Plant-derived oils reduce pathogens and gaseous emissions from stored cattle waste.

Carvacrol and thymol in combination at 6.7 mM each completely inhibited the production of short-chain volatile fatty acids and lactate from cattle waste in anoxic flasks over 23 days. Fecal coliforms were reduced from 4.6 3 10 6 to 2.0 3 10 3 cells per ml 2 days after treatment and were nondetectable within 4 days. Total anaerobic bacteria were reduced from 8.4 3 10 10 to 1.5 3 10 7 cells per ml after 2 days and continued to be suppressed to that level after 14 days. If the concentration of carvacrol or thymol were doubled (13.3 mM), either could be used to obtain the same inhibitory fermentation effect. We conclude that carvacrol or thymol may be useful as an antimicrobial chemical to control pathogens and odor in stored livestock waste. Odor emitted from wastes results from incomplete anoxic degradation of the carbohydrate, protein, and lipid components (11, 27). This incomplete degradation results in the formation of offensive short-chain volatile fatty acids (VFAs), aromatic chemicals, amines and other nitrogenous compounds, and sulfur-containing compounds. In most livestock production facilities it is not possible to control the environment in which complete degradation of the waste to methane and carbon dioxide occurs. Conventional anoxic digesters for production of methane were popular during the 1970s and 1980s; however, economics and the technical expertise required to operate these digestors have diminished their popularity (14). Similarly, oxic treatment is not economically feasible, and it does little to conserve nutrients. We have previously reported that chlorhexidine diacetate, in combination with iodoacetate or diphenyliodonium chloride, can be used to inhibit key anoxic-degradation pathways (26). Another approach to reduce emissions might be the use of naturally occurring antimicrobial chemicals such as plant-derived oils (1‐3, 7, 23). Numerous reports indicate that plant oils have antimicrobial activity; however, most of these studies evaluate one oil against one microorganism in an artificial medium (3). Few studies are available that determine the effect of the oils in natural highly diverse microbial ecosystems such as those found in the gastrointestinal tract (15), food products (7, 9, 21), or waste management systems. In this study we evaluated carvacrol (5-isopropyl-2-methylphenol) and thymol (5-methyl-2-isopropylphenol) for their ability to reduce the production of gas and short-chain VFAs and the viability of total anaerobic bacteria and fecal coliforms in stored cattle waste. Waste was collected and processed similarly to our previous study (26). The waste slurry was divided into 500-ml aliquots, and antimicrobial plant oils were added directly to the slurry at the desired concentration. The slurry was blended 1 min to provide a homogenous mixing of the antimicrobial oils and poured into a 1-liter Erlenmeyer flask, which was sealed with a rubber stopper and left stationary at ambient temperature (25°C). Treatments were in triplicate, and the contents in the flasks were gently swirled before being sampled at the times indicated (Fig. 1 and Table 1). Another study evaluated the effect of the antimicrobial plant oils on cattle waste under natural semioxic conditions (lagoon or basin simulation). In this study wide-mouth (10-cm) jars (17 cm tall, 13.5 cm in diameter, 1.6-liter volume) with a working volume of 1 liter each were used (Fig. 2). Plastic lids provided with the jars were used to cover approximately 90% of the jar opening to prevent moisture loss over the 56-day experimental period. The sampling procedure was similar to that described above, except that no stirring or mixing occurred before the contents were sampled. A wide-mouth pipette was inserted into the slurry from top to bottom at which time slurry contents were simultaneously withdrawn. Gas volume and composition, shortchain VFAs, and L-lactate in the flasks were measured as previously described (13, 18, 26). Total culturable anaerobic bacteria and fecal coliforms were enumerated from a 1-ml sample removed from each flask. Each sample was serially diluted in anaerobic buffer, and triplicate roll tubes from each of three dilutions containing 30% ruminal fluid-based medium were inoculated as previously described (25). Roll tubes were incubated at 35°C, and colonies were counted after 7 days. Fecal coliforms were enumerated with 3M Petrifilm Escherichia coli coliform count plates (3M Microbiology Products, St. Paul, Minn.). Triplicate plates for each of three dilutions were inoculated and incubated at 35°C, and colonies were counted using AOAC official methods as described in the literature provided with the plates. Briefly, total coliform numbers consisted of both red and blue colonies associated with gas at 24 h after inoculation. All chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.) with the exception of carvacrol, which was obtained from Aldrich (Milwaukee, Wisc.). Data were analyzed as a split-plot in time with the general linear models (GLM) procedure of SAS Inst., Inc. (Gary, N.C.) (19). A series of experiments using different batches of waste and