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Effect of endogenous hydrolytic enzymes pretreatment on the anaerobic digestion of sludge
Bioresource Technology 146 (2013) 758–761
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of endogenous hydrolytic enzymes pretreatment on the anaerobic digestion of sludge Shuyu Yu a, Guangming Zhang a,b,⇑, Jianzheng Li a, Zhiwei Zhao a,⇑, Xiaorong Kang a a b
School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China School of Environment & Resource, Renmin University of China, Beijing 100872, China
h i g h l i g h t s Endogenous enzymes from sludge are used for sludge lysis and anaerobic digestion enhancement. Enzyme pretreatment mainly reduces large size particle sludge. Enzyme solubilized EPS, which enhanced the subsequent anaerobic digestion.
a r t i c l e
i n f o
Article history: Received 18 June 2013 Received in revised form 17 July 2013 Accepted 19 July 2013 Available online 26 July 2013 Keywords: Sludge Endogenous enzyme Anaerobic digestion Pretreatment
a b s t r a c t In this study, the effects of endogenous amylase, endogenous protease and combined amylase/protease pretreatment of sludge were studied to enhance the efficiency of sludge anaerobic digestion. These enzymes were obtained from bacterial fermentation and bacteria were separated from the sludge. All treatments improved sludge solubilization and acidification but had little influence on the floc sizes. In terms of sludge solubilization and acidification amylase was better than protease or mixed enzyme. After 7 h endogenous amylase treatment, the supernatant soluble chemical oxygen demand and volatile fatty acids concentration increased by 78.2% and 129.6%, respectively. But, in terms of anaerobic biodegradability, the best result was obtained with combined enzyme treatment, biogas production increased by 23.1% compared to the control after 11 days of anaerobic digestion. Scanning electron micrographs observation and particle size analysis revealed that the most important mechanism for the enzyme treatment of sludge might be solubilization of extracellular polymeric substances. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction With the worldwide improvement of the municipal wastewater treatment quality, the rapid increase in sludge production is inevitable. The biological wastewater treatment leads to the result that pollutants are concentrated in the sludge. Therefore, sludge not only contains many organic matters, but also contains heavy metals, pathogens, and persistent organic pollutants to cause secondary environment pollution. For the past few years, the wastewater treatment plant (WWTP) has been under tremendous pressure and continuous to endeavor reducing the sludge disposal/management costs. It can be as high as 50% of the total WWTP operating cost. Therefore, much attention is being focused on: (1) reusing of the valuable components for production of value-added production such as biopesticides, industrial enzymes, ⇑ Corresponding authors. Tel.: +86 13520956445 (G. Zhang). Tel.: +86 15204679777 (Z. Zhao). E-mail addresses: [email protected] (G. Zhang), [email protected] (Z. Zhao). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.07.087
biopolymers, most of these reuses are at the research stage (Du Bois and Mercier, 2009). (2) Recovering energy from sludge treatment by incineration, pyrolysis, gasification, direct liquefaction, and anaerobic digestion (Appels et al., 2008). Anaerobic digestion is one of the most promising treatments for municipal sludge from the standpoint of energy recovery (Du Bois and Mercier, 2009). Meanwhile, it is also the main application at large- and medium-sized WWTP currently. Hydrolysis is recognized as rate-limiting step in the complex digestion process, therefore pretreatment is necessary to make organic matter bioavailable to the microorganisms during digestion (Carrere et al., 2010). Biological pretreatment offers unique advantages compared to chemical or physical processes as it is environmental friendly and neither causes pollution nor needs special equipment (Parawira, 2012). Biological pretreatment can be classified into 2 categories: (1) adding industrial (Yang et al., 2010) or endogenous enzymes prior to anaerobic digestion processes; (2) adding specific bacteria which can secret certain enzymes (Li et al., 2009; Tang et al., 2012). It has been shown to result in improved degradation,
S. Yu et al. / Bioresource Technology 146 (2013) 758–761
dewatering properties of the sludge and increase in methane production, and widely researched in lab- and in full-scale plants (Parmar et al., 2001). The high-cost of commercial enzymatic preparation make the pretreatment economically infeasible (Parmar et al., 2001). Thus, bioaugmentation (enhancement of the endogenous enzyme or enzyme-producing microorganisms from the municipal sludge extracellular hydrolases) or inoculation of the extraneous enzymeproducing stains is concerned as the proper way. While the effects of inoculation thermophilic microorganisms on sludge solubilization and subsequent thermophilic anaerobic digestion have been widely investigated (Li et al., 2009), mesophilic pretreatment by endogenous strain enzyme has not been sufficiently researched. The vast majority of the anaerobic processes applied in practice are still mesophilic (Appels et al., 2008). Therefore, in this study two strains isolated from municipal sludge were used to produce endogenous hydrolase. Then the effects of endogenous amylase, protease and the combination of amylase with protease addition on sludge characteristic were investigated. The performance of subsequence mesophilic anaerobic digestion was observed. Meanwhile, the possible mechanism of sludge disintegration during endogenous hydrolase treatment was discussed based on electron microscopes observations and particle size analysis.
2. Methods 2.1. Waste activated sludge Sludge used in this experiment was collected from a local wastewater treatment plant. The sludge had SCOD of 136– 211 mg/L, volatile solids (VS) of 24984–27566 mg/L, total suspended solid (TS) of 37991–38331 mg/L, total chemical oxygen demand (TCOD) of 26852–30074 mg/L, VFA of less than 30 mg/L, and pH of 6.9.
759
2.4. The measurement of enzyme activity Protease activity was estimated with casein as a substrate using modification of the method of Tang et al. (2012). The activity of amylase was determined at 37 °C as that reported by Sodhi et al. (2005). All experiments were carried out in triplicate. 2.5. Biochemical methane potential (BMP) test Batch anaerobic digestion tests were carried out to assess sludge biodegradability. 100 ml sample of pretreated sludge, seeded with 30 ml of anaerobic digestion sludge, was fed into a 250 ml serum bottles. The BMP tests were performed in a water bath at 37 °C. Blank tests contained only inoculums and control tests contained inoculums and inactivated endogenous enzyme (heated to 100 °C for 10 min).The reported value of biogas volume of untreated sludge was the measured value subtracting that of blank; and the reported biogas volume of treated sludge was the measured value subtracting that of control. Digestion experiment for each condition was duplicated. 2.6. Other analytical method All pretreated sludge samples were partially centrifuged at 6000 rpm for 10 min, and the supernatant reserved for the measurement of soluble chemical oxygen demand (SCOD), and volatile fatty acids (VFAs). SCOD and VFAs values obtained from the enzyme addition after well mixed samples were subtracted from all the measured results. The sludge TS, VS, pH, SCOD, and TCOD were measured following standard methods. Particle size distribution was determined using a laser diffraction sensor (Mastersizer 2000, Malvern Firm). The supernatant was passed through a 0.45 lm membrane filter for the analysis of VFAs. VFAs content was performed as described in our previous publication (Yu et al., 2013). 3. Results and discussion
2.2. Endogenous hydrolases harvest The amylase-producing- stain (Bacillus subtilis) and proteaseproducing stain (Aeromonas hydrophila), isolated from waste activated sludge in our previous study (Yu et al., 2013) were used in the present research. These two isolates were cultivated in liquid medium with shaking rate 120 rpm for 34 h at 35 °C. The fermentation liquor of strains were centrifuged at 8000g for 10 min at 4 °C, the supernatant was considered as endogenous hydrolases crude enzyme and it was stored at 4 °C for further study. The B. subtilis and A. hydrophila crude enzyme was, respectively named endogenous A and endogenous B. Actually both A and B were a mixture of multiple enzymes, but A was relatively riched in amylase and B was rich in protease.
2.3. Pretreatment 30 ml solution of enzyme A was added into 300 ml sludge sample to investigate effects of endogenous amylase on sludge. 30 ml enzyme solution of B was added into 300 ml sludge sample to investigate effects of endogenous protease on sludge. 15 ml A solution and 15 ml B solution were added into 300 ml sludge sample as a combinational application of protease and amylase. After the enzyme was added, the mixture was stir well immediately. Then these samples were kept with shaking rate 120 rpm for 28 h at 37 °C.
3.1. The effect of endogenous enzyme on sludge solubilization and acidification It is well known that hydrolysis of sludge solids is the rate-limiting step in the anaerobic digestion process. The sludge solubilization is often used to identify the occurrence of sludge floc lysis (Foladori et al., 2010). The increase of SCOD represents the sludge solubilization. Fig. 1 shows the effects of the endogenous enzymes on the sludge SCOD evolution with respect to the incubation time. As was expected, the SCOD release showed an increasing trend with the incubation time, because the interaction opportunities between microorganism, enzyme and substrates increased. Especially for the sample of endogenous enzyme A treatment, the SCOD increased rapidly with incubation time up to 7 h; after that the increase slowed down. SCOD release were 1229, 2823, 2162 and 2240 mg/L for control, treatment of endogenous enzyme A, B, and A + B, respectively, during 7 h of enzyme treatment at 37 °C. This corresponded to 78.2, 29.5, and 30.2% increase in SCOD release for treatment of endogenous enzyme A, B, A + B, respectively as compared to the control sample. These results indicated that enzyme A had strong ability of sludge hydrolysis, which containing a high level of amylase activity (0.47 U/mg VSS). This was probably due to the different of hydrolysis constants between carbohydrate and protein. Christ et al. (2000) reported the typical values of rate coefficients for carbohydrates and proteins in the activated sludge. In their study, kinetic coefficient of the first-order rate of hydrolysis for carbohydrate was higher than that for
760
S. Yu et al. / Bioresource Technology 146 (2013) 758–761
2000
3500
1800 VFA concentration (mg/L)
4000
2500 2000 1500 1000 500
1400
9h
7
9
28h
Fig. 1. Impacts of endogenous enzymes on the evolution of SCOD in the treated sludge, process temperature was 37 °C.
proteins. Shao et al. (2013) also pointed that the degradation efficiency of sludge VS was greater than that of protein. The maximum solubilization of organic matter was 11.07% (SCOD/TCOD) for treatment by endogenous enzyme A + B after 28 h incubation. The value was similar to the report of Wawrzynczyk (2007), in which SCOD/ TCOD was 7.5–17.1% after treatment by mixed enzymes. Although the net solubilization was not strong, 20% improvement in methane production was reported in that research. Sludge acidification is the result of soluble organic with low molecular weight fermentation by the associative acidogenic bacteria. VFAs as the main acidification products for the acidification of municipal sludge, and are the most important intermediates in the anaerobic digestion process. The VFAs concentration and composition in the supernatant during different enzymatic treatments and durations were investigated. As shown in Fig. 2, treatments with endogenous enzymes resulted in VFAs concentration increase. It suggested that endogenous enzymes were effective in acidifying the organic of the sludge. For the untreated sludge, VFAs concentration barely increased, during 7 h incubation; after that the increase accelerated with the incubation time. It was very likely that the rate of hydrolysis of sludge limited the subsequent acidification. Among the three treatments, VFAs concentration treated by enzyme A was significantly higher than the other two samples during the incubation. This might be because enzyme A was more conducive to the solubilization of polysaccharide, which was acidized prior to protein. Yu and Fang (2001) found that degradation of protein began only after carbohydrate became depleted. The VFAs concentration increased from 133 to 421, 1082, 1128 and 1719 mg/L, respectively at incubation durations of 0, 2, 5, 7, 9 and 28 h. However, acetic acid percentage decreased compared to other samples. Since acetic acid contains much lower carbon than do other VFAs produce, it needs lower enzymes or metabolic steps to in into convert biogas (Park et al., 2013). In short, the pretreatment of endogenous enzyme favored the hydrolysis and acidification of sludge, and changed the percentage of VFAs composition. 3.2. The effects of endogenous enzymes on floc size
7h
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28h
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2h
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Fig. 2. Effects of endogenous enzyme on VFAs (Volatile Fatty Acids) production and composition.
4.8% of the whole volume, whereas they occupied 4.5% in the untreated sludge. The mean particle size decreased from 87.4 lm (control) to 74.1, 85.4, and 65.8 lm, when sludge was pretreated by endogenous enzyme A, B, and A + B, respectively. The reduction of floc size was not significantly compared to physical (such as sonication) or chemical (such as ozonation) pretreatments. For example, Chu et al. (2001) obtained a mean particle size decreased from 99 lm to 22 lm, at sonication density of 0.33 W/ml and frequency of 20 kHz, during 20 min of sonication. In Fig. 1, we found that the solubilization of enzyme on sludge was limited, thus the reduction of particle was not caused by the direct conversion from solid to soluble particles. The possible reason was that enzyme acted against extracellular polymeric substances (EPS) which have a role in sludge aggregation. The reduction in the particle size imposed some solubilization of solid material, increased the surface area of floc. It would increase the surface area available for contact with the bacteria responsible for degradation, subsequently. As evident from Figs. 1 and 3, the endogenous enzymes could solubilize sludge effectively, but the capacity for sludge disintegration was limited. 3.3. Scanning electron micrographs (SEM) of sludge The effect of the applied endogenous enzymes on the sludge structure was investigated by SEM (The photos are not shown in this paper). Prior to pretreatment, the sludge with tight structure and microorganism coated in EPS were observed. Mechanisms for sludge solubilization in various pretreatment technologies are bacteria cell wall disruption, EPS solubilization, or combination of both. The SEM images showed that structure of the treated sludge became looser compared to the untreated 7 1.0
6
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5 volume (%)
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1000 Particle size
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The effects of different endogenous enzymes on floc size distribution after 28 h inoculation are represented in Fig. 3. For the treated sludge, volume occupied by particles bigger than 200 lm decreased. Especially for A + B treated sludge, it decreased from 7.3% (control) to 3.4%. Interestingly, for the treated sludge, the volume occupied by small particles did not increase, even showed a slight decrease. For A + B treated sludge, particle of 10 lm occupied
1 0 0.1
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10 Particle size
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Fig. 3. Particles size distribution for different endogenous enzymes addition.
S. Yu et al. / Bioresource Technology 146 (2013) 758–761
enzyme treatment. In terms of anaerobic biodegradability, endogenous enzyme led to an enhancement of biogas production. For the combined enzyme treatment, biogas production increased by 23.1% compared to control after 11 days of anaerobic digestion. SEM observation and particle size analysis revealed that enzyme was less effective for floc disruption, indicating the main mechanism of EPS solubilization.
800 Accumulated biogas production (ml)
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720 640 560 480 400 320
Acknowledgement
240 160
Authors thank the financial supports from the National Natural Science Foundation of China (51278489).
80 0 0
5
10
15
20
25
30
References
Time (days) Fig. 4. Biogas production in mesophilic anaerobic batch tests with raw and endogenous enzyme pretreated sludge.
sludge. However, endogenous enzymes did not cause significant lysis or disruption of filaments. This result indicated that the increasing soluble organic mainly derived from EPS and the effect of enzymatic treatment on sludge integrity was limited. Surface of the treated sludge presented much more filaments and scatter bacteria due to solubilization of EPS. Similar findings have been reported and Sesay et al. (2006) observed that enzymatic treatment did not cause any significant cell lysis as measured by viable cell counts. 3.4. The effects of endogenous enzyme pretreatment on sludge anaerobic digestion As shown in Fig. 4, sludge anaerobic digestion was affected by enzymatic pretreatment. Initial biogas production rate (indicated by the slope of carve) up to day 14 was similar, except for the endogenous hydrolyse A + B pretreated sludge. At day 22 the accumulated biogas production was nearly 660 ml for A and A + B pretreatment samples, whereas for the control and B treatment, they were around 560 ml, representing 17.8% increase. Although, as shown in Fig. 1, the maximum solubilization of organic matter was only 11.07% for treatment by endogenous enzyme A + B after 28 h incubation. The final biogas production was improved by 18.6%, 15.6%, and 20.2%, respectively when sludge was treated by enzyme A, B, and A + B. The difference between solubilization effect and biogas improvement might resulted from continuous hydrolyzing of sludge by residual endogenous enzyme during the process of digestion (Yao et al., 2013). 4. Conclusion Endogenous hydrolase pretreatments were favorable to the solubilization of municipal sludge, at a moderate temperature range. Moreover, the SCOD concentration and VFAs yields were higher after amylase treatment than those after protease or combined
Appels, L., Baeyens, J., Degreve, J., Dewil, R., 2008. Principles and potential of the anaerobic digestion of waste-activated sludge. Prog. Energ. Combust. 34, 755– 781. Carrere, H. et al., 2010. Pretreatment methods to improve sludge anaerobic degradability: a review. J. Hazard. Mater. 183, 1–15. Christ, O., Wilderer, P.A., Angerhofer, R., Faulstich, M., 2000. Mathematical modeling of the hydrolysis of anaerobic processes. Water Sci. Technol. 41, 61–65. Chu, C.P., Chang, B.V., Liao, G.S., Jean, D.S., Lee, D.J., 2001. Observations on changes in ultrasonically treated waste-activated sludge. Water Res. 35, 1038–1046. Du Bois, E., Mercier, A., 2009. Energy recovery. In: Xu, C.C., Lancaster, J. (Eds.), Treatment of Secondary Sludge for Energy Recovery. Nova Science Publishers Inc., New York, pp. 187–212. Foladori, P., Tamburini, S., Bruni, L., 2010. Bacteria permeabilisation and disruption caused by sludge reduction technologies evaluated by flow cytometry. Water Res. 44, 4888–4899. Li, X.S., Ma, H.Z., Wang, Q.H., Matsumoto, S., Maeda, T., Ogawa, H.I., 2009. Isolation, identification of sludge-lysing strain and its utilization in thermophilic aerobic digestion for waste activated sludge. Bioresour. Technol. 100, 2475–2481. Parawira, W., 2012. Enzyme research and applications in biotechnological intensification of biogas production. Crit. Rev. Biotechnol. 32, 172–186. Park, J., Kim, S., Park, H., Lim, D.J., Yoon, J., 2013. Feasibility of anaerobic digestion from bioethanol fermentation residue. Bioresour. Technol. 141, 177–183. Parmar, N., Singh, A., Ward, O.P., 2001. Enzyme treatment to reduce solids and improve settling of sewage sludge. J. Ind. Microbiol. Biotechnol. 26, 383–386. Sesay, M.L., Ozcengiz, G., Sanin, F.D., 2006. Enzymatic extraction of activated sludge extracellular polymers and implications on bioflocculation. Water Res. 40, 1359–1366. Shao, L., Wang, T., Li, T., Lü, F., He, P., 2013. Comparison of sludge digestion under aerobic and anaerobic conditions with a focus on the degradation of proteins at mesophilic temperature. Bioresour. Technol. 140, 131–137. Sodhi, H.K., Sharma, K., Gupta, J.K., Soni, S.K., 2005. Production of a thermostable alpha-amylase from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production. Process Biochem. 40, 525–534. Tang, Y., Yang, Y.L., Li, X.M., Yang, Q., Wang, D.B., Zeng, G.M., 2012. The isolation, identification of sludge-lysing thermophilic bacteria and its utilization in solubilization for excess sludge. Environ. Technol. 33, 961–966. Wawrzynczyk, J., 2007. Enzymatic Treatment of Wastewater Sludge: Solubilization, Improvement of Anaerobic Digestion and Extraction of Extracellular Polymeric Substances (Ph.D. thesis). Division of Pure and Applied Biochemistry, Lund University, Lund, Sweden. Yang, Q. et al., 2010. Enhanced efficiency of biological excess sludge hydrolysis under anaerobic digestion by additional enzymes. Bioresour. Technol. 101, 2924–2930. Yao, Y. et al., 2013. Anaerobic digestion of poplar processing residues for methane production after alkaline treatment. Bioresour. Technol. 134, 347–352. Yu, H.Q., Fang, H., 2001. Acidification of mid- and high-strength dairy wastewaters. Water Res. 35, 3697–3705. Yu, S.Y., Zhang, G.M., Zhao, Z.W., 2013. Effect of endogenous amylase on characterization and anaerobic digestibility of sludge. J. Harbin Inst. Tech. 45, 48–52.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Effect of endogenous hydrolytic enzymes pretreatment on the anaerobic digestion of sludge Shuyu Yu a, Guangming Zhang a,b,⇑, Jianzheng Li a, Zhiwei Zhao a,⇑, Xiaorong Kang a a b
School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China School of Environment & Resource, Renmin University of China, Beijing 100872, China
h i g h l i g h t s Endogenous enzymes from sludge are used for sludge lysis and anaerobic digestion enhancement. Enzyme pretreatment mainly reduces large size particle sludge. Enzyme solubilized EPS, which enhanced the subsequent anaerobic digestion.
a r t i c l e
i n f o
Article history: Received 18 June 2013 Received in revised form 17 July 2013 Accepted 19 July 2013 Available online 26 July 2013 Keywords: Sludge Endogenous enzyme Anaerobic digestion Pretreatment
a b s t r a c t In this study, the effects of endogenous amylase, endogenous protease and combined amylase/protease pretreatment of sludge were studied to enhance the efficiency of sludge anaerobic digestion. These enzymes were obtained from bacterial fermentation and bacteria were separated from the sludge. All treatments improved sludge solubilization and acidification but had little influence on the floc sizes. In terms of sludge solubilization and acidification amylase was better than protease or mixed enzyme. After 7 h endogenous amylase treatment, the supernatant soluble chemical oxygen demand and volatile fatty acids concentration increased by 78.2% and 129.6%, respectively. But, in terms of anaerobic biodegradability, the best result was obtained with combined enzyme treatment, biogas production increased by 23.1% compared to the control after 11 days of anaerobic digestion. Scanning electron micrographs observation and particle size analysis revealed that the most important mechanism for the enzyme treatment of sludge might be solubilization of extracellular polymeric substances. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction With the worldwide improvement of the municipal wastewater treatment quality, the rapid increase in sludge production is inevitable. The biological wastewater treatment leads to the result that pollutants are concentrated in the sludge. Therefore, sludge not only contains many organic matters, but also contains heavy metals, pathogens, and persistent organic pollutants to cause secondary environment pollution. For the past few years, the wastewater treatment plant (WWTP) has been under tremendous pressure and continuous to endeavor reducing the sludge disposal/management costs. It can be as high as 50% of the total WWTP operating cost. Therefore, much attention is being focused on: (1) reusing of the valuable components for production of value-added production such as biopesticides, industrial enzymes, ⇑ Corresponding authors. Tel.: +86 13520956445 (G. Zhang). Tel.: +86 15204679777 (Z. Zhao). E-mail addresses: [email protected] (G. Zhang), [email protected] (Z. Zhao). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.07.087
biopolymers, most of these reuses are at the research stage (Du Bois and Mercier, 2009). (2) Recovering energy from sludge treatment by incineration, pyrolysis, gasification, direct liquefaction, and anaerobic digestion (Appels et al., 2008). Anaerobic digestion is one of the most promising treatments for municipal sludge from the standpoint of energy recovery (Du Bois and Mercier, 2009). Meanwhile, it is also the main application at large- and medium-sized WWTP currently. Hydrolysis is recognized as rate-limiting step in the complex digestion process, therefore pretreatment is necessary to make organic matter bioavailable to the microorganisms during digestion (Carrere et al., 2010). Biological pretreatment offers unique advantages compared to chemical or physical processes as it is environmental friendly and neither causes pollution nor needs special equipment (Parawira, 2012). Biological pretreatment can be classified into 2 categories: (1) adding industrial (Yang et al., 2010) or endogenous enzymes prior to anaerobic digestion processes; (2) adding specific bacteria which can secret certain enzymes (Li et al., 2009; Tang et al., 2012). It has been shown to result in improved degradation,
S. Yu et al. / Bioresource Technology 146 (2013) 758–761
dewatering properties of the sludge and increase in methane production, and widely researched in lab- and in full-scale plants (Parmar et al., 2001). The high-cost of commercial enzymatic preparation make the pretreatment economically infeasible (Parmar et al., 2001). Thus, bioaugmentation (enhancement of the endogenous enzyme or enzyme-producing microorganisms from the municipal sludge extracellular hydrolases) or inoculation of the extraneous enzymeproducing stains is concerned as the proper way. While the effects of inoculation thermophilic microorganisms on sludge solubilization and subsequent thermophilic anaerobic digestion have been widely investigated (Li et al., 2009), mesophilic pretreatment by endogenous strain enzyme has not been sufficiently researched. The vast majority of the anaerobic processes applied in practice are still mesophilic (Appels et al., 2008). Therefore, in this study two strains isolated from municipal sludge were used to produce endogenous hydrolase. Then the effects of endogenous amylase, protease and the combination of amylase with protease addition on sludge characteristic were investigated. The performance of subsequence mesophilic anaerobic digestion was observed. Meanwhile, the possible mechanism of sludge disintegration during endogenous hydrolase treatment was discussed based on electron microscopes observations and particle size analysis.
2. Methods 2.1. Waste activated sludge Sludge used in this experiment was collected from a local wastewater treatment plant. The sludge had SCOD of 136– 211 mg/L, volatile solids (VS) of 24984–27566 mg/L, total suspended solid (TS) of 37991–38331 mg/L, total chemical oxygen demand (TCOD) of 26852–30074 mg/L, VFA of less than 30 mg/L, and pH of 6.9.
759
2.4. The measurement of enzyme activity Protease activity was estimated with casein as a substrate using modification of the method of Tang et al. (2012). The activity of amylase was determined at 37 °C as that reported by Sodhi et al. (2005). All experiments were carried out in triplicate. 2.5. Biochemical methane potential (BMP) test Batch anaerobic digestion tests were carried out to assess sludge biodegradability. 100 ml sample of pretreated sludge, seeded with 30 ml of anaerobic digestion sludge, was fed into a 250 ml serum bottles. The BMP tests were performed in a water bath at 37 °C. Blank tests contained only inoculums and control tests contained inoculums and inactivated endogenous enzyme (heated to 100 °C for 10 min).The reported value of biogas volume of untreated sludge was the measured value subtracting that of blank; and the reported biogas volume of treated sludge was the measured value subtracting that of control. Digestion experiment for each condition was duplicated. 2.6. Other analytical method All pretreated sludge samples were partially centrifuged at 6000 rpm for 10 min, and the supernatant reserved for the measurement of soluble chemical oxygen demand (SCOD), and volatile fatty acids (VFAs). SCOD and VFAs values obtained from the enzyme addition after well mixed samples were subtracted from all the measured results. The sludge TS, VS, pH, SCOD, and TCOD were measured following standard methods. Particle size distribution was determined using a laser diffraction sensor (Mastersizer 2000, Malvern Firm). The supernatant was passed through a 0.45 lm membrane filter for the analysis of VFAs. VFAs content was performed as described in our previous publication (Yu et al., 2013). 3. Results and discussion
2.2. Endogenous hydrolases harvest The amylase-producing- stain (Bacillus subtilis) and proteaseproducing stain (Aeromonas hydrophila), isolated from waste activated sludge in our previous study (Yu et al., 2013) were used in the present research. These two isolates were cultivated in liquid medium with shaking rate 120 rpm for 34 h at 35 °C. The fermentation liquor of strains were centrifuged at 8000g for 10 min at 4 °C, the supernatant was considered as endogenous hydrolases crude enzyme and it was stored at 4 °C for further study. The B. subtilis and A. hydrophila crude enzyme was, respectively named endogenous A and endogenous B. Actually both A and B were a mixture of multiple enzymes, but A was relatively riched in amylase and B was rich in protease.
2.3. Pretreatment 30 ml solution of enzyme A was added into 300 ml sludge sample to investigate effects of endogenous amylase on sludge. 30 ml enzyme solution of B was added into 300 ml sludge sample to investigate effects of endogenous protease on sludge. 15 ml A solution and 15 ml B solution were added into 300 ml sludge sample as a combinational application of protease and amylase. After the enzyme was added, the mixture was stir well immediately. Then these samples were kept with shaking rate 120 rpm for 28 h at 37 °C.
3.1. The effect of endogenous enzyme on sludge solubilization and acidification It is well known that hydrolysis of sludge solids is the rate-limiting step in the anaerobic digestion process. The sludge solubilization is often used to identify the occurrence of sludge floc lysis (Foladori et al., 2010). The increase of SCOD represents the sludge solubilization. Fig. 1 shows the effects of the endogenous enzymes on the sludge SCOD evolution with respect to the incubation time. As was expected, the SCOD release showed an increasing trend with the incubation time, because the interaction opportunities between microorganism, enzyme and substrates increased. Especially for the sample of endogenous enzyme A treatment, the SCOD increased rapidly with incubation time up to 7 h; after that the increase slowed down. SCOD release were 1229, 2823, 2162 and 2240 mg/L for control, treatment of endogenous enzyme A, B, and A + B, respectively, during 7 h of enzyme treatment at 37 °C. This corresponded to 78.2, 29.5, and 30.2% increase in SCOD release for treatment of endogenous enzyme A, B, A + B, respectively as compared to the control sample. These results indicated that enzyme A had strong ability of sludge hydrolysis, which containing a high level of amylase activity (0.47 U/mg VSS). This was probably due to the different of hydrolysis constants between carbohydrate and protein. Christ et al. (2000) reported the typical values of rate coefficients for carbohydrates and proteins in the activated sludge. In their study, kinetic coefficient of the first-order rate of hydrolysis for carbohydrate was higher than that for
760
S. Yu et al. / Bioresource Technology 146 (2013) 758–761
2000
3500
1800 VFA concentration (mg/L)
4000
2500 2000 1500 1000 500
1400
9h
7
9
28h
Fig. 1. Impacts of endogenous enzymes on the evolution of SCOD in the treated sludge, process temperature was 37 °C.
proteins. Shao et al. (2013) also pointed that the degradation efficiency of sludge VS was greater than that of protein. The maximum solubilization of organic matter was 11.07% (SCOD/TCOD) for treatment by endogenous enzyme A + B after 28 h incubation. The value was similar to the report of Wawrzynczyk (2007), in which SCOD/ TCOD was 7.5–17.1% after treatment by mixed enzymes. Although the net solubilization was not strong, 20% improvement in methane production was reported in that research. Sludge acidification is the result of soluble organic with low molecular weight fermentation by the associative acidogenic bacteria. VFAs as the main acidification products for the acidification of municipal sludge, and are the most important intermediates in the anaerobic digestion process. The VFAs concentration and composition in the supernatant during different enzymatic treatments and durations were investigated. As shown in Fig. 2, treatments with endogenous enzymes resulted in VFAs concentration increase. It suggested that endogenous enzymes were effective in acidifying the organic of the sludge. For the untreated sludge, VFAs concentration barely increased, during 7 h incubation; after that the increase accelerated with the incubation time. It was very likely that the rate of hydrolysis of sludge limited the subsequent acidification. Among the three treatments, VFAs concentration treated by enzyme A was significantly higher than the other two samples during the incubation. This might be because enzyme A was more conducive to the solubilization of polysaccharide, which was acidized prior to protein. Yu and Fang (2001) found that degradation of protein began only after carbohydrate became depleted. The VFAs concentration increased from 133 to 421, 1082, 1128 and 1719 mg/L, respectively at incubation durations of 0, 2, 5, 7, 9 and 28 h. However, acetic acid percentage decreased compared to other samples. Since acetic acid contains much lower carbon than do other VFAs produce, it needs lower enzymes or metabolic steps to in into convert biogas (Park et al., 2013). In short, the pretreatment of endogenous enzyme favored the hydrolysis and acidification of sludge, and changed the percentage of VFAs composition. 3.2. The effects of endogenous enzymes on floc size
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Fig. 2. Effects of endogenous enzyme on VFAs (Volatile Fatty Acids) production and composition.
4.8% of the whole volume, whereas they occupied 4.5% in the untreated sludge. The mean particle size decreased from 87.4 lm (control) to 74.1, 85.4, and 65.8 lm, when sludge was pretreated by endogenous enzyme A, B, and A + B, respectively. The reduction of floc size was not significantly compared to physical (such as sonication) or chemical (such as ozonation) pretreatments. For example, Chu et al. (2001) obtained a mean particle size decreased from 99 lm to 22 lm, at sonication density of 0.33 W/ml and frequency of 20 kHz, during 20 min of sonication. In Fig. 1, we found that the solubilization of enzyme on sludge was limited, thus the reduction of particle was not caused by the direct conversion from solid to soluble particles. The possible reason was that enzyme acted against extracellular polymeric substances (EPS) which have a role in sludge aggregation. The reduction in the particle size imposed some solubilization of solid material, increased the surface area of floc. It would increase the surface area available for contact with the bacteria responsible for degradation, subsequently. As evident from Figs. 1 and 3, the endogenous enzymes could solubilize sludge effectively, but the capacity for sludge disintegration was limited. 3.3. Scanning electron micrographs (SEM) of sludge The effect of the applied endogenous enzymes on the sludge structure was investigated by SEM (The photos are not shown in this paper). Prior to pretreatment, the sludge with tight structure and microorganism coated in EPS were observed. Mechanisms for sludge solubilization in various pretreatment technologies are bacteria cell wall disruption, EPS solubilization, or combination of both. The SEM images showed that structure of the treated sludge became looser compared to the untreated 7 1.0
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The effects of different endogenous enzymes on floc size distribution after 28 h inoculation are represented in Fig. 3. For the treated sludge, volume occupied by particles bigger than 200 lm decreased. Especially for A + B treated sludge, it decreased from 7.3% (control) to 3.4%. Interestingly, for the treated sludge, the volume occupied by small particles did not increase, even showed a slight decrease. For A + B treated sludge, particle of 10 lm occupied
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Fig. 3. Particles size distribution for different endogenous enzymes addition.
S. Yu et al. / Bioresource Technology 146 (2013) 758–761
enzyme treatment. In terms of anaerobic biodegradability, endogenous enzyme led to an enhancement of biogas production. For the combined enzyme treatment, biogas production increased by 23.1% compared to control after 11 days of anaerobic digestion. SEM observation and particle size analysis revealed that enzyme was less effective for floc disruption, indicating the main mechanism of EPS solubilization.
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Acknowledgement
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Authors thank the financial supports from the National Natural Science Foundation of China (51278489).
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References
Time (days) Fig. 4. Biogas production in mesophilic anaerobic batch tests with raw and endogenous enzyme pretreated sludge.
sludge. However, endogenous enzymes did not cause significant lysis or disruption of filaments. This result indicated that the increasing soluble organic mainly derived from EPS and the effect of enzymatic treatment on sludge integrity was limited. Surface of the treated sludge presented much more filaments and scatter bacteria due to solubilization of EPS. Similar findings have been reported and Sesay et al. (2006) observed that enzymatic treatment did not cause any significant cell lysis as measured by viable cell counts. 3.4. The effects of endogenous enzyme pretreatment on sludge anaerobic digestion As shown in Fig. 4, sludge anaerobic digestion was affected by enzymatic pretreatment. Initial biogas production rate (indicated by the slope of carve) up to day 14 was similar, except for the endogenous hydrolyse A + B pretreated sludge. At day 22 the accumulated biogas production was nearly 660 ml for A and A + B pretreatment samples, whereas for the control and B treatment, they were around 560 ml, representing 17.8% increase. Although, as shown in Fig. 1, the maximum solubilization of organic matter was only 11.07% for treatment by endogenous enzyme A + B after 28 h incubation. The final biogas production was improved by 18.6%, 15.6%, and 20.2%, respectively when sludge was treated by enzyme A, B, and A + B. The difference between solubilization effect and biogas improvement might resulted from continuous hydrolyzing of sludge by residual endogenous enzyme during the process of digestion (Yao et al., 2013). 4. Conclusion Endogenous hydrolase pretreatments were favorable to the solubilization of municipal sludge, at a moderate temperature range. Moreover, the SCOD concentration and VFAs yields were higher after amylase treatment than those after protease or combined
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