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Pathogen removal in farm-scale psychrophilic anaerobic digesters processing swine manure
Bioresource Technology 102 (2011) 641–646
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Pathogen removal in farm-scale psychrophilic anaerobic digesters processing swine manure Daniel Massé a,*, Yan Gilbert a, Edward Topp b a b
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, 2000, College Street, Sherbrooke, QC, Canada J1M 0C8 Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V 4T3
a r t i c l e
i n f o
Article history: Received 21 April 2010 Received in revised form 29 July 2010 Accepted 5 August 2010 Available online 10 August 2010 Keywords: Anaerobic digestion Pathogens Swine manure Psychrophilic
a b s t r a c t This study assessed the efficiency of commercial-scale psychrophilic anaerobic digestion in sequencing batch reactors (PADSBRs) for pathogen removal from pig manure. The impact of treatment cycle length and of hydraulic flow regimes on pathogen removal efficiency was investigated. Two conventionally operated SBRs (BR1 and BR2) and two SBRs simultaneously fed during the draw step (BR3 and BR4) were monitored over a two-year period. PADSBRs significantly decreased the concentration of coliforms, Salmonella, Campylobacter spp., and Y. enterocolitica, respectively from about 106, 103 CFU g1, 103, and 104 CFU g1 to undetectable levels in most samples. Densities of the gram-positive Clostridium perfringens and Enterococcus spp. remained high (105 CFU g1) in the digesters throughout treatment. The PADSBRs maintained the same level of pathogen removal when the treatment cycle length was reduced from 2 to 1 week. Mass balances on volatile fatty acids (VFAs) revealed short-circuits of inlet flow respectively averaging 6.3% and 6.4% for BR3 and BR4, significantly reducing the overall performance of these reactors regarding pathogens removal. The results obtained in this study show the ability of low temperature anaerobic digestion to remove or significantly reduce indicator and pathogen concentration from raw pig manure. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction Canada is among the ten most important hog producers worldwide, based on mass of pig meat produced (FAO, 2009). Moreover, during the past 20 years, the Canadian pig industry has changed to more specialized facilities and intensified production systems. In 2006, there was three times fewer farms than in 1986, while the average number of pigs increased by more than 400% in swine facilities (CCP, 2008). The quantity of pigs produced in the country during this period has more than doubled, raising concerns about the environmental impacts of the industry, particularly with respect to swine manure management (CCP, 2008). Manure is an excellent source of crop nutrients and will improve soil structure through provision of organic matter. However, over-application will increase the risk of movement of nitrogen and phosphorus to groundwater and adjacent surface water (Sharpley et al., 2002). Most environmental studies concerning swine manure management either have focused on the effects of nutrients on water quality, or odour problems and air quality. Nonetheless, the microbial quality of manure should not be neglected since many outbreaks of gastroenteritis related to livestock * Corresponding author. Tel.: +1 418 565 9174; fax: +1 418 564 5507. E-mail address: [email protected] (D. Massé).
operations have been reported (Guan and Holley, 2003; Pell, 1997; Spencer and Guan, 2004). The most prevalent pathogenic microorganisms from manure are the bacteria Salmonella, Escherichia coli, Yersinia, Campylobacter, and the protozoa Giardia and Cryptosporidium (Bicudo and Goyal, 2003; Hutchison et al., 2005), but Clostridium perfringens, Listeria monocytogenes, and Treponema hydrosenteriae have also been reported as causative agents of human infections related to livestock (Colleran, 2000). The persistence of enteric pathogens in manure will vary according to manure handling practices, storage management, ambient conditions and duration, type of slurry, and pathogen type (Bicudo and Goyal, 2003). There is an urgent need for cost-effective methods to address environmental issues of pig manure by reducing microbial and chemical contaminants. Côté et al. (2006) studied the efficiency of a low temperature anaerobic laboratory-scale digester to reduce viable populations of indicator microorganisms (total coliforms, E. coli) and selected pathogens (Salmonella, Yersinia enterocolitica, Cryptosporidium and Giardia) in swine slurries. Forty liter anaerobic intermittently-fed sequencing batch reactors (SBR) operated at 20 °C during 20 day cycles reduced total coliforms by 1.6 to 4.2 log10 units (CFU g1) (97.9–100%), and E. coli by 2.5–4.2 log10 units (99.7–100%). Salmonella, Cryptosporidium, and Giardia concentrations decreased to undetectable levels (<100 CFU g1). Even though the latter study
0960-8524/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.08.020
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showed the efficiency of lab-scale psychrophilic anaerobic digestion regarding pathogen and indicator removal, no study reports the performance of commercial-scale digesters for the removal of these microorganisms. In the study reported here, the long term performance and reliability of farm-scale (165 and 450 m3) low temperature digesters for the reduction of pathogens concentration in raw swine manure was investigated.
The average organic loading rate (OLR) was based on the amount of total chemical oxygen demand (TCOD) fed per volume of sludge present at the start of a cycle and the cycle length. It was calculated as follows:
Lf ¼
V f Cf V i tc
ð1Þ
where Lf is the loading rate based on total cycle length (kgTCOD m3 day1), Vf is the volume of feed (m3), Cf is TCOD concentration in the feed (kg m3), Vi is the volume of sludge in the reactor at the beginning of the cycle (m3), and tc is the total duration of the cycle (day).
2. Methods 2.1. Reactor operation Four farm-scale cylindrical digesters (BR1–BR4) were used for psychrophilic anaerobic digestion in sequencing batch reactors (PADSBRs) of pig manure, and monitored over a two years period (June 2007–2009). The effective volume was 165 m3 (Outside Diameter (OD) 7 m) for BR1 and BR2, and 450 m3 (OD 11 m) for BR3 and BR4. The outlet consisted of a flexible pipe (Inside Diameter (ID) = 0.101 m) attached to a steel wire, allowing sludge height control during the draw step. Feeding was performed through a polyvinyl chloride (PVC) pipe (ID = 0.101 m) at the bottom of the reactor. An inflatable device was located at the top of the reactor to allow for gas and liquid displacement without air entering the system. Three reactors (BR1, BR2, and BR3) in operation since 2001 were located on the same farm and operated under a 7 day cycle, while the fourth (BR4) in operation since 2004 was operated under a 14 day cycle on another farm. BR1 and BR2 were operated as conventional SBRs, i.e. after sludge settling, supernatant was removed before feeding with fresh manure. Rather than being removed before feeding, BR3 and BR4’s supernatant was forced outside the bioreactors by simultaneously feeding with fresh manure at the bottom of the digesters. Approximately from the 500th day to the end of the monitoring period, BR3 was operated as a conventional SBR due to operational constraints. Due to solids accumulation inside BR3, 46 m3 of sludge were removed at the 665th day of monitoring, before the feed step. The temperature of all reactors was maintained throughout the year at an average of 24 °C, with fluctuations ranging from 20 to 26.5 °C. Composite samples were collected from the effluent for analysis during the drawing step. Raw manure was sampled during the filling period. All samples were kept at 4 °C until the analysis, which was done less than 24 h after sampling. Table 1 summarizes the operating conditions during the study and manure characteristics.
2.2. Physico-chemical analysis Soluble chemical oxygen demand (SCOD) of fresh manure and effluent samples was determined by analyzing the supernatant of centrifuged slurry. Total and soluble COD were determined according to the method developed by Knechtel (1978). Alkalinity, pH, total solids (TS), volatile solids (VS), total Kjeldahl nitrogen (TKN) and ammonia nitrogen (NH4–N) were determined using standard methods (APHA, 1992). TKN and NH4–N were analyzed using a Kjeltec auto-analyzer model TECATOR 1030 (Tecator AB, Hoganas, Sweden). Before VFAs quantification, samples were conditioned. Ten grams of sample was centrifuged at 41,700g, 30 min at 15 °C. One microliter of 0.5 M H2SO4 was added to 5 mL of the supernatant and centrifuged at 21,800g, 15 min at 15 °C. An internal standard (2-ethylbutyrate at 2 g L1) was added (0.5 mL) to a tube with 0.5 mL of the acidified–centrifuged supernatant and 0.1 g of DOWEX 50WX8 resin (The Dow Chemical Company, Midland, MI) and vortexed. VFAs were measured with a Perkin Elmer gas chromatograph model 8310 (Perkin Elmer, Waltham, MA), mounted with a DB-FFAP high resolution column. Results were analyzed using TurboChrom version 6.2.1 software (Perkin Elmer). 2.3. Microbiological analysis For pathogen quantification, raw manure and anaerobic digester effluent samples were prepared by diluting 1:10 in sterile sodium metaphosphate buffer (2 g L1; pH 6.8). Total coliforms were enumerated by direct plating on mEndo–LES agar (Difco, Mississauga, ON, Canada) and incubated at 37 °C for 18–20 h. Colonies which produced a distinctive metallic green sheen were counted as total coliforms. Fecal coliforms were quantified by
Table 1 Operational conditions of full-scale PADSBRs and inlet characteristics of manure fed to bioreactors.
a
Parameters
BR1
BR2
BR3
BR4
Operational conditions Loading rate (kg TCOD m3 d1) Total cycle length (day) Reactor effective volume (m3)
1.47 (0.57)a 7 120
1.48 (0.59) 7 120
1.31 (0.45) 7 425
1.65 (0.64) 14 425
Manure characteristics TCOD (g O2 L1) SCOD (g O2 L1) pH Alkalinity (mg CaCO3 L1) Acetic acid (mg L1) Propionic acid (mg L1) Butyric acid (mg L1) TS (%) VS (%) TKN (mg N L1) NH4–N (mg N L1)
115 (44) 41 (12) 6.88 (0.14) 17,300 (4100) 9300 (3100) 3600 (1300) 5400 (2300) 7.20 (2.02) 5.63 (1.65) 6600 (1600) 4600 (1200)
106 (41) 38 (12) 6.90 (0.16) 16,300 (4100) 8500 (2900) 3300 (1100) 5400 (2200) 6.72 (2.01) 5.21 (1.64) 6300 (1700) 4400 (1300)
97 (34) 35 (10) 6.95 (0.17) 15,600 (3400) 7900 (2700) 3100 (1100) 4800 (1800) 6.20 (1.69) 4.77 (1.40) 5900 (1300) 4100 (1000)
98 (39) 33 (15) 7.33 (0.21) 20,300 (5000) 8600 (3700) 2900 (1000) 2600 (1700) 6.99 (2.23) 5.24 (1.86) 6600 (1900) 5100 (1500)
Values in parenthesis represent the standard deviation of observations (Number of observations for BR1 = 81, BR2 = 89, BR3 = 99, BR4 = 30).
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direct plating on mFC agar (Difco) and incubated for 18–20 h at 44.5 °C. Colonies producing a distinctive indigo blue color were enumerated as fecal coliforms. Enterococcus spp. were counted by direct plating onto mEnterococcus agar (Difco) and incubated at 37 °C for 48 h. Colonies producing a burgundy color were counted as Enterococcus sp. E. coli were enumerated by direct plating on mFC basal medium (Difco) supplemented with 3-bromo-4chloro-5-indolyl-b-D-glucuronide (100 mg L1, Med-Ox Diagnostics, Ottawa, ON) and incubated for 18–24 h at 44.5 °C. Colonies producing the characteristic blue color indicative of b-glucuronidase activity were enumerated as E. coli. C. perfringens was quantified on mCP agar (Med-Ox Diagnostics) incubated at 44.5 °C for 24 h under anaerobic conditions. Yellow colonies with a yellow halo and which turned magenta after exposure to ammonium hydroxide fumes were expected to be C. perfringens. Presumptive colonies were confirmed by inoculation into skim milk broth. Stormy fermentation of the broth after 24 h was considered a positive confirmation for C. perfringens. This protocol allowed the quantification of vegetative cells and spores together. Y. enterocolitica were enumerated by direct plating onto Cefsulodin–Irgasan–Novobiocin agar (CIN, Difco) and incubated for 18 h at 30 °C. Colonies that were less than 2 mm in diameter and formed a red bulls-eye were considered presumptive colonies for Y. enterocolitica. Five presumptive colonies were picked and inoculated into lysine arginine iron agar slants (LAIA) and considered positive if LAIA results were alkaline slant, acid butt, H2S negative and gas negative (Weagant, 1983). Campylobacter spp. were counted by direct plating onto Charcoal Cefoperazone Desoxycholate Agar (CCDA, Oxoid, Nepean, ON) supplemented with 0.032 mg mL1 of cefoperazone and 0.01 mg mL1 of amphotericin B (Oxoid) and Campy-Line Agar (CLA, (Line, 2001)). Plates were incubated at 42 °C in microaerophilic conditions for 48 h in an anaerobic jar with GasPakTM EZ Campy (6–16% O2, 2–10% CO2, BD, Oakville, ON). Campylobacterlike colonies were quantified on both media. On CLA agar, presumptive colonies of Campylobacter are smooth, convex, burgundy colored and are approximately 2–4 mm in diameter. On CCD agar, presumptive colonies of Campylobacter are smooth, convex, and translucent colorless to cream colored and are approximately 2– 4 mm in diameter. Suspected colonies were also observed under microscope and tested for Gram coloration, oxidase and catalase, (Sigma–Aldrich, Oakville, ON) after inoculation onto Columbia blood agar and incubate 24 h at 35 °C under microaerophilic conditions. Results were confirmed using Campylobacter Latex kit as described by the manufacturer (Oxoid). Salmonella spp. were quantified by direct plating onto Salmonella chromogenic agar (Oxoid) and XLD plates (Difco). Plates were incubated at 42 °C for 24 h before being enumerated. Salmonellalike colonies were quantified on both media. On XLD agar, presumptive colonies of Salmonella are smooth, convex, and red-col-
ored with a black bulls-eye and are approximately 2–4 mm in diameter. Colonies may also produce a red halo in the media. On Salmonella chromogenic agar, presumptive colonies of Salmonella are mauve to magenta in color and are approximately 1.5–3 mm in diameter. Suspected colonies were picked and streaked on BHI agar and plates were incubated at 35 °C for 24 h. A well isolated colony was inoculated into the Rapid Salmonella Latex test as described by manufacturer (Oxoid). When further identification was necessary, a suspect colony was inoculated onto a fresh nutrient agar and incubated 24 h at 35 °C. Gram stain, oxidase and catalase tests were performed and an API 20E strip was inoculated for ultimate identification. 2.4. Statistical analysis Statistical data analysis was carried out using the software JMP, version 7.0.1 (SAS Institute Inc., Cary, NC). Homogeneity of variances was analysed with the Levene test statistic. Means, standard deviations and frequency distributions of the data were determined. Differences between means were tested with comparative statistics, using one-way ANOVA. After obtaining F-ratios, Tukey’s post hoc tests and Student t-test were performed to determine significantly different pairs of data. The level of significance was set at P < 0.05. The value ‘‘99” was used for statistical analysis when pathogen and indicator concentrations were below the method detection limit (<100 CFU g1). 3. Results and discussion 3.1. Manure characteristics Four farm-scale psychrophilic anaerobic digesters were monitored over a two-year period to assess their reliability and stability with respect to removal of enteric pathogens. No significant difference (P > 0.05) was found between raw manure samples collected at bioreactor inlet for all tested parameters (Table 1). The average OLR applied to the bioreactors ranged from 1.31 to 1.65 kg TCOD m3 d1, with a TCOD concentration around 100,000 mg O2 L1. The pH of raw manure was near neutrality, even though high VFAs concentrations were observed, mainly because of the high amount of alkalinity in the manure. Pathogen and indicator concentrations in raw pig manure were highly variable (Table 2), but within the range of reported values (Hill and Sobsey, 1998; Hutchison et al., 2005; Kearney et al., 1993a). The mean concentration of total and fecal coliforms, Enterococcus spp. and E. coli was 2.7 105, 1.9 105, 3.8 105, and 8.8 104 CFU g1, respectively. Lower mean concentrations were observed for Y. enterocolitica, Salmonella and Campylobacter spp., with 2.0 104, 2.2 103, and 3.0 103 CFU g1, respectively. Y. enterocolitica, Salmonella and Campylobacter concentrations
Table 2 Performance of PADSBRs regarding pathogen and indicator removal. Parameters
Total coliforms Fecal coliforms Escherichia coli Enterococcus spp. Clostridium perfringens Yersinia enterocolitica Salmonella spp. Campylobacter spp. a
Concentration range (CFU g1)
6.0 102–1.5 106 5.0 102–1.6 106 4.0 102–5.8 105 1.8 104–1.7 106 <1.0 103–3.7 106 <1.0 102–3.3 105 <1.0 102–5.0 104 <1.0 102–5.2 104
Log10 Removala BR1
BR2
BR3
SBR
SBR
Non-SBR
SBR
BR4 Non-SBR
SBR
2.9 2.8 2.9 0.8 0.2 2.1 1.2 1.3
2.5 2.6 2.8 1.0 0.0 2.1 1.4 1.5
1.0 0.9 0.9 0.7 0.2 1.1 0.9 1.1
2.4 3.0 2.8 0.6 0.1 1.9 1.3 1.4
1.2 1.2 0.9 0.8 0.2 1.3 1.1 1.1
2.7 2.8 2.8 1.0 0.1 2.4 1.3 1.3
Values were calculated using mean inlet and outlet concentrations (Number of observations for BR1 = 48, BR2 = 48, BR3 = 45, BR4 = 45).
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equal of below the method detection limit (100 CFU g1) were detected in 10%, 43% and 24%, respectively of the samples from the inlets of BR1–BR3 inlet. For BR4 inlet, 10%, 45% and 53% of samples showed concentrations equal or below the detection limit, for these microorganisms, respectively. 3.2. Bioreactor performance Since biogas production was not monitored on a regular basis on these full-scale reactors during digestion, the overall bioreactor performance was verified by monitoring TCOD removal. Conventionally operated SBRs (BR1 and BR2) showed TCOD removal above 70%, while those being simultaneously fed with fresh manure during the drawing step (BR3 and BR4) only reached 61.6% and 56.3% (Fig. 1C and D), respectively. Near the end of the two-year monitoring period (500th day), BR3 performance decreased significantly as measured by TCOD removal. This was principally due to typical solid accumulations inside the reactor which increased the solids concentration at the outlet. This was indicated by an increase of the outlet VS/TS ratio (Fig. 1C). Similar results were obtained for BR4 at the end of the monitoring period (Fig. 1D). For BR3, satisfactory performance was regained after removing solids from the reactor. The low temperature AD treatment resulted in significantly lower concentrations of pathogens and indicators in BR1 and BR2 effluents. An average reduction of 2.6–2.9 log10 units and 1.2– 1.4 log10 units was observed for fecal coliforms and Salmonella spp. for an HRT of 7 days (Table 2), leading to undetectable levels in more than 81% and 96% of outlet samples, respectively. Other studies obtained longer pathogen and indicator removal times for similar or higher temperatures during digestion. Kumar et al. (1999) used an ampicillin-resistant E. coli strain to study the persistence of this organism in cattle dung slurry during anaerobic digestion. The survival was 25 days at room temperature (18–25 °C) and 15 days at 35 °C. In the same study, the complete elimination of Salmonella at 35 °C occurred on the 15th day
B
40
Short circuit (%)
Short circuit (%)
35
30 25 20 15 10 5 0
40
Conventional SBR
35
30 25 20 15 10 5
0
200
400
600
0
800
Time (d)
D
0.8
250000
0.6 0.5
150000
0.4 100000
0.3 0.2
50000
0.1 0
200
400
Time (d)
600
0 800
TCOD (mg O2 L-1)
0.7
200000
0
200
400
600
800
Time (d)
VS/TS
TCOD (mg O2 L-1)
C
0
0.8
250000
0.7
200000
0.6 0.5
150000
0.4 100000
0.3
VS/TS
A
whereas 25 days were necessary at room temperature. Berg and Berman (1980) reported a reduction of 2.0 and 1.8 log10 units of indigenous total and fecal coliforms, respectively, during the mesophilic digestion (35 °C; SRT 20 days) of municipal sludge. In another study, Salmonella and E. coli, which were added to liquid pig manure, and coliforms of the indigenous flora, were reduced by 1.0–2.1 log units when subjected to anaerobic filter treatment at 35 °C in two pilot-plant reactors operated at 0.8–4.2 days HRT (Olsen, 1988). In an investigation studying the survival of indigenous pathogens in a full-scale anaerobic digester which was daily fed with cattle farm wastes and operated at 28 °C, Kearney et al. (1993a) found that Campylobacter jejuni was the most resistant bacterium tested among E. coli, Salmonella enterica Typhimurium, Y. enterocolitica, and L. monocytogenes. Its mean T90 (time to reduce its concentration of 90%) value was 438.6 days, compared to Y. enterocolitica that showed a T90 of 18.2 days. Mean T90 observed under the operational conditions tested in their study suggest that the process did not allow the complete removal of tested pathogens. In comparison, undetectable levels of Y. enterocolitica and Campylobacter were obtained in our study in more than 87% of BR1 and BR2 outlet samples for only 7 days treatment. The other 13% showed an average concentration of 7.5 102 CFU g1 for these microorganisms. It seems that for the same species or genera, various removal efficiencies are observed, depending on the substrate being digested. For example, 90% of Salmonella spp. was removed in 34.5 days when digesting cattle slurry mixed with hen, pig and potato wastes at 28 °C, whereas it was completely removed in 15 days when digesting cattle dung slurry at 35 °C (Kumar et al., 1999), in 21 days when digesting vegetable, fruit, and garden waste in mesophilic conditions (Termorshuizen et al., 2003), and in only 7 days during the psychrophilic digestion (24 °C) of pig manure in this study. According to Smith et al. (2005), efficient mixing and organic matter stabilisation are the main factors controlling the rate of inactivation under mesophilic conditions and not a direct effect
0.2
50000
0.1
0
0
200
400
600
0 800
Time (d)
Fig. 1. Percentage of VFAs going through BR3 (A) and BR4 (B) by short circuiting of hydraulic flow, TCOD removal and VS–TS ratio in BR3 (C) and BR(4). Arrows indicate periods when bioreactors were operated as conventional SBRs. Dashed line represents the cycles when BR3 operation was switched toward conventional SBR. Feed (); Effluent ( ); VS/TS (N).
D. Massé et al. / Bioresource Technology 102 (2011) 641–646
of temperature. Thus, the observed differences regarding pathogen removal from various matrices could be due to the level of stabilization reached for each of the digested substrates. C. perfringens and Enterococcus spp. were not significantly removed by psychrophilic anaerobic digestion (Table 2). Negligible removal of 0.2 log10 unit was reported for C. perfringens spores during the treatment of swine manure in an anaerobic lagoon in North Carolina (Hill and Sobsey, 1998). Similarly, no significant C. perfringens removal was observed during the mesophilic digestion (HRT = 20 days) of sewage sludge in a full-scale anaerobic reactor, measured both as spores and total counts (Chauret et al., 1999). The same study also showed that Enterococcus spp. was not significantly removed from the sludge during digestion. Effenberger et al. (2006) also reported low removal efficiencies for fecal enterococci (about 1.0 log10 unit) after the first stage of a mesophilic– thermophilic–mesophilic anaerobic digestion process treating liquid dairy cattle manure. These data are in agreement with our results since Enterococcus spp. removal was at most 1.0 log10 unit with an average concentration in bioreactors’ outlet around 105 CFU g1. Enterococcus is a facultative anaerobic microorganism that is present in the intestinal tract of various animals, Enterococcus faecium and Enterococcus faecalis being the most frequently encountered species in pig’s (Klein, 2003). This genus is recognized as being able to survive a range of stresses and hostile environments, including those of extreme temperature (5–65 °C), pH (4.5–10.0) and high salt concentrations, enabling them to colonize a wide range of niches (Fisher and Phillips, 2009). C. perfringens is an anaerobic spore-forming bacterium that can withstand stresses such as low oxidation–reduction potential (ORP). Therefore, it is likely to survive anaerobic manure digestion. Many Clostridium species are commonly observed in anaerobic digesters, being responsible with other microorganisms for acetate production (Diekert and Wohlfarth, 1994). They can also produce lower fatty acids from acetate or ethanol when the concentration of hydrogen is high, thereby reversing the reactions of the syntropic bacteria. This may be an indicator of reactor instability (O’Flaherty et al., 2006). It is thus not surprising that high concentrations of Enterococcus and Clostridium are observed in outlets of anaerobic digesters. Temperature and retention time are critical parameters for indicator and pathogen survival during anaerobic digestion of wastes (Arthurson, 2008; Sahlström, 2003). The elimination of E. coli and Salmonella was faster at 35 °C than at room temperature during anaerobic digestion of cattle slurry (Kumar et al., 1999). S. typhimurium, Y. enterocolitica and L. monocytogenes also declined more rapidly at 17 °C than at 4 °C during anaerobic digestion of this substrate (Kearney et al., 1993b). According to Olsen and Larsen (1987), increasing retention time in mesophilic digesters enhanced pathogen removal efficiencies toward those encountered for anaerobic thermophilic treatment. However, Kearney (1991) reported that S. typhimurium had a T90 value of 2.1 days at a 25 day HRT, but after the HRT was reduced to 5 days the T90 value decreased to 1.2 days. It was hypothesized that higher concentrations of VFAs at shorter HRTs would be correlated with a decline in viable pathogens number. Since their results did not show significant correlation between VFAs and pathogen concentrations, they speculated that other factors such as the source and type of slurry, pH and temperature of the anaerobic digestion process could also have a significant effect on the pathogen populations. Regarding S. typhimurium removal, Gadre et al. (1986) reported a T90 of approximately 7.7 days during mesophilic (37 °C) anaerobic digestion of cattle dung, while Olsen and Larsen (1987) found a T90 of 2.0 days during the digestion of pig slurry in similar conditions. Even though T90 were not specifically calculated during our study, results suggest that T90 values of analyzed pathogens and indicator microorganisms were lower than 7 days under the conditions
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encountered in the psychrophilic anaerobic digesters. Since temperature was lower in these reactors than in mesophilic ones, parameters other than temperature are probably responsible for the elimination of pathogens. Côté et al. (2006) studied the removal of pathogens and indicator microorganisms during PADSBRs of swine manure in 28 day HRT laboratory-scale digesters (42 L) operated at 20 °C. At the end of the treatment, 47%, 75% and 100% of the collected samples were free of total coliforms, E. coli and Salmonella, respectively. They observed a diminution of 1.6–4.2 log10 units and of 2.5–4.2 log10 units in the residual samples still having detectable concentrations of total coliforms and E. coli, respectively. Farm-scale reactors operated in our study showed comparable performances with lab-scale regarding pathogen removal for a similar temperature, at a much lower HRT (7 days). This process has already proven successful for pig manure treatment in full-scale reactors, regarding biogas production and COD removal (Masse et al., 2009). Results obtained in this study show that scaled-up PADSBR was not only successful in stabilizing the organic matter, but was also able to decrease pathogen and indicator concentrations to undetectable levels. Lower performances regarding pathogen and indicator removal were also observed for BR3 and BR4 when operated as non-conventional SBRs (Table 2). BR1 and BR2 removed more than 2.5 log10 units of total and fecal coliforms, and E. coli, corresponding to more than 99.7% removal. Only 0.9–1.2 log10 unit of these microorganisms was removed by BR3 and BR4, corresponding to 86.1–94.0% removal. Y. enterocolitica removal averaged 2.1 log10 units for BR1 and BR2, and ranged from 1.1 to 1.3 log10 unit for the other reactors. Salmonella and Campylobacter spp. removal by BR1 and BR2 ranged from 1.2 to 1.5 log10 unit, corresponding to 94.1–96.6%, while their concentration decreased by 0.9–1.1 log10 unit in BR3 and BR4. Enterococcus spp. and C. perfringens concentration remained high in all effluents, with average concentrations of 5.6 104 and 2.7 105 CFU g1, respectively. Since filling was performed simultaneously with drawing in BR3 and BR4, it was hypothesized that there could be a short circuit in the hydraulic flow. Mass balances were performed to estimate which proportion of influent entering the reactor during filling was going out with the supernatant overflow. VFAs were chosen as short circuiting indicators, because of their low concentration after digestion and of a more precise and reliable quantification method compared to TCOD and bacteria. It appeared that an average of 6.42% (standard deviation (SD) = 5.50) and 6.33% (SD = 6.29) of VFAs entering the system were directly going to the bioreactor’s outlet while filling BR3 and BR4, respectively. Sporadically, for diverse operational constraints, BR3 and BR4 were fed after the drawing step, as for conventional SBRs, indicated by arrows in Fig. 1A and B. Moreover, BR3 operational conditions were switched toward those of a conventional SBR near the 500th day of this study (Fig. 1). Results obtained clearly show that the presence of VFAs in the bioreactor outlet is due to short circuiting, since negligible VFA concentrations were observed in the outlet when filling and drawing were performed separately. The increase of TCOD in BR3 and BR4 effluent (Fig. 1C and D) due to short circuiting was significant compared to conventionally operated SBRs (BR1 and BR2; ca. 25,000 mgTCOD L1). The presence of short circuits could lead to an incompletely stabilized digestate, causing odor problems and greenhouse gas production during storage of the effluents from the bioreactors. The difference was also significant for pathogens and indicators. Two hydraulic retention times (7 and 14 days) were tested in this study, in BR3 and BR4, respectively. Even though these reactors showed higher TCOD and pathogen and indicator concentrations in effluents compared to BR1 and BR2, mainly because of short-circuiting of hydraulic flow, no significant impact was ob-
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served on pathogen and carbon removal when the treatment cycle length was reduced from 2 to 1 week (P > 0.05). Moreover, as represented by arrows in Fig. 1, a return to conventional SBR operations for BR3 and BR4 allowed the elimination of hydraulic short circuiting and permitted the comparison of conventionally operated SBRs under two HRTs for some cycles. Under these operational conditions, similar pathogen and indicator removal efficiencies as for BR1 and BR2 were reached for all tested organisms (Table 2), again showing that these full-scale reactors performed similarly when the treatment cycle length was reduced from 2 to 1 week. 4. Conclusion Farm-scale psychrophilic anaerobic digestion in sequential batch reactors (PADSBRs), operated at 7 or 14 days HRT and 24 °C, significantly decreased the concentration of total and fecal coliforms, E. coli, Salmonella, Campylobacter spp., and Y. enterocolitica to undetectable levels in most samples. The concentration of C. perfringens did not decrease significantly during treatment while Enterococcus spp. concentration diminished by less than 1.0 log10 unit, still remaining above 105 CFU g1. All these results were similar to those obtained from laboratory-scale PADSBRs, which were 3000 to 10,000 times smaller in volume than the full-scale digesters. No significant impact was observed on pathogen and carbon removal when the treatment cycle length was reduced from 2 to 1 week. This allows the use of smaller reactors, significantly reducing installation and operational costs of this particular process. Acknowledgements We thank Bio-Terre for providing access to on-farm full-scale bioreactors and for samples collection. We are also grateful to Denis Deslauriers and Andrew Scott for their dedication to this project and for providing their technical expertise. References APHA, 1992. Standard methods for the examination of water and wastewater, 18th ed. American Public Health Association. Arthurson, V., 2008. Proper sanitization of sewage sludge: a critical issue for a sustainable society. Applied and Environmental Microbiology 74 (17), 5267– 5275. Berg, G., Berman, D., 1980. Destruction by anaerobic mesophilic and thermophilic digestion of viruses and indicator bacteria indigenous to domestic sludges. Applied and Environmental Microbiology 39 (2), 361–368. Bicudo, J.R., Goyal, S.M., 2003. Pathogens and manure management systems: a review. Environmental Technology 24 (1), 115–130. CCP, 2008. Statistics & Market Reports, Canadien Pork Concil. Ottawa, ON. Chauret, C., Springthorpe, S., Sattar, S., 1999. Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion. Canadian Journal of Microbiology 45 (3), 257–262. Colleran, E., 2000. Hygienic and sanitation requirements in biogas plants treating animal manures or mixtures of manures and other organic wastes. In: Ørtenblad, H. (Ed.), Anaerobic Digestion: Making energy and solving modern waste problems. Herning Municipal Authorities, Denmark, pp. 77–86. AD-NETT. Côté, C., Massé, D.I., Quessy, S., 2006. Reduction of indicator and pathogenic microorganisms by psychrophilic anaerobic digestion in swine slurries. Bioresource Technology 97 (4), 686–691. Diekert, G., Wohlfarth, G., 1994. Metabolism of homoacetogens. Antonie van Leeuwenhoek 66 (1), 209–221.
Effenberger, M., Bachmaier, J., Garcs, G., Gronauer, A., Wilderer, P., Lebuhn, M., 2006. Mesophilic–thermophilic–mesophilic anaerobic digestion of liquid dairy cattle manure. Water Science and Technology 53 (8), 253–261. FAO, 2009. FAO Statistical Yearbook 2007–2008, Food and Agriculture Organization of the United Nations. Fisher, K., Phillips, C., 2009. The ecology, epidemiology and virulence of Enterococcus. Microbiology 155 (6), 1749–1757. Gadre, R.V., Ranade, D.R., Godbole, S.H., 1986. A note on survival of salmonellas during anaerobic digestion of cattle dung. Journal of Applied Microbiology 60 (2), 93–96. Guan, T., Holley, R.A., 2003. Pathogen survival in swine manure environments and transmission of human enteric illness – a review. Journal of Environmental Quality 32 (2), 383–392. Hill, V., Sobsey, M., 1998. Microbial indicator reductions in alternative treatment systems for swine wastewater. Water Science and Technology 38 (12), 119– 122. Hutchison, M.L., Walters, L.D., Avery, S.M., Munro, F., Moore, A., 2005. Analyses of Livestock Production, Waste Storage, and Pathogen Levels and Prevalences in Farm Manures. Applied and Environmental Microbiology 71 (3), 1231–1236. Kearney, T., 1991. Survival of pathogenic bacteria in anaerobic digesters. The Queen’s University of Belfast, Belfast, UK. Kearney, T.E., Larkin, M.J., Frost, J.P., Levett, P.N., 1993a. Survival of pathogenic bacteria during mesophilic anaerobic digestion of animal waste. Journal of Applied Microbiology 75 (3), 215–219. Kearney, T.E., Larkin, M.J., Levett, P.N., 1993b. The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria. Journal of Applied Microbiology 74 (1), 86–93. Klein, G., 2003. Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract. International Journal of Food Microbiology 88 (2–3), 123–131. Knechtel, J.R., 1978. A more economical method for the determination of chemical oxygen demand. Water and Wastes Engineering 14 (4), 25–28. Kumar, R., Gupta, M.K., Kanwar, S.S., 1999. Fate of bacterial pathogens in cattle dung slurry subjected to anaerobic digestion. World Journal of Microbiology and Biotechnology 15 (3), 335–338. Line, J.E., 2001. Development of a selective differential agar for isolation and enumeration of Campylobacter spp.. Journal of Food Protection 64, 1711–1715. Masse, D.I., Masse, L., Xia, Y., Gilbert, Y., 2009. Potential of low temperature anaerobic digestion to address current environmental concerns on swine production. Journal of Animal Science 88 (13-electronic-suppl), E112–E120. O’Flaherty, V., Collins, G., Mahony, T., 2006. The microbiology and biochemistry of anaerobic bioreactors with relevance to domestic sewage treatment. Reviews in Environmental Science and Biotechnology 5 (1), 39–55. Olsen, J.E., 1988. Studies on the reduction of pathogenic and indicator bacteria in liquid pig manure treated by sedimentation and anaerobic filter digestion for methane generation. Biological Wastes 24 (1), 17–26. Olsen, J.E., Larsen, H.E., 1987. Bacterial decimation times in anaerobic digestions of animal slurries. Biological Wastes 21 (3), 153–168. Pell, A.N., 1997. Manure and microbes: public and animal health problem? Journal of Dairy Science 80 (10), 2673–2681. Sahlström, L., 2003. A review of survival of pathogenic bacteria in organic waste used in biogas plants. Bioresource Technology 87 (2), 161–166. Sharpley, A., Meisinger, J.J., Breeuwsma, A., Sims, J.T., Daniel, T.C., Schepers, J.S., 2002. Impacts of animal manure management on ground and surface water quality. In: Hatfiels, J.L., Stewart, B.A. (Eds.), Animal Waste Utilization: Effective Use of Manure as A Soil Resource. CRC Press. Smith, S.R., Lang, N.L., Cheung, K.H.M., Spanoudaki, K., 2005. Factors controlling pathogen destruction during anaerobic digestion of biowastes. Waste Management 25 (4), 417–425. Spencer, J.L., Guan, J., 2004. Public Health Implications Related to Spread of Pathogens in Manure From Livestock and Poultry Operations. In: Spencer, J.F.T., Ragout de Spencer, A.L. (Eds.), Public Health Microbiology: Methods and Protocols. Humana Press, Totowa, NJ, pp. 503–515. Termorshuizen, A.J., Volker, D., Blok, W.J., Brummeler, E.t., Hartog, B.J., Janse, J.D., Knol, W., Wenneker, M., 2003. Survival of human and plant pathogens during anaerobic mesophilic digestion of vegetable, fruit, and garden waste. European Journal of Soil Biology 39 (3), 165–171. Weagant, S.D., 1983. Medium for presumptive identification of Yersinia enterocolitica. Applied and Environmental Microbiology 45 (2), 472–473.
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Pathogen removal in farm-scale psychrophilic anaerobic digesters processing swine manure Daniel Massé a,*, Yan Gilbert a, Edward Topp b a b
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, 2000, College Street, Sherbrooke, QC, Canada J1M 0C8 Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V 4T3
a r t i c l e
i n f o
Article history: Received 21 April 2010 Received in revised form 29 July 2010 Accepted 5 August 2010 Available online 10 August 2010 Keywords: Anaerobic digestion Pathogens Swine manure Psychrophilic
a b s t r a c t This study assessed the efficiency of commercial-scale psychrophilic anaerobic digestion in sequencing batch reactors (PADSBRs) for pathogen removal from pig manure. The impact of treatment cycle length and of hydraulic flow regimes on pathogen removal efficiency was investigated. Two conventionally operated SBRs (BR1 and BR2) and two SBRs simultaneously fed during the draw step (BR3 and BR4) were monitored over a two-year period. PADSBRs significantly decreased the concentration of coliforms, Salmonella, Campylobacter spp., and Y. enterocolitica, respectively from about 106, 103 CFU g1, 103, and 104 CFU g1 to undetectable levels in most samples. Densities of the gram-positive Clostridium perfringens and Enterococcus spp. remained high (105 CFU g1) in the digesters throughout treatment. The PADSBRs maintained the same level of pathogen removal when the treatment cycle length was reduced from 2 to 1 week. Mass balances on volatile fatty acids (VFAs) revealed short-circuits of inlet flow respectively averaging 6.3% and 6.4% for BR3 and BR4, significantly reducing the overall performance of these reactors regarding pathogens removal. The results obtained in this study show the ability of low temperature anaerobic digestion to remove or significantly reduce indicator and pathogen concentration from raw pig manure. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction Canada is among the ten most important hog producers worldwide, based on mass of pig meat produced (FAO, 2009). Moreover, during the past 20 years, the Canadian pig industry has changed to more specialized facilities and intensified production systems. In 2006, there was three times fewer farms than in 1986, while the average number of pigs increased by more than 400% in swine facilities (CCP, 2008). The quantity of pigs produced in the country during this period has more than doubled, raising concerns about the environmental impacts of the industry, particularly with respect to swine manure management (CCP, 2008). Manure is an excellent source of crop nutrients and will improve soil structure through provision of organic matter. However, over-application will increase the risk of movement of nitrogen and phosphorus to groundwater and adjacent surface water (Sharpley et al., 2002). Most environmental studies concerning swine manure management either have focused on the effects of nutrients on water quality, or odour problems and air quality. Nonetheless, the microbial quality of manure should not be neglected since many outbreaks of gastroenteritis related to livestock * Corresponding author. Tel.: +1 418 565 9174; fax: +1 418 564 5507. E-mail address: [email protected] (D. Massé).
operations have been reported (Guan and Holley, 2003; Pell, 1997; Spencer and Guan, 2004). The most prevalent pathogenic microorganisms from manure are the bacteria Salmonella, Escherichia coli, Yersinia, Campylobacter, and the protozoa Giardia and Cryptosporidium (Bicudo and Goyal, 2003; Hutchison et al., 2005), but Clostridium perfringens, Listeria monocytogenes, and Treponema hydrosenteriae have also been reported as causative agents of human infections related to livestock (Colleran, 2000). The persistence of enteric pathogens in manure will vary according to manure handling practices, storage management, ambient conditions and duration, type of slurry, and pathogen type (Bicudo and Goyal, 2003). There is an urgent need for cost-effective methods to address environmental issues of pig manure by reducing microbial and chemical contaminants. Côté et al. (2006) studied the efficiency of a low temperature anaerobic laboratory-scale digester to reduce viable populations of indicator microorganisms (total coliforms, E. coli) and selected pathogens (Salmonella, Yersinia enterocolitica, Cryptosporidium and Giardia) in swine slurries. Forty liter anaerobic intermittently-fed sequencing batch reactors (SBR) operated at 20 °C during 20 day cycles reduced total coliforms by 1.6 to 4.2 log10 units (CFU g1) (97.9–100%), and E. coli by 2.5–4.2 log10 units (99.7–100%). Salmonella, Cryptosporidium, and Giardia concentrations decreased to undetectable levels (<100 CFU g1). Even though the latter study
0960-8524/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.08.020
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showed the efficiency of lab-scale psychrophilic anaerobic digestion regarding pathogen and indicator removal, no study reports the performance of commercial-scale digesters for the removal of these microorganisms. In the study reported here, the long term performance and reliability of farm-scale (165 and 450 m3) low temperature digesters for the reduction of pathogens concentration in raw swine manure was investigated.
The average organic loading rate (OLR) was based on the amount of total chemical oxygen demand (TCOD) fed per volume of sludge present at the start of a cycle and the cycle length. It was calculated as follows:
Lf ¼
V f Cf V i tc
ð1Þ
where Lf is the loading rate based on total cycle length (kgTCOD m3 day1), Vf is the volume of feed (m3), Cf is TCOD concentration in the feed (kg m3), Vi is the volume of sludge in the reactor at the beginning of the cycle (m3), and tc is the total duration of the cycle (day).
2. Methods 2.1. Reactor operation Four farm-scale cylindrical digesters (BR1–BR4) were used for psychrophilic anaerobic digestion in sequencing batch reactors (PADSBRs) of pig manure, and monitored over a two years period (June 2007–2009). The effective volume was 165 m3 (Outside Diameter (OD) 7 m) for BR1 and BR2, and 450 m3 (OD 11 m) for BR3 and BR4. The outlet consisted of a flexible pipe (Inside Diameter (ID) = 0.101 m) attached to a steel wire, allowing sludge height control during the draw step. Feeding was performed through a polyvinyl chloride (PVC) pipe (ID = 0.101 m) at the bottom of the reactor. An inflatable device was located at the top of the reactor to allow for gas and liquid displacement without air entering the system. Three reactors (BR1, BR2, and BR3) in operation since 2001 were located on the same farm and operated under a 7 day cycle, while the fourth (BR4) in operation since 2004 was operated under a 14 day cycle on another farm. BR1 and BR2 were operated as conventional SBRs, i.e. after sludge settling, supernatant was removed before feeding with fresh manure. Rather than being removed before feeding, BR3 and BR4’s supernatant was forced outside the bioreactors by simultaneously feeding with fresh manure at the bottom of the digesters. Approximately from the 500th day to the end of the monitoring period, BR3 was operated as a conventional SBR due to operational constraints. Due to solids accumulation inside BR3, 46 m3 of sludge were removed at the 665th day of monitoring, before the feed step. The temperature of all reactors was maintained throughout the year at an average of 24 °C, with fluctuations ranging from 20 to 26.5 °C. Composite samples were collected from the effluent for analysis during the drawing step. Raw manure was sampled during the filling period. All samples were kept at 4 °C until the analysis, which was done less than 24 h after sampling. Table 1 summarizes the operating conditions during the study and manure characteristics.
2.2. Physico-chemical analysis Soluble chemical oxygen demand (SCOD) of fresh manure and effluent samples was determined by analyzing the supernatant of centrifuged slurry. Total and soluble COD were determined according to the method developed by Knechtel (1978). Alkalinity, pH, total solids (TS), volatile solids (VS), total Kjeldahl nitrogen (TKN) and ammonia nitrogen (NH4–N) were determined using standard methods (APHA, 1992). TKN and NH4–N were analyzed using a Kjeltec auto-analyzer model TECATOR 1030 (Tecator AB, Hoganas, Sweden). Before VFAs quantification, samples were conditioned. Ten grams of sample was centrifuged at 41,700g, 30 min at 15 °C. One microliter of 0.5 M H2SO4 was added to 5 mL of the supernatant and centrifuged at 21,800g, 15 min at 15 °C. An internal standard (2-ethylbutyrate at 2 g L1) was added (0.5 mL) to a tube with 0.5 mL of the acidified–centrifuged supernatant and 0.1 g of DOWEX 50WX8 resin (The Dow Chemical Company, Midland, MI) and vortexed. VFAs were measured with a Perkin Elmer gas chromatograph model 8310 (Perkin Elmer, Waltham, MA), mounted with a DB-FFAP high resolution column. Results were analyzed using TurboChrom version 6.2.1 software (Perkin Elmer). 2.3. Microbiological analysis For pathogen quantification, raw manure and anaerobic digester effluent samples were prepared by diluting 1:10 in sterile sodium metaphosphate buffer (2 g L1; pH 6.8). Total coliforms were enumerated by direct plating on mEndo–LES agar (Difco, Mississauga, ON, Canada) and incubated at 37 °C for 18–20 h. Colonies which produced a distinctive metallic green sheen were counted as total coliforms. Fecal coliforms were quantified by
Table 1 Operational conditions of full-scale PADSBRs and inlet characteristics of manure fed to bioreactors.
a
Parameters
BR1
BR2
BR3
BR4
Operational conditions Loading rate (kg TCOD m3 d1) Total cycle length (day) Reactor effective volume (m3)
1.47 (0.57)a 7 120
1.48 (0.59) 7 120
1.31 (0.45) 7 425
1.65 (0.64) 14 425
Manure characteristics TCOD (g O2 L1) SCOD (g O2 L1) pH Alkalinity (mg CaCO3 L1) Acetic acid (mg L1) Propionic acid (mg L1) Butyric acid (mg L1) TS (%) VS (%) TKN (mg N L1) NH4–N (mg N L1)
115 (44) 41 (12) 6.88 (0.14) 17,300 (4100) 9300 (3100) 3600 (1300) 5400 (2300) 7.20 (2.02) 5.63 (1.65) 6600 (1600) 4600 (1200)
106 (41) 38 (12) 6.90 (0.16) 16,300 (4100) 8500 (2900) 3300 (1100) 5400 (2200) 6.72 (2.01) 5.21 (1.64) 6300 (1700) 4400 (1300)
97 (34) 35 (10) 6.95 (0.17) 15,600 (3400) 7900 (2700) 3100 (1100) 4800 (1800) 6.20 (1.69) 4.77 (1.40) 5900 (1300) 4100 (1000)
98 (39) 33 (15) 7.33 (0.21) 20,300 (5000) 8600 (3700) 2900 (1000) 2600 (1700) 6.99 (2.23) 5.24 (1.86) 6600 (1900) 5100 (1500)
Values in parenthesis represent the standard deviation of observations (Number of observations for BR1 = 81, BR2 = 89, BR3 = 99, BR4 = 30).
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direct plating on mFC agar (Difco) and incubated for 18–20 h at 44.5 °C. Colonies producing a distinctive indigo blue color were enumerated as fecal coliforms. Enterococcus spp. were counted by direct plating onto mEnterococcus agar (Difco) and incubated at 37 °C for 48 h. Colonies producing a burgundy color were counted as Enterococcus sp. E. coli were enumerated by direct plating on mFC basal medium (Difco) supplemented with 3-bromo-4chloro-5-indolyl-b-D-glucuronide (100 mg L1, Med-Ox Diagnostics, Ottawa, ON) and incubated for 18–24 h at 44.5 °C. Colonies producing the characteristic blue color indicative of b-glucuronidase activity were enumerated as E. coli. C. perfringens was quantified on mCP agar (Med-Ox Diagnostics) incubated at 44.5 °C for 24 h under anaerobic conditions. Yellow colonies with a yellow halo and which turned magenta after exposure to ammonium hydroxide fumes were expected to be C. perfringens. Presumptive colonies were confirmed by inoculation into skim milk broth. Stormy fermentation of the broth after 24 h was considered a positive confirmation for C. perfringens. This protocol allowed the quantification of vegetative cells and spores together. Y. enterocolitica were enumerated by direct plating onto Cefsulodin–Irgasan–Novobiocin agar (CIN, Difco) and incubated for 18 h at 30 °C. Colonies that were less than 2 mm in diameter and formed a red bulls-eye were considered presumptive colonies for Y. enterocolitica. Five presumptive colonies were picked and inoculated into lysine arginine iron agar slants (LAIA) and considered positive if LAIA results were alkaline slant, acid butt, H2S negative and gas negative (Weagant, 1983). Campylobacter spp. were counted by direct plating onto Charcoal Cefoperazone Desoxycholate Agar (CCDA, Oxoid, Nepean, ON) supplemented with 0.032 mg mL1 of cefoperazone and 0.01 mg mL1 of amphotericin B (Oxoid) and Campy-Line Agar (CLA, (Line, 2001)). Plates were incubated at 42 °C in microaerophilic conditions for 48 h in an anaerobic jar with GasPakTM EZ Campy (6–16% O2, 2–10% CO2, BD, Oakville, ON). Campylobacterlike colonies were quantified on both media. On CLA agar, presumptive colonies of Campylobacter are smooth, convex, burgundy colored and are approximately 2–4 mm in diameter. On CCD agar, presumptive colonies of Campylobacter are smooth, convex, and translucent colorless to cream colored and are approximately 2– 4 mm in diameter. Suspected colonies were also observed under microscope and tested for Gram coloration, oxidase and catalase, (Sigma–Aldrich, Oakville, ON) after inoculation onto Columbia blood agar and incubate 24 h at 35 °C under microaerophilic conditions. Results were confirmed using Campylobacter Latex kit as described by the manufacturer (Oxoid). Salmonella spp. were quantified by direct plating onto Salmonella chromogenic agar (Oxoid) and XLD plates (Difco). Plates were incubated at 42 °C for 24 h before being enumerated. Salmonellalike colonies were quantified on both media. On XLD agar, presumptive colonies of Salmonella are smooth, convex, and red-col-
ored with a black bulls-eye and are approximately 2–4 mm in diameter. Colonies may also produce a red halo in the media. On Salmonella chromogenic agar, presumptive colonies of Salmonella are mauve to magenta in color and are approximately 1.5–3 mm in diameter. Suspected colonies were picked and streaked on BHI agar and plates were incubated at 35 °C for 24 h. A well isolated colony was inoculated into the Rapid Salmonella Latex test as described by manufacturer (Oxoid). When further identification was necessary, a suspect colony was inoculated onto a fresh nutrient agar and incubated 24 h at 35 °C. Gram stain, oxidase and catalase tests were performed and an API 20E strip was inoculated for ultimate identification. 2.4. Statistical analysis Statistical data analysis was carried out using the software JMP, version 7.0.1 (SAS Institute Inc., Cary, NC). Homogeneity of variances was analysed with the Levene test statistic. Means, standard deviations and frequency distributions of the data were determined. Differences between means were tested with comparative statistics, using one-way ANOVA. After obtaining F-ratios, Tukey’s post hoc tests and Student t-test were performed to determine significantly different pairs of data. The level of significance was set at P < 0.05. The value ‘‘99” was used for statistical analysis when pathogen and indicator concentrations were below the method detection limit (<100 CFU g1). 3. Results and discussion 3.1. Manure characteristics Four farm-scale psychrophilic anaerobic digesters were monitored over a two-year period to assess their reliability and stability with respect to removal of enteric pathogens. No significant difference (P > 0.05) was found between raw manure samples collected at bioreactor inlet for all tested parameters (Table 1). The average OLR applied to the bioreactors ranged from 1.31 to 1.65 kg TCOD m3 d1, with a TCOD concentration around 100,000 mg O2 L1. The pH of raw manure was near neutrality, even though high VFAs concentrations were observed, mainly because of the high amount of alkalinity in the manure. Pathogen and indicator concentrations in raw pig manure were highly variable (Table 2), but within the range of reported values (Hill and Sobsey, 1998; Hutchison et al., 2005; Kearney et al., 1993a). The mean concentration of total and fecal coliforms, Enterococcus spp. and E. coli was 2.7 105, 1.9 105, 3.8 105, and 8.8 104 CFU g1, respectively. Lower mean concentrations were observed for Y. enterocolitica, Salmonella and Campylobacter spp., with 2.0 104, 2.2 103, and 3.0 103 CFU g1, respectively. Y. enterocolitica, Salmonella and Campylobacter concentrations
Table 2 Performance of PADSBRs regarding pathogen and indicator removal. Parameters
Total coliforms Fecal coliforms Escherichia coli Enterococcus spp. Clostridium perfringens Yersinia enterocolitica Salmonella spp. Campylobacter spp. a
Concentration range (CFU g1)
6.0 102–1.5 106 5.0 102–1.6 106 4.0 102–5.8 105 1.8 104–1.7 106 <1.0 103–3.7 106 <1.0 102–3.3 105 <1.0 102–5.0 104 <1.0 102–5.2 104
Log10 Removala BR1
BR2
BR3
SBR
SBR
Non-SBR
SBR
BR4 Non-SBR
SBR
2.9 2.8 2.9 0.8 0.2 2.1 1.2 1.3
2.5 2.6 2.8 1.0 0.0 2.1 1.4 1.5
1.0 0.9 0.9 0.7 0.2 1.1 0.9 1.1
2.4 3.0 2.8 0.6 0.1 1.9 1.3 1.4
1.2 1.2 0.9 0.8 0.2 1.3 1.1 1.1
2.7 2.8 2.8 1.0 0.1 2.4 1.3 1.3
Values were calculated using mean inlet and outlet concentrations (Number of observations for BR1 = 48, BR2 = 48, BR3 = 45, BR4 = 45).
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equal of below the method detection limit (100 CFU g1) were detected in 10%, 43% and 24%, respectively of the samples from the inlets of BR1–BR3 inlet. For BR4 inlet, 10%, 45% and 53% of samples showed concentrations equal or below the detection limit, for these microorganisms, respectively. 3.2. Bioreactor performance Since biogas production was not monitored on a regular basis on these full-scale reactors during digestion, the overall bioreactor performance was verified by monitoring TCOD removal. Conventionally operated SBRs (BR1 and BR2) showed TCOD removal above 70%, while those being simultaneously fed with fresh manure during the drawing step (BR3 and BR4) only reached 61.6% and 56.3% (Fig. 1C and D), respectively. Near the end of the two-year monitoring period (500th day), BR3 performance decreased significantly as measured by TCOD removal. This was principally due to typical solid accumulations inside the reactor which increased the solids concentration at the outlet. This was indicated by an increase of the outlet VS/TS ratio (Fig. 1C). Similar results were obtained for BR4 at the end of the monitoring period (Fig. 1D). For BR3, satisfactory performance was regained after removing solids from the reactor. The low temperature AD treatment resulted in significantly lower concentrations of pathogens and indicators in BR1 and BR2 effluents. An average reduction of 2.6–2.9 log10 units and 1.2– 1.4 log10 units was observed for fecal coliforms and Salmonella spp. for an HRT of 7 days (Table 2), leading to undetectable levels in more than 81% and 96% of outlet samples, respectively. Other studies obtained longer pathogen and indicator removal times for similar or higher temperatures during digestion. Kumar et al. (1999) used an ampicillin-resistant E. coli strain to study the persistence of this organism in cattle dung slurry during anaerobic digestion. The survival was 25 days at room temperature (18–25 °C) and 15 days at 35 °C. In the same study, the complete elimination of Salmonella at 35 °C occurred on the 15th day
B
40
Short circuit (%)
Short circuit (%)
35
30 25 20 15 10 5 0
40
Conventional SBR
35
30 25 20 15 10 5
0
200
400
600
0
800
Time (d)
D
0.8
250000
0.6 0.5
150000
0.4 100000
0.3 0.2
50000
0.1 0
200
400
Time (d)
600
0 800
TCOD (mg O2 L-1)
0.7
200000
0
200
400
600
800
Time (d)
VS/TS
TCOD (mg O2 L-1)
C
0
0.8
250000
0.7
200000
0.6 0.5
150000
0.4 100000
0.3
VS/TS
A
whereas 25 days were necessary at room temperature. Berg and Berman (1980) reported a reduction of 2.0 and 1.8 log10 units of indigenous total and fecal coliforms, respectively, during the mesophilic digestion (35 °C; SRT 20 days) of municipal sludge. In another study, Salmonella and E. coli, which were added to liquid pig manure, and coliforms of the indigenous flora, were reduced by 1.0–2.1 log units when subjected to anaerobic filter treatment at 35 °C in two pilot-plant reactors operated at 0.8–4.2 days HRT (Olsen, 1988). In an investigation studying the survival of indigenous pathogens in a full-scale anaerobic digester which was daily fed with cattle farm wastes and operated at 28 °C, Kearney et al. (1993a) found that Campylobacter jejuni was the most resistant bacterium tested among E. coli, Salmonella enterica Typhimurium, Y. enterocolitica, and L. monocytogenes. Its mean T90 (time to reduce its concentration of 90%) value was 438.6 days, compared to Y. enterocolitica that showed a T90 of 18.2 days. Mean T90 observed under the operational conditions tested in their study suggest that the process did not allow the complete removal of tested pathogens. In comparison, undetectable levels of Y. enterocolitica and Campylobacter were obtained in our study in more than 87% of BR1 and BR2 outlet samples for only 7 days treatment. The other 13% showed an average concentration of 7.5 102 CFU g1 for these microorganisms. It seems that for the same species or genera, various removal efficiencies are observed, depending on the substrate being digested. For example, 90% of Salmonella spp. was removed in 34.5 days when digesting cattle slurry mixed with hen, pig and potato wastes at 28 °C, whereas it was completely removed in 15 days when digesting cattle dung slurry at 35 °C (Kumar et al., 1999), in 21 days when digesting vegetable, fruit, and garden waste in mesophilic conditions (Termorshuizen et al., 2003), and in only 7 days during the psychrophilic digestion (24 °C) of pig manure in this study. According to Smith et al. (2005), efficient mixing and organic matter stabilisation are the main factors controlling the rate of inactivation under mesophilic conditions and not a direct effect
0.2
50000
0.1
0
0
200
400
600
0 800
Time (d)
Fig. 1. Percentage of VFAs going through BR3 (A) and BR4 (B) by short circuiting of hydraulic flow, TCOD removal and VS–TS ratio in BR3 (C) and BR(4). Arrows indicate periods when bioreactors were operated as conventional SBRs. Dashed line represents the cycles when BR3 operation was switched toward conventional SBR. Feed (); Effluent ( ); VS/TS (N).
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of temperature. Thus, the observed differences regarding pathogen removal from various matrices could be due to the level of stabilization reached for each of the digested substrates. C. perfringens and Enterococcus spp. were not significantly removed by psychrophilic anaerobic digestion (Table 2). Negligible removal of 0.2 log10 unit was reported for C. perfringens spores during the treatment of swine manure in an anaerobic lagoon in North Carolina (Hill and Sobsey, 1998). Similarly, no significant C. perfringens removal was observed during the mesophilic digestion (HRT = 20 days) of sewage sludge in a full-scale anaerobic reactor, measured both as spores and total counts (Chauret et al., 1999). The same study also showed that Enterococcus spp. was not significantly removed from the sludge during digestion. Effenberger et al. (2006) also reported low removal efficiencies for fecal enterococci (about 1.0 log10 unit) after the first stage of a mesophilic– thermophilic–mesophilic anaerobic digestion process treating liquid dairy cattle manure. These data are in agreement with our results since Enterococcus spp. removal was at most 1.0 log10 unit with an average concentration in bioreactors’ outlet around 105 CFU g1. Enterococcus is a facultative anaerobic microorganism that is present in the intestinal tract of various animals, Enterococcus faecium and Enterococcus faecalis being the most frequently encountered species in pig’s (Klein, 2003). This genus is recognized as being able to survive a range of stresses and hostile environments, including those of extreme temperature (5–65 °C), pH (4.5–10.0) and high salt concentrations, enabling them to colonize a wide range of niches (Fisher and Phillips, 2009). C. perfringens is an anaerobic spore-forming bacterium that can withstand stresses such as low oxidation–reduction potential (ORP). Therefore, it is likely to survive anaerobic manure digestion. Many Clostridium species are commonly observed in anaerobic digesters, being responsible with other microorganisms for acetate production (Diekert and Wohlfarth, 1994). They can also produce lower fatty acids from acetate or ethanol when the concentration of hydrogen is high, thereby reversing the reactions of the syntropic bacteria. This may be an indicator of reactor instability (O’Flaherty et al., 2006). It is thus not surprising that high concentrations of Enterococcus and Clostridium are observed in outlets of anaerobic digesters. Temperature and retention time are critical parameters for indicator and pathogen survival during anaerobic digestion of wastes (Arthurson, 2008; Sahlström, 2003). The elimination of E. coli and Salmonella was faster at 35 °C than at room temperature during anaerobic digestion of cattle slurry (Kumar et al., 1999). S. typhimurium, Y. enterocolitica and L. monocytogenes also declined more rapidly at 17 °C than at 4 °C during anaerobic digestion of this substrate (Kearney et al., 1993b). According to Olsen and Larsen (1987), increasing retention time in mesophilic digesters enhanced pathogen removal efficiencies toward those encountered for anaerobic thermophilic treatment. However, Kearney (1991) reported that S. typhimurium had a T90 value of 2.1 days at a 25 day HRT, but after the HRT was reduced to 5 days the T90 value decreased to 1.2 days. It was hypothesized that higher concentrations of VFAs at shorter HRTs would be correlated with a decline in viable pathogens number. Since their results did not show significant correlation between VFAs and pathogen concentrations, they speculated that other factors such as the source and type of slurry, pH and temperature of the anaerobic digestion process could also have a significant effect on the pathogen populations. Regarding S. typhimurium removal, Gadre et al. (1986) reported a T90 of approximately 7.7 days during mesophilic (37 °C) anaerobic digestion of cattle dung, while Olsen and Larsen (1987) found a T90 of 2.0 days during the digestion of pig slurry in similar conditions. Even though T90 were not specifically calculated during our study, results suggest that T90 values of analyzed pathogens and indicator microorganisms were lower than 7 days under the conditions
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encountered in the psychrophilic anaerobic digesters. Since temperature was lower in these reactors than in mesophilic ones, parameters other than temperature are probably responsible for the elimination of pathogens. Côté et al. (2006) studied the removal of pathogens and indicator microorganisms during PADSBRs of swine manure in 28 day HRT laboratory-scale digesters (42 L) operated at 20 °C. At the end of the treatment, 47%, 75% and 100% of the collected samples were free of total coliforms, E. coli and Salmonella, respectively. They observed a diminution of 1.6–4.2 log10 units and of 2.5–4.2 log10 units in the residual samples still having detectable concentrations of total coliforms and E. coli, respectively. Farm-scale reactors operated in our study showed comparable performances with lab-scale regarding pathogen removal for a similar temperature, at a much lower HRT (7 days). This process has already proven successful for pig manure treatment in full-scale reactors, regarding biogas production and COD removal (Masse et al., 2009). Results obtained in this study show that scaled-up PADSBR was not only successful in stabilizing the organic matter, but was also able to decrease pathogen and indicator concentrations to undetectable levels. Lower performances regarding pathogen and indicator removal were also observed for BR3 and BR4 when operated as non-conventional SBRs (Table 2). BR1 and BR2 removed more than 2.5 log10 units of total and fecal coliforms, and E. coli, corresponding to more than 99.7% removal. Only 0.9–1.2 log10 unit of these microorganisms was removed by BR3 and BR4, corresponding to 86.1–94.0% removal. Y. enterocolitica removal averaged 2.1 log10 units for BR1 and BR2, and ranged from 1.1 to 1.3 log10 unit for the other reactors. Salmonella and Campylobacter spp. removal by BR1 and BR2 ranged from 1.2 to 1.5 log10 unit, corresponding to 94.1–96.6%, while their concentration decreased by 0.9–1.1 log10 unit in BR3 and BR4. Enterococcus spp. and C. perfringens concentration remained high in all effluents, with average concentrations of 5.6 104 and 2.7 105 CFU g1, respectively. Since filling was performed simultaneously with drawing in BR3 and BR4, it was hypothesized that there could be a short circuit in the hydraulic flow. Mass balances were performed to estimate which proportion of influent entering the reactor during filling was going out with the supernatant overflow. VFAs were chosen as short circuiting indicators, because of their low concentration after digestion and of a more precise and reliable quantification method compared to TCOD and bacteria. It appeared that an average of 6.42% (standard deviation (SD) = 5.50) and 6.33% (SD = 6.29) of VFAs entering the system were directly going to the bioreactor’s outlet while filling BR3 and BR4, respectively. Sporadically, for diverse operational constraints, BR3 and BR4 were fed after the drawing step, as for conventional SBRs, indicated by arrows in Fig. 1A and B. Moreover, BR3 operational conditions were switched toward those of a conventional SBR near the 500th day of this study (Fig. 1). Results obtained clearly show that the presence of VFAs in the bioreactor outlet is due to short circuiting, since negligible VFA concentrations were observed in the outlet when filling and drawing were performed separately. The increase of TCOD in BR3 and BR4 effluent (Fig. 1C and D) due to short circuiting was significant compared to conventionally operated SBRs (BR1 and BR2; ca. 25,000 mgTCOD L1). The presence of short circuits could lead to an incompletely stabilized digestate, causing odor problems and greenhouse gas production during storage of the effluents from the bioreactors. The difference was also significant for pathogens and indicators. Two hydraulic retention times (7 and 14 days) were tested in this study, in BR3 and BR4, respectively. Even though these reactors showed higher TCOD and pathogen and indicator concentrations in effluents compared to BR1 and BR2, mainly because of short-circuiting of hydraulic flow, no significant impact was ob-
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served on pathogen and carbon removal when the treatment cycle length was reduced from 2 to 1 week (P > 0.05). Moreover, as represented by arrows in Fig. 1, a return to conventional SBR operations for BR3 and BR4 allowed the elimination of hydraulic short circuiting and permitted the comparison of conventionally operated SBRs under two HRTs for some cycles. Under these operational conditions, similar pathogen and indicator removal efficiencies as for BR1 and BR2 were reached for all tested organisms (Table 2), again showing that these full-scale reactors performed similarly when the treatment cycle length was reduced from 2 to 1 week. 4. Conclusion Farm-scale psychrophilic anaerobic digestion in sequential batch reactors (PADSBRs), operated at 7 or 14 days HRT and 24 °C, significantly decreased the concentration of total and fecal coliforms, E. coli, Salmonella, Campylobacter spp., and Y. enterocolitica to undetectable levels in most samples. The concentration of C. perfringens did not decrease significantly during treatment while Enterococcus spp. concentration diminished by less than 1.0 log10 unit, still remaining above 105 CFU g1. All these results were similar to those obtained from laboratory-scale PADSBRs, which were 3000 to 10,000 times smaller in volume than the full-scale digesters. No significant impact was observed on pathogen and carbon removal when the treatment cycle length was reduced from 2 to 1 week. This allows the use of smaller reactors, significantly reducing installation and operational costs of this particular process. Acknowledgements We thank Bio-Terre for providing access to on-farm full-scale bioreactors and for samples collection. We are also grateful to Denis Deslauriers and Andrew Scott for their dedication to this project and for providing their technical expertise. References APHA, 1992. Standard methods for the examination of water and wastewater, 18th ed. American Public Health Association. Arthurson, V., 2008. Proper sanitization of sewage sludge: a critical issue for a sustainable society. Applied and Environmental Microbiology 74 (17), 5267– 5275. Berg, G., Berman, D., 1980. Destruction by anaerobic mesophilic and thermophilic digestion of viruses and indicator bacteria indigenous to domestic sludges. Applied and Environmental Microbiology 39 (2), 361–368. Bicudo, J.R., Goyal, S.M., 2003. Pathogens and manure management systems: a review. Environmental Technology 24 (1), 115–130. CCP, 2008. Statistics & Market Reports, Canadien Pork Concil. Ottawa, ON. Chauret, C., Springthorpe, S., Sattar, S., 1999. Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion. Canadian Journal of Microbiology 45 (3), 257–262. Colleran, E., 2000. Hygienic and sanitation requirements in biogas plants treating animal manures or mixtures of manures and other organic wastes. In: Ørtenblad, H. (Ed.), Anaerobic Digestion: Making energy and solving modern waste problems. Herning Municipal Authorities, Denmark, pp. 77–86. AD-NETT. Côté, C., Massé, D.I., Quessy, S., 2006. Reduction of indicator and pathogenic microorganisms by psychrophilic anaerobic digestion in swine slurries. Bioresource Technology 97 (4), 686–691. Diekert, G., Wohlfarth, G., 1994. Metabolism of homoacetogens. Antonie van Leeuwenhoek 66 (1), 209–221.
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