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. Continuous experiments were carried out in up-flow anaerobic sludge bed 共UASB兲 reactors degrading a mixture of phenol and p-cresol as the main carbon and energy sources. The total chemical oxygen demand 共COD兲 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 共phenol, p-cresol, and o-cresol兲 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 共supplied at 132 mg/L兲 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/共ASCE兲0733-9372共2003兲129:11共999兲 CE Database subject headings: Anaerobic processes; Biodegradation; Phenol; Methane; Sludge; Cisterns; Wastewater treatment.
Introduction Phenolic compounds are present in the wastewaters of the chemical and petrochemical industries and represent an important source of environmental pollution 共Berne´ and Cordonnier 1995兲. Wastewaters from a refinery are a complex mixture of organic and inorganic compounds, often containing more than one type of phenolic compound. Phenol and cresols are major constituents found in refinery effluents 共Berne´ and Cordonnier 1995兲. Phenol has been recognized as being either toxic or lethal to fish at concentrations of 5 to 25 mg/L 共Hill and Robinson 1975兲. Many substituted phenols, including chlorophenols, nitrophenols, and cresols have been designated as priority pollut-ants by the U.S. Environmental Protection Agency 共Keith and Telliard 1979兲. 1 Research Group Leader, Programa de Biotecnologı´a, Instituto Mexicano del Petro´leo, Eje Central La´zaro Ca´rdenas 152, C.P. 07730, Me´xico, D.F. 共corresponding author兲. E-mail:
[email protected] 2 Research Fellow, Programa de Biotecnologı´a, Instituto Mexicano del Petro´leo. Eje Central La´zaro Ca´rdenas 152, C.P. 07730, Me´xico, D.F. 3 Associate Professor, Department of Chemical and Environmental Engineering, Univ. of Arizona. P.O. Box 210011 Tucson, AZ 85721-0011. E-mail:
[email protected] 4 Researcher, Programa de Biotecnologı´a, Instituto Mexicano del Petro´leo, Eje Central La´zaro Ca´rdenas 152, C.P. 07730, Me´xico, D.F. 5 Researcher, Programa de Biotecnologı´a, Instituto Mexicano del Petro´leo, Eje Central La´zaro Ca´rdenas 152, C.P. 07730, Me´xico, D.F. Note. Associate Editor: Spyros G. Pavlostathis. Discussion open until April 1, 2004. Separate discussions must be submitted for individual papers. To extend the closing date by one month, a written request must be filed with the ASCE Managing Editor. The manuscript for this paper was submitted for review and possible publication on March 12, 2002; approved on November 8, 2002. This paper is part of the Journal of Environmental Engineering, Vol. 129, No. 11, November 1, 2003. ©ASCE, ISSN 0733-9372/2003/11-999–1006/$18.00.
Among the possible treatment methods, anaerobic treatment is a viable option due to the anaerobic biodegradability of phenolic compounds. Many authors have studied the biodegradation of phenol and cresols in methanogenic consortia utilizing batch assays 共Fedorak and Hudrey 1984; Blum et al. 1986; Smolenski and Suflita 1987; Bisaillon et al. 1991, 1993; Razo-Flores et al. 1996a,b; Kennes et al. 1997兲. However, the majority of these studies were conducted using single phenolics, like phenol or p-cresol. The biological treatment of complex phenolic wastewaters, has often been studied utilizing activated carbon 共AC兲 in anaerobic reactors; where, the AC served to adsorb toxic phenolic pollutants and acted as a carrier for bacterial growth 共Nakhla et al. 1989; Nakhla et al. 1990兲. There are fewer references on the continuous anaerobic treatment of mixed phenolic compounds without any dose of AC 共Zhou and Fang 1997; Fang and Zhou 2000; Tawfiki et al. 2000兲. The bioreactor systems most commonly described for the anaerobic treatment of phenolics are the up-flow anaerobic sludge bed 共UASB兲 reactor 共Hwang and Cheng 1991; Chang et al. 1995; Zhou and Fang 1997; Kennes et al. 1997兲 and the fixed film anaerobic reactor 共Charest et al. 1999兲. The successful treatment of petroleum effluents will require that major phenolic substrates 共phenol and cresols兲 should be degraded simultaneously. Therefore, biodegradation studies should evaluate mixtures of phenols. In this study, continuous and batch experiments with anaerobic granular sludge were conducted in order to study the biodegradation of phenolic mixtures and investigate the effect of specific phenolic compounds 共p- and o-cresol兲 on the phenol biodegradation. Maximum volumetric loading rates were determined in continuous UASB reactors using phenol and p-cresol as the main carbon and energy sources. Specific biodegradation rates 共SBR兲 were determined for single and mixtures of phenolic compounds.
Materials and Methods Inoculum The methanogenic granular sludges used in this study were obtained from two different UASB reactors treating industrial effluents: Cuauhtemoc Brewery 关共CB兲 Puebla, Mexico兴 and Shell Nederland Chemie 关共SNC兲 Moerdijk, The Netherlands兴. The sludges were elutriated to remove the fines and predigested at 30°C during 30 days in order to deplete most of the endogenous substrates in the sludge. The characteristics of the sludges were: 8% and 9.5% volatile suspended solids 共VSS兲; 0.2 and 0.43 g chemical oxygen demand 共COD兲-CH4 /g VSS/day of acetoclastic methanogenic activity, for the CB and SNC sludges, respectively.
Basal Medium The basal medium used during the experiments contained the following compounds 共mg/L兲: NaHCO3 (5,000), NH4 Cl (280), CaCl2 •2H2 O (10), K2 HPO4 (250), MgSO4 (100), yeast extract 共100兲, and 1 mL of micronutrients stock solution which contained 共mg/L兲: FeCl2 •4H2 O (2,000), H3 BO3 (50), ZnCl2 (50), CuCl2 •2H2 O (38), MnCl2 •4H2 O (500), (NH4 ) 6 Mo7 O24 •4H2 O (50), AlCl3 •6H2 O (90), CoCl2 •6H2 O (2,000), NiCl2 •6H2 O (142), Na2 SeO•5H2 O (164), EDTA 共1,000兲, resazurin 共200兲, and HCl 36% 共1 mL兲. The final pH of the medium was 7.2.
Simultaneous Biodegradation of Phenol and p-Cresol in Up-Flow Anaerobic Sludge Bed Reactors The continuous experiments were performed in two separate glass UASB reactors 共0.145 m in length and 0.039 m of internal diameter兲 with liquid volumes of 160 mL, placed in a temperature controlled room at 30°C. Both reactors were inoculated with 40 g 共wet weight兲 of a mixture of granular sludge 共80% CB—sludge and 20% SNC—sludge兲. The reactors were operated at a hydraulic retention time 共HRT兲 of 0.5–0.6 days during the experiments. The influent was delivered to the reactors with a variable speed Masterflex Digital Pump 共Cole-Parmer, Vernon Hills, Ill.兲 at a flow rate of 270–320 mL/day. The reactors were started up with acetate 共1 g COD/L兲 and after 1 month of operation 共designated as day 0 in figures and tables兲, the reactors additionally received a mixture of phenol and p-cresol, 280 and 132 mg/L, respectively 共1 g COD/L兲, to provide a total organic loading rate 共OLR兲 of 4 kg COD/m3/day. The acetate concentration in the feeding was maintained until day 60, being reduced to half and completely removed from the feeding between days 61 to 64. Afterward, the inlet phenolics concentration was increased stepwise in order to determine the maximum OLR of the phenolic substrate that could be applied to the reactors. The methane production was measured by liquid displacement using a 4% 共weight/volume兲 NaOH solution to scrub out the carbon dioxide from the biogas. The performance of the reactors was monitored by measuring the pH, methane, and the concentration of the phenolic compounds and COD in the influent and effluent.
Effect of o-Cresol on the Phenol and p-Cresol Biodegradation in a Continuous Up-Flow Anaerobic Sludge Bed Reactor The operation of one of the reactors was extended for an additional period of 200 days inoculated with 25.6 g 共wet weight兲 of the adapted granular sludge. Phenol, p-cresol, and o-cresol were
introduced to the reactor at the following concentrations: 550 mg/L, 132 mg/L, and 132 mg/L, respectively 共1.98 g COD/L兲, as the main carbon and energy sources. The basal medium was modified for the second stage of operation, the sodium bicarbonate concentration was 2,000 mg/L and the concentration of the other elements was reduced ten times. The performance of the reactor was followed with the same parameters as in the first stage of operation.
Batch Biodegradability Assays The batch anaerobic biodegradability assays were conducted in 124 mL glass serum bottles. The assays with unadapted sludge were conducted with 0.95 g 共wet weight兲 of CB-granular sludge, whereas batch assays with adapted granular sludge were performed using 1 g 共wet weight兲 of phenol-p-cresol adapted granular sludge withdrawn from the UASB reactors at the end of the first stage of the continuous experiments 共after 135 days of operation兲. The inoculum was transferred to the serum bottles, which contained 50 mL of the basal medium. The serum bottles were sealed with 12 mm thick butyl rubber stoppers and flushed with a gas mixture of 30% CO2 and 70% N2 for 5 min. The serum bottles were incubated overnight at 30°C to allow the biological consumption of residual oxygen. One day later, the desired amount of the phenols tested was added with a syringe to triplicated serum bottles from a concentrated stock solution. Experiments with unadapted granular sludge were conducted at the following concentrations 共experiment number indicated in parenthesis兲: phenol, 200 mg/L 共1兲, p-cresol, 150 mg/L 共2兲, and mixtures of phenol at 200 mg/L plus p-cresol at 50 共3兲, 100 共4兲, and 150 mg/L 共5兲, respectively. Experiments with adapted granular were conducted at the concentrations described in Table 1. All phenols were used as the main carbon and energy sources. The serum bottles were incubated at 30°C. The concentration of the compounds and the accumulated methane production was monitored during the duration of the experiment. The SBR of the compounds was calculated from the slope of the phenols concentration versus time curve. The net cumulative methane production was expressed as a percentage of the theoretical methane production 共TMP兲 expected from the test chemical mineralization based on the Tarvin and Buswell 共1934兲 equation. Corrections were made for background levels of methane production monitored in controls lacking any added test compound. The concentrations of phenolic compounds are referred to in COD units, commonly expressed as its equivalent in COD per liter of liquid. Conversion factors utilized were: 2.38 g COD/g phenol, 2.515 g COD/g cresol, and 0.503 L CH4 /g COD at 0.77 atm and 30°C.
Analytical Methods The methane content in the gas samples was determined by gas chromatography 共Perkin Elmer 8500, Mexico City兲. The gas chromatograph was equipped with a steel column 共2.4 m by 3.2 mm兲 packed with Super Q 共80/100 mesh, Millipore Corp., Bedford Mass.兲. 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 content 共100 L兲 in the headspace were determined with a pressure-gas lock syringe 共Pressure-Lok series A-2, Dynatech Precision Sampling Corp., Baton Rouge, LA兲. An isobaric precise proportion of the known headspace volume could be analyzed. Phenolic compounds were analyzed in the centrifuged aqueous phase by reverse-phase high-pressure liquid chromatography
Table 1. Phenolics Concentration 共Single Compound or in Mixture兲 and Specific Biodegradation Rates Determined in Batch Experiments Using
Phenol/p-Cresol Adapted Granular Sludge 共2 g Volatile Suspended Solids/L兲 Withdrawn from Reactor 2 at End of Continuous Experiment 共Day 135兲 Compound 共mg/L兲/specific biodegradation rate 共mg chemical oxygen demand/g volatile suspended solids/day兲
Experiment 1 2 3 4 5 6
Phenol
p-Cresol
o-Cresol
Methane 共% theoretical methane production兲a
25/10.1⫾0.9 75/20.9⫾0.9 150/30.1⫾0.5
102⫾ 1.4 97⫾ 2 102⫾ 3 98⫾3b 95⫾2b 100⫾4b
200/135.4⫾0.7 200/156.3⫾2.5 200/132.5⫾6.6 200/116.6⫾4.8 200/121.4⫾5.7
100/92.5⫾3.9 100/75.1⫾4.1 100/73.9⫾2.8 100/71.1⫾2.3 100/55.6⫾3.1
Note: o-Cresol was completely transformed to an unidentified compound that was not further degraded. a Methane 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. b The theoretical methane production was estimated based on phenol and p-cresol degradation.
共HPLC Hewlett Packard 1100, Mexico City兲 using a Silica C-8 共ZORBAX兲 column. Absorbance was detected at 270 nm and the column temperature was 25°C. The solvent phase was methanol/ water 共60%/40%兲 with a flow of 1 mL/min. The size of the sample was 10 L. The retention time of the compounds were 3, 3.8, and 4 min for phenol, p-cresol, and o-cresol, respectively. The pH was determined immediately after sampling with an Accument 915 pH meter 共Fisher Scientific, Mexico City兲 and a Corning electrode 共Acton, Mass.兲. All the other analytical determinations 共VSS and COD兲 were performed as described in American Public Health Association 共1985兲. Phenol, p-cresol 共J. T. Baker, Mexico City兲, o-cresol 共Merck, Mexico City兲, and all the other chemicals were of the highest purity available.
Results Simultaneous Biodegradation of Phenol and p-Cresol in Continuous Up-Flow Anaerobic Sludge Bed Reactors Two laboratory scale UASB reactors, R1 and R2, were operated for 5 months in order to investigate the continuous anaerobic treatment of a mixture of phenol and p-cresol as the main carbon and energy sources. After acetate was completely consumed and removed from the feeding on day 64, the substrates of the reactors had been gradually replaced with phenol and p-cresol. During this period of acclimatization, phenol was removed more completely compared to the p-cresol as the phenol removal efficiency was 85%; whereas the p-cresol removal efficiency was less than 60%. The total COD removal efficiency reached 85% after 55 days of operation with the phenolic substrates which was considered to be the moment in time the biomass became adapted to the biodegradation of the two phenolic compounds. Thereafter, the concentration of phenols in the influent was increased periodically after at least 10 HRT and when greater than 85% COD removal was obtained. The operational parameters and treatment efficiencies obtained during the continuous operation of the two reactors are summarized in Table 2. The time course of the treatment performance of R1 is shown in Fig. 1. R1 was operated for more than 100 days with a COD removal as high as 94%, even when the OLR was increased stepwise to 6.8 kg COD/m3/day. The phenolic compounds concentration in the influent during these operation periods was increased
from 1 to 4 g COD/L at a phenol/p-cresol ratio of 2:1. Similar OLR 共7.7 kg COD/m3/day兲 applied to the reactor but at a phenol/ p-cresol ratio of 1:1 共800:800 mg/L兲, exhibited a decrease of the total COD removal efficiency below 50%. The phenol and p-cresol removal dropped to 40% and 20%, respectively. The efficiency drop suggested an inhibitory effect toward the anaerobic biomass. After the OLR was reduced to 1.7 kg COD/m3/day, R1 required 20 days to reach a COD removal of 80% 共Fig. 1兲. R2 was operated in parallel under similar operational conditions as R1, except during the last 30 days of operation at which time the ratio of phenol to p-cresol was increased instead of decreased. The time course of the treatment performance of R2 is shown in Fig. 2. Similarly to R1, R2 exhibited a high COD removal efficiency 共90%兲 even at phenolic substrate concentration of 4 g COD/L and an OLR of 7.2 kg COD/m3/day but at a phenol/ p-cresol ratio of 3:1 共1200:400 mg/L兲. On day 108, the OLR was raised to 9.2 kg COD/m3/day at an influent phenolic substrate concentration of 5 g COD/L, that produced a decrease in the total COD removal efficiency to less than 50%. In this period, the phenol and p-cresol removal efficiency dropped to 50% and 20%, respectively. As shown in Fig. 2, a high COD removal efficiency was restored within a couple of days after the OLR was reduced to 3.4 kg COD/m3/day. These results indicate that, the maximum OLR that could be applied to the reactors was 7 kg COD/m3/day. The average methane production (mL CH4 /day) in both reactors during steadystate operation is presented in Table 2. The measured methane production in both reactors accounted for an average of 85% of the COD removed during steady-state operation. No accumulation of aromatic intermediates was detected based on HPLC analyses of the effluent.
Anaerobic Batch Biodegradability of Phenol and Cresols The specific biodegradation rates of phenolic compounds mixtures were evaluated using nonadapted and adapted sludge under batch methanogenic conditions. Additionally, the effect of cresols on the phenol degradation was evaluated. The biodegradation under methanogenic conditions of phenol/p-cresol at different concentrations was observed as indicated by the decrease in the concentration of each compound and the cumulative methane production. The biodegradation was demonstrated as indicated by the high recovery of methane compared to the theoretical production
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 共days兲 Reactor Reactor 1 Influent phenols concentration 共g chemical oxygen demand/L兲 Phenol/p-cresol ratio Organic loading rate 共kg chemical oxygen demand/m3/day兲 Efficiencies Total chemical oxygen demand removal 共%兲 CH4 production 共mL/day兲 Reactor 2 Influent phenols concentration 共g chemical oxygen demand/L兲 Phenol/p-cresol ratio Organic loading rate 共kg chemical oxygen demand/m3/day兲 Efficiencies Total chemical oxygen demand removal 共%兲 CH4 production 共mL/day兲
49– 60
68 –73
77– 82
87–94
96 –104
108 –112
125–135
1
1.5
2
3
4
4
1
2:1 3.9
2:1 3.2
2:1 3.6
2:1 5.6
2:1 6.8
1:1 7.7
2:1 1.7
86.4⫾3.8
88.7⫾2.7
90.3⫾2.5
94.1⫾1.9
93.9⫾0.73
43⫾12
66.6⫾9.8
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
1
1.5
2
3
4
5
2
2:1 3.9
2:1 3.3
2:1 3.6
2:1 5.5
3:1 7.2
3:1 9.2
3:1 3.4
88.9⫾2.2
89.4⫾2.5
92.6⫾1.9
92.6⫾2.9
90.7⫾1.5
53⫾27
88.5⫾7.3
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.
共TMP兲 in those treatments amended with phenol and p-cresol as shown in Table 1 for adapted granular sludge. The lag phase prior to the onset of biodegradation using nonadapted granular sludge was 20 days. Phenol and p-cresol concentrations were completely depleted but at very low SBRs: 2.5 and 1.1 mg COD/g VSS/day for phenol and p-cresol, respectively, as single compounds, whereas the average SBR for phenol and p-cresol in mixture was 2.8 and 1.8 mg COD/g VSS/day, respectively. In the experiments performed with adapted granular sludge using a mixture of phenol, p-, and o-cresol, the lag phase was negligible and the SBR were approximately two orders of magnitude higher than that observed with nonadapted sludge 共Table 1兲, indicating enrichment of phenol- and p-cresol-degrading microorganisms during the continuous experiments. From evaluating the interaction of substrates, p- and o-cresol did not significantly affect phenol biodegradation. On the other hand, both phenol 共200 mg/l兲 and o-cresol 共150 mg/l兲 negatively affected p-cresol biodegradation as indicated by the decrease in the SBR of p-cresol. The impact of o-cresol was even more severe than that of phenol. o-cresol disappeared completely from the medium but was not biodegraded to methane. Instead, an unidentified metabolite detected by HPLC accumulated concomitantly with the disappearance of o-cresol.
Effect of the Presence of o-Cresol in the Mixture Phenol and p-Cresol in the Continuous Reactor The UASB reactor R2 was selected for continued operation for an additional experiment evaluating the role of o-cresol. The main objective of this experiment was to study the effect of o-cresol as a third compound introduced into the phenolic substrate mixture, toward the biodegradability of phenol and p-cresol. The operational conditions and performance of the reactor during this pe-
riod are illustrated in Fig. 3 and Table 3. The initial OLR was maintained at an average of 2.95 kg COD/m3/day at a phenol/ cresols ratio of 2:1. The COD removal efficiency was higher than 80%. The phenol and p-cresol were completely removed as was indicated by their very low concentrations in the effluent. Regarding o-cresol, interestingly, during a period of 50 days, this compound was removed with efficiencies around 60%. On day 192, the removal efficiency increased up to 80% and remained at that value for 80 more days 共days 192–272兲. Consequently, a steadystate was obtained with total COD removal greater than 80%. As no metabolite was detected based on HPLC analyses, possibly o-cresol was biodegraded to methane and CO2 . On day 272, the OLR was increased to an average of 5 kg COD/m3/day. After 10 days under these conditions, the removal efficiencies for p-cresol and o-cresol dropped to 20%. The phenol biodegradation was less affected as an efficiency of 60% was maintained, in agreement with the results obtained from batch experiments. At the time, the former OLR conditions were reestablished and the biodegradation of each phenolic component of the mixture recovered to the prior levels. The total COD removal obtained was greater than 70%. The average methane production (mL CH4 /day) in this reactor during steady-state operation is presented in Table 3. The methane production in this reactor accounted for an average of 96% of the COD removed during steady-state operation.
Discussion Effluents from the petroleum industry are expected to contain mixtures of phenol and cresols as the main COD bearing fractions. Thus, for successful anaerobic treatment of petroleum effluents, an important prerequisite is the simultaneous degradation of phenols and cresols. In this study, phenolic mixtures containing
Fig. 1. Operational efficiency during the continuous anaerobic treatment of a phenol/p-cresol mixture in the up-flow anaerobic sludge bed R1: 共A兲 organic loading rate, influent phenol, and p-cresol concentration; 共B兲 total chemical oxygen demand removal; and 共C兲 phenol and p-cresol removal determined by high-pressure liquid chromatography. Acetate was added to the feeding; 1 g chemical oxygen 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 scale UASB reactors under methanogenic conditions. The results of this study confirmed that OLR as high as 7 kg COD/m3/day can be applied with COD removal efficiencies greater than 90% with phenol-p-cresol mixture as the main carbon and energy sources. Approximately 40 and 50 days were required for the growth of p-cresol and phenol degrading bacteria, respectively, sufficient to accommodate near complete removal of the phenolics. Diverse examples of a single phenolic compound degradation under methanogenic conditions in continuous systems are reported in literature. However, information about the degradation of a mixture of phenolic compounds is not very common. Table 4 presents a comparison of some continuous anaerobic treatment results of phenolic compounds, single, or in mixture, from literature together with the ones obtained in this work. The results of this study indicate that the OLR applied to the UASB reactors fed with a phenolic mixture are similar to previous studies where only a single phenolic compound was used. Full biodegradation of phenol/p-cresol mixture to methane is thus possible in continuous UASB reactors. In order to clarify the effect of the phenol/p-cresol ratio on the reactor efficiencies, R1 and R2 were operated at phenol/p-cresol ratios of 1:1 and 3:1 during the last period of operation. At that time, the OLR was sharply increased, resulting in a 50% drop in the COD removal efficiency in both reactors. When subsequently the OLRs were reduced to 1.7 and 3.4 kg COD/m3/day, for R1
Fig. 2. Operational efficiency during the continuous anaerobic treatment of a phenol/p-cresol mixture in the up-flow anaerobic sludge bed R2: 共A兲 organic loading rate, influent phenol, and p-cresol concentration; 共B兲 total chemical oxygen demand removal; and 共C兲 phenol and p-cresol removal determined by high-pressure liquid chromatography. Acetate was added to the feeding; 1 g chemical oxygen demand/L from days 0– 60 and 0.5 g chemical oxygen demand/L from days 61– 64.
and R2, respectively, COD removal efficiencies higher than 65% were obtained in both reactors. However, when comparing the recovery times required for the reactors to return to high COD removal efficiencies, the reactor with the higher proportion of p-cresol 共R1兲 needed 3 weeks; whereas, the reactor with the lower proportion of p-cresol 共R2兲 required only a couple of days. These results indicated that p-cresol concentrations higher than 700 mg/L in the influent caused severe inhibition to the microbial consortium. Fang and Zhou 共2000兲 observed, in a UASB reactor operated for more than 200 days, a decrease of the removal efficiency when the p-cresol concentration which was in the range of 400–500 mg/L, and 1000 mg/L of phenol was present. PuigGrajales et al. 共2000兲, reported a 50% decrease in acetoclastic methanogenic activity for granular sludge at phenol and p-cresol concentrations of 1504 mg/L and 408 mg/L, respectively. Wang et al. 共1988兲, reported that in batch experiments using a phenolenriched methanogenic culture, concentrations of p-cresol between 500 and 700 mg/L dramatically reduced the phenol biodegradation rate 共200 mg/L兲. When present at high concentrations, p-cresol was biodegraded at very slow rate, despite the complete disappearance of phenol. In the same way, organic overloads in this study tended to first affect the p-cresol compared to phenol removal. Fang and Zhou 共2000兲 found that the effect of OLR was much more drastic in reactors with increasing phenolic concentrations than in reactors with constant phenolic concentrations but decreased the HRT. From these observations, we conclude that
Fig. 3. Operational efficiency during the continuous anaerobic treatment of a phenol/p-cresol/o-cresol mixture in the up-flow anaerobic sludge bed R2: 共A兲 organic loading rate, total, and individual; 共B兲 phenol, p-cresol, and o-cresol removal determined by high-pressure liquid chromatography
the residual p-cresol concentration in R1 was responsible for the prolonged inhibition effect observed in this study. Batch experiments conducted with the adapted granular sludge confirmed these results. In one report 共Wang et al. 1988兲, diauxic behavior was observed in which no biodegradation of p-cresol occurred until phenol was completely utilized. In our case, we did not observe any diauxic phenomenon. The batch assays indicated orders of magnitude increases in the phenol and p-cresol degradation activity of granular sludge during the continuous experiments, clearly indicating that the enrichment and growth of appropriate phenol and p-cresol degraders took place. Bioaugmentation of enrichment cultures to anaerobic reactors, therefore, can be utilized to decrease the lag periods for steady-state operation on phenolic wastewaters 共Guiot et al. 2000; Tawfiki et al. 2000兲.
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 共Schink et al. 2000兲; whereas, p-cresol degradation is initiated by fumurate addition to the methyl group forming benzyl succinate 共Mu¨ller et al. 2001兲. 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 contrast previous findings in which o-cresol has generally been observed to be recalcitrant to degradation under methanogenic conditions. The majority of the reports indicate that o-cresol was not mineralized 共Fedorak and Hudrey 1984; Blum et al. 1986; Wang et al. 1989; Razo-Flores et al. 1996b兲. 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 and p-cresol were completely removed from the influent and, after an adequate period of adaptation, 85% of o-cresol supplied 共110 mg/L兲 was also removed. Table 4 presents a comparison of literature reported data on the treatment of phenolic mixture of more than two compounds in continuous anaerobic reactors. Experiments carried out in fixed film anaerobic reactors with methanogenic-enriched consortia achieved the biodegradation for more than 90% for each element of the mixture phenol, p- and, o-cresol 共100, 35, and 35 mg/L兲 after a period of 350 days using two different enriched anaerobic consortia 共Tawfiki et al. 1999兲. Charest et al. 共1999兲, under the same conditions, eliminated 78% of o-cresol 共21 mg/L兲 and 97% 共144 mg/L兲 of phenol in an effluent from the petrochemical industry. However, in both cases, the consortia used required proteose peptone or whey as a cosubstrate, 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 共days兲 R2
231–275
279–314
320–350
Hydraulic retention time 共days兲 Organic loading rate 共kg chemical oxygen demand/m3/day兲 Phenol/cresols ratio
0.67⫾0.03 2.95⫾0.31 2:1
0.71⫾0.12 4.99⫾0.33 1:1
0.77⫾0.12 2.86⫾0.54 2:1
Efficiencies Total chemical oxygen demand removal 共%兲 CH4 production 共mL/day兲
81.8⫾4.8 194⫾26
60.8⫾17.7 238⫾46
70.5⫾12.7 157⫾29
Table 4. Comparison of Continuous Anaerobic Treatment Results of Phenolic Compounds, Single Compounds, or in Mixture, from Literature
Compound
Reactor
Phenol
Anaerobic fermentor Expanded bed Activated carbon anaerobic filter Up-flow anaerobic sludge bed Anaerobic activated carbon
Phenol Phenol Phenol Phenol Formaldehyde Methanol p-Cresol p-Cresol Phenol p-Cresol Phenol m-Cresol Phenol p-Cresol o-Cresol Phenol p-Cresol o-Cresol Phenol p-Cresol m-Cresol o-Cresol Phenol p-Cresol Phenol p-Cresol Phenol p-Cresol
Up-flow anaerobic sludge bed Up-flow anaerobic sludge bed Up-flow anaerobic sludge bed Up-flow anaerobic sludge bed Fixed film upflow anaerobic reactor Bioaugmented enriched consortium Up-flow anaerobic sludge bed
CH4 Organic loading rate Chemical oxygen 共kg chemical oxygen demand Compound 共% chemical oxygen removal/m3/day兲 removal 共%兲 removal 共%兲 demand removed兲 0.31 7.0 6.32
95.0 100.0 80.0– 84.0
100.0
—
Suidan et al. 共1988兲
100.0 —
— 42.0– 63.0
Wang et al. 共1986兲 Gardner et al. 共1988兲
6.0
97.7
3.4
95.0
7.2
99.9
99.0 — — —
2.5
80.0
80.0
8.12
85.0
95.0 65.0 98.0 20.0 100.0 91.0 100.0 100.0 100.0 77.0 97.0 10.0 5.0 78.0 100.0 93.0 100.0 85.0 100.0 100.0
4.3
—
2.12
94.0
0.66
85.0
Fixed film anaerobic Filter
2.0
—
Up-flow anaerobic sludge bed 共R1兲 Up-flow anaerobic sludge bed 共R2兲
7.0
94.0
7.1
91.0
Up-flow anaerobic sludge bed 共R2兲
2.95
81.8
Reference
99.9
94.7
Fang et al. 共1996兲
71.4
Goeddertz et al. 共1990兲
91.6
Hwang and Chen 共1991兲
— 93.6
Kennes et al. 共1997兲 Fang and Zhou 共2000兲
—
Zhou and Fang 共1997兲
—
Tawfiki et al. 共1999兲
—
Tawfiki et al. 共2000兲
—
Charest et al. 共1999兲
80.6
This work
85
This work
110
This work
80.0
o-Cresol
degrade p-cresol 共Bisaillon et al. 1993; Charest et al. 1999; Tawfiki et al. 1999兲.
SBR of the phenolic compounds and the capability to degrade mixtures of these compounds including the persistent o-cresol.
Conclusions
Acknowledgments
UASB reactors were successfully used to study the biodegradation of phenol and p-cresol mixtures at an OLR of 7 kg COD/m3/day. Also, tertiary mixtures, which included o-cresol, were treated with a global COD removal up to 82% at an OLR of 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 phenol/cresols mixtures representative of major substrates in chemical and petrochemical wastewaters. The most important control parameter was the toxicity of each element of the mixture. In the case of cresols, their concentration should not exceed 600 mg/L. The use of acclimated granular sludge, enriched with phenol and p-cresol degrading microorganisms, highly increased the
This work was supported by the IMP Project Nos. D.00021 and D.00037 and by the Consejo Nacional de Ciencia y Tecnologı´a 共CONACYT兲 from Mexico, Project No. 31537-B. The writers ˜ eda. acknowledge the technical assistance of Roma´n Castan
References American Public Health Association 共APHA兲. 共1985兲. Standard methods for examination of water and wastewater, 16th Ed., American Public Health Association, Washington, D.C. Berne´, F., and Cordonnier, J. 共1995兲. ‘‘Treatment of spent caustic.’’ Industrial water treatment: Refining petrochemicals and gas processing
techniques, Gulf Publishing Company, Paris, 124 –153. Bisaillon, J., Le´pine, F., Beaudet, R., and Sylvestre, M. 共1991兲. ‘‘Carboxylation of o-cresol by anaerobic consortium under methanogenic conditions.’’ Appl. Environ. Microbiol., 57, 2131–2134. Bisaillon, J., Le´pine, F., Beaudet, R., and Sylvestre, M. 共1993兲. ‘‘Potential for carboxilation-dehydroxylation of phenolic compounds by a methanogenic consortium.’’ Can. J. Microbiol., 39, 642– 648. Blum, D., Hergenroeder, R., Parkin, G., and Speece, R. 共1986兲. ‘‘Anaerobic treatment of coal conversion wastewater constituents: biodegradability and toxicity.’’ J. Water Pollut. Control Fed., 58, 122–131. Chang, Y., Nishio, N., and Nagai, S. 共1995兲. ‘‘Characteristics of granular methanogenic sludge grown on phenol synthetic medium and methanogenic fermentation of phenolic wastewater in a UASB reactor.’’ J. Ferment. Bioeng., 79, 348 –353. Charest, A., Bisaillon, J., Le´pine, F., and Beaudet, R. 共1999兲. ‘‘Removal of phenolic compounds from a petrochemical effluent with a methanogenic consortium.’’ Can. J. Microbiol., 45, 235–241. Fang, H., Chen, T., Li, Y., and Chui, H. 共1996兲. ‘‘Degradation of phenol in wastewater in a upflow anaerobic sludge blanket reactor.’’ Water Res., 30, 1353–1360. Fang, H., and Zhou, G. 共2000兲. ‘‘Degradation of phenol and p-cresol in reactors.’’ Water Sci. Technol., 42共5,6兲, 237–244. Fedorak, P. P., and Hrudey, S. E. 共1984兲. ‘‘The effects of phenol and some alkylphenolics on batch anaerobic methanogenesis.’’ Water Res., 18, 361–367. Gardner, D. A., Suidan, M. T., and Kobayashi, H. A. 共1988兲. ‘‘Role of GAC activity and particle size during the fluidized-bed anaerobic treatment of refinery sour water stripper bottoms.’’ J. Water Pollut. Control Fed., 60, 505–513. Goeddertz, J. G., Weber, A. S., and Ying, W. C. 共1990兲. ‘‘Startup and operation of an anaerobic biological activated carbon 共AnBAC兲 process for treatment of a high strength multicomponent inhibitory wastewater.’’ Envir. Prog., 9, 110–117. Guiot, S. R., Tawfiki-Hajji, K., and Lepine, F. 共2000兲. ‘‘Immobilization strategies for bioaugmentation of anaerobic reactors treating phenolic compounds.’’ Water Sci. Technol., 42共5,6兲, 245–250. Hill, G., and Robinson, C. W. 共1975兲. ‘‘Substrate inhibition kinetics: Phenol degradation by Pseudomonas putida.’’ Biotechnol. Bioeng., 17, 1599–1615. Hwang, P., and Cheng, S. 共1991兲. ‘‘Treatment of p-cresol with a recirculating UASB reactor using the concept of kinetic control.’’ Water Sci. Technol., 24共5兲, 133–140. Keith, L. H., and Telliard, W. A. 共1979兲. ‘‘Priority pollutants. I—a perspective view.’’ Environ. Sci. Technol., 13, 416 – 423. Kennes, C., Mendez, R., and Lema, J. M. 共1997兲. ‘‘Methanogenic degradation of p-cresol in batch and in continuous UASB reactors.’’ Water Res., 31, 1549–1554. Mu¨ller, J. A., Galushko, A. S., Kappler, A., and Schink, B. 共2001兲. ‘‘Initiation of anaerobic degradation of p-cresol by formation of
4-hydroxybenzylsuccinate in Desulfobacterium cetonicum.’’ J. Bacteriol., 183, 752–757. Nakhla, G. F., Suidan, M. T., and Pfeffer, J. T. 共1989兲. ‘‘Operational control of an anaerobic GAC reactor treating hazardous wastes.’’ Water Sci. Technol., 21, 167–173. Nakhla, G. F., Suidan, M. T., and Pfeffer, J. T. 共1990兲. ‘‘Control of anaerobic GAC reactors treating inhibitory wastewaters.’’ J. Water Pollut. Control Fed., 62, 65–72. ˜ a-Arguelles, M. E., and RazoPuig-Grajales, L., Olguı´n-Lora, P., Acun Flores, E. 共2000兲. ‘‘Acetoclastic methanogenic toxicity produced by alkyl-substituted phenolic compounds.’’ Proc., 6th Latin-American Workshop on Anaerobic Digestion, Univ. Federal de Pernanbuco, Recife, Brasil, 2, 155–158 共in Spanish兲. Razo-Flores, E., Svitelskaya, A., Donlon, B., Field, J., and Lettinga, G. 共1996a兲. ‘‘The effect of granular sludge source on the anaerobic biodegradability of aromatic compounds.’’ Bioresour. Technol., 56, 215– 220. Razo-Flores, E., Donlon, B., Field, J., and Lettinga, G. 共1996b兲. ‘‘Biodegradability of N-substituted aromatics and alkylphenols under methanogenic conditions using granular sludge.’’ Water Sci. Technol., 33共3兲, 47–57. Schink, B., Phillips, B., and Mu¨ller, J. A. 共2000兲. ‘‘Anaerobic degradation of phenolic compounds.’’ Naturwissenschaften, 87, 12–23. Smolenski, W. J., and Suflita, J. M. 共1987兲. ‘‘Biodegradation of cresol isomers in anoxic aquifers.’’ Appl. Environ. Microbiol., 53, 710–716. Suidan, M. T., Najm, I. N., Pfeffer, J. T., and Wang, Y.-T. 共1988兲. ‘‘Anaerobic biodegradation of phenol: Inhibition kinetics and system stability.’’ J. Environ. Eng., 114共6兲, 1359–1376. Tarvin, D., and Buswell, A. M. 共1934兲. ‘‘The methane fermentation of organic acids and carbohydrates.’’ J. Am. Chem. Soc., 56, 1751–1755. Tawfiki, K., Le´pine, F., Bisaillon, J., and Beaudet, R. 共1999兲. ‘‘Simultaneous removal of phenol, o- and p-cresol by mixed anaerobic consortia.’’ Can. J. Microbiol., 45, 318 –325. Tawfiki, K. et al. 共2000兲. ‘‘Effects of bioaugmentation strategies in UASB reactors with methanogenic consortium for removal of phenolic compounds.’’ Biotechnol. Bioeng., 67, 419– 423. Wang, Y., Suidan, M. T., and Rittman, B. E. 共1986兲. ‘‘Anaerobic treatment of phenol by an expanded-bed reactor.’’ J. Water Pollut. Control Fed., 58, 227–233. Wang, Y., Suidan, M. T., Pfeffer, J., and Najm, I. 共1988兲. ‘‘Effects of some alkyl phenols on methanogenic degradation of phenol.’’ Appl. Environ. Microbiol., 54, 1277–1279. Wang, Y., Suidan, M. T., Pfeffer, J., and Najm, I. 共1989兲. ‘‘The effect of concentrations of phenols on their batch methanogenesis.’’ Biotechnol. Bioeng., 33, 1352–1357. Zhou, G., and Fang, H. 共1997兲. ‘‘Codegradation of phenol and m-cresol in an UASB reactor.’’ Bioresour. Technol., 61, 47–52.
