Biodegradation of Mixtures of Phenolic Compounds in an Upward-Flow Anaerobic Sludge Blanket Reactor

Elı´as Razo-Flores1; Margarita Iniestra-Gonza´lez2; Jim A. Field3; Patricia Olguı´n-Lora4; and Laura Puig-Grajales5

Abstract: The anaerobic biodegradability of mixtures of phenolic compounds was studied under continuous and batch systems. Con- tinuous experiments were carried out in up-flow anaerobic sludge bed ͑UASB͒ reactors degrading a mixture of phenol and p-cresol as the main carbon and energy sources. The total chemical oxygen demand ͑COD͒ removal above 90% was achieved even at organic loading rates as high as 7 kg COD/m3/day. Batch experiments were conducted with mixtures of phenolic compounds ͑phenol, p-cresol, and o-cresol͒ to determine the specific biodegradation rates using unadapted and adapted anaerobic granular sludge. Phenol and p-cresol were mineralized by adapted sludge with rates several orders of magnitude higher than unadapted sludge. Additionally, an UASB reactor was operated with the mixture phenol, p-cresol, and o-cresol. After 54 days of operation, 80% of o-cresol ͑supplied at 132 mg/L͒ was eliminated. The phenol biodegradation was not affected by the presence of o-cresol. These results demonstrate that major phenolic components in petrochemical effluents can be biodegraded simultaneously during anaerobic treatment. DOI: 10.1061/͑ASCE͒0733-9372͑2003͒129:11͑999͒ CE Database subject headings: Anaerobic processes; Biodegradation; Phenol; Methane; Sludge; Cisterns; Wastewater treatment.

Introduction Among the possible treatment methods, anaerobic treatment is a viable option due to the anaerobic biodegradability of phenolic Phenolic compounds are present in the wastewaters of the chemi- compounds. Many authors have studied the biodegradation of cal and petrochemical industries and represent an important phenol and cresols in methanogenic consortia utilizing batch as- source of environmental pollution ͑Berne´ and Cordonnier 1995͒. says ͑Fedorak and Hudrey 1984; Blum et al. 1986; Smolenski and Wastewaters from a refinery are a complex mixture of organic and Suflita 1987; Bisaillon et al. 1991, 1993; Razo-Flores et al. inorganic compounds, often containing more than one type of 1996a,b; Kennes et al. 1997͒. However, the majority of these phenolic compound. Phenol and cresols are major constituents studies were conducted using single phenolics, like phenol or ͑ ´ ͒ found in refinery effluents Berne and Cordonnier 1995 . Phenol p-cresol. The biological treatment of complex phenolic wastewa- has been recognized as being either toxic or lethal to fish at con- ters, has often been studied utilizing activated carbon ͑AC͒ in centrations of 5 to 25 mg/L ͑Hill and Robinson 1975͒. Many anaerobic reactors; where, the AC served to adsorb toxic phenolic substituted phenols, including chlorophenols, nitrophenols, and pollutants and acted as a carrier for bacterial growth ͑Nakhla cresols have been designated as priority pollut-ants by the U.S. ͒ Environmental Protection Agency ͑Keith and Telliard 1979͒. et al. 1989; Nakhla et al. 1990 . There are fewer references on the continuous anaerobic treatment of mixed phenolic compounds ͑ 1 without any dose of AC Zhou and Fang 1997; Fang and Zhou Research Group Leader, Programa de Biotecnologıá, Instituto ͒ Mexicano del Petro´leo, Eje Central La´zaro Ca´rdenas 152, C.P. 07730, 2000; Tawfiki et al. 2000 . The bioreactor systems most com- Me´xico, D.F. ͑corresponding author͒. E-mail: [email protected] monly described for the anaerobic treatment of phenolics are the 2Research Fellow, Programa de Biotecnologıá, Instituto Mexicano del up-flow anaerobic sludge bed ͑UASB͒ reactor ͑Hwang and Cheng Petro´leo. Eje Central La´zaro Ca´rdenas 152, C.P. 07730, Me´xico, D.F. 1991; Chang et al. 1995; Zhou and Fang 1997; Kennes et al. 3Associate Professor, Department of Chemical and Environmental 1997͒ and the fixed film anaerobic reactor ͑Charest et al. 1999͒. Engineering, Univ. of Arizona. P.O. Box 210011 Tucson, AZ 85721-0011. The successful treatment of petroleum effluents will require that E-mail: jimfi[email protected] major phenolic substrates ͑phenol and cresols͒ should be de- 4Researcher, Programa de Biotecnologıá, Instituto Mexicano del Petro´leo, Eje Central La´zaro Ca´rdenas 152, C.P. 07730, Me´xico, D.F. graded simultaneously. Therefore, biodegradation studies should 5Researcher, Programa de Biotecnologıá, Instituto Mexicano del evaluate mixtures of phenols. In this study, continuous and batch Petro´leo, Eje Central La´zaro Ca´rdenas 152, C.P. 07730, Me´xico, D.F. experiments with anaerobic granular sludge were conducted in Note. Associate Editor: Spyros G. Pavlostathis. Discussion open until order to study the biodegradation of phenolic mixtures and inves- April 1, 2004. Separate discussions must be submitted for individual tigate the effect of specific phenolic compounds ͑p- and o-cresol͒ papers. To extend the closing date by one month, a written request must on the phenol biodegradation. Maximum volumetric loading rates be filed with the ASCE Managing Editor. The manuscript for this paper were determined in continuous UASB reactors using phenol and was submitted for review and possible publication on March 12, 2002; approved on November 8, 2002. This paper is part of the Journal of p-cresol as the main carbon and energy sources. Specific biodeg- Environmental Engineering, Vol. 129, No. 11, November 1, 2003. radation rates ͑SBR͒ were determined for single and mixtures of ©ASCE, ISSN 0733-9372/2003/11-999–1006/$18.00. phenolic compounds.

JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / NOVEMBER 2003 / 999 Materials and Methods introduced to the reactor at the following concentrations: 550 mg/L, 132 mg/L, and 132 mg/L, respectively ͑1.98 g COD/L͒,as the main carbon and energy sources. The basal medium was Inoculum modified for the second stage of operation, the sodium bicarbon- The methanogenic granular sludges used in this study were ob- ate concentration was 2,000 mg/L and the concentration of the tained from two different UASB reactors treating industrial efflu- other elements was reduced ten times. The performance of the ents: Cuauhtemoc Brewery ͓͑CB͒ Puebla, Mexico͔ and Shell reactor was followed with the same parameters as in the first stage Nederland Chemie ͓͑SNC͒ Moerdijk, The Netherlands͔. The slud- of operation. ges were elutriated to remove the fines and predigested at 30°C during 30 days in order to deplete most of the endogenous sub- Batch Biodegradability Assays strates in the sludge. The characteristics of the sludges were: 8% and 9.5% volatile suspended solids ͑VSS͒; 0.2 and 0.43 g chemi- The batch anaerobic biodegradability assays were conducted in ͑ ͒ cal oxygen demand COD -CH4 /g VSS/day of acetoclastic 124 mL glass serum bottles. The assays with unadapted sludge methanogenic activity, for the CB and SNC sludges, respectively. were conducted with 0.95 g ͑wet weight͒ of CB-granular sludge, whereas batch assays with adapted granular sludge were per- ͑ ͒ Basal Medium formed using 1 g wet weight of phenol-p-cresol adapted granular sludge withdrawn from the UASB reactors at the end of the The basal medium used during the experiments contained the first stage of the continuous experiments ͑after 135 days of op- ͑ ͒ following compounds mg/L : NaHCO3 (5,000), NH4Cl (280), eration͒. The inoculum was transferred to the serum bottles, CaCl2•2H2O (10), K2HPO4 (250), MgSO4 (100), yeast extract which contained 50 mL of the basal medium. The serum bottles ͑100͒, and 1 mL of micronutrients stock solution which contained were sealed with 12 mm thick butyl rubber stoppers and flushed ͑ ͒ mg/L : FeCl2•4H2O (2,000), H3BO3 (50), ZnCl2 (50), with a gas mixture of 30% CO2 and 70% N2 for 5 min. The serum CuCl2•2H2O (38), MnCl2•4H2O (500), (NH4)6Mo7O24 bottles were incubated overnight at 30°C to allow the biological 4H2O (50), AlCl3 6H2O (90), CoCl2 6H2O (2,000), NiCl2 consumption of residual oxygen. One day later, the desired • • • ͑ ͒ •6H2O (142), Na2SeO•5H2O (164), EDTA 1,000 , resazurin amount of the phenols tested was added with a syringe to tripli- ͑200͒, and HCl 36% ͑1mL͒. The final pH of the medium was 7.2. cated serum bottles from a concentrated stock solution. Experi- ments with unadapted granular sludge were conducted at the following concentrations ͑experiment number indicated in Simultaneous Biodegradation of Phenol and p-Cresol parenthesis͒: phenol, 200 mg/L ͑1͒, p-cresol, 150 mg/L ͑2͒, and in Up-Flow Anaerobic Sludge Bed Reactors mixtures of phenol at 200 mg/L plus p-cresol at 50 ͑3͒, 100 ͑4͒, The continuous experiments were performed in two separate glass and 150 mg/L ͑5͒, respectively. Experiments with adapted granu- UASB reactors ͑0.145 m in length and 0.039 m of internal diam- lar were conducted at the concentrations described in Table 1. All eter͒ with liquid volumes of 160 mL, placed in a temperature phenols were used as the main carbon and energy sources. The controlled room at 30°C. Both reactors were inoculated with 40 g serum bottles were incubated at 30°C. The concentration of the ͑wet weight͒ of a mixture of granular sludge ͑80% CB—sludge compounds and the accumulated methane production was moni- and 20% SNC—sludge͒. The reactors were operated at a hydrau- tored during the duration of the experiment. The SBR of the com- lic retention time ͑HRT͒ of 0.5–0.6 days during the experiments. pounds was calculated from the slope of the phenols concentra- The influent was delivered to the reactors with a variable speed tion versus time curve. The net cumulative methane production Masterflex Digital Pump ͑Cole-Parmer, Vernon Hills, Ill.͒ at a was expressed as a percentage of the theoretical methane produc- flow rate of 270–320 mL/day. The reactors were started up with tion ͑TMP͒ expected from the test chemical mineralization based acetate ͑1 g COD/L͒ and after 1 month of operation ͑designated on the Tarvin and Buswell ͑1934͒ equation. Corrections were as day 0 in figures and tables͒, the reactors additionally received a made for background levels of methane production monitored in mixture of phenol and p-cresol, 280 and 132 mg/L, respectively controls lacking any added test compound. The concentrations of ͑1 g COD/L͒, to provide a total organic loading rate ͑OLR͒ of 4 phenolic compounds are referred to in COD units, commonly kg COD/m3/day. The acetate concentration in the feeding was expressed as its equivalent in COD per liter of liquid. Conversion maintained until day 60, being reduced to half and completely factors utilized were: 2.38 g COD/g phenol, 2.515 g COD/g removed from the feeding between days 61 to 64. Afterward, the cresol, and 0.503 L CH4 /g COD at 0.77 atm and 30°C. inlet phenolics concentration was increased stepwise in order to determine the maximum OLR of the phenolic substrate that could Analytical Methods be applied to the reactors. The methane production was measured by liquid displacement using a 4% ͑weight/volume͒ NaOH solu- The methane content in the gas samples was determined by gas tion to scrub out the carbon dioxide from the biogas. The perfor- chromatography ͑Perkin Elmer 8500, Mexico City͒. The gas chro- mance of the reactors was monitored by measuring the pH, meth- matograph was equipped with a steel column ͑2.4 m by 3.2 mm͒ ane, and the concentration of the phenolic compounds and COD packed with Super Q ͑80/100 mesh, Millipore Corp., Bedford in the influent and effluent. Mass.͒. The temperatures of the column, the injector port, and the flame ionization detector were 250°C. The carrier gas was helium at a flow rate of 40 mL/min. Samples for measuring methane Effect of o-Cresol on the Phenol and p-Cresol content ͑100 ␮L͒ in the headspace were determined with a Biodegradation in a Continuous Up-Flow Anaerobic pressure-gas lock syringe ͑Pressure-Lok series A-2, Dynatech Sludge Bed Reactor Precision Sampling Corp., Baton Rouge, LA͒. An isobaric precise The operation of one of the reactors was extended for an addi- proportion of the known headspace volume could be analyzed. tional period of 200 days inoculated with 25.6 g ͑wet weight͒ of Phenolic compounds were analyzed in the centrifuged aqueous the adapted granular sludge. Phenol, p-cresol, and o-cresol were phase by reverse-phase high-pressure liquid chromatography

1000 / JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / NOVEMBER 2003 Table 1. Phenolics Concentration ͑Single Compound or in Mixture͒ and Specific Biodegradation Rates Determined in Batch Experiments Using Phenol/p-Cresol Adapted Granular Sludge ͑2 g Volatile Suspended Solids/L͒ Withdrawn from Reactor 2 at End of Continuous Experiment ͑Day 135͒ Compound ͑mg/L͒/specific biodegradation rate ͑mg chemical oxygen demand/g volatile suspended solids/day͒ Experiment Phenol p-Cresol o-Cresol Methane ͑% theoretical methane production͒a 1 200/135.4Ϯ0.7 102Ϯ 1.4 2 100/92.5Ϯ3.9 97Ϯ 2 3 200/156.3Ϯ2.5 100/75.1Ϯ4.1 102Ϯ 3 4 200/132.5Ϯ6.6 100/73.9Ϯ2.8 25/10.1Ϯ0.9 98Ϯ3b 5 200/116.6Ϯ4.8 100/71.1Ϯ2.3 75/20.9Ϯ0.9 95Ϯ2b 6 200/121.4Ϯ5.7 100/55.6Ϯ3.1 150/30.1Ϯ0.5 100Ϯ4b Note: o-Cresol was completely transformed to an unidentified compound that was not further degraded. aMethane production was corrected for the values in the sludge blank controls, which were used for comparison to the theoretical methane production of the test compounds. The theoretical methane production was determined after 5 days. bThe theoretical methane production was estimated based on phenol and p-cresol degradation.

͑HPLC Hewlett Packard 1100, Mexico City͒ using a Silica C-8 from 1 to 4 g COD/L at a phenol/p-cresol ratio of 2:1. Similar ͑ZORBAX͒ column. Absorbance was detected at 270 nm and the OLR ͑7.7 kg COD/m3/day͒ applied to the reactor but at a phenol/ column temperature was 25°C. The solvent phase was methanol/ p-cresol ratio of 1:1 ͑800:800 mg/L͒, exhibited a decrease of the water ͑60%/40%͒ with a flow of 1 mL/min. The size of the total COD removal efficiency below 50%. The phenol and sample was 10 ␮L. The retention time of the compounds were 3, p-cresol removal dropped to 40% and 20%, respectively. The ef- 3.8, and 4 min for phenol, p-cresol, and o-cresol, respectively. ficiency drop suggested an inhibitory effect toward the anaerobic The pH was determined immediately after sampling with an Ac- biomass. After the OLR was reduced to 1.7 kg COD/m3/day, R1 cument 915 pH meter ͑Fisher Scientific, Mexico City͒ and a required 20 days to reach a COD removal of 80% ͑Fig. 1͒. Corning electrode ͑Acton, Mass.͒. All the other analytical deter- R2 was operated in parallel under similar operational condi- minations ͑VSS and COD͒ were performed as described in tions as R1, except during the last 30 days of operation at which American Public Health Association ͑1985͒. Phenol, p-cresol ͑J. time the ratio of phenol to p-cresol was increased instead of de- T. Baker, Mexico City͒, o-cresol ͑Merck, Mexico City͒, and all creased. The time course of the treatment performance of R2 is the other chemicals were of the highest purity available. shown in Fig. 2. Similarly to R1, R2 exhibited a high COD removal efficiency ͑90%͒ even at phenolic substrate concentration of 4 g COD/L and an OLR of 7.2 kg COD/m3/day but at a phenol/ Results p-cresol ratio of 3:1 ͑1200:400 mg/L͒. On day 108, the OLR was raised to 9.2 kg COD/m3/day at an influent phenolic substrate Simultaneous Biodegradation of Phenol and p-Cresol concentration of 5 g COD/L, that produced a decrease in the total in Continuous Up-Flow Anaerobic Sludge Bed COD removal efficiency to less than 50%. In this period, the Reactors phenol and p-cresol removal efficiency dropped to 50% and 20%, respectively. As shown in Fig. 2, a high COD removal efficiency Two laboratory scale UASB reactors, R1 and R2, were operated was restored within a couple of days after the OLR was reduced for 5 months in order to investigate the continuous anaerobic to 3.4 kg COD/m3/day. treatment of a mixture of phenol and p-cresol as the main carbon These results indicate that, the maximum OLR that could be and energy sources. After acetate was completely consumed and applied to the reactors was 7 kg COD/m3/day. The average meth- removed from the feeding on day 64, the substrates of the reactors ane production (mL CH4 /day) in both reactors during steady- had been gradually replaced with phenol and p-cresol. During this state operation is presented in Table 2. The measured methane period of acclimatization, phenol was removed more completely production in both reactors accounted for an average of 85% of compared to the p-cresol as the phenol removal efficiency was the COD removed during steady-state operation. No accumula- 85%; whereas the p-cresol removal efficiency was less than 60%. tion of aromatic intermediates was detected based on HPLC The total COD removal efficiency reached 85% after 55 days of analyses of the effluent. operation with the phenolic substrates which was considered to be the moment in time the biomass became adapted to the biodegra- Anaerobic Batch Biodegradability of Phenol and dation of the two phenolic compounds. Thereafter, the concentra- Cresols tion of phenols in the influent was increased periodically after at least 10 HRT and when greater than 85% COD removal was The specific biodegradation rates of phenolic compounds mix- obtained. The operational parameters and treatment efficiencies tures were evaluated using nonadapted and adapted sludge under obtained during the continuous operation of the two reactors are batch methanogenic conditions. Additionally, the effect of cresols summarized in Table 2. on the phenol degradation was evaluated. The biodegradation The time course of the treatment performance of R1 is shown under methanogenic conditions of phenol/p-cresol at different in Fig. 1. R1 was operated for more than 100 days with a COD concentrations was observed as indicated by the decrease in the removal as high as 94%, even when the OLR was increased step- concentration of each compound and the cumulative methane pro- wise to 6.8 kg COD/m3/day. The phenolic compounds concentra- duction. The biodegradation was demonstrated as indicated by the tion in the influent during these operation periods was increased high recovery of methane compared to the theoretical production

JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / NOVEMBER 2003 / 1001 Table 2. Operational Parameters and Treatment Efficiency During Continuous Operation of Up-Flow Anaerobic Sludge Bed Reactors Treating Phenol and p-Cresol Mixture as Main Carbon and Energy Sources Operation period ͑days͒ Reactor 49–60 68–73 77–82 87–94 96–104 108–112 125–135 Reactor 1 Influent phenols concentration 1 1.5 2 3 4 4 1 ͑g chemical oxygen demand/L͒ Phenol/p-cresol ratio 2:1 2:1 2:1 2:1 2:1 1:1 2:1 Organic loading rate 3.9 3.2 3.6 5.6 6.8 7.7 1.7 ͑kg chemical oxygen demand/m3/day͒ Efficiencies Total chemical oxygen 86.4Ϯ3.8 88.7Ϯ2.7 90.3Ϯ2.5 94.1Ϯ1.9 93.9Ϯ0.73 43Ϯ12 66.6Ϯ9.8 demand removal ͑%͒ ͑ ͒ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ CH4 production mL/day 248 11 205 6 252 23 364 20 454 20 150 20 119 18 49–60 68–73 77–82 83–96 101–107 108–112 119–135 Reactor 2 Influent phenols concentration 1 1.5 2 3 4 5 2 ͑g chemical oxygen demand/L͒ Phenol/p-cresol ratio 2:1 2:1 2:1 2:1 3:1 3:1 3:1 Organic loading rate 3.9 3.3 3.6 5.5 7.2 9.2 3.4 ͑kg chemical oxygen demand/m3/day͒ Efficiencies Total chemical oxygen 88.9Ϯ2.2 89.4Ϯ2.5 92.6Ϯ1.9 92.6Ϯ2.9 90.7Ϯ1.5 53Ϯ27 88.5Ϯ7.3 demand removal ͑%͒ ͑ ͒ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ CH4 production mL/day 300 13 213 13 268 26 358 32 486 18 370 66 265 16 Note: Acetate was added to the feeding; 1 g chemical oxygen demand/L fromdays 0–60 and 0.5 g chemical osygen demand/L fromdays 61–64. The reactors were operated at a hydraulic retention time of 0.5–0.6 days.

͑TMP͒ in those treatments amended with phenol and p-cresol as riod are illustrated in Fig. 3 and Table 3. The initial OLR was shown in Table 1 for adapted granular sludge. maintained at an average of 2.95 kg COD/m3/day at a phenol/ The lag phase prior to the onset of biodegradation using non- cresols ratio of 2:1. The COD removal efficiency was higher than adapted granular sludge was 20 days. Phenol and p-cresol con- 80%. The phenol and p-cresol were completely removed as was centrations were completely depleted but at very low SBRs: 2.5 indicated by their very low concentrations in the effluent. Regard- and 1.1 mg COD/g VSS/day for phenol and p-cresol, respectively, ing o-cresol, interestingly, during a period of 50 days, this com- as single compounds, whereas the average SBR for phenol and pound was removed with efficiencies around 60%. On day 192, p-cresol in mixture was 2.8 and 1.8 mg COD/g VSS/day, respec- the removal efficiency increased up to 80% and remained at that tively. In the experiments performed with adapted granular sludge value for 80 more days ͑days 192–272͒. Consequently, a steady- using a mixture of phenol, p-, and o-cresol, the lag phase was state was obtained with total COD removal greater than 80%. As negligible and the SBR were approximately two orders of mag- no metabolite was detected based on HPLC analyses, possibly ͑ nitude higher than that observed with nonadapted sludge Table o-cresol was biodegraded to methane and CO2 . On day 272, the 1͒, indicating enrichment of phenol- and p-cresol-degrading mi- OLR was increased to an average of 5 kg COD/m3/day. After 10 croorganisms during the continuous experiments. From evaluat- days under these conditions, the removal efficiencies for p-cresol ing the interaction of substrates, p- and o-cresol did not signifi- and o-cresol dropped to 20%. The phenol biodegradation was less cantly affect phenol biodegradation. On the other hand, both affected as an efficiency of 60% was maintained, in agreement phenol ͑200 mg/l͒ and o-cresol ͑150 mg/l͒ negatively affected with the results obtained from batch experiments. At the time, the p-cresol biodegradation as indicated by the decrease in the SBR former OLR conditions were reestablished and the biodegradation of p-cresol. The impact of o-cresol was even more severe than of each phenolic component of the mixture recovered to the prior that of phenol. o-cresol disappeared completely from the medium levels. The total COD removal obtained was greater than 70%. but was not biodegraded to methane. Instead, an unidentified me- The average methane production (mL CH4 /day) in this reactor tabolite detected by HPLC accumulated concomitantly with the during steady-state operation is presented in Table 3. The methane disappearance of o-cresol. production in this reactor accounted for an average of 96% of the COD removed during steady-state operation. Effect of the Presence of o-Cresol in the Mixture Phenol and p-Cresol in the Continuous Reactor Discussion The UASB reactor R2 was selected for continued operation for an additional experiment evaluating the role of o-cresol. The main Effluents from the petroleum industry are expected to contain objective of this experiment was to study the effect of o-cresol as mixtures of phenol and cresols as the main COD bearing frac- a third compound introduced into the phenolic substrate mixture, tions. Thus, for successful anaerobic treatment of petroleum ef- toward the biodegradability of phenol and p-cresol. The opera- fluents, an important prerequisite is the simultaneous degradation tional conditions and performance of the reactor during this pe- of phenols and cresols. In this study, phenolic mixtures containing

1002 / JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / NOVEMBER 2003 Fig. 1. Operational efficiency during the continuous anaerobic treatment of a phenol/p-cresol mixture in the up-flow anaerobic sludge Fig. 2. Operational efficiency during the continuous anaerobic treat- bed R1: ͑A͒ organic loading rate, influent phenol, and p-cresol con- ment of a phenol/p-cresol mixture in the up-flow anaerobic sludge centration; ͑B͒ total chemical oxygen demand removal; and ͑C͒ phe- bed R2: ͑A͒ organic loading rate, influent phenol, and p-cresol con- nol and p-cresol removal determined by high-pressure liquid chroma- centration; ͑B͒ total chemical oxygen demand removal; and ͑C͒ phe- tography. Acetate was added to the feeding; 1 g chemical oxygen nol and p-cresol removal determined by high-pressure liquid chroma- demand/L from days 0–60 and 0.5 g chemical oxygen demand/L tography. Acetate was added to the feeding; 1 g chemical oxygen from days 61–64. demand/L from days 0–60 and 0.5 g chemical oxygen demand/L from days 61–64. phenol and cresols were successfully mineralized in laboratory and R2, respectively, COD removal efficiencies higher than 65% scale UASB reactors under methanogenic conditions. The results were obtained in both reactors. However, when comparing the of this study confirmed that OLR as high as 7 kg COD/m3/day recovery times required for the reactors to return to high COD can be applied with COD removal efficiencies greater than 90% removal efficiencies, the reactor with the higher proportion of with phenol-p-cresol mixture as the main carbon and energy p-cresol ͑R1͒ needed 3 weeks; whereas, the reactor with the lower sources. Approximately 40 and 50 days were required for the proportion of p-cresol ͑R2͒ required only a couple of days. These growth of p-cresol and phenol degrading bacteria, respectively, results indicated that p-cresol concentrations higher than 700 sufficient to accommodate near complete removal of the pheno- mg/L in the influent caused severe inhibition to the microbial lics. Diverse examples of a single phenolic compound degrada- consortium. Fang and Zhou ͑2000͒ observed, in a UASB reactor tion under methanogenic conditions in continuous systems are operated for more than 200 days, a decrease of the removal effi- reported in literature. However, information about the degradation ciency when the p-cresol concentration which was in the range of of a mixture of phenolic compounds is not very common. Table 4 400–500 mg/L, and 1000 mg/L of phenol was present. Puig- presents a comparison of some continuous anaerobic treatment Grajales et al. ͑2000͒, reported a 50% decrease in acetoclastic results of phenolic compounds, single, or in mixture, from litera- methanogenic activity for granular sludge at phenol and p-cresol ture together with the ones obtained in this work. The results of concentrations of 1504 mg/L and 408 mg/L, respectively. Wang this study indicate that the OLR applied to the UASB reactors fed et al. ͑1988͒, reported that in batch experiments using a phenol- with a phenolic mixture are similar to previous studies where only enriched methanogenic culture, concentrations of p-cresol be- a single phenolic compound was used. Full biodegradation of tween 500 and 700 mg/L dramatically reduced the phenol biodeg- phenol/p-cresol mixture to methane is thus possible in continuous radation rate ͑200 mg/L͒. When present at high concentrations, UASB reactors. p-cresol was biodegraded at very slow rate, despite the complete In order to clarify the effect of the phenol/p-cresol ratio on the disappearance of phenol. In the same way, organic overloads in reactor efficiencies, R1 and R2 were operated at phenol/p-cresol this study tended to first affect the p-cresol compared to phenol ratios of 1:1 and 3:1 during the last period of operation. At that removal. Fang and Zhou ͑2000͒ found that the effect of OLR was time, the OLR was sharply increased, resulting in a 50% drop in much more drastic in reactors with increasing phenolic concen- the COD removal efficiency in both reactors. When subsequently trations than in reactors with constant phenolic concentrations but the OLRs were reduced to 1.7 and 3.4 kg COD/m3/day, for R1 decreased the HRT. From these observations, we conclude that

JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / NOVEMBER 2003 / 1003 Based on the results of Table 1, a mixture of three phenolic compounds can be treated by anaerobic methods. Phenol SBR was not affected by the presence of p-cresol, and even the biodegradation rate increased. On the other hand, p-cresol SBR was significatively affected by the presence of both phenol and o-cresol. In the continuous experiments, different lag periods were observed for high levels of phenolic removal efficiency. Complete p-cresol degradation was established prior to that of phenol. Sludge which was already adapted to the degradation of these two phenolics, also degraded o-cresol at greater than 80% removal efficiency after 60 days. These results could indicate that phenol and p-cresol are anaerobically biodegraded by different kinds of microorganisms possessing different pathways. Phenol degradation is initiated by phosphorylation of an OH group followed by carboxylation of the ring in the paraposition ͑Schink et al. 2000͒; whereas, p-cresol degradation is initiated by fumu- rate addition to the methyl group forming benzyl succinate ͑Mu¨ller et al. 2001͒. Regarding o-cresol, this compound was completely transformed to an unidentified compound in the batch assays with unadapted sludge. In the continuous columns exposed to o-cresol for extended periods of time, an o-cresol-degrading population had developed. The evidence is based on high levels of o-cresol removal in the adapted columns and by the ability of adapted sludge to convert o-cresol to methane. These observations con- trast previous findings in which o-cresol has generally been ob- Fig. 3. Operational efficiency during the continuous anaerobic treat- served to be recalcitrant to degradation under methanogenic con- ment of a phenol/p-cresol/o-cresol mixture in the up-flow anaerobic ditions. The majority of the reports indicate that o-cresol was not sludge bed R2: ͑A͒ organic loading rate, total, and individual; ͑B͒ mineralized ͑Fedorak and Hudrey 1984; Blum et al. 1986; Wang phenol, p-cresol, and o-cresol removal determined by high-pressure et al. 1989; Razo-Flores et al. 1996b͒. liquid chromatography Consequently, the observation that o-cresol could be biodegraded in the continuous UASB reactor treating a mixture of three phenolic compounds is of technological significance. The phenol the residual p-cresol concentration in R1 was responsible for the and p-cresol were completely removed from the influent and, prolonged inhibition effect observed in this study. Batch experi- after an adequate period of adaptation, 85% of o-cresol supplied ments conducted with the adapted granular sludge confirmed ͑110 mg/L͒ was also removed. Table 4 presents a comparison of these results. In one report ͑Wang et al. 1988͒, diauxic behavior literature reported data on the treatment of phenolic mixture of was observed in which no biodegradation of p-cresol occurred more than two compounds in continuous anaerobic reactors. Ex- until phenol was completely utilized. In our case, we did not periments carried out in fixed film anaerobic reactors with observe any diauxic phenomenon. methanogenic-enriched consortia achieved the biodegradation for The batch assays indicated orders of magnitude increases in more than 90% for each element of the mixture phenol, p- and, the phenol and p-cresol degradation activity of granular sludge o-cresol ͑100, 35, and 35 mg/L͒ after a period of 350 days using during the continuous experiments, clearly indicating that the en- two different enriched anaerobic consortia ͑Tawfiki et al. 1999͒. richment and growth of appropriate phenol and p-cresol degraders Charest et al. ͑1999͒, under the same conditions, eliminated 78% took place. Bioaugmentation of enrichment cultures to anaerobic of o-cresol ͑21 mg/L͒ and 97% ͑144 mg/L͒ of phenol in an efflu- reactors, therefore, can be utilized to decrease the lag periods for ent from the petrochemical industry. However, in both cases, the steady-state operation on phenolic wastewaters ͑Guiot et al. 2000; consortia used required proteose peptone or whey as a cosub- Tawfiki et al. 2000͒. strate, and the consortia removing phenol and o-cresol cannot

Table 3. Operational Parameters and Treatment Efﬁciency During Continuous Operation of Up-flow Anaerobic Sludge Bed R2 Treating a Phenol, p-Cresol, and o-Cresol Mixture as Main Carbon and Energy Sources Operation period ͑days͒ R2 231–275 279–314 320–350 Hydraulic retention time ͑days͒ 0.67Ϯ0.03 0.71Ϯ0.12 0.77Ϯ0.12 Organic loading rate ͑kg chemical oxygen demand/m3/day͒ 2.95Ϯ0.31 4.99Ϯ0.33 2.86Ϯ0.54 Phenol/cresols ratio 2:1 1:1 2:1

Efﬁciencies Total chemical oxygen demand removal ͑%͒ 81.8Ϯ4.8 60.8Ϯ17.7 70.5Ϯ12.7 ͑ ͒ Ϯ Ϯ Ϯ CH4 production mL/day 194 26 238 46 157 29

1004 / JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / NOVEMBER 2003 Table 4. Comparison of Continuous Anaerobic Treatment Results of Phenolic Compounds, Single Compounds, or in Mixture, from Literature

Organic loading rate Chemical oxygen CH4 ͑kg chemical oxygen demand Compound ͑% chemical oxygen Compound Reactor removal/m3/day͒ removal ͑%͒ removal ͑%͒ demand removed͒ Reference Phenol Anaerobic 0.31 95.0 100.0 — Suidan et al. ͑1988͒ fermentor Phenol Expanded bed 7.0 100.0 100.0 — Wang et al. ͑1986͒ Phenol Activated carbon 6.32 80.0–84.0 — 42.0–63.0 Gardner et al. ͑1988͒ anaerobic filter Phenol Up-flow anaerobic 6.0 97.7 99.9 94.7 Fang et al. ͑1996͒ sludge bed Phenol Anaerobic 99.0 Formaldehydeactivated carbon 3.4 95.0 — 71.4 Goeddertz et al. ͑1990͒ Methanol — p-Cresol Up-flow anaerobic 7.2 99.9 — 91.6 Hwang and Chen ͑1991͒ sludge bed p-Cresol Up-flow anaerobic 2.5 80.0 80.0 — Kennes et al. ͑1997͒ sludge bed Phenol Up-flow anaerobic 8.12 85.0 95.0 93.6 Fang and Zhou ͑2000͒ p-Cresol sludge bed 65.0 Phenol Up-flow anaerobic sludge bed 4.3 — 98.0 — Zhou and Fang ͑1997͒ m-Cresol 20.0 Phenol Fixed film upflow 100.0 p-Cresol anaerobic reactor 2.12 94.0 91.0 — Tawfiki et al. ͑1999͒ o-Cresol 100.0 Phenol Bioaugmented 100.0 p-Cresol enriched consortium 0.66 85.0 100.0 — Tawfiki et al. ͑2000͒ o-Cresol Up-flow anaerobic sludge bed 77.0 Phenol 97.0 p-Cresol Fixed film anaerobic 2.0 — 10.0 — Charest et al. ͑1999͒ m-Cresol Filter 5.0 o-Cresol 78.0 Phenol Up-flow anaerobic 7.0 94.0 100.0 80.6 This work p-Cresol sludge bed ͑R1͒ 93.0 Phenol Up-flow anaerobic 7.1 91.0 100.0 85 This work p-Cresol sludge bed ͑R2͒ 85.0 Phenol 100.0 p-Cresol Up-flow anaerobic 2.95 81.8 100.0 110 This work sludge bed ͑R2͒ o-Cresol 80.0 degrade p-cresol ͑Bisaillon et al. 1993; Charest et al. 1999; Taw- SBR of the phenolic compounds and the capability to degrade fiki et al. 1999͒. mixtures of these compounds including the persistent o-cresol.

Conclusions Acknowledgments

UASB reactors were successfully used to study the biodegrada- This work was supported by the IMP Project Nos. D.00021 and tion of phenol and p-cresol mixtures at an OLR of 7 kg D.00037 and by the Consejo Nacional de Ciencia y Tecnologıá 3 COD/m /day. Also, tertiary mixtures, which included o-cresol, ͑CONACYT͒ from Mexico, Project No. 31537-B. The writers were treated with a global COD removal up to 82% at an OLR of acknowledge the technical assistance of Romań Castan˜eda. 3 kg COD/m3/day and o-cresol was biodegraded at an average concentration of 110 mg/L. These results indicate that methanogenic treatment can be successfully used for biodegradation of References phenol/cresols mixtures representative of major substrates in chemical and petrochemical wastewaters. The most important American Public Health Association ͑APHA͒. ͑1985͒. Standard methods control parameter was the toxicity of each element of the mixture. for examination of water and wastewater, 16th Ed., American Public In the case of cresols, their concentration should not exceed 600 Health Association, Washington, D.C. mg/L. The use of acclimated granular sludge, enriched with phe- Berne´, F., and Cordonnier, J. ͑1995͒. ‘‘Treatment of spent caustic.’’ In- nol and p-cresol degrading microorganisms, highly increased the dustrial water treatment: Refining petrochemicals and gas processing

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