Enzyme and Microbial Technology 29 (2001) 417 427

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Anaerobic granule formation and tetrachloroethylene (TCE) removal in an upow anaerobic sludge blanket (UASB) reactor
Delia Teresa Sponza*
zmir, Turkey Dokuz Eylu l University Engineering Faculty Environmental Engineering Department, Buca Kaynaklar Campus, I Received 26 March 2001; received in revised form 11 June 2001; accepted 21 June 2001

Abstract The granulation process was examined using synthetic wastewater containing tetrachloroethylene (TCE) in a 2 liters laboratory upow anaerobic sludge blanket (UASB) reactor. The anaerobic biotransformation of TCE was investigated during the granulation process by reducing the HRT and increasing the chemical oxygen demand (COD) and TCE loadings. Anaerobic unacclimated sludge and glucose were used as seed and primary substrate, respectively. Massive initial granules were developed after 1.5 months of start-up, which grew at an accelerated pace for 7 months and then became fully grown. The effect of operational parameters such as inuent TCE concentrations, COD and TCE loading, food to microorganism (F/M) ratio and specic methanogenic activity (SMA) were also considered during granulation. The granular sludge cultivated had a maximum diameter of 2.5 mm and SMA of 1.32 g COD (gTSS.day)1. COD and TCE removal efciencies of 92% and 88% were achieved when the reactor was operating at TCE and COD loading rates of 30 mg (l.day)1 and 10.5 g (l.day)1, respectively. This corresponds to HRT of 0.40 day and F/M ratio of 1.28 gCOD (gTSS.day)1. Kinetic coefcients of k (maximum specic substrate utilization rate), Ks (half velocity coefcient), Y (growth yield coefcient) and b (decay coefcient) were determined to be 2.38 mgCOD (mgTSS.day)1, 108 mgCOD l1, 0.17 mgTSS (mgCOD)1 and 0.015 day1, respectively for TCE biotransformation together with glucose as carbon and energy source during granulation. 2001 Elsevier Science Inc. All rights reserved.
Keywords: UASB reactor; Granulation; Granule; TCE treatment

1. Introduction Anaerobic treatment of toxic and refractory industrial wastewater has become a viable technology and has been most commonly used. Since its introduction 15 years ago, UASB reactors containing granular sludge have become popular worldwide and have been commonly used for wastewater from agricultural industries. Recent studies have demonstrated that the UASB technology is applicable to treating aromatic and aliphatic chlorinated chemicals such as trichloro ethylene, carbon tetrachloride and chlorophenol. Furthermore, the UASB granules exhibited higher resistance to the toxicity of chlorinated aromatic and aliphatics than the occulent digester sludge [1,2]. Granulation of methanogenic bacteria in UASB reactors is important in the treatment of various industrial wastewater containing toxic substances due to their compact struc-

* Fax: 90 232-4531153. E-mail address: delya.sponza@deu.edu.tr (D.T. Sponza).

ture which protect the bacteria from inhibitory and toxic pollutants. The ability of the granules to resist toxicity could be attributed to their layered microstructure. However, these granules occasionally disintegrate in industrial reactors which results in loss of activity when they are developed in carbohydrate containing wastewaters before being used as seed [3]. This extends the granulation period which is generally known to be a very slow process. The anaerobic granules developed in organic wastewaters which do not contain chlorinated organic compounds normally do not have signicant dechlorinating activity. Biomass in the reactor took a long time to become adapted to the new substrate. Only after about 90 days of acclimation did the biogas production rates and COD removal efciency begin to recover. Wu et al. determined that volatile fatty acid (VFA) degrading granules did not exhibit degrading activity for pentachlorophenol (PCP) [3]. During an operational period of 225 days was spent on the development of PCP degrading granules and one year after start-up 80% PCP removal efciency was obtained. PCP degrading granules had a much higher tolerance to PCP

0141-0229/01/$ see front matter 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 4 0 2 - 1

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Abbreviations specic substrate utilization rate (day)1 concentration of microorganisms in UASB reactor (mg l1) dS/dt microbial substrate utilization rate [mg (l.day)1] k maximum specic substrate utilization rate [mgCOD (mgTSS.day)1] Ks half velocity coefcient (mgCOD l1) S TCE or COD concentrations surrounding the microorganisms (mg l1) Q inuent ow rate (l day1) So inuent TCE or COD concentration (mg l1) V reactor volume (l) c solid retention time (SRT) (day) Y growth yield coefcient [mgTSS(mgCOD)1] b bacterial decay coefcient (day1) Qw waste ow rate (l day1) Xw waste microbial concentration (mg l1) Xe efuent microbial concentration (mg l1) Lr X

concentrations than VFA degrading granules. The methane production potential decreased 40% in VFA granules at a PCP concentrations of 5 mg l1 while PCP degrading granules completely dechlorinated and mineralized [3]. On the contrary, granules from industrial reactors, when fed with carbohydrate and VFAs such as acetic and propionic acid, changed their structure. Studies showed that granules from the UASB reactor treating the pulp and paper mill wastewater when fed with VFA in continuous UASB reactors can change their color, elemental composition, are not so densely packed and may oat [4]. Kosaric et al. observed that three months after start-up granules appeared in the efuents. Granules over 2 mm in diameter did not settle, since a hollow core within the granules resulted in poor settling [4]. Higher loading rates and upow velocities of wastewater containing toxic compounds results in mortalities in granules during granulation process in UASB reactors. In a study performed by Wu et al. PCP degrading granules were used as seed material to start-up a (pentachloroethylene) PCE dechlorinating UASB reactor [4]. Lower PCE removal efciencies were observed at loading rates as high as 79 mg (l day)1. Ninety-ve percent PCE removal efciency was achieved at an HRT 4 h and a PCE loading rate of 36 mg (l day)1. Fang et al. observed that benzoate acclimated granules showed better resistance to chlorinated aromatics than starch degrading and distillery granules since they did not have prior exposure to aromatic chemicals during granulation in UASB reactors [5]. For instance, acclimated and benzoate-degrading granules exhibited better resistance to cresol, phenol, cathetol and PCE toxicity. The inhibition of methanogenic activity via these substances was not severe

for UASB reactors containing benzoate-degrading granules [5]. Hydrophobic functional group such as aromatic and aliphatic chemicals were very toxic and showed a higher inhibition effect on methanogenesis [6]. Anaerobic granules with special dechlorinating activities can be developed by using microbial consortia which are able to form granular structures together with additional dechlorinating organisms [7]. On the other hand, the actual values of efciency may vary when slowly or rapidly degrading primary substrates are used together with acclimated culture. When rapidly degrading substrates such as lactate, methanol, sugar and acetate are used the dechlorination fraction of substrate may increase during acclimation and granulation. Anaerobic sludge should be acclimated to toxic organics at the beginning of granulation or during these processes [8]. Tetrachloroethylene (C2Cl4) is widely used as a soil and grain fumigant, a industrial solvent and a dry-cleaning or degreasing uid. Biotransformation of TCE has been studied several times by different researchers. TCE was transformed by reductive dehalogenation to trichloroethylene and dichloroethylene [9], to ethylene [10], to ethane [11,12], and vinyl chloride [13] under anaerobic conditions. In addition, TCE was at least partially mineralized to CO2 [14]. In a study performed by Distefano et al. high concentrations of TCE such as 550 mM were routinely dechlorinated to 80% ethylene and 20% vinyl chloride in 160 ml serum bottles [15]. High concentrations of TCE (550 mM) inhibited the methanogenic transformations. Eighty percent and 85% TCE and COD removal efciencies were obtained up to 39 mg (l.day)1 of TCE loading rate in a UASB reactor at a HRT of 4.5 h and inuent TCE concentrations varied between 6.59.3 mgl1 by using butyrate and glucose as carbon source. The intermediates of TCE were found to be trichloroethylene, dichloroethylene, vinyl chloride and ethylene [3]. Christiansen et al., studied the reductive dechlorination of TCE in a UASB reactor operating in batch mode [16]. It was found that TCE was reductively dechlorinated to dichloroethylene (DCE). When the TCE concentration was increased from 4.6 mM to 27 mM, the transformation rate decreased [16]. 80 and 75% removal efciencies for TCE were obtained in CSTR reactor at loading rates varying between 35 and 110 mg TCE (l.day)1 at a HRT of 10 days and average biomass concentration of 2300 mg l1 when acetic acid and propionic acid were used as primary substrate, respectively. Various substrates including acetate and glucose can serve as electron donors for dechlorination of TCE [3]. The effects of different types of supplemented substrates such as acetate, glucose, lactate and methanol on the biotransformation of TCE were demonstrated [17,18]. TCE degradation in wastewater was studied by Tatsumoto et al. in reactors containing an anaerobic sludge and granular biologic activated carbon [19]. It was found that TCE was degraded as adsorption and biotransformation, the microbial activity of which signied an 80% decrease of TCE. Prakash and Gupta studied the biodegradation of TCE in an UASB reactor containing 550 mg l1

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Fig. 1. Schematic conguration of UASB reactor.

of TCE to develop granular sludge [20]. 92% TCE and 94% COD removal efciencies were obtained and trichloroethylene, 1,2-dichloroethylene, vinyl chloride and ethylene were formed on dehalogenation of TCE. Granules of 2.3 mm sizes were bioaugmented 82 days following the start-up period. In spite of the studies mentioned above, granule formation and the effectiveness of operational factors for UASB reactor containing TCE has not been fully investigated. The granulation process was not extensively examined using TCE containing wastewaters. The studies showed that granules with TCE proceed more slowly than in a UASB reactor fed with readily biodegraded carbohydrates. This can be attributed to the use of granules developed in carbonaceous wastewater for wastewater treatment. Furthermore, studies involving granule formation in UASB reactors containing TCE are limited. Investigations concerning the anaerobic granulation process in TCE containing wastewater, inuence of operational parameters such as HRT, F/M ratio and organic loading rate have not been adequately researched in recent studies. In this study, anaerobic granular sludge was developed for the treatment of TCE in a UASB reactor. The performance of anaerobic granules bio-augmented for the removal of TCE was monitored with SMA, COD, and TCE treatment efciencies during granulation period. The effect of operational parameters such as F/M ratio, HRT and organic loading on treatment efciency were examined during granulation.

taining acidogenic and methanogenic phase biomass of a Yeast Beaker Factory in Izmir-Turkey. This seed was not granulated or acclimated to TCE before the start-up of the UASB reactor. Anaerobic sludge was acclimated and granulated together during the start-up period in a continuous operation of the UASB reactor by increasing the inuent concentration and loadings of TCE gradually due to the low growth and long start-up period of anaerobic microorganisms. The properties of anaerobic sludge was as follows: Suspended solid (TSS) concentrations of 33 g l1, volatile suspended solid concentrations (VSS) of 21 g l1 and sludge volume index (SVI) of 90 ml g1 TSS. The specic methanogenic activity (SMA) and median bioparticle diameter of the sludge were 0.07 g CH4-COD (g TSS.day)1 and 0.02 mm, respectively. 2.2. UASB reactor and experimental methodology Fig. 1 illustrates the 2 liter stainless-steel UASB reactor used in this study, which had an internal diameter of 90 mm and a height of 1000 mm. Five evenly distributed sampling ports were installed over the top of the reactor. The studies were conducted at 35 2C by means of a temperature controlled heater in the reactor. The reactor was lled with 350 ml of settled anaerobic sludge resulting in a seed concentration of 8 g l1 of TSS fed in continuous mode with glucose and TCE containing Vanderbilt Mineral Medium. The operational conditions including inuent concentrations of TCE, COD, TSS concentrations were measured in the reactor and upow rates applied throughout 230 days of operation are depicted in Table 1. During the operation period, pH and gas production were measured daily, COD, TCE and VFA concentrations in efuents were monitored

2. Materials and methods 2.1. Seed The occulent anaerobic microorganisms used in this study were obtained from an anaerobic CSTR reactor con-

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Table 1 Operational parameters in UASB reactor throughout 230 days of operation Days COD inuent (mg 11) 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 TCE inuent (mg 11) 2 2 2 5 5 10 10 14 14 25 14 20 14 14 25 25 30 30 40 30 40 40 40 Upow rate (1 day1) 1 1 1 2 2 3 3 4 4 2 5 5 2 2 5 6 6 5 7 5 8 8 8 TSS inside reactor (g 11) 8 6.5 6 7 9.5 10 10.5 11 12 12.5 13 14 16 17 18.5 19 20 21 22.5 23.2 23.3 23.5 24

010 1020 2030 3040 4050 5060 6070 7080 8090 90100 100110 110120 120130 130140 140150 150160 160170 170180 180190 190200 200210 210220 220230

ranging between 3000 and 6000 mg l1 was used to maintain the neutral pH throughout the continuous feed. The composition of the Vanderbilt mineral medium that was used as the basal medium in all batches (anaerobic toxicity assay-ATA and specic methanogenic activity testSMA) and continuous experiments was as follows (in milligrams per liter of basal medium): NH4Cl (1200), MgSO4 (400), KCl (400), Na2S.9H2O (300), CaCl2.2H2O (50), (NH4)2HPO4 (80), FeCl2. 4H2O (40), CoCl2.6H2O (10), Kl (10), (NaPO3)6 (10), MnCl2.4H2O (0.5), NH4VO3 (0.5), CuCl2.2H2O (0.5), ZnCl2 (0.5), AlCl3.6H2O (0.5), NaMoO4.2H2O (0.5), H3BO3 (0.5), NiCl2.6H2O (0.5), NaWO4.2H2O (0.5), Na2SeO3 (0.5), Cystein (10), NaHCO3 (6000). The medium was made up in demineralized water. 2.4. Analytical procedures 2.4.1. Routine analyses Cumulative gas and methane gas were measured by liquid displacement method. Volatile fatty acids(VFA), were measured by a two stage titration method developed by Anderson and Yang [22]. Biomass was measured as total suspended solids (TSS) by following Standard Methods [23]. COD was measured by closed Reux colorimetric spectrophotometric method numbered 5220 D [23]. GC-MS PurgeTrap capillary column method (6210 D) was used in the determination of TCE concentrations in water [23]. HP 6890 GC system (Hewlett Packard, Avondale, Pa) with micro-cell electron capture (ECD) detector and a Column DB-624 (25 m*0.32 mm*0.52 m, Supelco Inc. Bellefonte, PA) were used. Helium was the carrier gas with a ow rate of 1 ml (min)1. Column temperature was kept at 35C for 5 min then programmed at 6C (min)1 to 160C and at 20C (min)1 to 220C. Mass range was maintained between 45 and 180 amu. Injector and detector temperature were adjusted to 200 and 280C, respectively. 2.4.2. Determination of granule diameter Sludge samples taken from the reactor were prepared on a slide for size measurement in a light microscope (Prior, England). Granule diameter was measured by stage and ocular micrometers. Approximately six granule diameters were measured and the average granule size was calculated for every sampling by determining how many units of the ocular micrometer superimposed a known magnitude on the stage micrometer on overall magnication 1500. 2.4.3. Anaerobic toxicity assay (ATA) ATAs were performed at 35C using serum bottles with a capacity of 150 ml as described by Owen [24]. Serum bottles were lled with 35 ml of Vanderbilt mineral medium containing glucose. The liquid volume of the serum bottles was 50 ml and these were seeded with 15 ml of anaerobic granules which were enriched in yeast industry wastewater and unacclimated to TCE. The glucose COD in the serum bottles was stoichiometrically restored to 3500 mg l1 daily

weekly. TSS concentrations in the reactor and efuent samples were monitored biweekly. 2.3. Substrate and mineral medium The feed solution of the UASB reactor consisted of glucose as a primary carbon source and TCE dissolved in Vanderbilt mineral medium. During the st two months, for rapid granulation S/Fe2 and Cl/Fe2 ratios were maintained between 1.6 1.8 and 0.3 0.5, respectively, by using a suitable amount of Na2S, FeCl2 and chlorinated salts as given in the composition of Vanderbilt Mineral Medium. 800 mg l1 of NH4-N, 100 mg l1 of Ca2 and 100 mg l1 of PO4-P concentrations were adjusted by adding 3000 mg l1 of NH4Cl, 370 mg l1 of CaCl2. 2H2O and 415 mg l1 of (NH4)2 HPO4, respectively, to the feed medium during the start-up of granulation. S/P and Ca/Fe2 ratios were maintained between 0.4 0.6 and 2.12.3, respectively, during the rst two months of continuous operation of the UASB [3,21]. COD/N/P ratios were adjusted between 105108, 2.6 3.2 and 0.8 1.2, respectively. Furthermore, every day two milliliters of stock trace metal solution was added to the reactor feed in continuous operation for growth stimulatory purpose. The ingredients of this solution consisted of: (gram per liter) FeCl2.6H2O (28.5), NiCl2.6H2O (8), CoCl2.6H2O (8). 0.01 g l1 of sodium thioglicollate was added to reduce the redox potential and sustain the anaerobic conditions. NaHCO3

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based on the volume of the methane gas produced the previous day. The glucose COD level of 3500 mg l1 ensured the presence of non limiting substrate conditions in the serum bottles. The measured bicarbonate alkalinity in the test bottles ranged between 3500 and 4000 mg l1 as CaCO3. Gas production was measured daily using a glass gas displacement device which was lled with a salt saturated 5% sulfuric acid and water solution colored with methyl orange [23]. The total gas produced passed through a 2 N NaOH solution to absorb the CO2, the remaining CH4 gas was collected and measured daily. Before the toxicity experiments, serum bottles were operated until the variation in daily gas production was less than 15% for at least 7 consecutive days. All the control samples and the serum bottles were run in triplicate. After observing the steadystate conditions, TCE concentrations of 5 40 mg l1 were administered to serum bottles as slug-doses from concentrated stock solutions of this chemical. This chemical was reagent grade and was obtained from Merck Chemical Co., Inc. The effect of TCE on glucose utilization was compared to the control samples. Inhibition was dened as a decrease in cumulative methane compared to the control sample. 2.4.4. Specic methanogenic activity (SMA) The anaerobic granulated sludge samples taken from the bottom sampling port of the UASB reactor were measured during granulation for SMA. The SMA test was conducted in 150 ml serum bottles at 35C under anaerobic conditions. Wastewater samples taken from feed during operations of the UASB reactor containing glucose, minerals and suitable TCE doses were added to the serum bottles in the same manner as those used in the ATA. 2.5. Start-up Anaerobic bacterial biomass was acclimated and granulated with gradually increasing concentrations of TCE in the inuent in a step-wise manner with decreasing the HRT. Unless there was a considerable transformation and a stable level of COD and TCE concentrations in efuent, the organic loadings were not raised. To accelerate the acclimation and shorten the granulation period, TCE doses, COD loadings and upow rates were gradually increased depending on COD and TCE removal efciencies. The HRT in this study was initially 2 days but was decreased to 0.25 days after 230 days of operation. The corresponding loading rates for COD and applied HRT are illustrated in Fig. 2 while TCE loadings are given in Fig. 3 for 230 day of operation period. The COD in inuent was kept at constant concentrations of 3500 mg l1 by adding glucose to the feed as a primary substrate. Synthetic wastewater comprising glucose and increased concentrations of TCE plus balanced minerals, nutrients and alkalinity were added to the feed of the reactor using a peristaltic pump. NaHCO3 was added consistently at 5000 500 mg l1 to maintain proper pH and buffering capacity. Addition of 0.01 g l1 of sodium thio-

Fig. 2. COD loading rates and HRT changes in TCE fed UASB reactor.

glicollate ensured the anaerobic condition and reduced the redox potential in the reactor.

3. Results and discussion 3.1. ATA tests The purpose of the batch ATA experiments was to obtain information on the toxicity of TCE in order to develop a spike pattern strategy of this compound in the UASB operation. In this study, inhibition is dened as a reduction in the activity, in terms of gas production, of a batch reactor relative to its activity before the addition of the TCE. The initial biomass concentration of anaerobic unacclimated sludge and daily gas production were measured to be 2300 mg l1 and 60 ml (day)1, respectively. The average cumulative methane production was not signicantly affected by 5 mg l1 of TCE as compared to the control samples. The 10 mg l1 of TCE spike demonstrated an inhibitory effect compared to the control sample from day 7 to 10. TCE concentrations of 10 mg l1 and above resulted in inhibition (Fig. 4). The concentrations of TCE resulting in 50% inhibition of the rate of production (IC50) of methane gas in a 2-day contact time period were found to be 18 mg l1 by extrapolation of the results in Fig. 5. This result was lower than the ndings of Blum and Speece1 (IC50 22 mg

Fig. 3. TCE loading rates in TCE fed UASB reactor.

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Fig. 6. F/M and SMA changes in TCE fed UASB.

Fig. 4. Toxicity effects of TCE on anaerobic unacclimated sludge.

l1) [25], Chiu (IC50 28 mg l1) [14], and Freedman and Gossett (IC50 20 mg l1) [10]. 3.2. Performance of UASB experiments In the beginning of start-up and acclimation during granulation, F/M ratios were continuously increased based on the SMA measured, with the F/M to SMA ratio maintained consistently at about 0.75, on average. The effect of F/M ratio applied to SMA is depicted in Fig. 6. High substrate efciency has been proved appropriate to fast granulation [26,27,28,29,30,31]. Herbert et al. reported that a high organic loading may be applied when there is difculty in developing granulation [32]. Lettinga et al. suggested that the if sludge granulation at the beginning stage of the reactor were well developed, a very high organic loading could be applied to the UASB reactor [33]. For this reason, the COD and TCE loading rates were slowly increased when the reactor removed over 75% of soluble COD and TCE from the wastewater. The stepwise increase of the F/M ratios was achieved through the decrease of hydraulic retention time (HRT) coupled with the increase of COD and TCE loading rates. See Figs. 2, 3 and 6.

The smooth operation was interrupted four times on days 75, 110, 160 and 180. The interruptions were due to the deterioration of the reactor performance as reected by the lowering of SMA and the COD removal efciency on each of these occasions, the TCE loading rates being reduced from 28, 50, 90, 40 mg (l.day)1 to 10, 25, 75, 75 mg (l.day)1, respectively. This corresponded to reduced COD loading rates from 7, 8.75, 10.5, 12.25 g (l.day)1 to 3.5, 3.5, 8.75, 8.75 g (l.day)1 and was kept at that level until COD removal efciency and SMA were fully recovered before the loading rates were increased once more. The substrate removal was not apparent during the rst month of operation and thereafter until day 80. During this period, the efuent COD and TCE removal efciencies in the efuent were between 40%70% and 35% 85% respectively. Figs. 7 and 8 show the COD and TCE concentrations in inuent and efuent samples. As can be seen from Figs. 2, 3 and 8, until day 30, the efuent TCE concentration was 1 mg l1 corresponding to TCE conversion of around 50% at TCE loading rates ranging between 15 mg TCE (l.day)1 and corresponding HRTs varied between 12 days. After day 30, the efuent TCE concentration started to decrease and went down to 1 mg l1 from 10 mg l1 between days 50 and 60 at a loading rate of 15 mg TCE(l.day)1 a HRT of 0.66 days. The COD removal efciency was measured at between 80 and 95%. In this period the efuent total volatile fatty acid (TVFA)

Fig. 5. IC50 value for TCE (IC50 18 mg/liter).

Fig. 7. Inuent and efuent COD concentrations in TCE fed UASB reactor.

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Fig. 8. Inuent and efuent TCE concentrations.

concentrations varied between 25 and 42 mg l1 (Fig. 9). Although the inuent characteristics were altered (increasing in TCE, COD loading and decreasing in HRT) this change in the efuent quality was attributed to acclimation of anaerobic sludge to TCE. On day 80, after observing 30% efuent quality with an inuent TCE loading of 28 mg TCE (l.day)1, the inuent TCE loading was decreased to 10 mg (l.day)1 by day 90. On the same date, TVFA concentrations in efuent were measured to be 670 mg l1, corresponding to about 40% removal of COD. As can be seen from Figs. 7 and 8, the efuent COD and TCE concentrations were 2000 and 7 mg l1, respectively, on the same days. The TCE and COD loadings increased any further after observing a steady state efuent quality. On day 100, the efuent TCE concentrations dropped to less than 2 mg l1. The percentage transformation of TCE vas found to be 98%, while the percentage of COD removal varied between 35 and 42%. This low COD removal was due to the formation and difculty in biotransformation of intermediates of glucose, namely acetic and propionic acids which were measured as TVFA(890 mg l1). On day 110, the TCE and COD loading rates were raised to 50 mg (l.day)1 and 8.75 g COD (l.day)1, respectively, after reaching 90% treatment efciencies in the efuent TCE and COD from earlier days. Under these conditions the percentage COD and TCE removals decreased to 50 and

Fig. 9. TVFA concentrations in efuent.

60%. The TVFA concentrations also increased to 850 mg l1 on the same day. This can be explained by the accumulation of both glucose intermediates such as propionic and acetic acid and TCE intermediates even though these were not measured. This indicated some difculties in the biotransformation of both intermediates due to high COD loading and TCE toxicity to biomass. Under these conditions the COD and TCE loadings were dropped to 3.5 g (l.day)1 and 14 mg (l.day)1, respectively, on day 120. Loading rates were gradually increased when COD and TCE removal efciencies reached 80% in the efuent. The TCE and COD loading rates still increased up to 80 mg (l.day)1 9.5 g (l.day)1, respectively, until day 160. On day 150, 89% TCE removal efciency was obtained for inuent TCE concentrations of 25 mg l1 at TCE loading rates of 75 mg (l.day)1. This result is signicantly better than those obtained by Wu et al. [8] in an acetic acid fed UASB reactor. They reported treatment efciencies of about 69% and 90% for inuent TCE concentrations of 5 and 20 mg l1 at TCE loading rates of 42 and 49 mg (l.day)1, respectively, [8]. When TCE loading rate of 90 mg (l.day)1 was maintained in the inuent which corresponded to COD loading of 10.5 g COD (l.day)1, the efuent TCE concentration was raised to 17 mg l1 from 3 mg l1 on day 160. The efuent TVFA concentrations were increased to 580 mg l1 after the introduction of 90 mg (l.day)1 of TCE loading. On day 180, a HRT of 0.28 day was maintained in the UASB reactor with the TCE and COD loading rate in the feed kept at 140 mg (l.day)1 and 14 g (l.day)1, respectively. The efuent COD and TCE concentrations were increased to 1500 and 20 mg l1, respectively. Even though the percentage of removal efciency (50%) was not relatively good, the efuent TVFA concentrations were not signicantly increased (180 mg l1) This suggested that there was still residual toxicity damage from TCE to the anaerobic biomass from the previous TCE feed or new COD loading shocks to microorganisms. Under these operating conditions, the TCE loading rate was dropped to 75 mg (l.day)1 by day 190. This change was reected immediately in the efuent quality of the system. TVFA concentrations in the efuent dropped to 20 mg l1 and the percentage of TCE and COD removal increased to around 96 and 94%, respectively. After day 190, even though the loadings were raised the efuent TCE and COD concentrations started to decrease and went down to 300 and 5 mg l1 between days 210 and 230, respectively. In this period the efuent TVFA concentrations were measured to be 30 mg l1. Because none of the operating parameters or inuent characteristics were not altered during the 40 days, this change in the efuent quality was attributed to the acclimation of granular sludge to TCE concentrations of 40 mg l1 in inuent at TCE, COD loadings of 160 mg (l.day)1 and 14 g (l.day)1, respectively. Approximately 90% TCE and 94% COD mean removal efciencies were obtained. The results obtained in this study were signicantly better than the study of Christiansen et al.

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where an UASB reactor was fed with methanol at maximum TCE loading rate of 35 mg (l.day)1 with 90% TCE biotransformation performance in efuent [34]. 3.2.1. Relationships between SMA and F/M ratio during granulation process The SMA, bioparticle size and TSS concentrations in the reactor were used to characterize granulation. The SMA is an indicator of the methanogenic activity of the biomass forming granules. The SMA started increasing almost immediately after start-up as an increasing F/M ratio was applied. After just 1.5 months of operation, the SMA had increased from 0.29 g COD (g TSS.day)1 initially to 0.52 g COD (g TSS.day)1 as the F/M ratio was increased to 0.44 g COD (g TSS.day)1 from 0.2 g COD (g TSS.day)1. This can be attributed to the high primary substrate sufciency applied and biodegradability of the TCE with the appropriate minerals and nutrients maintained, all of which led to the rapid subsequent development of bacterial acclimation. The SMA sharply decreased 4 times during the operation period as a resulting of high TCE and COD loadings applied on days 75, 110, 160 and 180. Until day 90, increasing the F/M ratio had a pronounced effect on the increase in SMA. After this day the SMA ratio was not inuenced by F/M ratio (Fig. 6). After day 110, the F/M ratio in the reactor remained stable around 0.64 or slightly decreased due to high TSS concentrations measured in the reactor(14 16 g l1), although TCE and COD loadings were increased. In this case, increased substrate concentrations might have had a greater impact than reduced F/M on the increase in SMA. The SMA increased from 0.44 g COD (g TSS.day)1 to 0.68 g COD (g TSS.day)1 70 days following start-up and acclimation period with the change in the F/M ratio applied. After day 70, the F/M ratio was increased from 0.27 gCOD (g TSS.day)1 to 0.65 gCOD (g TSS.day)1. On day 80, when the F/M ratio was raised to 0.65 gCOD (g TSS.day)1 the SMA decreased to 0.28 g COD (g TSS.day)1 from earlier levels of 0.68 g COD (g TSS.day)1. This corresponded to high TCE loading of 28 mg TCE (l.day)1 maintained in the inuent on the same day. The efuent COD and TCE concentrations also increased to 2000 and 7 mg l1 from earlier levels of 150 and 2 mg l1, respectively. This indicates accumulation of some intermediates since the VFA concentrations were measured to be 810 mg l1. The SMA decreased on days 160 and 180 depending on high TCE and COD loadings such as 90 mg (l.day)1, 10.5 g (l.day)1, and 140 mg (l.day)1 and 12.25 g (l.day)1, respectively. After day 200, SMA remained stable as a maximum value of 1.32 g COD (g TSS.day)1. This result is comparable with those obtained by Yan and Tay in an UASB reactor treating only glucose, peptone and meat extract without any chlorinated toxicant (1.72 g COD (g TSS.day)1 [28].

Fig. 10. Granule diameter and TSS concentrations in TCE fed UASB reactor.

3.2.2. Granule size Divalent cations (Ca2, Fe2) are reported to bridge negatively charged bacteria together faster for initiated granulation [28]. Furthermore, S/Fe and Cl/Fe ratio inuences the composition of granules and have been shown to be benecial for granule formation [3]. Supporting these statements the Ca2, Fe2, S2 and Cl1 amounts were adjusted as mentioned in the Materials and Methods section for rapid granulation together with reducing HRTs and increasing organic loadings. The bioparticle size increased slowly during the initial month of operation. At around 11.5 months, massive initial granules were being formed in the lower part of the UASB reactor and began to grow during acclimation and pregranulation. Fig. 10 shows the variations of granule size and TSS concentrations during granulation. The mean granule diameter was measured to be 1.0 mm at the end of 1 months of operation. The initiated granules continued to grow rapidly for 3 months until large granules were formed with mean diameters of 1.5 mm. The granule size reached 1.8 mm after 100 days of operation period. After 220 days the maximum granule diameter was measured to be 2.5 mm. The bioparticles had grown to about 100 times their initial original size of 0.02 mm. 3.2.3. TSS concentration in and in the efuent of the UASB reactor The proles of TSS concentrations in and in the efuent of the reactor showed a gradual and progressive change during start-up and differed signicantly before and after granulation. As mentioned above, an increase in TSS concentration during granulation was as a result of biomass synthesis in the UASB reactor (Fig. 10). Initially, the biomass synthesis was low due to the low F/M ratio applied. On the other hand, the washout was higher due to the poor settleability of seed sludge until day 40. The sludge concentration in the reactor slightly decreased with the mini-

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425

Fig. 12. TSS concentration through reactor height. Fig. 11. Granule size through reactor height.

mum of 7 g l1 achieved after about 1 month of operation. By day 20, with the progressive improvement of granule initiation, the TSS concentration gradually increased due to higher granulated biomass and lower washout from the efuent. After 200 days of operation the biomass concentration in the reactor reached 24 g l1. The TSS concentrations in the efuent increased to 900 mg l1 during the start-up of the pre-granulation period due to the disintegration of microbial aggregates and washout. On days 130 and 200, TSS concentrations in efuent were measured to be 400 and 250 mg l1, respectively (data were given elsewhere). This can be explained by high upow rates applied and consequently high gas production even though the data were not given. The gas produced inside in the granules gets more difcult to release by increasing upow rates. This result causes rising gas bubbles and disintegration of the granule and can split the large granules. 3.2.4. Proles of TSS concentrations and granule size at different reactor heights The sludge stayed loose initially and expanded easily like a blanket. As the granulation process proceeded, the sludge bioparticles were progressively stratied with the larger ones settling down in the lower part of the reaction zone and the smaller ones expanded or suspended in the upper part of the reaction zone. Fig. 11 illustrates the maximum granule diameter at different reactor heights on different days. The larger particles settled in the lower part of the sludge bed. The smaller ones were also suspended due to mixing and increasing of upow rates and the release of more gas bubbles during anaerobic treatment. The maximum sludge diameter at the bottom part of the reactor was found to be 1 mm and 2.05 mm at the end of 30 and 180 days of operation periods, respectively. During the 230 days of full granulation, the TSS concentrations were monitored two times along the reactor height (Fig. 12). As can be seen from this gure on day 30, the maximum TSS concentrations were measured to be 5 and 10 g l1 at reactor heights of 40 and 5 cm, respectively. On day 180, the measured TSS concentrations varied between 18 and 20 g l1 at the same heights.

3.3. Determination of kinetic coefcients The rate of substrate utilization is directly related to the concentration of microorganisms in the granules and concentration of the growth limiting substrate surrounding the granule microorganisms. This relationship can be described by an expression similar to Monods equation which is widely used to describe the relationship between bacterial growth rate and the concentration of growth limiting substrate: Lr 1/X (dS/dt) kS/(Ks S) Lr Q(So S)/VX (1)

At quasi steady-state conditions, Lr may be expressed as: (2)

Microbial substrate utilization rate is related to solids retention time(SRT) as follows: 1/c YLr b (3)

By denition, c is the total quantity of active biomass in the reactor divided by the total quantity of active biomass withdrawn daily and was determined as follows;

c VX/[QwXw (Q Qw)Xe]

(4)

The term Qw Xw only makes sense if there is a waste sludge stream, and there is no mention here of sludge wastage. Therefore c can be expressed as follows,

c VX/(QeXe)

(5)

The kinetic constant of Y, b, k and Ks were determined by using the experimental data obtained during granulation on days 40, 50, 70, 100, 140, 150, 170 and 200 when it had reached a steady-state condition. The kinetic coefcients mentioned above were calculated based on the COD since the glucose was the primary substrate and energy source used by the anaerobic microorganisms for growth. The y intercept and slope of the line from the graph plotted between Lr and 1/c (1/SRT) gave the values of b(decay coefcient) and Y(growth yield coefcient), respectively. Y was determined to be 0.17 mg TSS (mg COD)1 while b was found to be 0.015 day1 (1/SRT 0.2523* Lr

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0.0163, R2 0.78). From the plot between 1/S versus 1/literr, k (maximum specic substrate utilization rate) and Ks (half velocity coefcient) were determined to be 2.38 mg COD (mg TSS.day1) and 108 mg COD l1 (1/literr 5.571*1/S 0.3161, R2 0.98). The maximum COD utilization rate (k), b and Y values determined in this study were compared with similar coefcients as reported by other researchers. k is signicantly higher than the ndings nal [36] and slightly lower than Sponza of Guiot et al. [35] U [37,38] in continuous UASB reactors containing only glucose as a carbon source which did not contain chlorinated toxic organic substrate (1.1, 0.91, 3.11 and 2.99 mgCOD (mg TSS.day)1, respectively). In some other studies performed by de Bruin et al., [11] and Ho rber et al. [39], the TCE dechlorination rate (k) of 0.4 mgTCE (mg TSS.day)1 and 0.6 mgTCE (mg TSS.day)1, respectively were obtained during continuous operation of UASB reactor fed with sucrose, format and acetate. These values are about 60% of the maximum substrate utilization rate observed in our study. The b value found in this study appears to be on the lower side as compared to suspended type sludge, while the Y value seems to be signicantly higher even if TCE was treated with glucose in the case of granular sludge. This can be attributed to high COD utilization by dense biomass in granules. Lower Ks values indicate the afnity of granular sludge to substrate and shows that the TCE and glucose were not accumulated in the reactor.

COD (g TSS.day)1 at the end of acclimation and granulation. The results of this study showed that TCE degrading granules can be efciently developed by appropriate HRT, TCE, COD loading rates and F/M ratios even at the beginning of granulation if the start-up period is well operated. The development of granules was inuenced by the wastewater itself, HRT, organic loadings and the F/M ratio applied, particularly for toxic chemicals containing wastewaters.

Acknowledgments The author would like to express her thanks to BI TAK (The Turkish Scientic and Technical Research TU Council) for nancial support under Grant YDABC AG-485 and to Dokuz Eylu l University Support Foundation, Grant no 0908. 97.02.03.

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4. Conclusions TCE was inhibitory to unacclimated anaerobic bacteria in the batch reactor at concentrations as low as 5 and 10 mg/liter. TCE in synthetic wastewater was effectively degraded in continuous operation of a laboratory scale UASB reactor at loading rates ranging between 28 and 75 mg TCE (l.day)1 during TCE degrading granule formation from unacclimated anaerobic culture. Over 87% TCE was removed at 37C and an HRT of 0.28 day. This corresponds to 92% of COD removal efciency at COD loading rates varying between 7 and 10.5 g (l.day)1. Thereafter, at TCE loading rates ranging between 80 and 160 mgTCE (l.day)1 the TCE removal efciency decreased to 80%. When the loading rates were lowered; the COD, and TCE removal efciencies recovered gradually. At high loading rates the COD, TC and TCE removal efciencies dropped to 50% on average but they readily recovered to 88% in about 1 or 2 weeks by reducing the loading rates and maintaining the F/M ratio at 75% of SMA during start-up and maturation period. Under these conditions, biomass in the reactor took a short time to become adapted to the new loading rate. The granulation process was examined through TCE removal in UASB reactor by measurements of granular size and SMA. TCE degrading granules were developed having a maximum diameter of 2.5 mm and SMA of 1.32 g

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