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Agriculture and Agri-Food Canada, Research Centre, 5403 1st Ave. S., Lethbridge, Alberta, Canada T1J 4B1
* Corresponding author (larney{at}agr.gc.ca)
Received for publication September 10, 2002.
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: CFU, colony forming units • MDL, minimum detection level • TAH, total aerobic heterotrophs • TC, total coliforms
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Escherichia coli O157:H7 is one of the many strains of the bacterium E. coli. In Canada, E. coli O157:H7–contaminated water caused seven deaths and made more than 2000 people ill in Walkerton, Ontario in May 2000. The outbreak was linked to contamination of the town's water supply from land application of livestock manure on a nearby farm (O'Connor, 2002). Even land application of stockpiled manure poses a threat, since Kudva et al. (1998) reported that E. coli O157:H7 survived for more than one year in a nonaerated ovine manure pile that was exposed to environmental conditions.
A rapid increase in the number of intensive livestock operations in Alberta over the past decade has compelled many farmers to seek alternative methods to direct land application of raw manure. By 2002, the adoption of manure composting had increased to at least 12 feedlots in Lethbridge County, in southern Alberta. Composting allows transportation of nutrients (especially nitrogen and phosphorus) from high nutrient-loading areas and also reduces odor complaints at land application (Rynk, 1992). It eliminates the parasitic protozoa Giardia and Cryptosporidium (McAllister, unpublished data, 1999) and reduces weed seed viability (Larney and Blackshaw, 2003). The principal mode of disinfection during composting is based on time–temperature relationships that destroy pathogens, although antagonistic microorganisms and ammonia may also play a role (Epstein, 1997; Himathongkham and Riemann, 1999). For pathogen elimination during windrow composting of biosolids, temperatures should be maintained at >55°C for 15 d or longer (USEPA, 1992). During this period, the windrow should be turned a minimum of five times. Similar guidelines exist in Canada, which also state that final composts should contain <log10 3 cells g-1 dry wt. of fecal coliforms (Canadian Council of Ministers of the Environment, 1996).
However, even though the elimination of pathogens by composting has been well documented (Déportes et al., 1998; Krogmann et al., 2002; Tiquia et al., 2002), composting regimes (time, temperature) required to achieve elimination of TC, E. coli, or other pathogens vary widely. Turner (2002) demonstrated inactivation of E. coli in farmyard manure, pig (Sus scrofa) feces, and cereal straw after only 2 h at 55°C. In contrast, Schleiff and Dorn (1997) reported that E. coli could be cultured from dry poultry (Gallus gallus domesticus) manure after 88 d of composting. Pathogen reduction with increasing time was shown by Himathongkham and Riemann (1999), who reported that while E. coli O157:H7 was able to grow for 2 d in fresh chicken manure at 20°C, with a resulting 1 to 2 log10 unit increase in colony forming units (CFU), increasing storage time to 6 d decreased populations by 3 to 4 log10 units. The effect of higher temperatures on reducing the time required for pathogen reduction was illustrated by Himathongkham et al. (1999), who reported a 105–fold reduction in E. coli O157:H7 after 105 d at 4°C or 45 d at 37°C in a laboratory incubation with cattle manure. Also, Droffner and Brinton (1995) found that E. coli survived for 59 d at 60°C, compared with only 9 d at 60 to 70°C in an inoculated laboratory food waste compost.
The mechanism for removal of pathogens during aerobic composting may not simply be a result of the thermal environment. Turner (2002) indicated that pathogen inactivation was not merely dependent on temperature but was also affected by water content and nature of the substrate. If incomplete inactivation occurred due to low temperatures, recovery and regrowth of the damaged pathogenic populations may be possible. In a study on composted dairy waste solids as recycled bedding, Mote et al. (1988) found that even though there was an initial decline or even a disappearance of TC due to composting, the bacteria reestablished in large numbers without reinocculation. Droffner et al. (1995) presented evidence for the survival of E. coli in active compost that indicated that, although classified as a mesophile, it had a mechanism for survival and perhaps replication at elevated temperatures (>60°C).
Although pathogen elimination is a recognized benefit of composting, the lack of definitive relationships between elimination and composting duration, substrates, and temperature conditions prompted a study on the fate of coliform bacteria during open-air windrow composting of beef feedlot manure in southern Alberta. Since there has been a recent increase in the use of byproducts from Alberta's forest industry (wood chips, shavings, sawdust) as alternative bedding to traditional cereal straw for feedlot cattle (McAllister et al., 1998), we compared manure from feedlot pens bedded with straw or wood chips. There is some evidence to suggest that wood products contain antimicrobial compounds that may inhibit bacterial levels (Allison and Anderson, 1951; Kudva et al., 1998).
This is the first study of its kind in the intensive feedlot area of southern Alberta, where manure management practices are coming under increased public and media scrutiny. It sought to quantify the duration of composting and accompanying windrow temperatures required to achieve coliform population reductions under typical field conditions.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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In 1998, the windrows were turned 16 times (Days 3, 7, 10, 14, 17, 21, 24, 28, 31, 38, 45, 52, 66, 73, 94, and 108 of composting) with a tractor-pull EarthSaver windrow turner (Fuel Harvesters Equipment, Midland, TX). This turning frequency was higher than at most commercial feedlots in southern Alberta (approximately five to seven times over a 90-d period). In 1999, the windrows were turned seven times (Days 7, 14, 21, 29, 42, 56, and 70), which was closer to the turning frequency at commercial feedlots. Turning was more frequent in the early stages of composting to stimulate thermophilic (>40°C) microbial activity and became less so as composting progressed and microbial activity diminished. After the active composting phase (turning) the material entered a "curing" phase (no turning) for a further 90 to 100 d until windrow temperature approached ambient.
Compost Sampling
Compost sampling for bacterial enumeration coincided with turning and sampling for chemical and physical properties (Larney et al., 2001). In 1998, windrows were sampled eight times: at establishment (Day 0) and just before turning on Days 7, 14, 21, 28, 45, 66, and 94. There were nine sampling times in 1999: at Day 0 and just before turning on Days 7, 14, 21, 28, 42, 56, and 70, as well as Day 91 (no turning event).
In 1998, the sampling protocol involved cutting each of the compost windrows in three places (east, center, and west of windrow) with a skid-steer loader to expose six vertical faces. Three of the faces (one each from east, center, and west) were sampled at three vertical locations (top, middle, bottom). The vertical location samples from each face were composited to give three subsamples (east, west, center) per windrow. Each sampling time had a total of 12 samples (two bedding types x two replicates x three horizontal locations). In 1999, the sampling protocol varied in that the vertical locations were kept separate. Two faces exposed in the center of the windrow were sampled at three vertical locations (top, middle, bottom). The vertical location samples from each face were then composited to give three samples (top, middle, bottom). Each sampling time had a total of 12 samples (two bedding types x two replicates x three vertical locations).
Bacterial Enumeration
Total coliforms, E. coli, and total aerobic heterotrophs (TAH) were enumerated in our study. Total coliform bacteria are excreted in high numbers in animal and human feces, and are often used as indicators of fecal contamination in water and food, even though not all TC bacteria are of fecal origin. We did not use TC data as an indicator of fecal contamination, since clearly, fecal contamination is inapplicable when working with cattle manure. The TC data were used to examine persistence and potential regrowth during the composting process, since the behavior of pathogenic groups may be better represented by TC than by more specific E. coli data. We feel that following TC populations in a known experimental sample (manure) as it changes over time (to compost) gives relevant data on environmental fate. Escherichia coli is a member of the fecal coliform group, a subset of the TC group, and its presence can be used as a surrogate for specific pathogenic E. coli strains. Ogden et al. (2001) reported that since the die-off rate of E. coli O157 was the same as that of the commensal E. coli population in a laboratory study, the field behavior of E. coli O157 in a cattle slurry application study could be approximated by monitoring the population of E. coli. Total aerobic heterotroph levels indicate the overall magnitude of indigenous microbial activity in decomposing organic matter.
Fresh compost samples (10 g wet wt.) were added to 90 mL of sodium phosphate buffer (pH 6.5, 0.05 M) and mixed in a Stomacher blender (Model 400; Seward Medical, Mississauga, ON, Canada) for 2 min. The suspension was serially diluted to the appropriate levels in sodium phosphate buffer. The dilutions were inoculated (1 mL) into triplicate Fluorocult LMX broth tubes (9 mL; Merck KGaA, Darmstadt, Germany) and incubated aerobically at 37°C for enumeration of TC and E. coli by the most probable number (MPN) method. Total coliforms were enumerated after 48 h as those tubes with hydrolysis of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal). Escherichia coli was enumerated after 24 and 48 h as those tubes with hydrolysis of both X-Gal and 4-methylumbelliferyl-ß-D-glucuronide (MUG). Selected colonies of presumptive TC and E. coli were isolated from positive tubes on Fluorocult LMX agar plates for identity confirmation using membrane fatty acid composition, cellular morphology, and biochemical characteristics (Smibert and Krieg, 1994; Paisley 1996; Garthwright, 1998). Samples positive for coliforms were confirmed positive by isolation in 96.7% of the cases. In a few cases coliform bacteria were not isolated, while enterococci were. The data were not corrected for these false positive samples since the TC results would change only marginally. All samples positive for E. coli were confirmed positive by pure culture isolation and identification.
The TC and E. coli enumerations are presented as log10 cells g-1 dry wt. Water content of compost subsamples (oven-drying at 60°C for 48 h) was used to express values on a dry weight basis. For both enumerations, the minimum detection level (MDL) was log10 0.56 cells g-1 wet wt. We assumed an average compost water content of 0.5 kg kg-1 to maintain a constant MDL across all sampling dates and present data on a dry weight basis. The MDL of log10 0.56 cells g-1 wet wt. is equivalent to log10 0.86 cells g-1 dry wt. at a compost water content of 0.5 kg kg-1. For determining treatment means, values < MDL were assigned a value of 50% of the MDL (after converting to dry weight), which explains the presence of data points < MDL in the figures presented.
The serial dilutions were also spread-plated (100 µL) in triplicate onto tryptic soy agar plates for enumeration of TAH (as log10 CFU g-1 dry wt.) after incubation at 39°C for 48 h.
Compost Temperature and Water Content
Thermocouples and a datalogger (Sciemetric, Nepean, ON, Canada) were installed as soon as the windrows were formed. They were removed just before each turning and reinstalled immediately after turning. Temperatures were logged every 20 min and averaged to give mean daily values. In 1998, three thermocouples (bottom, middle, top) were attached to each of three stainless steel pipes. The pipes were then placed vertically in one replicate of each bedding treatment near the center of the windrow, and about 2 m from the east and west ends. The bottom location was approximately 0.3 m, the center location approximately 0.6 m, and the top location approximately 0.9 m above ground level. Datalogger malfunction resulted in missing temperature data for Days 13 and 14, 30 through 33, and 50. In 1999, temperatures were measured at three vertical locations (as in 1998: bottom, middle, and top) in three windrow positions: the east and west ends of the one replicate of each manure bedding treatment (approximately 25% and 75% along its length), and the center of the other replicate of each treatment. These nine temperatures were averaged to give the bedding treatment means. The bedding x vertical location (n = 3) means were also calculated to examine the location effect.
Although water content was determined on the bacterial compost samples (to convert enumeration data to a dry weight basis), larger compost samples (approximately 0.6 kg) were taken to track water content (after oven-drying at 60°C for 48 h) during composting. In 1998, the six vertical faces were sampled at three (top, middle, and bottom) locations, giving 18 samples per replicate. In 1999, samples were taken at the top, middle, and bottom locations of the two exposed vertical faces giving six samples per replicate.
Statistical Analyses
In 1998 and 1999, the effect of bedding on TC, E. coli, and TAH populations was examined using the general linear models procedure (SAS Institute, 1990). Additionally, in 1999, the vertical location effect (bottom, middle, top) was examined as a subtreatment in a split-plot design with bedding as the main treatment.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The regrowth of TC between Day 45 and Day 94 in 1998 agrees with the findings of Hassen et al. (2001), who reported a phase of resurgent growth of fecal coliforms after 9 wk of MSW composting. They attributed this secondary growth to recontamination or redistribution during windrow turning. Krogmann et al. (2002) found that after an initial population decrease in fecal streptococci (Enterococcus spp.), a slight increase (approximately 10-fold) was observed in later composting stages of horse (Equus caballus) manure. Their explanation of this included (i) normal data variability, (ii) pile contamination by turning with dirty equipment or by vermin between sampling dates, or (iii) recontamination of sanitized compost by unsanitized material from the outer mesophilic area of the pile during turning. Any, or all, of these reasons may explain regrowth in our study.
The time–temperature profiles in our study were typical for composting of organic materials and similar to those reported for open-windrow composting of feedlot manure in Nebraska (Eghball et al., 1997). There was an early rapid rise to thermophilic conditions, slight temperature decreases followed by partial recovery associated with each turning event, and then a gradual cooling of the windrows toward the end of active composting. However, there was little benefit of prolonged exposure above the recognized thermal kill limit of 55°C, as most TC and E. coli were eliminated in the first 7 d of composting before attaining 55°C. Additionally, the higher residual levels of TC (Fig. 1) observed toward the end of composting in 1998 compared with 1999 were not due to cooler conditions, as there were warmer temperatures in 1998 compared with 1999 (Fig. 5).
Since most of the coliform population decline occurred in the first 7 d of composting, temperature conditions during this period were examined more closely (Table 2) . In 1998, the average temperature was 39.5°C in the straw-bedded and 41.5°C in the wood chip–bedded compost during the first 7 d. Maximum temperatures were 50.1°C for straw-bedded and 53.5°C for wood chip–bedded compost. In 1999, average temperatures for the first 7 d were 35°C for straw-bedded and 33.5°C for wood chip–bedded and the maxima were 40.7°C for straw-bedded and 38.5°C for wood chip–bedded compost (Table 2). The coolest temperature regime in either year was at the bottom windrow location in 1999, which had an average temperature of 30.2°C and a maximum temperature of 35.8°C during the first 7 d. This regime led to a decrease in TC from log10 7.08 to log10 3.66 cells g-1 (dry wt.) and a decrease in E. coli from log10 6.7 to log10 3.41 cells g-1. Hassen et al. (2001) reported a decrease from log10 7.40 cells g-1 (dry wt.) to log10 3.90 cells g-1 in municipal solid waste compost at 55 to 60°C over 15 wk. Our E. coli levels declined a similar magnitude at much lower temperatures in just 7 d. Our findings were closer to those of Lung et al. (2001), who found that E. coli O157:H7 at levels of log10 7.0 CFU g-1 in raw cow manure was not detected after 72 h of composting at 45°C.
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Lack of nutrients caused by high populations of indigenous microorganisms in manure or the production of compounds detrimental to coliforms may also play a role in the decline of pathogens during composting (Himathongkham et al., 1999). Pietronave et al. (2002) demonstrated that indigenous microflora suppressed seeded E. coli growth in a nonsterilized finished compost while E. coli grew rapidly in sterilized compost. Sidhu et al. (2001) reported an antagonistic effect of indigenous microorganisms on Salmonella in composted biosolids. However, the antagonistic effect declined with duration of storage and hence long-term storage was not recommended. In our study, the maintenance of microbial activity and competition for nutrients by TAH throughout the composting period may have played a role in suppressing TC and E. coli. However, since the significant bedding effects on TAH populations did not translate into significant effects on TC or E. coli levels, the role of this inactivation mechanism may have been small.
Another mechanism of pathogen reduction is pH change. Ugwuanyi et al. (1999) found that E. coli was more sensitive to elimination at pH 7 than pH 8 during aerobic digestion at 55°C. Our straw-bedded manure had a pH 8.1, while wood chip–bedded manure had a pH 7.3 on Day 0 in 1998 (Larney et al., 2001). The pH values decreased to 7.3 on Day 14 for the straw-bedded manure and pH 6.8 on Day 14 for the wood chip–bedded manure, which may have enhanced the pathogen reduction effect.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Total coliforms and E. coli were not significantly affected by bedding type (straw vs. wood chips) at any time. Total coliforms were detectable until Day 94 in 1998 (log10 1.69 cells g-1 dry wt.). Although this level is well below established guidelines (<log10 3.0 cells g-1 dry wt. of fecal coliforms [Canadian Council of Ministers of the Environment, 1996]), it shows that there may be some potential for regrowth of coliforms at the later stages of composting. Escherichia coli was <MDL after Day 45 in 1998 and after Day 7 in 1999. Dessication probably played a minor role in coliform elimination, since water loss was low (<0.07 kg kg-1) in the first 7 d of composting when most of the elimination occurred. Total aerobic heterotroph populations remained high (>7.0 log10 CFU g-1 dry wt.) throughout the composting period and their competition for nutrients may have caused an antagonistic effect on pathogens.
In reducing coliform populations by >99.9%, land application of compost instead of raw manure should significantly reduce the risk of water quality degradation in areas of intensive livestock production, like southern Alberta. Although pathogen reduction is a recognized benefit of composting, our quantification of its magnitude, as well as the duration and temperature regimes required for typical feedlot manures under southern Alberta conditions, adds to our knowledge base on composting as a manure management alternative in the region.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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REFERENCES |
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