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Inactivation of Salmonella spp. in cow manure composts formulated to different initial C:N ratios
Bioresource Technology 100 (2009) 5898–5903
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Inactivation of Salmonella spp. in cow manure composts formulated to different initial C:N ratios Marilyn C. Erickson a,*, Jean Liao a, Li Ma a, Xiuping Jiang b, Michael P. Doyle a a b
Center for Food Safety, University of Georgia, Griffin, GA 30223, United States Department of Food Science and Human Nutrition, Clemson University, Clemson, SC, United States
a r t i c l e
i n f o
Article history: Received 6 February 2009 Received in revised form 19 June 2009 Accepted 19 June 2009 Available online 28 July 2009 Keywords: Salmonella Compost Volatile acids pH Thermal inactivation
a b s t r a c t Aerobic composting is a common management practice to inactivate pathogens in manure; however, additional research on the role of compost composition in pathogen inactivation is needed. The objective of this study was therefore to determine the effect of the carbon:nitrogen (C:N) ratio and the presence of ammonium sulfate on inactivation of Salmonella spp. in cow manure-based mixtures composted in a bioreactor under controlled conditions. Compost preparations with an initial C:N ratio of 20:1 required a maximum of 4 days of storage before Salmonellae were inactivated by 7 log10, whereas preparations with C:N ratios of 30:1 and 40:1 C:N required more than 5 and 7 days of storage, respectively. The pH values of both the 20:1 and 30:1 C:N preparations decreased during the onset of composting before increasing to >8. In contrast, pH values of 40:1 C:N preparations increased immediately to >8, generally within the first day of storage. Maximum temperatures observed in 20:1 C:N preparations for inactivation of pathogens were less than 50 °C, and the cumulative heat exposure required for pathogen inactivation in 20:1 C:N preparations was 15-fold less than in 40:1 C:N preparations. Supplementation of compost mixtures with 0.08% ammonium sulfate resulted in slightly higher temperatures; however, these higher temperatures did not translate into more rapid rates of pathogen inactivation. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Animal agriculture has been an important contributor to the United States’ economic well-being; however, it also provides an abundance of manure that must be managed. Estimates indicate that animal waste produced in the US is somewhere between 3 and 20 times greater than human waste with 90% of this waste being generated by cattle and 5% by poultry (Rogers and Haines, 2005). Animal manure is a well-documented source of zoonotic pathogens, such as Escherichia coli O157:H7, Salmonella spp., and Campylobacter spp. (Zhao et al., 1995; Pell, 1997). These pathogens can survive in manure and manure-amended soils for extended periods of time (Wang et al., 1996; Himathongkham and Riemann, 1999; Himathongkham et al., 2000; Jiang et al., 2002; You et al., 2006) and subsequently contaminate vegetables grown in those soils (Natvig et al., 2002; Islam et al., 2004a,b, 2005). Since the number of reported produce-related outbreaks per year doubled between the period of 1973–1987 and 1988–1992, manure as a source of contamination could be an important contributor to this statistic (NACMCF, 1999). Manures may be subjected to aerobic composting to minimize the occurrence of manure as a source of zoonotic pathogens. To prepare compost, nitrogen (N)-rich manures are mixed with one * Corresponding author. Tel.: +1 770 412 4742; fax: +1 770 229 3216. E-mail address: [email protected] (M.C. Erickson). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.06.083
or more carbon (C) amendments to produce a nutrient-rich environment favorable for the metabolism of thermophilic microorganisms. Heat generated from the metabolic activity of these microbes plays a major role in the inactivation of zoonotic pathogens. Hence, the primary process criteria used worldwide for ensuring the microbiological safety of composts have been narrowly defined time–temperature conditions. In the US, process conditions for composting manures is based on EPA’s 503 regulations for composting biosolids and include either a minimum temperature of 55 °C for 3 days in aerated static piles or in-vessel systems, or 55 °C for 15 days in windrow systems (US EPA, 1999). In other countries, there is general agreement that temperatures higher than 55 °C and below 65 °C have the desired effect, although differences exist on the duration for which these temperatures must be achieved (Hogg et al., 2002). Despite these recommended process conditions, a time–temperature approach may not always be sufficient to ensure complete inactivation of pathogens within the entire compost mass. For example, Droffner and Brinton (1995) reported that Salmonella typhimurium Q survived for at least 9 days at 60–70 °C in compost prepared from food biowaste and for at least 5 days in a compost prepared from waste water sludge. Hutchison et al. (2005b) has also reported extended survival of pathogens in field studies of static compost piles. In that study, Salmonella, E. coli O157:H7, and Listeria survived in poultry manure-based compost piles when exposed to
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temperatures above 55 °C for more than 8 days. Similarly, E. coli O157:H7 survived in dairy cow and pig manure compost mixtures when exposed to temperatures above 60 °C for more than 3 and 8 days, respectively. In light of these studies, it is proposed that chemical or biological factors contribute significantly to inactivation variations within these systems. The involvement of factors in pathogen inactivation other than heat is supported by the composting study in which Salmonellae were more readily inactivated when inoculated into the compost than when held in sealed glass ampules within the compost (Platz, 1978). Raw material composition is one chemical parameter that has potential to affect both direct and indirect pathogen inactivation during thermal composting. For example, a review on the thermal resistance of Salmonellae noted that some amino acids and sugars increase heat resistance, whereas other compounds such as cysteine and glutathione decrease heat resistance (Doyle and Mazzotta, 2000). Qualitative and quantitative compositional differences between manures (Lynch, 1987) could be responsible for pathogens surviving longer in beef cattle waste compared to dairy cattle, pig, poultry layer, or sheep waste (Hutchison et al., 2005a). Different exogenous C sources (sawdust versus straw) have also contributed to different patterns of heating (i.e., peak heating temperature and length of peak heating) and could therefore contribute to variations in pathogen inactivation, although the feedstocks appeared not to have a significant effect on the microbial profile of the mature compost (Green et al., 2004). Relative proportions of manure and C amendments in the compost formulations may also affect the metabolic response of indigenous compost microflora and in turn the survival of zoonotic pathogens. Microorganisms use C for both energy and growth, whereas N is essential for protein and reproduction. Raw materials blended to provide a C:N ratio of 25:1 to 30:1 are considered ideal for compost degradation, although initial C:N ratios from 20:1 to 40:1 are considered acceptable (Rynk, 1992). At C:N ratios close to 20:1, however, C not bound in complex carbohydrates is utilized but is insufficient to stabilize all of the N leading to the production of ammonia, a known bactericidal agent in inactivation of S. typhimurium, E. coli O157:H7, and Listeria monocytogenes in manures (Himathongkham and Riemann, 1999; Himathongkham et al., 2000; Park and Diez-Gonzalez, 2003). C:N ratios of 40:1 and above, on the other hand, lead to elevated microbial activities and heat generation during initial stages of composting but decreased microbial activity and heat generation as composting progresses and N becomes limiting. There is only one study reported to date addressing the fate of pathogens or their surrogates in response to different C:N formulations. In that study, fecal streptococci and total coliforms were eliminated in a system having an initial C:N ratio of 32.9, whereas their populations were only partially reduced at a C:N ratio of 67.5 (Lafond et al., 2002). The objective of our study was to determine the influence of C:N on Salmonella spp. inactivation in compost mixtures containing dairy cow manure and initial C:N ratios ranging from 20:1 to 40:1. In addition, ammonium sulfate, an ingredient sometimes added to compost mixtures to stimulate biological activity in early stages of composting, was evaluated for its potential effect on inactivation of Salmonella. Bench-scale composting systems were utilized for these investigations in order to provide control over environmental factors (i.e., air flow, ambient temperatures) that could otherwise introduce variation in treatment responses.
2. Methods 2.1. Preparation of Salmonella strains for experimental trials Three strains of Salmonella enteritidis (ME-18, H4639, and H3353) and one strain of Salmonella newport (11590) were labeled
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with jellyfish green-fluorescent protein (GFP) and an ampicillinresistant marker according to the method described by Sambrook et al. (1989). When viewed under a handheld UV light (365 nm), the resulting transformed colonies emitted bright green fluorescence. Each strain of Salmonella was thawed from a frozen culture and individually streaked onto tryptic soy agar (TSA; Difco Laboratories, Detroit, Mich.) containing 100 lg/ml ampicillin (TSA-A) for incubation at 37 °C for 20–24 h. Individual colonies were subsequently streaked onto a second plate for another incubation at 37 °C for 20–24 h. Individual colonies were selected from these plates and inoculated into 10 ml of tryptic soy broth (Becton Dickinson, Sparks, MD) containing 100 lg/ml of ampicillin (TSB-A). These inoculated broths were incubated at 37 °C for 20–24 h with agitation (150 rpm). The bacteria were harvested by centrifugation (4050g, 15 min, 4 °C) and washed three times in 0.1% peptone water (Difco). The individual isolates were reconstituted with 0.1% peptone water to an optical density at 630 nm of 0.5 (109 cfu/ml). Equal volumes of each Salmonella strain were combined to give one four-strain mixture. Cell populations of the Salmonella mixture were determined by plating on TSA-A. 2.2. Compost ingredients and preparation For each trial, fresh cow manure was collected from a dairy farm near Griffin, Ga., and frozen for at least 24 h to kill insect eggs. Wheat straw, cottonseed meal, and ammonium sulfate were purchased from a local feed store. Manure, straw, and cottonseed meal were analyzed for N, C, and moisture content (MC) prior to their use. The wheat straw was shredded using a Flowtron Leaf Eater (Malden, MA) in order to provide greater homogeneity to the compost mixture and hence better temperature distributions and less heat loss (Gotaas, 1956). GFP-labeled pathogens were sprayed onto the manure (107 cfu/g) held in a 28-l sanitized mixing bowl. The material was mixed thoroughly with a Hobart mixer (Model D320; 3/4 h.p.) prior to adding the other raw ingredients. The proportions of the raw ingredients used in the mixture were adjusted to give initial MC of 60% and C:N ratios in the range of 20:1 to 40:1. Immediately after mixing, the compost mixtures were loaded into the composting apparatus. 2.3. Composting apparatus Two 15-l compost bioreactors were constructed based on a design described by Jiang et al. (2003). The bioreactor body was made up of PVC plastic pipe (48 cm height and 32 cm diameter). The bottom and top of each pipe was tightly fitted with PVC covers. Both covers had holes drilled in the center with the bottom hole serving as a connection to a condensate bottle and the top cover hole connected to the air inlet system. Within the bioreactor body, a perforated PVC rack was placed at a height of 5 cm from the bottom. The compost mixture (4 kg) was placed on this rack and material added to an initial height of 35 cm. Two sampling ports (3 cm diameter each) were drilled into the sides of the bioreactor body at heights of 6–9 and 10–13 cm above the PVC rack. Small holes were also drilled at 6.5 cm above the PVC rack for insertion of type T thermocouple wires into the compost mixture. One wire was inserted 3 cm into the mixture (bottom-edge location) and another wire was inserted 16 cm into the mixture (bottom-center location). In addition, thermocouples were inserted 10 cm into the mixture from the top of the bioreactor at edge and center locations (3 and 16 cm from bioreactor wall, respectively). All wires were connected to a multiple-channel HotMux data logger (DCC Corp., Pennsauken, NJ) and temperatures were recorded at 30-min intervals during the experimental trials. The bioreactors were placed inside a Precision
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30 Mechanical Convection incubator (Thermo Fisher Scientific, Waltham, Ma.) set at 40 °C. This incubator had been modified to vent trapped air to a filtered exhaust system. Compressed air was connected to each bioreactor to give an airflow rate of 155 ml/min. 2.4. Experimental design Ten trials were conducted over a period of 5 months. Each trial consisted of two bioreactors, one of which was formulated with 0.08% ammonium sulfate and the other formulated without ammonium sulfate. Four replicate trials were conducted with compost materials formulated to a C:N ratio of 30:1 whereas three replicate trials each were conducted with compost materials formulated to C:N ratios of 20:1 and 40:1. Compost samples (25 g) were removed in duplicate from the bioreactor on a daily basis over a period of 10 days using an autoclaved grabbing tool (Mechanical Pick-up No. 396, General Tools Mfg., NY, NY) placed inside a sterile tube that had been inserted into the compost at the defined sampling location (bottom-edge, bottom-center, topedge, and top-center). 2.5. Microbiological analyses Sample portions (5 g) were mixed with 45 ml of 0.1% peptone water in a sterile Whirl-PakÒ bag and macerated in a stomacher. Serial dilutions of each sample were then spread onto TSA-A plates and incubated at 37 °C for 24 h for enumeration by direct plate count. At the same time, a selective enrichment method was used that consisted of adding compost sample (5 g) to 45 ml of TSB-A and incubating at 37 °C for 24 h. Portions of these enriched samples were then streaked on TSA-A plates to elucidate the presence or absence of pathogen in the sample (limit of detection: 1 cell/ 5 g). Salmonella identification was confirmed using a Salmonella latex agglutination kit (Oxoid Limited, Hampshire, England). 2.6. Chemical analyses Oxygen levels in the bioreactors were determined with a Demista OT-21 oxygen probe (Arlington Heights, IL) just prior to removal of compost samples for chemical and microbial assays. The pH of compost samples was determined by adding 5 g to 250 ml of deionized water followed by measurement with an Accumet Basic pH meter (Fisher Scientific, Pittsburgh, PA). C content of ingredients and compost mixtures was determined on the basis of ash content obtained at 550 °C. MC was determined by drying a 5 g sample for 24 h in a vacuum oven and weighing. N content of compost mixtures and ingredients was determined by the University of Georgia Soil Testing Laboratory wherein samples were first ground and screened through a 10-mesh sieve before analysis by a macro-Kjeldahl method. 2.7. Statistical analyses Bacterial populations were converted to log10cfu/g before statistical analysis. Data were analyzed by analysis of variance (ANOVA) using the StatGraphics Centurion XV software package (StatPoint, Inc., Herndon, Virg.). When statistical differences were observed (P < 0.05) with ANOVA, differences among sample means were determined using the least significant difference test.
3. Results and discussion 3.1. C:N content of initial compost mixtures For each mixture formulated, a fresh batch of dairy cow manure was collected from the farm, analyzed for MC, C and N contents,
and mixed with wheat straw and cottonseed meal. Due to differences in composition of these manure samples, the amounts of the raw materials used varied slightly for each of the target C:N formulations. Initial C:N ratios of the compost mixtures were within a narrow range of the target values: 19.7 ± 2.6, 29.5 ± 2.1, and 42.0 ± 2.2, for the 20:1, 30:1, and 40:1 C:N formulations, respectively. 3.2. Moisture and oxygen levels during composting All treatments were formulated to an initial value of 60% MC. MC remained above 50% during composting in the bioreactors and thus were not considered a limiting factor for bacterial activity (Golueke, 1994). Similarly, oxygen levels throughout the composting period of evaluation were in a range (10–20%) considered adequate for aerobic composting (Finstein and Hogan, 1993). 3.3. Bioreactor sample location Continuous aeration using a positive pressure system has previously led to stratified temperatures within the bioreactor (Jiang et al., 2003). Four locations were therefore identified as sampling sites (bottom-edge, bottom-center, top-edge, and top-center) as representative of the diversity of conditions within the bioreactor. While edge locations had lower temperatures than center locations (data not shown), these differences were not statistically significant. In addition, sample location was not found to be a significant variable in pathogen inactivation or change in pH levels (data not shown). Hence, sample location data were merged into one data set for evaluation of other study variables. 3.4. Heat generation in response to ammonium sulfate amendment Although not a common amendment, the addition of ammonium sulfate to compost mixtures is recommended when N may not be readily available in the system for microbial activity. To evaluate the response of the systems in the presence and absence of this amendment, the accumulation of metabolic heat within each of the bioreactors was calculated and expressed as cumulative heat exposure (degree days) above the ambient incubator temperature of 40 °C (Fig. 1). In 40:1 C:N formulations, where readily available N may be a limiting factor, a slight increase in heat generation was noted during the first 3 days of composting when ammonium sulfate was added to a level of 0.08%. Differences became negligible after 3 days that is likely attributable to depletion of the ammonium sulfate. In 20:1 and 30:1 C:N formulations, no differences in heat generated during the first 3 days of storage occurred upon addition of ammonium sulfate indicating that N was not a limiting factor during that time. When the generation of heat started to rapidly increase for these formulations, however, cumulative heat exposure was slightly higher in formulations containing ammonium sulfate. Statistically, these differences in heat accumulation did not significantly affect the inactivation of Salmonella in the compost formulations. Hence, the ammonium sulfate data were consolidated for subsequent evaluation of the systems’ responses to C:N ratio. 3.5. Temperature profiles of bioreactor systems in different C:N formulations Within one day of composting, temperatures in the bioreactors had increased to the incubator temperature (40 °C) and above (Fig. 2). In compost preparations formulated to a C:N ratio of 40:1, temperatures increased to near maximum within the first day of composting and began to decline after only 2 days of composting. In 30:1 C:N formulations, temperature increased gradually
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3.6. Inactivation of Salmonella during composting of different C:N formulations
Cumulative Heat Exposure (Degree-Days)
60
40
20
0
0
2
4 Days
6
8
20:1 w/ AS
30:1 w/ AS
40:1 w/ AS
20:1 w/o AS
30:1 w/o AS
40:1 w/o AS
Temperature (ºC)
60
50
40
30 20:1 0
1
2
3
4
30:1 5
40:1 6
7
Days
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pH
8
7
6 20:1 0
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2
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Distinctly different pH profiles were observed during composting of formulations with different initial C:N ratios (Fig. 3). In both the 20:1 and 30:1 C:N formulations, the pH decreased but the reduction was greater and occurred for a longer time in the 20:1 than the 30:1 C:N formulation. The pH of the 40:1 C:N formulations, in contrast, increased during the first 2 days of composting. Accumulation of organic acids such as acetic acid has been associated with decreases in pH values (Reinhardt, 2002) and have also been held responsible for inhibition of thermophilic microorganisms (Sundberg et al., 2004) and delays in temperature development. Typically, formation of organic acids is ascribed to anaerobic conditions and it is possible that anaerobic microenvironments could have occurred in the bioreactors in which the compost formulations were not sufficiently mixed with the straw and cottonseed (Steniford, 1993). Alternatively, Reinhardt (2002) reported that many bacteria produce acetic acid even when grown aerobically in the presence of easily degradable substrates. 3.8. Comparison of bioreactor conditions for Salmonella inactivation
Fig. 2. Temperature profiles of cow manure compost mixtures formulated to different C:N ratios.
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During the first 2 days of composting, C:N formulation significantly affected the rate of Salmonella inactivation with populations in 20:1 and 40:1 C:N formulations being reduced by more than 7 log10 and populations in 30:1 C:N formulations being reduced by only 5 log10. Salmonella could still be detected by enrichment culture in 40:1 C:N formulated samples after 7 days of composting in bioreactors whereas the last day it was detected by enrichment culture in 20:1 and 30:1 C:N formulated samples was days 3 and 5, respectively. Given that heat generation in field conditions would likely be slower than occurred in these optimally controlled bioreactors, the differences in time to inactivation in the field would also likely be magnified with the different C:N formulations. 3.7. pH response during composting of different C:N formulations
Fig. 1. Cumulative heat (>40 °C) generated in dairy cow manure compost mixtures formulated to different C:N ratios with and without 0.08% ammonium sulfate (AS).
20
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4
30:1 5
40:1 6
7
Days Fig. 3. pH values of cow manure compost mixtures formulated to different C:N ratios.
from day 1 to day 4 of composting, whereas temperature increases in 20:1 C:N formulations did not occur until after the second day of composting and then increased to temperatures approximating those in 30:1 C:N formulations. In a previous study examining aerobic pig manure static compost piles, C:N formulations of 15:1 also exhibited a slower increase in temperature than piles formulated to a 30:1 C:N ratio (Huang et al., 2004).
Some investigators have suggested that it is unrealistic to ensure pathogen-free compost, but instead the compost should constitute an acceptable risk based on a significant reduction or inactivation of indicator microbes (Pereira Neto et al., 1987). Under that same pretext, Jones and Martin (2003) advocated that the absolute time to extinction of an organism is less important than the rate at which the cell numbers decline because the former is affected by the initial population level. It is our contention, on the other hand, that pathogen-free composts should be the target due to the potential for regrowth of Salmonella in composted materials that have not been exposed to sufficient heat (Hess et al., 2004) or contain insufficient levels of competing microflora (Hussong et al., 1985). Under that pretext and with the intent of incorporating a large margin of safety, a more suitable criterion for comparison of the effectiveness of composting treatments would be the time for inactivation of high doses (>107 cfu/g) of Salmonella. The bioreactor conditions we determined necessary for complete inactivation of Salmonella (not detectable by enrichment culture) when different C:N formulations were composted are summarized in Table 1. Due to the different pH and temperature profiles encountered with the different C:N formulations, Salmonella inactivation occurred under strikingly different conditions and reflect the involvement of different mechanisms. In 20:1 C:N formulations, complete inactivation of Salmonella occurred at low pH with little heat exposure, whereas in 40:1 C:N formulations, a higher maximum temperature and pH were required. Moreover, the time at which the conditions for complete Salmonella inactivation occurred during composting was statistically much earlier in
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Table 1 Bioreactor conditions for inactivation of Salmonella (not detectable by enrichment culture) during composting of cow manure/straw/cottonseed mixtures formulated to different C:N ratios.A,B C:N
Number of days to achieve complete inactivation of SalmonellaC
Maximum temperature (°C)D
Cumulative metabolic heat exposure (degree days)E
pHF
20 30 40
2.8 a 2.9 ab 3.6 b
44.6 a 52.9 b 56.8 c
2.1 a 12.9 b 31.9 b
5.69 a 8.00 b 9.15 b
A
Initial populations in compost formulations averaged 7.5 log10cfu/g. Values within a column followed by different letters are significantly different (P 6 0.05). C Based on average days for inactivation at each sample location within the bioreactors for all replicate trials. D Maximum temperature encountered during composting when Salmonella was still detected. E Accumulated product of time (days) and temperature (°C above the ambient incubator temperature of 40 °C) for the period when Salmonella could be detected. F pH on first day that pathogen was no longer detected in compost mixture. B
20:1 than in 40:1 C:N formulations. Several advantages to formulating initial compost mixtures to a 20:1 than a 40:1 C:N ratio are apparent. Shorter composting times for pathogen inactivation are better as there would be fewer opportunities for pathogens to be disseminated, such as through seepage water (Knop et al., 1996). More importantly, formulating compost mixtures to create slightly acidic conditions may ensure inactivation of Salmonella under conditions when composting does not generate sufficient heat for that purpose (i.e., at surface of unturned compost piles or under winter conditions when metabolic heat generated is quickly lost to the ambient air). In turn, lower composting temperatures could accelerate the rate of pathogen inactivation by supporting higher populations of antagonistic proteobacteria and fungi (Tang et al., 2007). Before such process conditions are advocated for the composting industry, other zoonotic pathogens associated with manure should be evaluated for their fate during composting of different C:N formulations. It will also be important to evaluate other types of animal manures to determine if similar pathogen inactivation results are obtained with the 20:1 C:N formulations as well as if the responses would be exhibited under field conditions. 4. Conclusions This study sought to determine if the C:N ratio or the presence of ammonium sulfate affected the inactivation of Salmonella spp. within bioreactors containing cow manure-based compost mixtures. Slightly higher temperatures encountered in compost systems supplemented with 0.08% ammonium sulfate did not consistently translate into more rapid rates of pathogen inactivation. In 20:1 C:N compost formulations, Salmonella spp. was inactivated with maximal temperatures less than 50 °C and the cumulative heat exposure required for pathogen inactivation was 15-fold less than required in 40:1 C:N preparations. The slightly acidic pH’s encountered in the 20:1 C:N compost systems may be due to generation of organic acids that in turn contributed to inactivation of the pathogen. Inactivation of Salmonella spp. under these conditions is advantageous as the pathogens can be inactivated at surface sites of static piles where temperatures are typically only slightly higher than ambient. Acknowledgements This research was supported by a USDA-Cooperative State Research, Education, and Extension Service National Integrated Food Safety Initiative Grant (Project 2004-51110-02162).
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M.C. Erickson et al. / Bioresource Technology 100 (2009) 5898–5903 Pell, A.N., 1997. Manure and microbes: public and animal health problem. Journal of Dairy Science 80, 2673–2681. Pereira Neto, J.T., Stentiford, E.I., Mara, D.D., 1987. Comparative survival of pathogenic indicators in windrow and static pile. In: de Bertoldi, M., Ferranti, M.P., L’Hermite, P., Zucconi, F. (Eds.), Compost: Production, Quality, and Use. Elsevier, New York, pp. 276–295. Platz, S., 1978. Survival of pathogenic bacteria and protozoa after short-time composting of poultry manure. In: Kelly, W.R. (Ed.), Animal and Human Health Hazards Associated with the Utilization of Animal Effluent. Office for Official Publications of the European Communities, Luxembourg, Belgium, pp. 209–215. Reinhardt, T., 2002. Organic acids as a decisive limitation to process dynamics during composting of organic matter. In: Insan, H., Riddech, N., Klammer, S. (Eds.), Microbiology of Composting. Springer, New York, pp. 177–188. Rogers, S., Haines, J., 2005. Detecting and Mitigating the Environmental Impact of Fecal Pathogens Originating from Confined Animal Feeding Operations: Review. EPA/600/R-06/021. Available at: (accessed 3.06.08). Rynk, R., 1992. On-farm Composting Handbook. Northeast Regional Agricultural Engineering Service, Ithaca, NY. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Steniford, E.I., 1993. Diversity of composting systems. In: Hoitink, H.A.J., Keener, H.M. (Eds.), Science and Engineering of Compost: Design, Environmental, Microbiological and Utilization Aspects. Renaissance Publications, Worthington, Ohio, pp. 95–110. Sundberg, C., Smårs, S., Jönsson, H., 2004. Low pH as an inhibiting factor in the transition from mesophilic to thermophilic phase in composting. Bioresource Technology 95, 145–150. Tang, J.-C., Shibata, A., Zhou, Q., Katayama, A., 2007. Effect of temperature on reaction rate and microbial community in composting of cattle manure with rice straw. Journal of Bioscience and Bioengineering 104, 321–328. US EPA, 1999. Standards for the Use or Disposal of Sewage Sludge (40 CFR Parts 403 and 503). Revised August 4, 1999. Available at: (accessed 20.05.08). Wang, G., Zhao, T., Doyle, M.P., 1996. Fate of enterohemorrhagic Escherichia coli O157:H7 in bovine feces. Applied and Environmental Microbiology 62, 2567– 2570. You, Y., Rankin, S.C., Aceto, H.W., Benson, C.E., Toth, J.D., Dou, Z., 2006. Survival of Salmonella enterica serovar Newport in manure and manure-amended soils. Applied and Environmental Microbiology 72, 5777–5783. Zhao, T., Doyle, M.P., Shere, J., Garber, L., 1995. Prevalence of enterohemorrhagic Escherichia coli O157:H7 in a survey of dairy herds. Applied and Environmental Microbiology 61, 1290–1293.
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Inactivation of Salmonella spp. in cow manure composts formulated to different initial C:N ratios Marilyn C. Erickson a,*, Jean Liao a, Li Ma a, Xiuping Jiang b, Michael P. Doyle a a b
Center for Food Safety, University of Georgia, Griffin, GA 30223, United States Department of Food Science and Human Nutrition, Clemson University, Clemson, SC, United States
a r t i c l e
i n f o
Article history: Received 6 February 2009 Received in revised form 19 June 2009 Accepted 19 June 2009 Available online 28 July 2009 Keywords: Salmonella Compost Volatile acids pH Thermal inactivation
a b s t r a c t Aerobic composting is a common management practice to inactivate pathogens in manure; however, additional research on the role of compost composition in pathogen inactivation is needed. The objective of this study was therefore to determine the effect of the carbon:nitrogen (C:N) ratio and the presence of ammonium sulfate on inactivation of Salmonella spp. in cow manure-based mixtures composted in a bioreactor under controlled conditions. Compost preparations with an initial C:N ratio of 20:1 required a maximum of 4 days of storage before Salmonellae were inactivated by 7 log10, whereas preparations with C:N ratios of 30:1 and 40:1 C:N required more than 5 and 7 days of storage, respectively. The pH values of both the 20:1 and 30:1 C:N preparations decreased during the onset of composting before increasing to >8. In contrast, pH values of 40:1 C:N preparations increased immediately to >8, generally within the first day of storage. Maximum temperatures observed in 20:1 C:N preparations for inactivation of pathogens were less than 50 °C, and the cumulative heat exposure required for pathogen inactivation in 20:1 C:N preparations was 15-fold less than in 40:1 C:N preparations. Supplementation of compost mixtures with 0.08% ammonium sulfate resulted in slightly higher temperatures; however, these higher temperatures did not translate into more rapid rates of pathogen inactivation. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Animal agriculture has been an important contributor to the United States’ economic well-being; however, it also provides an abundance of manure that must be managed. Estimates indicate that animal waste produced in the US is somewhere between 3 and 20 times greater than human waste with 90% of this waste being generated by cattle and 5% by poultry (Rogers and Haines, 2005). Animal manure is a well-documented source of zoonotic pathogens, such as Escherichia coli O157:H7, Salmonella spp., and Campylobacter spp. (Zhao et al., 1995; Pell, 1997). These pathogens can survive in manure and manure-amended soils for extended periods of time (Wang et al., 1996; Himathongkham and Riemann, 1999; Himathongkham et al., 2000; Jiang et al., 2002; You et al., 2006) and subsequently contaminate vegetables grown in those soils (Natvig et al., 2002; Islam et al., 2004a,b, 2005). Since the number of reported produce-related outbreaks per year doubled between the period of 1973–1987 and 1988–1992, manure as a source of contamination could be an important contributor to this statistic (NACMCF, 1999). Manures may be subjected to aerobic composting to minimize the occurrence of manure as a source of zoonotic pathogens. To prepare compost, nitrogen (N)-rich manures are mixed with one * Corresponding author. Tel.: +1 770 412 4742; fax: +1 770 229 3216. E-mail address: [email protected] (M.C. Erickson). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.06.083
or more carbon (C) amendments to produce a nutrient-rich environment favorable for the metabolism of thermophilic microorganisms. Heat generated from the metabolic activity of these microbes plays a major role in the inactivation of zoonotic pathogens. Hence, the primary process criteria used worldwide for ensuring the microbiological safety of composts have been narrowly defined time–temperature conditions. In the US, process conditions for composting manures is based on EPA’s 503 regulations for composting biosolids and include either a minimum temperature of 55 °C for 3 days in aerated static piles or in-vessel systems, or 55 °C for 15 days in windrow systems (US EPA, 1999). In other countries, there is general agreement that temperatures higher than 55 °C and below 65 °C have the desired effect, although differences exist on the duration for which these temperatures must be achieved (Hogg et al., 2002). Despite these recommended process conditions, a time–temperature approach may not always be sufficient to ensure complete inactivation of pathogens within the entire compost mass. For example, Droffner and Brinton (1995) reported that Salmonella typhimurium Q survived for at least 9 days at 60–70 °C in compost prepared from food biowaste and for at least 5 days in a compost prepared from waste water sludge. Hutchison et al. (2005b) has also reported extended survival of pathogens in field studies of static compost piles. In that study, Salmonella, E. coli O157:H7, and Listeria survived in poultry manure-based compost piles when exposed to
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temperatures above 55 °C for more than 8 days. Similarly, E. coli O157:H7 survived in dairy cow and pig manure compost mixtures when exposed to temperatures above 60 °C for more than 3 and 8 days, respectively. In light of these studies, it is proposed that chemical or biological factors contribute significantly to inactivation variations within these systems. The involvement of factors in pathogen inactivation other than heat is supported by the composting study in which Salmonellae were more readily inactivated when inoculated into the compost than when held in sealed glass ampules within the compost (Platz, 1978). Raw material composition is one chemical parameter that has potential to affect both direct and indirect pathogen inactivation during thermal composting. For example, a review on the thermal resistance of Salmonellae noted that some amino acids and sugars increase heat resistance, whereas other compounds such as cysteine and glutathione decrease heat resistance (Doyle and Mazzotta, 2000). Qualitative and quantitative compositional differences between manures (Lynch, 1987) could be responsible for pathogens surviving longer in beef cattle waste compared to dairy cattle, pig, poultry layer, or sheep waste (Hutchison et al., 2005a). Different exogenous C sources (sawdust versus straw) have also contributed to different patterns of heating (i.e., peak heating temperature and length of peak heating) and could therefore contribute to variations in pathogen inactivation, although the feedstocks appeared not to have a significant effect on the microbial profile of the mature compost (Green et al., 2004). Relative proportions of manure and C amendments in the compost formulations may also affect the metabolic response of indigenous compost microflora and in turn the survival of zoonotic pathogens. Microorganisms use C for both energy and growth, whereas N is essential for protein and reproduction. Raw materials blended to provide a C:N ratio of 25:1 to 30:1 are considered ideal for compost degradation, although initial C:N ratios from 20:1 to 40:1 are considered acceptable (Rynk, 1992). At C:N ratios close to 20:1, however, C not bound in complex carbohydrates is utilized but is insufficient to stabilize all of the N leading to the production of ammonia, a known bactericidal agent in inactivation of S. typhimurium, E. coli O157:H7, and Listeria monocytogenes in manures (Himathongkham and Riemann, 1999; Himathongkham et al., 2000; Park and Diez-Gonzalez, 2003). C:N ratios of 40:1 and above, on the other hand, lead to elevated microbial activities and heat generation during initial stages of composting but decreased microbial activity and heat generation as composting progresses and N becomes limiting. There is only one study reported to date addressing the fate of pathogens or their surrogates in response to different C:N formulations. In that study, fecal streptococci and total coliforms were eliminated in a system having an initial C:N ratio of 32.9, whereas their populations were only partially reduced at a C:N ratio of 67.5 (Lafond et al., 2002). The objective of our study was to determine the influence of C:N on Salmonella spp. inactivation in compost mixtures containing dairy cow manure and initial C:N ratios ranging from 20:1 to 40:1. In addition, ammonium sulfate, an ingredient sometimes added to compost mixtures to stimulate biological activity in early stages of composting, was evaluated for its potential effect on inactivation of Salmonella. Bench-scale composting systems were utilized for these investigations in order to provide control over environmental factors (i.e., air flow, ambient temperatures) that could otherwise introduce variation in treatment responses.
2. Methods 2.1. Preparation of Salmonella strains for experimental trials Three strains of Salmonella enteritidis (ME-18, H4639, and H3353) and one strain of Salmonella newport (11590) were labeled
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with jellyfish green-fluorescent protein (GFP) and an ampicillinresistant marker according to the method described by Sambrook et al. (1989). When viewed under a handheld UV light (365 nm), the resulting transformed colonies emitted bright green fluorescence. Each strain of Salmonella was thawed from a frozen culture and individually streaked onto tryptic soy agar (TSA; Difco Laboratories, Detroit, Mich.) containing 100 lg/ml ampicillin (TSA-A) for incubation at 37 °C for 20–24 h. Individual colonies were subsequently streaked onto a second plate for another incubation at 37 °C for 20–24 h. Individual colonies were selected from these plates and inoculated into 10 ml of tryptic soy broth (Becton Dickinson, Sparks, MD) containing 100 lg/ml of ampicillin (TSB-A). These inoculated broths were incubated at 37 °C for 20–24 h with agitation (150 rpm). The bacteria were harvested by centrifugation (4050g, 15 min, 4 °C) and washed three times in 0.1% peptone water (Difco). The individual isolates were reconstituted with 0.1% peptone water to an optical density at 630 nm of 0.5 (109 cfu/ml). Equal volumes of each Salmonella strain were combined to give one four-strain mixture. Cell populations of the Salmonella mixture were determined by plating on TSA-A. 2.2. Compost ingredients and preparation For each trial, fresh cow manure was collected from a dairy farm near Griffin, Ga., and frozen for at least 24 h to kill insect eggs. Wheat straw, cottonseed meal, and ammonium sulfate were purchased from a local feed store. Manure, straw, and cottonseed meal were analyzed for N, C, and moisture content (MC) prior to their use. The wheat straw was shredded using a Flowtron Leaf Eater (Malden, MA) in order to provide greater homogeneity to the compost mixture and hence better temperature distributions and less heat loss (Gotaas, 1956). GFP-labeled pathogens were sprayed onto the manure (107 cfu/g) held in a 28-l sanitized mixing bowl. The material was mixed thoroughly with a Hobart mixer (Model D320; 3/4 h.p.) prior to adding the other raw ingredients. The proportions of the raw ingredients used in the mixture were adjusted to give initial MC of 60% and C:N ratios in the range of 20:1 to 40:1. Immediately after mixing, the compost mixtures were loaded into the composting apparatus. 2.3. Composting apparatus Two 15-l compost bioreactors were constructed based on a design described by Jiang et al. (2003). The bioreactor body was made up of PVC plastic pipe (48 cm height and 32 cm diameter). The bottom and top of each pipe was tightly fitted with PVC covers. Both covers had holes drilled in the center with the bottom hole serving as a connection to a condensate bottle and the top cover hole connected to the air inlet system. Within the bioreactor body, a perforated PVC rack was placed at a height of 5 cm from the bottom. The compost mixture (4 kg) was placed on this rack and material added to an initial height of 35 cm. Two sampling ports (3 cm diameter each) were drilled into the sides of the bioreactor body at heights of 6–9 and 10–13 cm above the PVC rack. Small holes were also drilled at 6.5 cm above the PVC rack for insertion of type T thermocouple wires into the compost mixture. One wire was inserted 3 cm into the mixture (bottom-edge location) and another wire was inserted 16 cm into the mixture (bottom-center location). In addition, thermocouples were inserted 10 cm into the mixture from the top of the bioreactor at edge and center locations (3 and 16 cm from bioreactor wall, respectively). All wires were connected to a multiple-channel HotMux data logger (DCC Corp., Pennsauken, NJ) and temperatures were recorded at 30-min intervals during the experimental trials. The bioreactors were placed inside a Precision
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30 Mechanical Convection incubator (Thermo Fisher Scientific, Waltham, Ma.) set at 40 °C. This incubator had been modified to vent trapped air to a filtered exhaust system. Compressed air was connected to each bioreactor to give an airflow rate of 155 ml/min. 2.4. Experimental design Ten trials were conducted over a period of 5 months. Each trial consisted of two bioreactors, one of which was formulated with 0.08% ammonium sulfate and the other formulated without ammonium sulfate. Four replicate trials were conducted with compost materials formulated to a C:N ratio of 30:1 whereas three replicate trials each were conducted with compost materials formulated to C:N ratios of 20:1 and 40:1. Compost samples (25 g) were removed in duplicate from the bioreactor on a daily basis over a period of 10 days using an autoclaved grabbing tool (Mechanical Pick-up No. 396, General Tools Mfg., NY, NY) placed inside a sterile tube that had been inserted into the compost at the defined sampling location (bottom-edge, bottom-center, topedge, and top-center). 2.5. Microbiological analyses Sample portions (5 g) were mixed with 45 ml of 0.1% peptone water in a sterile Whirl-PakÒ bag and macerated in a stomacher. Serial dilutions of each sample were then spread onto TSA-A plates and incubated at 37 °C for 24 h for enumeration by direct plate count. At the same time, a selective enrichment method was used that consisted of adding compost sample (5 g) to 45 ml of TSB-A and incubating at 37 °C for 24 h. Portions of these enriched samples were then streaked on TSA-A plates to elucidate the presence or absence of pathogen in the sample (limit of detection: 1 cell/ 5 g). Salmonella identification was confirmed using a Salmonella latex agglutination kit (Oxoid Limited, Hampshire, England). 2.6. Chemical analyses Oxygen levels in the bioreactors were determined with a Demista OT-21 oxygen probe (Arlington Heights, IL) just prior to removal of compost samples for chemical and microbial assays. The pH of compost samples was determined by adding 5 g to 250 ml of deionized water followed by measurement with an Accumet Basic pH meter (Fisher Scientific, Pittsburgh, PA). C content of ingredients and compost mixtures was determined on the basis of ash content obtained at 550 °C. MC was determined by drying a 5 g sample for 24 h in a vacuum oven and weighing. N content of compost mixtures and ingredients was determined by the University of Georgia Soil Testing Laboratory wherein samples were first ground and screened through a 10-mesh sieve before analysis by a macro-Kjeldahl method. 2.7. Statistical analyses Bacterial populations were converted to log10cfu/g before statistical analysis. Data were analyzed by analysis of variance (ANOVA) using the StatGraphics Centurion XV software package (StatPoint, Inc., Herndon, Virg.). When statistical differences were observed (P < 0.05) with ANOVA, differences among sample means were determined using the least significant difference test.
3. Results and discussion 3.1. C:N content of initial compost mixtures For each mixture formulated, a fresh batch of dairy cow manure was collected from the farm, analyzed for MC, C and N contents,
and mixed with wheat straw and cottonseed meal. Due to differences in composition of these manure samples, the amounts of the raw materials used varied slightly for each of the target C:N formulations. Initial C:N ratios of the compost mixtures were within a narrow range of the target values: 19.7 ± 2.6, 29.5 ± 2.1, and 42.0 ± 2.2, for the 20:1, 30:1, and 40:1 C:N formulations, respectively. 3.2. Moisture and oxygen levels during composting All treatments were formulated to an initial value of 60% MC. MC remained above 50% during composting in the bioreactors and thus were not considered a limiting factor for bacterial activity (Golueke, 1994). Similarly, oxygen levels throughout the composting period of evaluation were in a range (10–20%) considered adequate for aerobic composting (Finstein and Hogan, 1993). 3.3. Bioreactor sample location Continuous aeration using a positive pressure system has previously led to stratified temperatures within the bioreactor (Jiang et al., 2003). Four locations were therefore identified as sampling sites (bottom-edge, bottom-center, top-edge, and top-center) as representative of the diversity of conditions within the bioreactor. While edge locations had lower temperatures than center locations (data not shown), these differences were not statistically significant. In addition, sample location was not found to be a significant variable in pathogen inactivation or change in pH levels (data not shown). Hence, sample location data were merged into one data set for evaluation of other study variables. 3.4. Heat generation in response to ammonium sulfate amendment Although not a common amendment, the addition of ammonium sulfate to compost mixtures is recommended when N may not be readily available in the system for microbial activity. To evaluate the response of the systems in the presence and absence of this amendment, the accumulation of metabolic heat within each of the bioreactors was calculated and expressed as cumulative heat exposure (degree days) above the ambient incubator temperature of 40 °C (Fig. 1). In 40:1 C:N formulations, where readily available N may be a limiting factor, a slight increase in heat generation was noted during the first 3 days of composting when ammonium sulfate was added to a level of 0.08%. Differences became negligible after 3 days that is likely attributable to depletion of the ammonium sulfate. In 20:1 and 30:1 C:N formulations, no differences in heat generated during the first 3 days of storage occurred upon addition of ammonium sulfate indicating that N was not a limiting factor during that time. When the generation of heat started to rapidly increase for these formulations, however, cumulative heat exposure was slightly higher in formulations containing ammonium sulfate. Statistically, these differences in heat accumulation did not significantly affect the inactivation of Salmonella in the compost formulations. Hence, the ammonium sulfate data were consolidated for subsequent evaluation of the systems’ responses to C:N ratio. 3.5. Temperature profiles of bioreactor systems in different C:N formulations Within one day of composting, temperatures in the bioreactors had increased to the incubator temperature (40 °C) and above (Fig. 2). In compost preparations formulated to a C:N ratio of 40:1, temperatures increased to near maximum within the first day of composting and began to decline after only 2 days of composting. In 30:1 C:N formulations, temperature increased gradually
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3.6. Inactivation of Salmonella during composting of different C:N formulations
Cumulative Heat Exposure (Degree-Days)
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Temperature (ºC)
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Distinctly different pH profiles were observed during composting of formulations with different initial C:N ratios (Fig. 3). In both the 20:1 and 30:1 C:N formulations, the pH decreased but the reduction was greater and occurred for a longer time in the 20:1 than the 30:1 C:N formulation. The pH of the 40:1 C:N formulations, in contrast, increased during the first 2 days of composting. Accumulation of organic acids such as acetic acid has been associated with decreases in pH values (Reinhardt, 2002) and have also been held responsible for inhibition of thermophilic microorganisms (Sundberg et al., 2004) and delays in temperature development. Typically, formation of organic acids is ascribed to anaerobic conditions and it is possible that anaerobic microenvironments could have occurred in the bioreactors in which the compost formulations were not sufficiently mixed with the straw and cottonseed (Steniford, 1993). Alternatively, Reinhardt (2002) reported that many bacteria produce acetic acid even when grown aerobically in the presence of easily degradable substrates. 3.8. Comparison of bioreactor conditions for Salmonella inactivation
Fig. 2. Temperature profiles of cow manure compost mixtures formulated to different C:N ratios.
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During the first 2 days of composting, C:N formulation significantly affected the rate of Salmonella inactivation with populations in 20:1 and 40:1 C:N formulations being reduced by more than 7 log10 and populations in 30:1 C:N formulations being reduced by only 5 log10. Salmonella could still be detected by enrichment culture in 40:1 C:N formulated samples after 7 days of composting in bioreactors whereas the last day it was detected by enrichment culture in 20:1 and 30:1 C:N formulated samples was days 3 and 5, respectively. Given that heat generation in field conditions would likely be slower than occurred in these optimally controlled bioreactors, the differences in time to inactivation in the field would also likely be magnified with the different C:N formulations. 3.7. pH response during composting of different C:N formulations
Fig. 1. Cumulative heat (>40 °C) generated in dairy cow manure compost mixtures formulated to different C:N ratios with and without 0.08% ammonium sulfate (AS).
20
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4
30:1 5
40:1 6
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Days Fig. 3. pH values of cow manure compost mixtures formulated to different C:N ratios.
from day 1 to day 4 of composting, whereas temperature increases in 20:1 C:N formulations did not occur until after the second day of composting and then increased to temperatures approximating those in 30:1 C:N formulations. In a previous study examining aerobic pig manure static compost piles, C:N formulations of 15:1 also exhibited a slower increase in temperature than piles formulated to a 30:1 C:N ratio (Huang et al., 2004).
Some investigators have suggested that it is unrealistic to ensure pathogen-free compost, but instead the compost should constitute an acceptable risk based on a significant reduction or inactivation of indicator microbes (Pereira Neto et al., 1987). Under that same pretext, Jones and Martin (2003) advocated that the absolute time to extinction of an organism is less important than the rate at which the cell numbers decline because the former is affected by the initial population level. It is our contention, on the other hand, that pathogen-free composts should be the target due to the potential for regrowth of Salmonella in composted materials that have not been exposed to sufficient heat (Hess et al., 2004) or contain insufficient levels of competing microflora (Hussong et al., 1985). Under that pretext and with the intent of incorporating a large margin of safety, a more suitable criterion for comparison of the effectiveness of composting treatments would be the time for inactivation of high doses (>107 cfu/g) of Salmonella. The bioreactor conditions we determined necessary for complete inactivation of Salmonella (not detectable by enrichment culture) when different C:N formulations were composted are summarized in Table 1. Due to the different pH and temperature profiles encountered with the different C:N formulations, Salmonella inactivation occurred under strikingly different conditions and reflect the involvement of different mechanisms. In 20:1 C:N formulations, complete inactivation of Salmonella occurred at low pH with little heat exposure, whereas in 40:1 C:N formulations, a higher maximum temperature and pH were required. Moreover, the time at which the conditions for complete Salmonella inactivation occurred during composting was statistically much earlier in
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Table 1 Bioreactor conditions for inactivation of Salmonella (not detectable by enrichment culture) during composting of cow manure/straw/cottonseed mixtures formulated to different C:N ratios.A,B C:N
Number of days to achieve complete inactivation of SalmonellaC
Maximum temperature (°C)D
Cumulative metabolic heat exposure (degree days)E
pHF
20 30 40
2.8 a 2.9 ab 3.6 b
44.6 a 52.9 b 56.8 c
2.1 a 12.9 b 31.9 b
5.69 a 8.00 b 9.15 b
A
Initial populations in compost formulations averaged 7.5 log10cfu/g. Values within a column followed by different letters are significantly different (P 6 0.05). C Based on average days for inactivation at each sample location within the bioreactors for all replicate trials. D Maximum temperature encountered during composting when Salmonella was still detected. E Accumulated product of time (days) and temperature (°C above the ambient incubator temperature of 40 °C) for the period when Salmonella could be detected. F pH on first day that pathogen was no longer detected in compost mixture. B
20:1 than in 40:1 C:N formulations. Several advantages to formulating initial compost mixtures to a 20:1 than a 40:1 C:N ratio are apparent. Shorter composting times for pathogen inactivation are better as there would be fewer opportunities for pathogens to be disseminated, such as through seepage water (Knop et al., 1996). More importantly, formulating compost mixtures to create slightly acidic conditions may ensure inactivation of Salmonella under conditions when composting does not generate sufficient heat for that purpose (i.e., at surface of unturned compost piles or under winter conditions when metabolic heat generated is quickly lost to the ambient air). In turn, lower composting temperatures could accelerate the rate of pathogen inactivation by supporting higher populations of antagonistic proteobacteria and fungi (Tang et al., 2007). Before such process conditions are advocated for the composting industry, other zoonotic pathogens associated with manure should be evaluated for their fate during composting of different C:N formulations. It will also be important to evaluate other types of animal manures to determine if similar pathogen inactivation results are obtained with the 20:1 C:N formulations as well as if the responses would be exhibited under field conditions. 4. Conclusions This study sought to determine if the C:N ratio or the presence of ammonium sulfate affected the inactivation of Salmonella spp. within bioreactors containing cow manure-based compost mixtures. Slightly higher temperatures encountered in compost systems supplemented with 0.08% ammonium sulfate did not consistently translate into more rapid rates of pathogen inactivation. In 20:1 C:N compost formulations, Salmonella spp. was inactivated with maximal temperatures less than 50 °C and the cumulative heat exposure required for pathogen inactivation was 15-fold less than required in 40:1 C:N preparations. The slightly acidic pH’s encountered in the 20:1 C:N compost systems may be due to generation of organic acids that in turn contributed to inactivation of the pathogen. Inactivation of Salmonella spp. under these conditions is advantageous as the pathogens can be inactivated at surface sites of static piles where temperatures are typically only slightly higher than ambient. Acknowledgements This research was supported by a USDA-Cooperative State Research, Education, and Extension Service National Integrated Food Safety Initiative Grant (Project 2004-51110-02162).
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