, since there is no need for external oxygen supply.
The achievement of such high removal efficiencies using mixed cultures from olive fruit pulp indicates a feasible, economical, and efficient technique for OMW biodegradation. Acknowledgements The authors would like to acknowledge the support of Ch. Theodorou for his financial support at the pilot-scale packed bed reactor, A. Tatsis for his technical support, S. Siozios and Prof. K. Bourtzis for their help in the identification of bacterial strains. This project has been accomplished for the General Secretariat of Research and Technology in the frame of the Framework Program (PEP) of Western Greece and has been co-funded by the European Fund for Rural Development (ETPA) and the Prefecture of Western Greece. References APHA, AWWA, WEF, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. American Public Health Association, Washington, DC, USA. Assas, N., Ayed, L., Marouani, L., Hamdi, M., 2002. Decolorization of fresh and stored-black olive mill wastewaters by Geothricum candidum. Process Biochemistry 38, 361–365. Azbar, N., Bayram, A., Filibeli, A., Muezzinoglu, A., Sengul, F., Ozer, A., 2004. A review of wastes management options in olive oil production. Critical Reviews on Environmental Science and Technology 34, 209–247.
Benitez, J., Beltran-Heredia, J., Torregrosa, J., Acero, J.L., Cercas, V., 1997. Aerobic degradation of olive mill wastewaters. Applied Microbiology and Biotechnology 47, 185–188. Borja, R., Martin, A., Maestro, R., Alba, J., Fiestas, J.A., 1992. Enhancement of the anaerobic digestion of oil mill waste by removal of phenolic inhibitors. Process Biochemistry 27, 231–237. D’Annibale, A., Ricci, M., Quaratino, D., Federici, F., Fenice, M., 2004. Panus tigrinus efficiently removes phenols, color and organic load from olive-mill wastewater. Research in Microbiology 155, 596–603. Fadil, K., Chahlaoui, A., Ouahbi, A., Zaid, A., Borja, R., 2003. Aerobic biodegradation and detoxification of wastewaters from the olive oil industry. International Biodeterioration & Biodegradation 51, 37–41. Fountoulakis, M.S., Dokianakis, S.N., Kornaros, M.E., Aggelis, G.G., Lyberatos, G., 2002. Removal of phenolics in olive mill wastewaters using the white-rot fungus Pleurotus ostreatus. Water Research 36, 4735–4744. Garcı´ a Garcı´ a, I., Pen˜a Jime´nez, P.R., Venceslada Bonilla, J.L., Martı´ n Martı´ n, A., Santos Martı´ n, M.A., Go´mez Ramos, E., 2000. Removal of phenol compounds from olive mill wastewater using Phanerochaete chrysosporium, Aspergillus niger, Aspergillus terreus and Geotrichum candidum. Process Biochemistry 35, 751–758. Greco Jr., G., Toscanoa, G., Cioffi, M., Gianfreda, L., Sannino, F., 1999. Dephenolisation of olive mill waste-waters by olive husk. Water Research 33, 3046–3050. Hamdi, M., 1996. Anaerobic digestion of olive mill wastewaters. Process Biochemistry 31, 105–110. Niaounakis, M., Halvadakis, C.P., 2004. Olive-Mill Waste Management: Literature Review and Patent Survey. Typothito-George Dardanos Publications, Athens. Ramos-Cormenzana, A., Juarez-Jimenez, B., Garcı´ a-Pareja, M.P., 1996. Antimicrobial activity of olive mill wastewaters (alpechı´ n) and biotransformed olive oil mill wastewater. International Biodeterioration & Biodegradation 38, 283–290. Roig, A., Cayuela, M.L., Sa´nchez-Monedero, M.A., 2006. An overview on olive mill wastes and their valorisation methods. Waste Management 26, 960–969. Scioli, C., Vollaro, L., 1997. The use of Yarrowia lilolytica to reduce pollution in olive mill wastewaters. Water Research 31, 2520–2524. Tomati, U., Galli, E., Di Lena, G., Buffone, R., 1991. Induction of laccase in Pleurotus ostreatus mycelium grown in olive oil wastewaters. Agrochimica 35, 273–279. Tziotzios, G., Teliou, M., Kaltsouni, V., Lyberatos, G., Vayenas, D.V., 2005. Biological phenol removal using suspended growth and packed bed reactors. Biochemical Engineering Journal 26, 65–71. Waterman, P.G., Mole, S., 1994. Analysis of phenolic plant metabolites. In: Lawton, J.H., Likens, G.E. (Eds.), Methods in Ecology. Blackwell Scientific Publications, Oxford.
International Biodeterioration & Biodegradation 60 (2007) 209–214 www.elsevier.com/locate/ibiod
Aerobic biological treatment of olive mill wastewater by olive pulp bacteria G. Tziotzios, S. Michailakis, D.V. Vayenas Department of Environmental and Natural Resources Management, University of Ioannina, Seferi 2, 30100 Agrinio, Greece Received 7 February 2007; received in revised form 28 February 2007; accepted 2 March 2007 Available online 16 April 2007
Abstract The capability of olive fruit bacteria to remove chemical oxygen demand (COD) and phenolic compounds from olive mill wastewater (OMW) using flasks and packed bed reactors was tested. Batch aerobic experiments were performed in flask reactors, with OMW at different dilutions (20%, 50%, and 100%). The maximum phenolic and dissolved COD removal reached up to 82–90% for the dilutions of 20%, 50%, and 100%, in 11, 23, and 30 days, respectively. Experiments were also conducted in a bench and a pilot-scale packed bed reactor, operated in the laboratory and at an olive mill, respectively. At 28 1C the maximum phenolic and dissolved COD removal, recorded at the bench-scale reactor, reached about 60% and 70%, respectively, for a time period of only 27 h. At the pilot-scale reactor, where the maximum daily temperature varied between 6 and 20 1C, the maximum phenolic and d-COD removal reached about 55% and 70% in 5 and 3 days, respectively. These results indicate an economically feasible method for OMW biodegradation. r 2007 Elsevier Ltd. All rights reserved. Keywords: Olive mill wastewaters; Biodegradation; Phenolic compounds; Packed bed reactors
1. Introduction The olive oil industry is very important in the Mediterranean countries, both in terms of wealth and tradition (Roig et al., 2006). Spain is the main world producer followed by Italy, Greece, Turkey, Syria, and Tunisia. Hence, this area is especially affected by olive mill waste pollution. However, many other countries such as Argentina, Australia, and South Africa are becoming emergent producers since they are promoting intensive olive tree cultivation. The extraction and manufacture of olive oil are carried out in numerous agro-industrial units in the Mediterranean countries, which generate an aqueous phase formed by the water content of the fruit combined with what was used to wash and process the olives: the combination is the socalled olive mill wastewater (OMW) and the annual production is estimated to an amount over 107 m3 (Benitez et al., 1997). Typically, the weight composition of OMW is Corresponding author. Tel.: +30 2641074117; fax: +30 2641074172.
E-mail address: [email protected] (D.V. Vayenas). 0964-8305/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2007.03.003
83–96% water, 3.5–15% organics, and 0.5–2% mineral salts. The organic fraction is composed of sugars (1.0–8.0%), N-compounds (0.5–2.4%), organic acids (0.5–1.5%), fats (0.02–1%) as well as phenols and pectins (1.0–1.5%) (Greco et al., 1999). The maximum biological oxygen demand (BOD) and chemical oxygen demand (COD) reach concentrations of 100 and 220 kg/m3, respectively. As regards to phenols, low-molecular weight compounds (hydroxytyrosol, tyrosol, catechol, methylcatechol, caffeic acid) are usually present in OMW, along with catechol-melaninic polymers. Owing to the content of these effluents, the environmental problems and potential hazards caused by OMW has prompted many countries to limit its discharge and to develop new technologies for reducing the pollutant power, such as the different chemical and biological treatments that have been investigated lately. Moreover, some extensive and detailed reviews have been recently published (Roig et al., 2006; Niaounakis and Halvadakis, 2004; Azbar et al., 2004; Hamdi, 1996). OMW is currently concentrated by evaporation in open pools, but this method is not satisfactory as a black
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foul-smelling sludge, difficult to remove, is produced. Various physicochemical methods have been proposed and tested but the high cost of these methods deteriorated their wide application. Biological methods used for OMW treatment are aerobic-activated sludge and anaerobic digestion. Although aerobic biological treatments succeed higher efficiencies, these processes are limited by the unbearable cost of the continuously provided mechanical aeration. A major limitation of anaerobic digestion of OMWs is inhibition of methanogenic bacteria by simple phenolic compounds and certain organic acids (Hamdi, 1996). The use of filamentous fungi for OMW pretreatment has been shown to reduce OMW toxicity and improve the biodegradability in anaerobic digestion (Assas et al., 2002). Many microorganisms have been tested: the fungi Pleurotus ostreatus, Bacillus pumilus, Panus tigrinus, the yeast Yarrowia Lipolytica, etc. (Fadil et al., 2003; Fountoulakis et al., 2002; Tomati et al., 1991; Ramos-Cormenzana et al., 1996; D’Annibale et al., 2004; Garcı´ a Garcı´ a et al., 2000; Scioli and Vollaro, 1997). The application of fungi on a large scale is limited by the difficulty of achieving sterilised conditions in open-air reactors. The aim of the present work was the utilisation of olive fruit bacteria for the removal of COD and phenolic compounds from OMWs using flask, bench and pilotscale packed bed reactors, under non-sterilised aerobic conditions. 2. Materials and methods 2.1. OMW OMW were obtained from two olive oil factories (three-phase centrifugal) located at Agrinio (Western Greece). Immediately after sampling, the OMWs were maintained at 20 1C throughout the experimentation period. Their composition is shown in Table 1. OMWa refers to the wastewater used for the experimental series in the flask reactors, and OMWb refers to the wastewater used in the bench and pilotscale packed bed reactors. The difference in the composition of the two OMWs is mainly attributed to different water addition amounts during the extraction process.
2.2. Media Olive fruit bacteria were grown in a phenol-based mineral media previously used by Tziotzios et al. (2005), consisting of 1 g K2HPO4, 0.644 g Na2HPO4, 1.3375 g NH4Cl, 0.2 g MgSO4 7H2O, 0.2 g KCl, and Table 1 Composition of OMW Parameter
OMWa
OMWb
Total suspended solids, TSS (g/L) Volatile suspended solids, VSS (g/L) Dissolved phenols (g syringic acid/L) Dissolved chemical oxygen demand, d-COD (g/L) Biological oxygen demand, BOD (g O2/L) pH
59.69 55.65 6.7 50.72 35.79 5.18
41.01 34.03 2.9 15.05 10.64 5.5
0.02 g yeast extract per litre of tap water. Phenol was added as the sole carbon and energy source. The addition of readily grown bacteria into the flask and bench-scale packed bed reactor containing OMW was accompanied by the addition of NH4Cl, K2HPO4, and MgSO4 7H2O, keeping the proportion of dissolved-COD:N:P:Mg ¼ 100:5:1:1 steady. In the case of the pilot-scale packed bed reactor operated nearby an olive mill, the same proportion was kept steady using common agricultural fertilisers.
2.3. Analytical methods Various parameters were measured during the experiments: total suspended solids (TSS), volatile suspended solids (VSS), dissolved (after filtration of OMW) COD, BOD were determined as proposed by Standard Methods (APHA, 1995). Phenol and d-phenolic (with respect to syringic acid) concentrations were determined spectrophotometrically according to the Folin–Ciocalteu method (Waterman and Mole, 1994), using a JASCO V-530 UV/vis spectrophotometer. Dissolved oxygen (D.O.) and pH were measured with a HANNA HI-9143 Microprocessor D.O. meter and a HANNA pH-211 Microprocessor pH meter, respectively.
2.4. pH, temperature Throughout the experiments in the flask and bench-scale packed bed reactors, the temperature was fairly constant at about 28 1C and the pH ranged from 5.570.2 (initial pH) to 6.570.2 (end of the experiment). The maximum daily temperature in the pilot-scale packed bed reactor operated in an olive mill ranged between 6 and 20 1C, and the pH ranged from 5.570.3 to 7.470.3 at the end of the experiment.
2.5. Selection of the bacterial community The original inoculum of bacteria was taken from olive pulp. This source was selected, since olives and OMW contain a significant amount of phenolics (Fountoulakis et al., 2002; Borja et al., 1992). At first, sodium acetate was used as a carbon source. Enrichment and acclimation of phenol-degrading bacteria was achieved in a flask reactor by the gradual replacement of sodium acetate by phenol up to a concentration of 100 ppm. Bacterial samples taken at this point showed, using 16S rRNA sequencing, that the dominant bacterial gena of the mixed culture were Alcaligenes and Acinetobacter. When complete phenol (100 ppm) degradation was recorded, an inoculum was transferred to the reactors, containing OMW and the appropriate nutrient medium.
2.6. Flask, bench and pilot-scale packed bed reactors Erlenmeyer flasks of 1 L were used as flask reactors. Different dilutions of OMW (20%, 50%, and 100%) were tested. The inoculum used in the flask reactors was 50 mL. Aquarium pumps were used to provide aeration. The bench-scale packed bed reactor established in the laboratory consisted of a Plexiglas tube, 160 cm high and with a 9 cm internal diameter. This filter height is typical of a full-scale industrial filter. Since it is the loadings (hydraulic and phenolics) per unit cross-sectional area that matter, no scale-up was necessary. Two different sizes of rippled plastic hollow tubes were tested as the support material of this reactor. This particular support material was chosen, since it is a cheap electrological material and commercially easy to find. The small-sized plastic tubes were of 1.6 cm internal diameter and 3.02 cm in length, with a specific surface area of 500 m2/m3, and filter porosity 0.8. The large-sized plastic tubes were of 2.65 cm internal diameter and 3.95 cm in length, with a specific surface area of 301.9 m2/m3, and filter porosity 0.95. The depth of support media in the filter was 143 cm. Filter backwash due to pore clogging from biomass growth was necessary and was performed at the end of each batch experiment using high water and air upward velocities. The produced sludge was subsequently treated in a composting unit.
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The pilot-scale packed bed reactor operated in the olive mill consisted of a cylindrical polyethylene tank with a conical base, 1.5 m in height and 1.8 m of internal diameter. Along the lower parts of the tank there were 11 ventilation ports, through which air could circulate. Inside the tank, a basket of 1.2 m in height and 1.75 m in diameter was placed. The basket was filled with the support material up to 1 m. The support material of this reactor was rippled plastic hollow tubes (commercial electrical tube), 3.85 cm internal diameter and 4 cm in length, with a specific surface area of 207.8 m2/m3, and porosity 0.93. OMW (500 L) was kept in a nearby tank, and was continuously recirculated in the reactor, from the top, by a rotary distributor.
2.7. Packed bed reactor startup During bench-scale packed bed reactor startup, inoculum (50 mL/L of working volume) was taken from the flask reactor with acclimated phenol bacteria and added to the packed bed reactor with appropriate amounts of nutrient medium, i.e., a concentrated phenol solution mixed with the influent feed to ensure bacterial growth. The initial phenol concentration in the filter was 500 ppm. Sufficient aeration was provided in the base of the packed bed reactor through an air pump. When complete phenol degradation was recorded, the filter was recharged with 500 ppm phenol and nutrients. The inoculum (50 mL/L of working volume) used in the recharges came from the bulk liquid of the filter, just before unloading. The inoculum volume was gradually reduced as bacterial attachment on the support medium was observed. When no further inoculum addition was required, and the filter reached a steady cycle-to-cycle performance after approximately 30 days, the experimental series with OMW took place. At the startup period of the pilot-scale packed bed reactor, inoculum (10 L) was added to the reactor in the beginning of each operating cycle. Inoculum addition was continued until biofilm formation was observed attached to the support material. The reactor reached steady cycle-to-cycle performance at approximately 40 days.
Fig. 1. d-Phenols concentrations in flask reactors with different dilutions of OMWa (20%, 50%, and 100%).
3. Results and discussion 3.1. Flask reactors with OMWa Experiments with OMWa were conducted in 1 L flask reactors (0.5 L working volume) with different OMW dilutions (20%, 50%, and 100%). Even though aquarium pumps were used to provide aeration, the D.O. concentration in the flasks hardly reached 1.5 ppm due to the high dissolved COD content. Three replicates of each dilution were performed and 50 mL inoculum of phenol-degrading bacteria was added to each flask. Duration of the operating cycles was determined by the time that no further reduction was observed. The concentrations of dissolved phenols and dissolved COD at specific time intervals for the three replicates of each dilution are shown in Figs. 1 and 2, respectively. Significant reduction in both d-phenolic and d-COD concentration was recorded. Particularly, for the dilutions of 20%, 50%, and 100%, it took 11, 23, and 30 days, respectively, to record a final 82.62%, 86.01%, and 90.44% reduction of d-phenols concentration. The d-COD reduction, for the same dilutions and time periods, reached 86.14%, 87.42%, and 91.11%, respectively. Such high performance strongly suggests that olive pulp bacteria are capable of degrading the d-phenol and d-COD of OMW.
Fig. 2. d-COD concentrations in flask reactors with different dilutions of OMWa (20%, 50%, and 100%).
3.2. Bench-scale packed bed reactor with OMWb The bench-scale packed bed reactor was initially operated in a draw-fill mode with plain phenol at a concentration of 500 ppm. When it finally reached steady state, the operation mode was switched to draw-fill mode with recirculation, and the experimental series with OMWb took place. The working volume of OMWb was 6 L, held in a bucket under the reactor, and was continuously
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Fig. 3. Schematic drawing of the bench-scale packed bed reactor.
recirculated (Fig. 3). The liquid in the reactor was trickled, so that air could circulate through it, thus the reactor was never flooded. Thus, D.O. concentration was maintained above 4.5 ppm during the experiments. This was precisely the benefit of using such an operating mode in the reactor. Since no external use of oxygen supply was required, the operational cost of the reactor was reduced drastically. Four operating cycles with 6 L of OMWb were performed for the two different sizes of support material tested. At the end of each operating cycle, the filter was backwashed using 5 L of water and high air upward velocity. The d-phenols concentration and d-COD concentration of the four operating cycles for each support media are shown in Figs. 4 and 5, respectively. A comparison between Figs. 1, 2 and Figs. 4, 5 shows clearly the effect of bacteria immobilisation on inert supports. The duration of the operating cycles in the case of the packed bed reactor is reduced to only 27 h, when compared to the duration of several days in the case of the flask reactors with suspended bacteria. The support material provides a large surface for bacterial attachment, thus increasing biomass concentration in the system. Furthermore, the use of larger tubes as support material does not seem to have a particular effect on the reactor’s performance. Excessive biomass growth due to high organic content, as well as the suspended solid concentration of OMW, led to rapid occupation of the extra empty space of the reactor. The d-phenols and d-COD degradation, for the two kinds of support media tested, reached about 60% and 70% for a time period of only 27 h. 3.2.1. BOD of treated and untreated OMWb in bench-scale packed bed reactor BOD measurements of OMWb were performed before and just after the wastewater treatment. Duplicates of each
Fig. 4. d-Phenols concentration in the bench-scale packed bed reactor with OMWb, loaded with different support media.
Fig. 5. d-COD concentration in the bench-scale packed bed reactor with OMWb, loaded with different support media.
sample were undertaken. At the initial BOD measurement, it was added in the BOD test bottle phenol seed from acclimatised phenol bacteria, while a seed control BOD test bottle was also prepared containing only phenol bacteria seed. BOD concentrations of the initial and the posttreatment OMWb were 10636 and 1192 mg O2/L, respectively. This corresponds to an approximately 90% BOD reduction after the treatment of OMWb, indicating that no further biological degradation is possible.
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3.3. Pilot-scale packed bed reactor with OMWb The pilot-scale packed bed reactor was operated at a draw-fill mode with recirculation. The OMWb working volume was 500 L. Also in this case, no external oxygen supply was used. In the startup period, inoculum of readily grown phenol bacteria was added at the beginning of each operating cycle. Inoculum addition was continued until were observed biofilm formations at the support material. Sampling during the draw-fill cycles was performed on a daily basis and not at shorter time intervals, due to the long distance from the laboratory to the olive mill. All samples were taken at the same time of the day, when the maximum temperature of the sample was recorded. During the day, the wastewater temperature in the reactor dropped down to 7–10 1C of the maximum. The d-phenols and d-COD concentrations of the samples are shown in Fig. 6. The sample’s pH and temperature are shown in Fig. 7. It was observed that, for each operating cycle, d-COD removal reached about 70% in 3 days, while d-phenols removal reached about 55% in 5 days. The temperature during these operating cycles ranged between 16 and 20 1C. A sudden temperature drop to 6 1C, on day 37, slowed down the filter’s performance until temperature reached its previous values. This result indicates that packed bed reactor is a useful equipment in OMW biodegradation at controlled temperature conditions. Sludge generation should also be considered to avoid limitation of process applicability. Experimental studies for sludge generation and handle are under way. However, kinetic experiments of olive pulp bacteria in batch cultures using phenol as carbon source (data not shown) revealed
Fig. 7. pH and temperature variation in the pilot-scale packed bed reactor, during draw-fill operating cycles with recirculation.
that about 67% of phenol is converted to biomass. It is anticipated that a similar bacterial yield would be recorded for OMW. Thus, sludge production and handle would be a problem for the proposed process, as well as for all biological methods. By default, attached growth processes produce less sludge than activated sludge processes and this is an advantage of the proposed method. In order to proceed with the produced sludge, a subsequent composting process could be designed. The particular wastewater used in the bench- and pilotscale packed bed reactors had low COD and phenolic content, mainly due to the increased water addition during the extraction process. Dilution of a typical (more concentrated) wastewater is not proposed in any case, since it is against the Greek legislation. It is expected that the packed bed reactors performance would remain almost the same even for more concentrated OMW, since sufficient aeration is the limiting step of the process and the particular operating mode ensures sufficient aeration. However, the produced sludge would be increased significantly, and for this reason sludge composting should be mandatory. 4. Conclusions Based on the experiments conducted in this study, the following conclusions are drawn:
Fig. 6. d-Phenols and d-COD removal (%) in the pilot-scale packed bed reactor, during draw-fill operating cycles with recirculation.
The use of indigenous bacteria populations from olive pulp provides a certain advantage and ensures durability under various operating conditions. Packed bed reactors proved very promising equipment for d-COD and phenolic removal from OMW, providing a support material for biofilm structure development and consequently high biomass concentration.
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The time needed for each operating cycle at the packed reactors is greatly reduced compared to that in the flask reactors. Draw-fill operation with recirculation of the packed bed reactors proved to be a very effective operating mode, since it ensures high d-COD phenolic removal efficiencies (60% phenolic and 70% d-COD removal). Draw-fill operation with recirculation reduces the operational cost of the reactor, since there is no need for external oxygen supply.
The achievement of such high removal efficiencies using mixed cultures from olive fruit pulp indicates a feasible, economical, and efficient technique for OMW biodegradation. Acknowledgements The authors would like to acknowledge the support of Ch. Theodorou for his financial support at the pilot-scale packed bed reactor, A. Tatsis for his technical support, S. Siozios and Prof. K. Bourtzis for their help in the identification of bacterial strains. This project has been accomplished for the General Secretariat of Research and Technology in the frame of the Framework Program (PEP) of Western Greece and has been co-funded by the European Fund for Rural Development (ETPA) and the Prefecture of Western Greece. References APHA, AWWA, WEF, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. American Public Health Association, Washington, DC, USA. Assas, N., Ayed, L., Marouani, L., Hamdi, M., 2002. Decolorization of fresh and stored-black olive mill wastewaters by Geothricum candidum. Process Biochemistry 38, 361–365. Azbar, N., Bayram, A., Filibeli, A., Muezzinoglu, A., Sengul, F., Ozer, A., 2004. A review of wastes management options in olive oil production. Critical Reviews on Environmental Science and Technology 34, 209–247.
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