- Email: [email protected]
Survival of Salmonella spp. in a simulated acid-phase anaerobic digester treating sewage sludge
Bioresource Technology 86 (2003) 53–57
Survival of Salmonella spp. in a simulated acid-phase anaerobic digester treating sewage sludge K. Fukushi
a,*
, S. Babel b, S. Burakrai
c
a Environmental Science Center, The University of Tokyo, Tokyo, 113-0033, Japan Sirindhorn International Institute of Technology, Thammasat University, Pathumthani 12121, Thailand School of Environment, Resources and Development, Asian Institute of Technology, Pathumthani 12120, Thailand b
c
Received in revised form 17 April 2002; accepted 24 April 2002
Abstract The presence of pathogenic microorganisms in municipal waste sludge may create a serious outbreak of water borne diseases if the sludge is used for agricultural purpose. An attempt to decrease the number of pathogenic microorganisms, Salmonella spp. using a simulated acid-phase anaerobic digester was tested in a laboratory-scale batch experiment. Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of a two-phase anaerobic digestion process. A high concentration of organic acid at a pH value of 5.5–6.0 prevents a decrease in Salmonella spp. concentration. Almost complete destruction of Salmonella spp. is observed within two days if the pH value is maintained below 5.5. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Two-phase anaerobic digestion; Salmonella spp.; Acid digester; Sludge; Pathogen
1. Introduction As a result of urbanization, many parts of the world have become densely populated. These highly populated areas produce various kinds of wastes, including industrial hazardous materials, landfill leachate, waste spill, and biological hazardous substances. Pathogenic microorganisms are produced mainly from ordinary households with infected individuals. It is well known that domestic sewage streams may contain high concentrations of pathogenic microorganisms. The activated sludge process has been used to treat domestic wastewater in various areas of the world. Many aerobic wastewater treatment processes remove a majority of pathogenic microorganisms in the original waste stream by adsorption, coagulation, and precipitation, or trap them in a sludge floe matrix (Surampalliry et al., 1994). Most of the pathogens removed from the stream are transferred into the primary and secondary sludge. Consequently, the sludge contains a high number of
*
Corresponding author. Tel.: +81-3-5841-2974; fax: +81-3-58028887. E-mail address: [email protected] (K. Fukushi).
pathogenic microorganisms that need to be treated and disposed of in a proper manner. Anaerobic digestion is known to obtain a good volume reduction of sludge with a considerable removal of pathogenic microorganisms. Although the conventional anaerobic digester is appropriate to achieve the requirement of class B sludge (Stukenberg et al., 1994), a costly process, such as thermophilic/mesophilic two-phase anaerobic process, may be required for the production of class A sludge determined by the USEPA (Huyard et al., 2000). The optimum pH for the growth of Salmonella species is about 6.2–7.2 (Fields, 1979), and Lin et al. (1995) and Foster and Hall (1991) reported Salmonella typhimurium showed a significant inhibition of growth at pH value below 4. These reports indicate that a low pH in the acid phase digester (less than 6.0) in the two-phase anaerobic digestion process makes it feasible to reduce the number of pathogens, including Salmonella spp. Considerable research reports have been issued on Salmonella destruction in sludge (Gantzer et al., 2001; Cheubarn and Pagilla, 2000). Thermophilic digestion is effective to decrease the number of Salmonella (Watanabe et al., 1997). Barrios et al. (2001) reported the effect of acetic acid concentration on Salmonella spp. survival. Destruction of pathogens by the two-phase anaerobic
0960-8524/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 1 1 2 - 8
54
K. Fukushi et al. / Bioresource Technology 86 (2003) 53–57
digestion process has not been fully investigated experimentally. The effect of pH and acid concentration warrants particular attention. In the two-phase process, pH value is linked with acid concentration, which makes investigation of a separate effect of pH and acid concentration on pathogen survival difficult. In this paper, the separate effect of pH and organic acid concentration on the survival of Salmonella spp. was investigated. A simulated acid digester was used for this investigation.
Table 1 Batch run condition for testing the effect of organic acid concentration on Salmonella spp. survival Run Total acid Acetic concentra- acid contion (mg/l) centration (mg/l)
Propionic acid concentration (mg/l)
Butyric acid concentration (mg/l)
Adjusted initial pH
A1 A2 A3 AC
2976 2976 0 0
2397 0 0 0
5.5 5.5 5.5 5.6
9306 6909 3933 0
3933 3933 3933 0
The reactor volume was 400 ml.
2. Methods The acid digester environment has a high organic acid concentration and a low pH. These two parameters are usually inversely correlated, and their individual effect on the survival of pathogenic microorganisms is not fully understood. In this paper, we controlled pH or organic acid concentration at a certain value to determine the individual effect of pH and organic acid concentration. In order to study the effect of organic acid concentration on the survival of Salmonella spp., sample sludge with Salmonella spp. was mixed with various concentrations of organic acids mixture (consisting of acetic, propionic, and butyric acids). In order to study the effect of pH on survival of Salmonella spp., sample sludge was spiked with the known amount of organic acid mixture, and the values of pH were varied from 3.5 to 5.5 by sodium hydrooxide and hydrochloric acid. All batch experiments were carried out in reactors maintained at an anaerobic condition at a mesophilic temperature of 37 °C. 2.1. Sample sludge The sludge used in this experiment was collected from a return sludge line after being passed through the secondary clarifier of Siphraya Domestic Wastewater Treatment Plant located in Bangkok, Thailand. The treatment plant employs a standard activated sludge process. The sample was kept at a temperature below 10 °C during transportation from the plant to the laboratory. Original population of Salmonella spp. was about 105 MPN/l00 ml in the collected sludge with volatile solids concentration of about 5500 mg/l. 2.2. Batch experiment procedure A 500-ml Erlenmeyer flask was used as a batch reactor. The batch experiment was started by flushing the flasks with nitrogen gas for 3 min to remove oxygen, closing the flasks with rubber stoppers tightly, and transferring sludge and chemicals through sampling ports on the rubber cap with a syringe. Plastic gassampling bags were installed with all flasks to collect digester gas and to prevent high pressure in the digesters. The identification of runs is summarized in Tables 1
Table 2 Batch run condition for testing the effect of pH on Salmonella spp. survival Run
Total acid concentration (designed) (mg/l)
Total acid concentration (measured) (mg/l)
Adjusted initial pH
P1 P2 P3 P4 P5 PC
9306 9306 9306 9306 9306 0
7068 9223 7256 7033 7633 117
3.5 4.0 4.5 5.0 5.5 No adjust
The reactor volume was 400 ml.
and 2. The composition of organic acids was decided from the information in the literature (Ghosh et al., 1975). All batch reactors were incubated in a walk-in constant-temperature chamber kept at 37 °C. Samples for the enumeration of Salmonella spp. were withdrawn by syringe through the sampling port located on the rubber cap of the flasks. 2.3. Enumeration of Salmonella spp. in sludge In order to increase the sensitivity of Salmonella spp. enumeration, a novel enumeration method was developed. The procedure was a modification of a method outlined in Standard Methods (part 9260D, APHA et al., 1995). Since the concentration of Salmonella spp. in sludge is not high, and since sludge includes many heterogeneous microorganisms, a three-step preliminary incubation of samples was performed to increase sensitivity of the analysis. The sludge samples withdrawn from reactors were treated with a 200-W ultrasonic generator (UD-200, Tomy Seiko Co. Ltd., Tokyo, Japan) for 1 min to disintegrate flock in the sludge, and then diluted with a 0.1% peptone solution for 10 2 –10 5 . One milliliter of diluted samples was transferred into five incubation tubes with 10 ml of EEM-broth medium (EMA23, Eiken, Tokyo, Japan), a preincubation medium for Salmonella spp. The EEM-broth medium is used to amplify the number of bacteria in the sample. After 24 h of incubation at 37 °C, 1 ml of the tube’s contents was transferred to incubation tubes with 10 ml of Rappa-
K. Fukushi et al. / Bioresource Technology 86 (2003) 53–57
port-broth medium (E-MB25, Eiken, Tokyo, Japan), a selective medium for Salmonella spp. The tubes were incubated at 37 °C for 48 h. Then, using a sterilized platinum loop, samples were taken from the tubes and streaked on MLCB-agar plates (Code 05041, Nissui, Tokyo, Japan). The plates were incubated at 37 °C for 18–24 h. The existence of Salmonella spp. can be determined by examining the color of the colony on the plate. Black colonies are formed if Salmonella spp. is in the sample. Confirmation of the determination was done by selecting a black-colored colony, and culturing as previously described, and streaking the selected microbes on the MLCB-agar plate. Using the MPN table provided in Standard Methods (APHA et al., 1995), the number of Salmonella spp. in the original sample was determined. Although this method is quite laborious, it provides very high sensitivity to the presence of Salmonella spp. The minimum concentration which can be measured with this method is 0.01 MPN/100 ml. Results presented in this paper is an average value of duplicated measurement of Salmonella spp.
3. Results and discussion 3.1. Effect of organic acid concentration at the same initial pH on the survival of Salmonella spp. In order to investigate the survival of pathogens (Salmonella spp., in this study) in an anaerobic acid digester, sludge obtained from the aeration tank of a standard activated sludge process was mixed with organic acids at various compositions and concentrations. Activated sludge obtained in Bangkok contains a considerable amount of Salmonella spp. Concentration of Salmonella spp. as well as other environmental parameters of sludge slurry was measured at the beginning of and during the experiment. Four runs of experiments were conducted. Each run was different in terms of concentration of total organic acid at the same initial pH of about 5.5. Organic acid concentrations during the batch experiment are shown in Fig. 1. The concentration of organic acids during the batch experiment varied significantly. Acid concentrations tended to decrease during A2 and A3 runs, whereas the control run, AC, showed almost zero acid concentration during the experiment. The decrease of acid concentration in A2 and A3 was believed to be due to a conversion of acid to methane gas. Unfortunately, the methane production was not confirmed. Values of pH during the batch runs are shown in Fig. 2. As mentioned in Section 2, only initial pH was adjusted. No pH adjustment during the batch experiment was performed. The pH in all runs increased during the batch experiment. Organic acids in the batch reactor were probably consumed by methane formers, and the
55
Fig. 1. Total organic acid concentration profile. Acid concentration was artificially adjusted at day 0.
pH was increased. It should be noted that the pH change was very small (about 0.5 pH unit) up to day 5, a typical acid digester retention time. Fig. 3 shows the concentration profile of Salmonella spp. during the batch experiment. The concentration of Salmonella spp. in the batch reactor generally decreased after the fifth day. However, the concentration of Salmonella spp. fluctuated between day 0 and day 5. Although the environmental conditions in the batch reactor were not optimal for Salmonella spp., they were not harmful. It is known that organic acid can be a substrate for Salmonella spp. that may assist the survival of Salmonella spp. in the acid digester. The ideal pH for the acid digester is considered to be about 5.5 (Ghosh et al., 1975), and typical acid concentration is between 3000 and 8000 mg/l. A high acid concentration in the acid digester is desirable for optimal operation of twophase anaerobic digestion since such an operational
Fig. 2. Profile of pH. Adjustment of pH was not performed during the experiment.
56
K. Fukushi et al. / Bioresource Technology 86 (2003) 53–57
Fig. 3. Profile of Salmonella spp. concentration. Initial total acid concentrations were 9306, 6909, 3933, and 0 mg/l for Al, A2, A3, and AC (control), respectively. Value of pH was shown in Fig. 2.
condition gives a better methane conversion and a high solid reduction rate. This result indicates Salmonella spp. survives better in a high acid concentration, which is good for the digestion process. In other words, a good acid digester produces more pathogens than a poor acid digester provided the pH is about 5.5–6.0. 3.2. Effect of pH at the same initial organic acid concentration on the survival of Salmonella spp. In the last section, we noted the general tendency for Salmonella spp. to decrease at various concentrations of acid at a similar pH condition. However, the time required to lower the concentration to an undetectable level was about 10 days. Holding sludge in the acid digester for 10 days is impractical. In this section, six batch experiments were conducted to test the effect of pH on the survival of Salmonella spp. Organic acid concentration was controlled at about 7500–9500 mg/l for all batch experiments, except the control run, during the experimental period of five days. Fig. 4 shows total organic acid concentrations during the batch experiments. All batch reactors initially were adjusted for the same organic acid concentration and various pH conditions, except the control run (PC). The acid concentration during the five days of the experiment was kept nearly constant. This result agrees with those shown in Fig. 1. The acid concentration of Al in Fig. 1 was an exception. However, if the data of days 3 and 4 are ignored, the concentration of acid in Al was almost constant for 10 days. Unfortunately, we were unable to determine the reason for a temporal decrease in acid observed in day 3 and day 4. Fig. 5 shows pH values during the batch experiments. Fig. 5 results also indicates that the pH and acid conditions were almost constant throughout the batch experiment period.
Fig. 4. Total organic acid concentration profile. Acid concentration and pH was artificially adjusted at day 0.
Fig. 6 exhibits the concentration of Salmonella spp. during the batch experiments. Salmonella spp. in batch runs at initial pH values of 5.0 or lower (P1, P2, P3, and P4) were killed within two days. Salmonella spp. survived longer in the batch reactor at a pH value of 5.5 (P5). However, Salmonella spp. in the P5 run were killed in five days. In the control run, PC, Salmonella spp. remained even after the fifth day. This result indicates that a low pH provides an adverse environment for Salmonella spp. survival. As demonstrated in Fig. 3, Salmonella spp. survived on the fifth day although the acid concentration was kept quite high. Himathongkham et al. (1999) reported that it takes 2.52 days to lower the concentration of Salmonella typhimurium 1 decimal in fresh cow manure at 37 °C in an anaerobic condition. The pH values of manure in their work were neutral or alkaline due to the high ammonium concentration. The result of Fig. 6
Fig. 5. Profile of pH. Acid concentration and pH was artificially adjusted at day 0.
K. Fukushi et al. / Bioresource Technology 86 (2003) 53–57
57
Acknowledgements The authors thank Dr. Sambhunath Ghosh for inspiring this work. This work was supported by the Japan International Cooperation Agency and the Royal Thai Government through the Asian Institute of Technology, Thailand. References
Fig. 6. Profile of Salmonella spp. concentration. Values of pH were adjusted 3.5, 4.0, 4.5, 5.0, 5.5 for P1, P2, P3, P4, and P5, respectively at day 0. Total acid concentration was adjusted to 9306 mg/l for all runs except PC (control).
shows that it took two days to lower the concentration of Salmonella spp. 5 decimals at a pH range between 3.5 and 5.0. At a pH value of 5.5, a typical acid digester pH, the time required to lower the Salmonella spp. concentration 1 decimal was about 1 day. Lin et al. (1995) and Foster and Hall (1991) reported Salmonella typhimurium showed a significant inhibition of growth at pH value below 4. Lowering the pH of waste leads to significant destruction of pathogens. From the viewpoint of a digester design engineer, complete pathogen elimination in a few days is very attractive in order to satisfy USEPA’s class A sludge requirements.
4. Conclusions Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of the two-phase anaerobic digestion process. High concentration of organic acid (5000–7000 mg/l of total organic acid) at a pH 5.5–6.0 prevented a decrease of Salmonella spp. concentration. If the pH value was maintained below 5.5, the number of Salmonella spp. decreased to the undetectable level. The acid digester of the two-phase anaerobic digestion process should be operated at a retention time of two days or longer at a pH below 5.5 for effective pathogen destruction.
American Public Health Association, American Water Works Association, Water Environment Federation, 1995. Standard methods for examination of water and wastewater, 19th edition, Washington DC. Barrios, J.A., Jimenez, B., Salgado, G., Garibay, A., Castrejon, A., 2001. Growth of fecal coliform and Salmonella spp. in physicochemical sludge treated with acetic acid. Water Sci. Technol. 44 (10), 85–90. Cheubarn, T., Pagilla, K.R., 2000. Anaerobic thermophilic/mesophilic dual-stage sludge treatment. J. Environ. Eng. ASCE 126 (9), 796– 801. Fields, M.L., 1979. Factor influencing growth of spoilage microorganisms. In: Fundamental of food microbiology. AVI Publishing Co., Westport, pp. 77–92. Foster, J.W., Hall, H.K., 1991. Inducible pH homeostasis and acid tolerance response of Salmonella typhimurium. J. Bacterial. 173 (16), 5129–5135. Gantzer, C., Gaspard, P., Galvez, L., Huyard, A., Dumouthier, N., Schwartzbrod, J., 2001. Monitoring of bacterial and parasitological contamination during various treatment of sludge. Water Res. 35 (16), 3763–3770. Ghosh, S., Conard, R.J., Klass, D.L., 1975. Anaerobic acidogenesis of wastewater sludge. J. Water Pollut. Con. Fed. 47 (1), 30–35. Himathongkham, S., Bahari, S., Riemann, H., Cliver, D., 1999. Survival of Escherichia coli O-157:H7 and Salmonella typhimurium in cow manure and cow manure slurry. FEMS Microbiol. Lett. 178 (2), 251–257. Huyard, A., Ferran, B., Audic, J.M., 2000. The two phase anaerobic digestion process: sludge stabilization and pathogen reduction. Water Sci. Technol. 42 (9), 41–47. Lin, J.S., Lee, I.S., Frey, J., Slonczewski, J.L., Foster, J.W., 1995. Comparative-analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J. Bacteriaol. 177 (14), 4097–4104. Stukenberg, Jr., Shimp, G., Sandino, J., Clark, J.H., Crosse, J.T., 1994. Compliance outlook-meeting 40-CFR Part-503, class-B pathogen reduction criteria with anaerobic-digestion. Water Environ. Res. 66 (3), 255–263. Surampalliry, R.Y., Banerjy, S.K., Chen, J.C., 1994. Microbiological stability of waste-water sludges from activated-sludge systems. Bioresource Technol. 49 (3), 203–207. Watanabe, H., Kitamura, T., Ochi, S., Ozaki, M., 1997. Inactivation of pathogenic bacteria under mesophilic and thermophilic conditions. Water Sci. Technol. 36 (6–7), 25–32.
Survival of Salmonella spp. in a simulated acid-phase anaerobic digester treating sewage sludge K. Fukushi
a,*
, S. Babel b, S. Burakrai
c
a Environmental Science Center, The University of Tokyo, Tokyo, 113-0033, Japan Sirindhorn International Institute of Technology, Thammasat University, Pathumthani 12121, Thailand School of Environment, Resources and Development, Asian Institute of Technology, Pathumthani 12120, Thailand b
c
Received in revised form 17 April 2002; accepted 24 April 2002
Abstract The presence of pathogenic microorganisms in municipal waste sludge may create a serious outbreak of water borne diseases if the sludge is used for agricultural purpose. An attempt to decrease the number of pathogenic microorganisms, Salmonella spp. using a simulated acid-phase anaerobic digester was tested in a laboratory-scale batch experiment. Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of a two-phase anaerobic digestion process. A high concentration of organic acid at a pH value of 5.5–6.0 prevents a decrease in Salmonella spp. concentration. Almost complete destruction of Salmonella spp. is observed within two days if the pH value is maintained below 5.5. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Two-phase anaerobic digestion; Salmonella spp.; Acid digester; Sludge; Pathogen
1. Introduction As a result of urbanization, many parts of the world have become densely populated. These highly populated areas produce various kinds of wastes, including industrial hazardous materials, landfill leachate, waste spill, and biological hazardous substances. Pathogenic microorganisms are produced mainly from ordinary households with infected individuals. It is well known that domestic sewage streams may contain high concentrations of pathogenic microorganisms. The activated sludge process has been used to treat domestic wastewater in various areas of the world. Many aerobic wastewater treatment processes remove a majority of pathogenic microorganisms in the original waste stream by adsorption, coagulation, and precipitation, or trap them in a sludge floe matrix (Surampalliry et al., 1994). Most of the pathogens removed from the stream are transferred into the primary and secondary sludge. Consequently, the sludge contains a high number of
*
Corresponding author. Tel.: +81-3-5841-2974; fax: +81-3-58028887. E-mail address: [email protected] (K. Fukushi).
pathogenic microorganisms that need to be treated and disposed of in a proper manner. Anaerobic digestion is known to obtain a good volume reduction of sludge with a considerable removal of pathogenic microorganisms. Although the conventional anaerobic digester is appropriate to achieve the requirement of class B sludge (Stukenberg et al., 1994), a costly process, such as thermophilic/mesophilic two-phase anaerobic process, may be required for the production of class A sludge determined by the USEPA (Huyard et al., 2000). The optimum pH for the growth of Salmonella species is about 6.2–7.2 (Fields, 1979), and Lin et al. (1995) and Foster and Hall (1991) reported Salmonella typhimurium showed a significant inhibition of growth at pH value below 4. These reports indicate that a low pH in the acid phase digester (less than 6.0) in the two-phase anaerobic digestion process makes it feasible to reduce the number of pathogens, including Salmonella spp. Considerable research reports have been issued on Salmonella destruction in sludge (Gantzer et al., 2001; Cheubarn and Pagilla, 2000). Thermophilic digestion is effective to decrease the number of Salmonella (Watanabe et al., 1997). Barrios et al. (2001) reported the effect of acetic acid concentration on Salmonella spp. survival. Destruction of pathogens by the two-phase anaerobic
0960-8524/03/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 1 1 2 - 8
54
K. Fukushi et al. / Bioresource Technology 86 (2003) 53–57
digestion process has not been fully investigated experimentally. The effect of pH and acid concentration warrants particular attention. In the two-phase process, pH value is linked with acid concentration, which makes investigation of a separate effect of pH and acid concentration on pathogen survival difficult. In this paper, the separate effect of pH and organic acid concentration on the survival of Salmonella spp. was investigated. A simulated acid digester was used for this investigation.
Table 1 Batch run condition for testing the effect of organic acid concentration on Salmonella spp. survival Run Total acid Acetic concentra- acid contion (mg/l) centration (mg/l)
Propionic acid concentration (mg/l)
Butyric acid concentration (mg/l)
Adjusted initial pH
A1 A2 A3 AC
2976 2976 0 0
2397 0 0 0
5.5 5.5 5.5 5.6
9306 6909 3933 0
3933 3933 3933 0
The reactor volume was 400 ml.
2. Methods The acid digester environment has a high organic acid concentration and a low pH. These two parameters are usually inversely correlated, and their individual effect on the survival of pathogenic microorganisms is not fully understood. In this paper, we controlled pH or organic acid concentration at a certain value to determine the individual effect of pH and organic acid concentration. In order to study the effect of organic acid concentration on the survival of Salmonella spp., sample sludge with Salmonella spp. was mixed with various concentrations of organic acids mixture (consisting of acetic, propionic, and butyric acids). In order to study the effect of pH on survival of Salmonella spp., sample sludge was spiked with the known amount of organic acid mixture, and the values of pH were varied from 3.5 to 5.5 by sodium hydrooxide and hydrochloric acid. All batch experiments were carried out in reactors maintained at an anaerobic condition at a mesophilic temperature of 37 °C. 2.1. Sample sludge The sludge used in this experiment was collected from a return sludge line after being passed through the secondary clarifier of Siphraya Domestic Wastewater Treatment Plant located in Bangkok, Thailand. The treatment plant employs a standard activated sludge process. The sample was kept at a temperature below 10 °C during transportation from the plant to the laboratory. Original population of Salmonella spp. was about 105 MPN/l00 ml in the collected sludge with volatile solids concentration of about 5500 mg/l. 2.2. Batch experiment procedure A 500-ml Erlenmeyer flask was used as a batch reactor. The batch experiment was started by flushing the flasks with nitrogen gas for 3 min to remove oxygen, closing the flasks with rubber stoppers tightly, and transferring sludge and chemicals through sampling ports on the rubber cap with a syringe. Plastic gassampling bags were installed with all flasks to collect digester gas and to prevent high pressure in the digesters. The identification of runs is summarized in Tables 1
Table 2 Batch run condition for testing the effect of pH on Salmonella spp. survival Run
Total acid concentration (designed) (mg/l)
Total acid concentration (measured) (mg/l)
Adjusted initial pH
P1 P2 P3 P4 P5 PC
9306 9306 9306 9306 9306 0
7068 9223 7256 7033 7633 117
3.5 4.0 4.5 5.0 5.5 No adjust
The reactor volume was 400 ml.
and 2. The composition of organic acids was decided from the information in the literature (Ghosh et al., 1975). All batch reactors were incubated in a walk-in constant-temperature chamber kept at 37 °C. Samples for the enumeration of Salmonella spp. were withdrawn by syringe through the sampling port located on the rubber cap of the flasks. 2.3. Enumeration of Salmonella spp. in sludge In order to increase the sensitivity of Salmonella spp. enumeration, a novel enumeration method was developed. The procedure was a modification of a method outlined in Standard Methods (part 9260D, APHA et al., 1995). Since the concentration of Salmonella spp. in sludge is not high, and since sludge includes many heterogeneous microorganisms, a three-step preliminary incubation of samples was performed to increase sensitivity of the analysis. The sludge samples withdrawn from reactors were treated with a 200-W ultrasonic generator (UD-200, Tomy Seiko Co. Ltd., Tokyo, Japan) for 1 min to disintegrate flock in the sludge, and then diluted with a 0.1% peptone solution for 10 2 –10 5 . One milliliter of diluted samples was transferred into five incubation tubes with 10 ml of EEM-broth medium (EMA23, Eiken, Tokyo, Japan), a preincubation medium for Salmonella spp. The EEM-broth medium is used to amplify the number of bacteria in the sample. After 24 h of incubation at 37 °C, 1 ml of the tube’s contents was transferred to incubation tubes with 10 ml of Rappa-
K. Fukushi et al. / Bioresource Technology 86 (2003) 53–57
port-broth medium (E-MB25, Eiken, Tokyo, Japan), a selective medium for Salmonella spp. The tubes were incubated at 37 °C for 48 h. Then, using a sterilized platinum loop, samples were taken from the tubes and streaked on MLCB-agar plates (Code 05041, Nissui, Tokyo, Japan). The plates were incubated at 37 °C for 18–24 h. The existence of Salmonella spp. can be determined by examining the color of the colony on the plate. Black colonies are formed if Salmonella spp. is in the sample. Confirmation of the determination was done by selecting a black-colored colony, and culturing as previously described, and streaking the selected microbes on the MLCB-agar plate. Using the MPN table provided in Standard Methods (APHA et al., 1995), the number of Salmonella spp. in the original sample was determined. Although this method is quite laborious, it provides very high sensitivity to the presence of Salmonella spp. The minimum concentration which can be measured with this method is 0.01 MPN/100 ml. Results presented in this paper is an average value of duplicated measurement of Salmonella spp.
3. Results and discussion 3.1. Effect of organic acid concentration at the same initial pH on the survival of Salmonella spp. In order to investigate the survival of pathogens (Salmonella spp., in this study) in an anaerobic acid digester, sludge obtained from the aeration tank of a standard activated sludge process was mixed with organic acids at various compositions and concentrations. Activated sludge obtained in Bangkok contains a considerable amount of Salmonella spp. Concentration of Salmonella spp. as well as other environmental parameters of sludge slurry was measured at the beginning of and during the experiment. Four runs of experiments were conducted. Each run was different in terms of concentration of total organic acid at the same initial pH of about 5.5. Organic acid concentrations during the batch experiment are shown in Fig. 1. The concentration of organic acids during the batch experiment varied significantly. Acid concentrations tended to decrease during A2 and A3 runs, whereas the control run, AC, showed almost zero acid concentration during the experiment. The decrease of acid concentration in A2 and A3 was believed to be due to a conversion of acid to methane gas. Unfortunately, the methane production was not confirmed. Values of pH during the batch runs are shown in Fig. 2. As mentioned in Section 2, only initial pH was adjusted. No pH adjustment during the batch experiment was performed. The pH in all runs increased during the batch experiment. Organic acids in the batch reactor were probably consumed by methane formers, and the
55
Fig. 1. Total organic acid concentration profile. Acid concentration was artificially adjusted at day 0.
pH was increased. It should be noted that the pH change was very small (about 0.5 pH unit) up to day 5, a typical acid digester retention time. Fig. 3 shows the concentration profile of Salmonella spp. during the batch experiment. The concentration of Salmonella spp. in the batch reactor generally decreased after the fifth day. However, the concentration of Salmonella spp. fluctuated between day 0 and day 5. Although the environmental conditions in the batch reactor were not optimal for Salmonella spp., they were not harmful. It is known that organic acid can be a substrate for Salmonella spp. that may assist the survival of Salmonella spp. in the acid digester. The ideal pH for the acid digester is considered to be about 5.5 (Ghosh et al., 1975), and typical acid concentration is between 3000 and 8000 mg/l. A high acid concentration in the acid digester is desirable for optimal operation of twophase anaerobic digestion since such an operational
Fig. 2. Profile of pH. Adjustment of pH was not performed during the experiment.
56
K. Fukushi et al. / Bioresource Technology 86 (2003) 53–57
Fig. 3. Profile of Salmonella spp. concentration. Initial total acid concentrations were 9306, 6909, 3933, and 0 mg/l for Al, A2, A3, and AC (control), respectively. Value of pH was shown in Fig. 2.
condition gives a better methane conversion and a high solid reduction rate. This result indicates Salmonella spp. survives better in a high acid concentration, which is good for the digestion process. In other words, a good acid digester produces more pathogens than a poor acid digester provided the pH is about 5.5–6.0. 3.2. Effect of pH at the same initial organic acid concentration on the survival of Salmonella spp. In the last section, we noted the general tendency for Salmonella spp. to decrease at various concentrations of acid at a similar pH condition. However, the time required to lower the concentration to an undetectable level was about 10 days. Holding sludge in the acid digester for 10 days is impractical. In this section, six batch experiments were conducted to test the effect of pH on the survival of Salmonella spp. Organic acid concentration was controlled at about 7500–9500 mg/l for all batch experiments, except the control run, during the experimental period of five days. Fig. 4 shows total organic acid concentrations during the batch experiments. All batch reactors initially were adjusted for the same organic acid concentration and various pH conditions, except the control run (PC). The acid concentration during the five days of the experiment was kept nearly constant. This result agrees with those shown in Fig. 1. The acid concentration of Al in Fig. 1 was an exception. However, if the data of days 3 and 4 are ignored, the concentration of acid in Al was almost constant for 10 days. Unfortunately, we were unable to determine the reason for a temporal decrease in acid observed in day 3 and day 4. Fig. 5 shows pH values during the batch experiments. Fig. 5 results also indicates that the pH and acid conditions were almost constant throughout the batch experiment period.
Fig. 4. Total organic acid concentration profile. Acid concentration and pH was artificially adjusted at day 0.
Fig. 6 exhibits the concentration of Salmonella spp. during the batch experiments. Salmonella spp. in batch runs at initial pH values of 5.0 or lower (P1, P2, P3, and P4) were killed within two days. Salmonella spp. survived longer in the batch reactor at a pH value of 5.5 (P5). However, Salmonella spp. in the P5 run were killed in five days. In the control run, PC, Salmonella spp. remained even after the fifth day. This result indicates that a low pH provides an adverse environment for Salmonella spp. survival. As demonstrated in Fig. 3, Salmonella spp. survived on the fifth day although the acid concentration was kept quite high. Himathongkham et al. (1999) reported that it takes 2.52 days to lower the concentration of Salmonella typhimurium 1 decimal in fresh cow manure at 37 °C in an anaerobic condition. The pH values of manure in their work were neutral or alkaline due to the high ammonium concentration. The result of Fig. 6
Fig. 5. Profile of pH. Acid concentration and pH was artificially adjusted at day 0.
K. Fukushi et al. / Bioresource Technology 86 (2003) 53–57
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Acknowledgements The authors thank Dr. Sambhunath Ghosh for inspiring this work. This work was supported by the Japan International Cooperation Agency and the Royal Thai Government through the Asian Institute of Technology, Thailand. References
Fig. 6. Profile of Salmonella spp. concentration. Values of pH were adjusted 3.5, 4.0, 4.5, 5.0, 5.5 for P1, P2, P3, P4, and P5, respectively at day 0. Total acid concentration was adjusted to 9306 mg/l for all runs except PC (control).
shows that it took two days to lower the concentration of Salmonella spp. 5 decimals at a pH range between 3.5 and 5.0. At a pH value of 5.5, a typical acid digester pH, the time required to lower the Salmonella spp. concentration 1 decimal was about 1 day. Lin et al. (1995) and Foster and Hall (1991) reported Salmonella typhimurium showed a significant inhibition of growth at pH value below 4. Lowering the pH of waste leads to significant destruction of pathogens. From the viewpoint of a digester design engineer, complete pathogen elimination in a few days is very attractive in order to satisfy USEPA’s class A sludge requirements.
4. Conclusions Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of the two-phase anaerobic digestion process. High concentration of organic acid (5000–7000 mg/l of total organic acid) at a pH 5.5–6.0 prevented a decrease of Salmonella spp. concentration. If the pH value was maintained below 5.5, the number of Salmonella spp. decreased to the undetectable level. The acid digester of the two-phase anaerobic digestion process should be operated at a retention time of two days or longer at a pH below 5.5 for effective pathogen destruction.
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