The effect of vinyl acetate in acetoclastic methanogenesis

Bioresource Technology 102 (2011) 1644–1648

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The effect of vinyl acetate in acetoclastic methanogenesis U. Durán ⇑, J. Gómez, O. Monroy, F. Ramírez ⇑⇑ Universidad Autónoma Metropolitana, Biotechnology Dept., P.A. 55-535, 09340 Iztapalapa, México D.F., Mexico

a r t i c l e

i n f o

Article history: Received 4 June 2010 Received in revised form 8 September 2010 Accepted 9 September 2010 Available online 17 September 2010 Keywords: Methanogenesis Vinyl acetate Inhibition UASB Anaerobic sludges

a b s t r a c t The influence of vinyl acetate (VA) in the methanogenesis was evaluated, by using an upflow anaerobic sludge blanket reactor of 1.5 L. The reactor was operated at 33.5 g/L volatile suspended solids to 30 ± 2 °C, a hydraulic residence time of 1 day, an organic loading rate of 1 kgCOD/m3/d of two different mixtures of VA and glucose. The VA was methanized to 81% when its proportion was of 10% into reactor loading rate, when VA proportion increased to 25%, the methane production rate decreased to 62% and the acetate production rate increased almost 8 times. These results indicated that VA was only hydrolyzed and glucose was not used as a co-substrate. The effect of glucose on VA methanogenic degradation was evaluated through batch reactors of 60 mL, concluding that the glucose supported the methanogenesis without favoring the VA elimination. On the other hand, the results of the sludge from the reactor in the presence of VA demonstrated that VA caused an irreversibly inhibition of acetoclastic methanogenesis when the anaerobic sludge was exposed to this compound. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The vinyl acetate (VA) is a volatile chemical widely being used (4.4 million tons in 2004) in the manufacture of adhesives, textiles, emulsions and resins for paints. The VA pollutes soils, air and water. At concentrations greater than 100 mg/L, it can be lethal for many organisms (ATSDR, 2008; Lara-Mayorga et al., 2010). Wastewaters from the polymeric resin industry that contain VA have been treated by anaerobic digestion. The treatment of acrylates, styrene and VA in upflow anaerobic sludge blanket (UASB) reactors at loading rates of chemical oxygen demand (COD) between 100 and 1250 mg COD/L/d, caused inhibition of the methanogenic activity. A 42% decrement was registered (Araya et al., 1999, 2000). Durán et al. (2008) showed that when the VA loading rate was increased from 100 to 135 mg COD/L/d, the biogas production rate decreased to 60%. They showed that in batch reactors methanogenesis is only possible at VA concentrations lower than 69 mg COD/L; on balance Stuckey et al. (1980) and Gren´ et al. (2009) showed that there was a 100% VA transformation to acetate, acetaldehyde and ethanol at concentrations between 67 and 2675 mg COD/L. However, the VA was methanized at 67 mg COD/L. All these results suggest that VA inhibited methanogenesis spite of the fact that the VA inhibition has not been determined yet. Nieder et al. (1990) suggested that the VA methanogenic pathway starts out with its own hydrolysis in order to form vinyl alco⇑ Corresponding author. Tel./fax: +52 555804723. ⇑⇑ Corresponding author. Tel./fax: +52 555804723. E-mail addresses: [email protected] (U. Durán), [email protected] (F. Ramírez). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.09.039

hol and acetate (Eq. (1)). First, the vinyl alcohol is oxidized and turns out into acetaldehyde (Eq. (2)). Second, this acetaldehyde is oxidized and turns out into acetate (Eq. (3)) which by the way is catalyzed by the enzyme acetaldehyde dehydrogenase that coupled to the NAD+ coenzyme reduction. According to Dwyer and Tiedje (1983,1986) there are reports about the acetate kinase and phosphotranferase, both key enzymes in acetoclastic methanogenesis, which are inhibited by the acetaldehyde (Frimmer and Widdel, 1989; Schink, 1997). Finally, the acetate is consumed by the Methanogenic archaea (Eq. (4)). The Methanogenic archaea involves only Methanosarcina and Methanosaeta, with different affinity constants for the acetate (Ks), though. The Methanosarcina affinity constant is lower that the Methanosaeta one (Gonzalez-Gil et al., 2001a,2001b; Díaz et al., 2006).

CH3 CO2 CH : CH2 þ H2 O ! CH2 : CHOH þ CH3 COO þ Hþ

DGo0 ¼ 55:0 kJ=reaction CH2 : CHOH ! CH3 CHO DGo0 ¼ þ3:80 kJ=reaction

ð1Þ ð2Þ

CH3 CHO þ H2 O ! CH3 COO þ Hþ þ H2

DGo0 ¼ 32:2 kJ=reaction

ð3Þ

2CH3 COO þ 2H2 O ! 2CH4 þ 2HCO3

DGo0 ¼ 28:3 kJ=reaction

ð4Þ

CH6 H12 O6 þ 3H2 O ! 3CH4 þ 3HCO3 þ 3Hþ

DGo0 ¼ 372:9 kJ=reaction

ð5Þ

U. Durán et al. / Bioresource Technology 102 (2011) 1644–1648

A large electron flow is produced during the glucose anaerobic digestion (Chidthaisong and Conrad, 2000). It is believed that the glucose acts out as a positive co-substrate in the anaerobic removal of double bond recalcitrant chemicals (Culubret et al., 2001; Somasiri et al., 2008), because its anaerobic digestion generates a huge electron flow (Eq. (5)). The glucose can act out as electron donor in order to methanize chemicals such as vinyl acetate, acrylates, styrene and dye Maxilon Yellow GL (Garibay-Orijel et al., 2005; Sarioglu and Bisgin, 2007; Durán et al., 2008). 1.1. Appendice 1 The purposes of this research are two. First, the VA effect on the anaerobic digestion with glucose as a co-substrate, both in continuous reactors (UASB) as well as in batch reactors. Second, we want to find out why there is an accumulation of the acetate. In order to reach this aim the acetoclastic methanogenenic capacity of the anaerobic sludge was assessed after being exposed to VA. 2. Methods All the water samples were analyzed following standard methods (APHA, 2005). Total suspended (TSS) and volatile suspended solids (VSS) in the anaerobic sludge were measured according to procedure 2710, COD by the closed reflux technique (procedure 5220). The carbon in gas phase (methane and carbon dioxide) was measured by displacement of a brine column (300 g NaCl/L) at pH 2 in order to avoid carbon dioxide dissolution (CO2). The acetate (Ac), the acetaldehyde (AcCHO) and the VA were quantified by flame ionization gas chromatography using an AT-1000 (0.53 mm  1.2 mm  10 m) at temperatures of 120 °C in the oven, 130 °C in the injector and 150 °C in the detector by using nitrogen as a carrier at 6.6 mL/min. The biogas composition was determined by thermal conductivity gas chromatography (TCD) using a Carbosphere 80/100 column, at temperatures of 140 °C in the oven, 170 °C in the injector, 190 °C in the detector by using helium at 25 mL/min (Durán et al., 2008). 2.1. Assays in a UASB reactor These tests were carried out by using a UASB reactor of 1.5 L, which was inoculated with 0.5 L of sludge taken from a reactor fed with wastewater from a chosen polymeric resins industry (Durán et al., 2008). This reactor was operated at 30 ± 2 °C, at one day hydraulic residence time (HRT) and an organic loading rate (BV) of 1.0 kg COD/m3/d. The reactor was fed with the following mineral medium (g/L): NH2PO4H2O, 0.703; NH4Cl, 0.028; KCl, 0.5; MgSO47H2O, 0.111; CaCl22H2O, 0.1 and NaCl, 0.297. Glucose (G) and vinyl acetate (VA) with three loading rates (BV), as described in Table 1, were used as carbon sources. 2.2. Assays in batch reactors The batch assays were performed twice at the same time after the first and the third load in 40 mL of the same media used in the UASB reactor. First they were inoculated with anaerobic sludge Table 1 Composition of the influent fed to the UASB reactor at BV of 1 g COD/L/d. Compound

BV1a (mg/L/d)

BV2 (mg/L/d)

BV3 (mg/L/d)

VACODb GCODc

0 1000 0

100 900 10

250 750 25

VA proportion (%) a b c

BV; loading rate. VACOD; Chemical oxygen VA demand. GCOD; Chemical oxygen Glucose demand.

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from UASB reactor. Then, they were incubated at 30 ± 2 °C, in 60 ml serological bottles. Assays in batch reactor were performed to the following studies: (a) VA methanogenesis kinetics were carried out in both, without glucose and with glucose (250 mg GCOD/L). In each kinetic five VA concentrations were tested: 100, 250, 500, 750 and 1000 mg VACOD/L. (b) The physiological VA effect on the acetoclastic methanogenesis was assessed by using samples of both, unexposed and exposed to VA (BV3, 25% of VA) UASB sludge at different acetate concentrations: 40, 75, 160, 240, 320, 400 and 530 (mg AcCOD/L). 2.3. Response variables COD removal efficiency (eCOD (%) = [mg removed COD/mg fed COD], product yields (Yp/s = mg COD product/mg COD consumed substrate), substrate uptaken rates (rS = mg COD substrate/L/d), product formation rates (rP = mg COD product/L/d), methanogenic specific activity (qCH4 = g COD methane/g VSS d) and specific rates of substrate uptaken (qs = g COD substrate/g VSS d) were measured in both, the batch and continuous reactors as response variables. 2.4. Statistical analysis The statistical difference among the efficiencies of individual consumption, the rates of substrate consumption, product formation and yields values for each loading rate, were subjected to a one-way ANOVA analysis. In order to determine the steady state in the methanogenesis, the variation coefficients for each of the previous variables happened to be under 10%. 3. Results and discussion 3.1. Continuous reactor The UASB reactor was fed at a loading rate of 1000 mg COD/L/d every time (Table 1). Two results were obtained when VACOD was not added: the COD removal efficiency (eCOD) was 97.6 ± 4% and the methane formation rate (rCH4) was 981 ± 42 mg CODCH4/L/d with the second load a mixture of 10% VACOD and 90% of GCOD was used as substrate at the same organic loading rate (BV2, Table 1). At a steady state (6% coefficient variation) a high quantity of methane was found as of an important glucose and VA consumption (792 ± 11 mg CODCH4/L/d) (Fig. 1a). The product yields for CH4 was 0.915 and for acetate (Ac) was 0.072. The methanogenesis was only registered under the 10% VACOD condition, as also reported by Stuckey et al. (1980) as well as Durán et al. (2008). With the third load a mixture of 25% VACOD and 75% GCOD was used as substrate at the same organic loading rate (BV3, Table 1). The methane formation rate (rCH4) decreased up to 62.4%. The acetate formation rate (rAc) increased from 61.3 ± 5 to 390.7 ± 7 mg COD/L/d. There was also acetaldehyde (AcCHO) formation at an acetaldehyde formation rate (rAcCHO) of 82.8 ± 11 mg COD/L/d (Fig. 1b). The main products obtained from this load were 0.487 acetate (Ac), 0.373 CH4 and 0.106 AcCHO. These results showed that a 2.5 higher VA concentration made the yields decreased. The methanogenesis also decreased and VA was hydrolyzed to acetate and acetaldehyde with no methane formation as reported by Nieder et al. (1990) and Gren´ et al. (2009). Supported by the COD stoichiometric balance at a 10% VACOD (BV2) concentration the methanogenesis is not inhibited. However with a 25% VACOD (BV3) the methanogenesis takes place only from

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B v (mg COD/L/d)

1200

VA influent VA effluent G influent G effluent

900

a

600 300 0 0

50

100

150

200 250 time (days)

300

350

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b

rP (mg COD/L/d)

1000 CH4

800

Ac AcCHO

600 400 200 0 0

50

100

150

200 250 time (days)

300

350

400

Fig. 1. UASB reactor substrate (a) and products (b) with three VA loads (BV1, BV2 and BV3).

1200

100

1000

80

800

εCOD (%)

rP (mg COD/L/d)

glucose. The acetate and acetaldehyde accumulated concentration comes only from VA hydrolysis. It was also found that glucose does not promote VA elimination. The Fig. 2 shows that the VA concentration which inhibits the acetoclastic methanogenesis up to 50% (IC50) in the UASB reactor ended up at 180 mg VACOD/L/d. Although rate of methane production (rCH4) decrease the production rates of acetate (rAc) and acetaldehyde (rAc,CHO) increased due to the VA enlargement from 10% to 25%. Nieder et al. (1990) observed that both the rate of VA hydrolysis and its concentration increase and they produce acetate as the main product. This suggests that VA inhibits the acetoclastic methanogenesis. Ince et al. (2003) and Xu et al. (2010) reported that in the acetoclastic archaea, the methanogenic activity disappear due to long exposures to toxic substances. Batch tests were carried out to obtain more evidence about the respiratory process of VA elimination as well as causes of acetate accumulation.

60 600

40

400

20

200 0

0 0

5

10

15

20

25

VACOD in feed (%) r C H4

r Ac

r AcC HO

ε COD (%)

Fig. 2. VACOD in feed effect oven the production rate of (rCH4), acetate (rAc) and acetaldehyde (rAcCHO) and COD removal efficiency (eCOD) in the UASB reactor.

3.2. Batch reactor assays 3.2.1. Effect of glucose on the VA methanogenesis Fig. 3 shows the time-concentration profiles of VA transformation with and without glucose as co-substrate. The rate of AV hydrolysis depends only of its own concentration. The presence of glucose does not affect the AV hydrolysis. The fact that glucose has not any effect on neither the methane production or over the COD removal rates was observed. Only at 100 mg AVCOD/L methane is registered coming from both the AV and glucose. Many phenomena were observed. The methane production is inhibited by VA in both situations with and without glucose presence. The methanogenic activity decreased with the VA concentration. The presence of glucose caused IC50 of 257 ± 3.1 mg VACOD/L. The COD removal rate of glucose-VA mixtures decreased to 60 ± 4.2% and a methanogenic activity was also observed because of the glucose methanization. However, a constant removal in the VA assays was registered because of an acetate and acetaldehyde methanization. So, it can be stated that glucose is not a cosubstrate for VA methanization. These results differ from those ones reported by Garibay-Orijel et al. (2005), Sarioglu and Bisgin (2007) and Durán et al. (2008) who observed an enlargement in the removal rate of recalcitrant compounds with glucose presence. In assays with VA as a sole carbon source, the methanogenesis was lower and the methane production maximum rate was 13.3 ± 1.7 mg COD/L/d. This record was obtained exclusively with 100 mg VACOD/L. The COD was slowly consumed and it always took place at the same rate 22 ± 2.2 mg/L/d. Since the rate of VA hydrolysis was much higher than the acetate consumption rate, the VA methanogenesis observed was very slowly. These results are very alike to those from Stuckey et al. (1980), who mentioned that the VA concentrations above 300 mg VACOD/L cause a 50% methanogenic activity decrement. In all the tests a methane production decrement was caused by a VA concentration increment. The acetate accumulation was probably observed due to the selective action on the acetoclastic Methanogenic archaea. Gonzalez-Gil et al. (2001a,2001b) and Díaz et al. (2006) have reported that anaerobic sludges with high affinity constant (KS) values for acetate contain larger members of Methanosarcina than Methanosaeta archaea. 3.2.2. VA effect over anaerobic sludge In order to determine the VA effect on the acetoclastic populations, the acetoclastic methanogenesis was assayed in both, VA exposed and VA unexposed sludges. The VA unexposed sludge had a qCH4 = 0.62 g CH4–COD/g VSS/d. On balance, the VA exposed sludge (10% and 25% VA) considerably decreased the rates of acetate consumption (70%) and methane production (90%). This shows that a long VA exposure irreversibly affected the sludge acetoclastic capacity. It can be though that the VA methanogenesis is a spontaneous reaction. However the acetate kinase and the phosphotransacetylase enzymes are needed to acetate activation. The substrate of acetaldehyde dehydrogenase, active enzyme during the VA hydrolysis, inhibits the initial stage of acetate activation (Frimmer and Widdel, 1989; Nieder et al., 1990). The acetate consumption specific rate profile (qAc) versus the acetate concentration shows a Monod profile in both assays. The correlation coefficients were 0.978 before VA exposure and 0.985 after VA exposure. Eqs. 6 and 7 show that the acetate consumption maximum rate (qAc max) decreased 69% and the affinity constant (KS) increased 18%, after the UASB reactor sludge was exposed to the VA. The sludges methanogenic activity VA effect was a noncompetitive inhibition, likely irreversible. There was an intake rate decrement without affecting significantly the acetate affinity. Our values were compared with the one reported by Schmidt and Ahring (1999)

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Concentration (mg COD/L)

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Ac

AcCHO

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CH 4

Fig. 3. Substrate consumption profiles with (+G) and without glucose as a co-substrate.

(95.4 mg AcCOD/L) under similar operating conditions. It was observed that the acetate affinity was not modified. The over 350 days sludge VA exposure affects the acetate consumption rate.

Sludge acetate consumption Monod profile before VA exposure:

qAc ðmg AcCOD =g VSS=dÞ ¼

0:136½AcCOD  86:2 mg AcCOD =L þ ½AcCOD 

ð6Þ

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Sludge acetate consumption Monod profile after VA exposure:

qAc ðmg AcCOD =g VSS=dÞ ¼

0:0423½AcCOD  101:9 mg AcCOD =L þ ½AcCOD 

ð7Þ

The VA possibly affects the methanogenesis in an enzymatic level. According to Nieder et al. (1990) the last stage of the VA hydrolysis (Eq. (3)) involves the aldehyde dehydrogenase. Evidence shows that this enzyme inhibits the initial stage of acetate activation in the Methanosarcina archaea (Frimmer and Widdel, 1989). So, the elimination pathway of this compound remained steady. By using a denaturing gradient gel electrophoresis (DGGE) analysis on the UASB sludge reactor the Methanosarcina archaea remained while Methanosaeta archaea disappeared, when the VACOD proportion increased from 10% to 25% (data not shown). 4. Conclusions Methanogenesis took place as the main process when the UASB reactor was fed with 10% VACOD, while when being fed with 25% VACOD the methane production rate decreased 62%. The acetoclastic methanogenesis was inhibited by 50% and the glucose did not promoted VA elimination. Under anaerobic conditions with different VA concentrations in the presence and absence of glucose, the batch assays showed that the VA hydrolysis rate was much higher than the acetate consumption rate. The VA effect assays on the acetoclastic methanogenesis showed that the VA affected irreversibly the acetoclastic methanogenesis. Acknowledgement This work was made with CONACyT economic support number 181014. References Agency for Toxic Substances and Disease (ATSDR), 2008. Public Health Statement for Vinyl Acetate. Encyclopedia of Earth. Eds. Cutler J. Cleveland. Washington, D.C. APHA, AWWA and WPCF, 2005. Standard methods for the examination of water and wastewater. 21st edition. American public health association. Washington, DC. Araya, P., Aroca, G., Chamy, R., 1999. Anaerobic treatment of the effluents from an industrial polymers synthesis plant. Waste Manag. 19, 141–146. Araya, P., Chamy, R., Mota, M., Alves, M., 2000. Biodegradability and toxicity of styrene in the anaerobic digestion process. Biotech. Lett. 22, 1477–1481.

Chidthaisong, A., Conrad, R., 2000. Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil. FEMS Microbiol. Ecol. 31, 73–86. Culubret, E.N., Luz, M., Amils, R., Sanz, J.L., 2001. Biodegradation of 1,1,1,2 tetrachloroethane under methanogenic conditions. Water Sci. Technol. 44 (4), 117–122. Díaz, E.E., Stams, A.J.M., Amils, R., Sanz, J.L., 2006. Phenotypic properties and microbial diversity of methanogenic granules from a full-scale upflow anaerobic sludge bed reactor treating brewery wastewater. Appl. Environ. Microbiol. 72 (7), 4942–4949. Durán, U., Monroy, O., Gómez, J., Ramírez, F., 2008. Biological wastewater treatment for removal resins in UASB reactor: influence of oxygen. Water Sci. Technol. 57 (7), 1047–1052. Dwyer, D.F., Tiedje, J.M., 1983. Degradation of ethylene glycol and polyethylene glycols by methanogenic consortia. Appl. Environ. Microbiol. 46 (1), 185–190. Dwyer, D.F., Tiedje, J.M., 1986. Metabolism of polyethylene glycol by two anaerobic bacteria, desulfovibrio desulfuricans and a bacteroides sp. Appl. Environ. Microbiol. 52 (4), 852–856. Frimmer, U., Widdel, F., 1989. Oxidation of ethanol by methanogenic bacteria: growth experiments and enzymatic studies. Arch. Microbiol. 152, 479–483. Garibay-Orijel, C., Ríos-Leal, E., García-Mena, J., Poggi-Varaldo, H.M., 2005. 2,4,6Trichlorophenol and phenol removal in methanogenic and partially-aerated methanogenic conditions in a fluidized bed bioreactor. J. Chem. Tech. Biotechnol. 80, 1180–1187. _ Gren´, I., Gaszczak, A., Szczyrba, E., Kabuzek, S., 2009. Enrichment, isolation and susceptibility profile of the growth substrate of bacterial strains able to degrade vinyl acetate. Pol. J. Environ. Stud. 18 (3), 383–390. Gonzalez-Gil, G., Seguezzo, L., Lettinga, G., Kleerebezem, R., 2001a. Kinetics and mass-transfer phenomena in anaerobic granular sludge. Biotechnol. Bioeng. 73 (2), 125–134. Gonzalez-Gil, G., Lens, P.N., Van Aelst, A., Van As, H., Versprille, A.I., Lettinga, G., 2001b. Cluster structure of anaerobic aggregates of an expanded granular sludge bed reactor. Appl. Environ. Microbiol. 67 (8), 3683–3692. Ince, B., Ince, O., Ayman Oz, N., 2003. Changes in acetoclastic methanogenic activity and microbial composition in an upflow anaerobic filter. Water Air Soil Pollut. 144, 301–315. Lara-Mayorga, I., Durán-Hinojosa, U., Arana-Cuenca, A., Monroy-Hermosillo, O., Ramírez-Vives, F., 2010. Vinyl acetate degradation by Brevibacillus agri isolated from a slightly aerated methanogenic reactor. Environ. Technol. 31 (1), : 1–6. Nieder, M., Sunarko, B., Meyer, O., 1990. Degradation of vinyl acetate by soil, sewage, sludge, and the newly isolated aerobic bacterium V2. Appl. Environ. Microbiol. 56 (10), 3023–3028. Sarioglu, M., Bisgin, T., 2007. Removal of Maxilon Yellow GL in a mixed methanogenic anaerobic culture. Dye Pygment. 75, 544–549. Schink, B., 1997. Energetics of syntrophic cooperation in methanogenic degradation. Microbiol. Mol. Biol. Rev. 61 (2), 262–280. Schmidt, J.E., Ahring, B.K., 1999. Immobilization patterns and dynamics of acetateutilizing methanogens immobilized in sterile granular sludge in upflow anaerobic sludge blanket reactors. Appl. Environ. Microbiol. 65 (3), 1050–1054. Somasiri, W., Li, X., Ruan, W., Jian, C., 2008. Evaluation of the efficacy of upflow anaerobic sludge blanket reactor in removal of colour and reduction of COD in real textile wastewater. Bioresour. Technol. 99 (9), 3692–3699. Stuckey, D., Owen, W., McCarty, P.L., 1980. Anaerobic toxicity evaluation by batch and semi-continuous assays. J. Water Pollut. Control Fed. 52 (4), 720–729. Xu, K., Liu, H., Chen, J., 2010. Effect of classic methanogenic inhibitors on the quantity and diversity of archaeal community and the reductive homoacetogenic activity during the process of anaerobic sludge digestion. Bioresour. Technol. 101 (8), 2600–2607.