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a Agriculture and Agri-Food Canada, P.O. Box 3000, Lethbridge, AB, Canada T1J 4B1
b Alberta Agriculture, Food and Rural Development, Lethbridge, AB, Canada T1J 4C7
c Department of Biological Sciences, 4401 University Drive, University of Lethbridge, Lethbridge, AB, Canada T1K 3M4
d Department of Renewable Resources, General Services Building, University of Alberta, Edmonton, AB, Canada T6G 2H1
e Chinook Regional Health Authority, 960 19th St. S., Lethbridge, AB, Canada T1J 1W5
* Corresponding author (millerjj{at}agr.gc.ca).
Received for publication September 17, 2002.
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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0.05) affected by season (SO4, Na, Mg, K, Ca, sodium adsorption ratio [SAR], total C, NO3–N, NH4–N, total P, and available P) than by bedding (K, pH, total C, C to N ratio, NH4–N, and available P). Bedding had no significant (P > 0.05) effect on the four bacterial groups whereas season affected all four groups. Numbers of E. coli and total coliforms (TC) were significantly higher by 1.72 to 2.02 log10 units in the summer than the other three seasons, which was consistent with a strong positive correlation of E. coli and TC with air temperature. The low ratio of bedding to manure in the pens was probably the major cause of the lack of significant bedding effects. Bedding material and seasonal timing of cleaning feedlot pens and land application of manure may be a potential tool to manage nutrients, soluble salts, and pathogens in manure.
Abbreviations: EC, electrical conductivity • SAR, sodium adsorption ratio • TAH, total aerobic heterotrophs • TC, total coliforms
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Most feedlots in Alberta are bedded with barley straw, but increasing numbers of feedlots are using wood-chip bedding in their operations. Wood chips generally have a higher carbon content, lower N and P values, greater acidity, and lower amounts of soluble salts than straw (Allison and Anderson, 1951). Although the higher C to N ratios of softwoods (>400:1) compared with straw (100:1) suggest a greater potential for N immobilization and nitrate depression under wood chips (Brady, 1974; Sommerfeldt and MacKay, 1987), this effect may be less pronounced because wood has a higher lignin content and therefore decomposes more slowly (Bollen and Lu, 1957). The chemical differences in straw and wood-chip bedding suggest there is a potential for differences in feedlot manure chemistry when these two bedding materials are used.
Wood, particularly the bark, contains many different organic chemicals such as phenols, organic acids, tars, tannins, ethyl alcohol, resins, and terpentine (Goldstein, 1982). These organic chemicals in wood have the potential to be natural antibacterial inhibitors (Allison and Anderson, 1951). Research has reported evidence for (Gibbs and Werkman, 1922; Nimenya et al., 2000) and against (Gibbs and Batchelor, 1927; Allison and Anderson, 1951) inhibition of bacteria by wood in different media (soil, manure, and urine). Allison and Anderson (1951) reported that when these antibacterial compounds are present in wood at typical concentrations, they have minimal toxicity on bacteria in soil. Further, the latter authors suggested that any toxicity that may exist initially disappears within a few weeks, because the toxic substances are destroyed by soil bacteria and fungi. Kudva et al. (1998) reported that small amounts of wood-chip bedding in cattle manure may have contributed to shorter survival times for Escherichia coli O157:H7. Nimenya et al. (2000) found that spruce sawdust inhibited urease-producing bacteria from converting urea to NH4 in dairy-cattle urine, and attributed the inhibitory effect to the tar contents of the wood. We are unaware of any studies that have determined E. coli or total coliform populations in beef-cattle manure with straw versus wood-chip bedding.
It is important to examine the effects of season on manure properties since feedlot operators may apply manure at different times of the year. Current guidelines in Alberta allow manure application at any time of the year (Province of Alberta, 2001), but most feedlot operators apply manure in the late summer or fall after silage harvest. Since many of the factors (diet, age, and condition of animal, water consumption, climate, etc.) that affect manure are time-dependent throughout the year, it seems reasonable to conclude that season may have a significant effect on the properties of feedlot-pen manure. We are unaware of any studies that have examined the effect of season on the chemical properties of feedlot manure; and few studies have examined the effect of season on the bacterial properties of feedlot manure (Rhodes and Hrubant, 1972). The latter authors found that microbial flora in feedlot manure were relatively constant throughout the year, although absolute numbers varied somewhat with seasonal conditions. Numbers of total coliform bacteria ranged between 106 and 108 colony forming units (CFU) g-1 (dry wt.) throughout the year.
The objective of this study was to determine the effect of bedding material and season on the chemical and bacterial properties of feedlot-pen manure in southern Alberta.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Chemical Analyses
All manure samples were oven-dried at 60°C for seven days, ground to pass a 2-mm sieve, and then extracted for chemical analyses. The exceptions were nitrate and ammonium, where extractions were conducted on fresh manure samples within two hours after sampling. Nitrate and ammonium in the manure were extracted using a 1:20 ratio of 10 g of manure and 200 mL of 2 M KCL after shaking at low speed for one hour. Ammonium N was determined using the Berthelot reaction on the autoanalyzer (Technicon Industrial Systems, 1973). Nitrate N was determined on the autoanalyzer using the copper-cadmium method (Technicon Industrial Systems, 1978). Electrical conductivity (EC), pH, and soluble salts (SO4, Cl, Na, Mg, K, and Ca) in manure were determined on 1:5 manure and water extracts (20 g manure and 100 mL distilled water) after shaking at low speed for one hour. Soluble Ca and Mg were analyzed using atomic absorption spectroscopy, and Na and K were determined using flame emission spectroscopy (Model AA5; PerkinElmer, Wellesley, MA) (Wright and Stuczynski, 1996). Soluble Cl was determined on the autoanalyzer using the mercuric thiocyanate method (Technicon Industrial Systems, 1974a). Sulfate was analyzed on the autoanalyzer using the barium chloride method (Technicon Industrial Systems, 1972). Available phosphorus (ortho-P) was extracted using a 1:25 ratio of 1 g of manure and 25 mL of Kelowna extract (Van Lierop, 1988) after shaking at low speed for one hour. Samples for total P, C, and N analyses were also finely ground to pass a 150-µm sieve. Total P was determined by a wet-oxidation procedure (Parkinson and Allen, 1975), and ortho-P was analyzed on the autoanalyzer using the ascorbic acid method (Technicon Industrial Systems, 1974b). Total C and N were determined using the Dumas automated combustion technique (McGill and Fiqueiredo, 1993) using a CNS analyzer (Carla Erba, Milan, Italy).
Barley straw and wood-chip bedding were oven-dried at 55°C for five days and then ground to pass a 2-mm sieve. Soluble anions, cations, EC, and pH of bedding were analyzed in this study, whereas C, N, and P analyses of the same bedding were taken from Larney et al. (2001). Inorganic N (NH4–N and NO3–N) and available P were analyzed using the same methods as described for manure. Soluble salts (Ca, Mg, Na, K, SO4, and Cl), EC, and pH were determined on 1:10 water extracts after shaking for one hour. Subsamples were further fine-ground to pass a 1-mm sieve for analyses of total N, C, and P using methods as described for manure. Total phenol content of the bedding material was determined by manual distillation using acidified deionized water, online distillation to concentrate the phenol in solution, followed by analysis of total phenolics using the 4-aminoantipyrine (4-AAP) colorimetric method (USEPA, 1979). All chemical properties are reported on a dry weight basis.
Bacterial Analyses
Manure samples were analyzed for total aerobic heterotrophs (TAH) at 27 and 39°C, total coliforms (TC), and Escherichia coli (E. coli). Manure samples were transported to the laboratory within 1 to 2 h and then plated within four hours. Manure was weighed out in 10-g samples and added to 90 mL of sodium phosphate buffer (pH 6.5, 0.05 M). The samples were then blended for 2 min at a medium setting in a stomacher blender and were then serially diluted to the appropriate levels in sodium phosphate buffer. The dilutions were spread-plated (100 µL) in triplicate onto Fluorcult LMX (Merck, Darmstadt, Germany) plates for presumptive enumeration of total coliform and E. coli and onto tryptic soy agar plates for enumeration of TAH. The Fluorocult LMX plates were incubated aerobically at 37°C for 48 h with presumptive E. coli enumerated after 24 h as those colonies with the ability to hydrolyze both 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-GAL) and 4-methylumbelliferly-ß-D-glucuronide (MUG). Presumptive TC were enumerated after 48 h as those colonies hydrolyzing X-GAL. The TAH were incubated at 27 and 39°C and enumerated after 48 h. Bacteria numbers on a wet weight basis were converted to a dry weight basis using the water content of the manure. The results are presented as colony forming units (CFU) per gram dry weight of manure with the standard deviation given for the replicate samples. Selected colonies of presumptive TC and E. coli were isolated for confirmation of identity through membrane fatty acid composition (Paisley, 1996), cellular morphology, and biochemical characteristics (Smibert and Kreig, 1994; Garthwright, 1998).
Measurement of the E. coli populations is the best indicator of the fecal contamination of both water (Edberg et al., 2000) and foods (Dogan et al., 2002). Since total commensal E. coli is also an accurate indicator of E. coli O157:H7 environmental persistence (Ogden et al., 2001), we measured the total E. coli population. In addition, since E. coli is the most numerous coliform in cattle manure, the TC population was determined as a check on the E. coli results, as well as to allow comparison with other studies. The TAH population was measured to give an indication of the magnitude of overall microbial activity in decomposing organic matter. The TAH incubated at 27°C are more indicative of environmental isolates whereas incubation at 39°C is more indicative of intestinal isolates.
Statistical Analyses
The chemical and bacteria data were analyzed using SAS-PC (SAS Institute, 1989) and a mixed model analyses (Littell et al., 1998). The main treatment effects of bedding and season were assessed for the chemical and bacterial parameters. A repeated statement with season was used when season was a factor in the model. Bacterial numbers were transformed to log10 values before statistical analyses. The seasons were defined based on equinoxes and solstices. The start of spring was 20 March (1998, 1999, 2000) and the start of summer was 20 (2000) or 21 June (1998, 1999). The start of fall was 22 (1998, 2000) or 23 September (1999) and the start of winter was 21 (1998, 2000) or 22 December (1999).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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0.05) affected by season (SO4, Na, Mg, K, Ca, SAR, total C, NO3–N, NH4–N, total P, and available P) than by bedding (K, pH, total C, C to N ratio, NH4–N, and available P) (Tables 2 and 3). For the bedding effects, least-squares mean (LSM) values for K were 26% higher for pen manure with straw (5.9 g kg-1) than with wood chips (4.7 g kg-1). The mean K content of straw was 13 times that of wood chips (Table 1). The pH was 7% lower for pen-manure with wood than straw (Table 2), which was consistent with a lower pH value of wood (Table 1). Total C and the C to N ratio were 9 and 13% higher, respectively, for manure with wood chips than with straw (Table 3). This was consistent with higher total C values and C to N ratios for wood (Table 1). Even though straw bedding had 44 times more NH4–N than wood chips (Table 1), the NH4–N content of manure with wood chips was 133% higher than with straw (Table 3). This may be related to the lower pH of manure with wood chips than with straw (Table 1). In comparison, Larney et al. (2001) reported no significant difference in the NH4–N content of pen-floor manure with straw or wood chips from this same feedlot. In contrast, Nimenya et al. (2000) found that spruce sawdust inhibited NH4 production from urea in cattle urine, and this was attributed to higher tar contents in the wood. They also proposed that the NH4 already produced could be absorbed by wheat-straw bedding. Available P in pen manure was 35% higher with wood than straw bedding (Table 3), even though available P was six times higher for straw than wood bedding (Table 1). The lower available P in pen manure with straw may be related to the higher Ca content of the straw, which could cause increased phosphate precipitation. Larney et al. (2001) also reported higher available P in pen manure with wood (2.1 g kg-1) than with straw (1.7 g kg-1). In contrast, SO4, Cl, Na, Mg, Ca, EC, SAR, total N, NO3–N, and total P were not significantly affected by bedding (Tables 2 and 3), even though values for these parameters were generally higher for straw than wood bedding (Table 1).
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For the seasonal effects, sulfate in the summer was lower by 50 to 93% than in the other three seasons; and Na in the spring was higher by 22 to 157% than in the other three seasons (Table 2). Magnesium in the fall and winter was higher by 33 to 55% than in the spring and summer; and K in the fall was higher by 32 to 67% than in the other three seasons (Table 2).
Calcium in the spring was lower by 75 to 175% than in the other three seasons. The SAR in the spring was higher by 67 to 231% than in the other three seasons, indicating a higher potential risk of soil sodicity at this time. In contrast, there was no significant seasonal effect on total salt content or EC (Table 2). Increasing salinity and sodicity of soils is a serious concern with long-term application of beef cattle manure (Chang et al., 1991). Although our results indicated that bedding cannot be used to manage the EC and SAR of the pen manure, season has potential as a tool to manage the SAR values of the manure. Total C in the winter was higher by 15% than in the spring and summer (Table 3), which may be related to more bedding used in winter. Nitrate N was lower in the spring by 114 to 269% than in the other three seasons, and NH4–N in the fall and winter were higher by 57 to 186% than in the spring and summer. Total P in the summer was higher by 20 to 71% than in the other three seasons, and available P in the summer was higher by 54 to 153% than in the other three seasons. In contrast, Cl, EC, pH, total N, and C to N values were not affected by season (Tables 2 and 3).
We believe that the significant seasonal differences in certain chemical properties may be related to a number of complex factors that have the potential to vary throughout the year. These may include the age, condition, and size of the cattle, whether cattle were present or absent in the pens, changes in the manure pack (depth and water content), amount of bedding used, water consumed, and climatic conditions. For example, we found a significant negative correlation (r = -0.70, P = 0.0004, n = 21) between mean daily air temperature and NH4–N content of the pen-floor manure, which may be related to greater volatilization losses of NH3 during the warmer summer season.
Bacterial Properties of Pen-Floor Manure
Throughout the study, 150 of 150 presumptive E. coli isolates were confirmed as E. coli. Of 158 presumptive coliform isolates, 136 were confirmed as coliform but not E. coli; the other 22 isolates were confirmed as E. coli when put back onto LMX plates and retested. Bedding had no significant (P > 0.05) effect on any of the four bacterial groups, but season had a significant effect on all groups (Table 4). The populations of E. coli and TC in the summer were higher by 1.72 to 3.37 log10 units than in the other three seasons (Table 4). In comparison, TAH at 27°C in the spring were higher by 0.36 to 0.83 log10 units than in the fall and winter; and TAH at 39°C in the summer were higher by 0.53 to 1.23 log10 units than in the other three seasons (Table 4). Overall, seasonal fluctuations in bacterial populations were much wider for E. coli and TC than for the TAH populations, indicating a greater seasonal effect on coliform bacteria than the overall bacterial populations. Rhodes and Hrubant (1972) found that the highest numbers of TC in cattle manure of a feedlot in Illinois, USA occurred in July and November, and the lowest counts were in January when air temperatures were coldest. Stoddard et al. (1998) also reported that fecal coliforms in soils (Kentucky, USA) declined most rapidly after fall application of manure, and attributed this to onset of freezing conditions.
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We expected that the bacteria levels would be lower in pens bedded with wood chips because of the greater antimicrobial properties of wood compared with straw. This trend may not have occurred because of the low absolute concentrations of total phenols produced by the bedding material, the high ratio (5:1 straw, 4:1 wood chips) of manure to bedding, and the high quantities of organic material. The barley straw, wood sawdust, and bark peelings contained 1.30, 1.28, and 3.76 mg kg-1 (dry-matter basis) total phenols, respectively. Because of the high ratio of manure to bedding, the concentration of bacteria may have been so high that the low concentrations of organic acids produced by the wood may not have had an antimicrobial effect. Larney et al. (2003) also reported that bedding material had no significant effect on E. coli and TC during windrow composting of manure from the same feedlot used in our study.
We assessed the relationship between selected environmental variables (water content of manure, air temperature, and relative humidity) related to season and numbers of bacteria in each of the four groups. There was a trend for a negative relationship between the water content of manure and numbers of the bacteria in the four groups (data not shown). However, water content of the manure accounted for only 25, 23, 9, and 23% of the variation in E. coli, TC, and TAH at 27°C and TAH at 39°C, respectively. Therefore, our results indicated that the water content of manure had a low potential to influence bacteria numbers in pen-floor manure. Wang et al. (1996) reported that E. coli O157:H7 persisted in cattle manure for considerable periods of time, even at water contents of <0.10 cm cm-3. In contrast, other researchers have reported lower coliform or E. coli O157:H7 numbers in the top layer of stockpiled cattle manure, and attributed this to drying and lower water contents (Thayer et al., 1974; Kudva et al., 1998).
There was a stronger positive relationship between mean daily air temperature (at nearby weather station) and mean numbers of the four bacterial groups (Fig. 1) . Air temperature explained 47, 47, 50, and 34% of the variation in E. coli, TC, and TAH at 27°C and TAH at 39°C, respectively. Second-degree polynomial (quadratic) equations gave the best regression fit to the data. Populations of E. coli and TC increased dramatically with an increase in air temperature. In contrast, TAH at 27 and 39°C increased more gradually with temperature and then leveled off. This indicated that coliform bacteria responded to air temperature differently that the overall bacterial population. Some studies have reported that die-off of microbial pathogens in soil or water generally increased with a rise in temperature (Reddy et al., 1981; Thelin and Gifford, 1983; Flint, 1987), with microbial competition playing a major role (Jiang et al., 2002). Fukushima et al. (1999) reported decreased persistence of inoculated E. coli strains in cattle manure at 25°C as compared with 5 and 15°C. In contrast, Kudva et al. (1998) reported no correlation between incubation temperatures and survival of E. coli O157:H7 in cattle feces inoculated with this strain of bacteria. We found no relationship between the relative humidity of air and bacteria numbers (data not shown).
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Manure application in Alberta is currently based on the total N and NH4–N content of manure. Our results indicated that neither bedding or season affected the total N content of pen manure. In contrast, higher NH4–N in manure with wood than straw and higher levels in the fall and winter than the spring and summer indicate that the available N content of manure might be managed using bedding or season. Allowable manure application rates in the future may be based on phosphorus (i.e., total P) instead of N. Although total P in our study was not affected by bedding, higher concentrations in the summer than other three seasons suggests that season might be a tool to manage P application to cropland.
Season had the strongest effect on populations of E. coli, TC, and TAH at 27 and 39°C in the pen manure. Bedding had no effect on the bacterial populations, and may be due to the high manure to bedding ratio in our pen-floor manure. Significantly higher populations of E. coli and TC in the pen manure during the summer and a positive correlation of populations with mean daily air temperature indicate that this warm season is of the most concern in terms of potential environmental contamination. All bacterial populations were lower in the winter than the other three seasons. However, we believe applying manure to frozen soils is not a good practice because of potential runoff of manure over frozen soils into surface water. If manure was applied in the spring or fall, there is a potential for fecal-indicator populations to be significantly lower than in the summer, and this may minimize environmental contamination by these bacteria. Bedding material and seasonal timing of cleaning feedlot pens and land application of manure may be a potential tool to manage certain nutrients, soluble salts, and pathogens in the manure. This could increase nutrient use by crops and decrease potential environmental contamination.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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