Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: ANAEROBIC MICROBIOLOGICAL PROCESSES IN ANIMAL MANURE MANAGEMENT

Location: Bioenergy Research Unit

2006 Annual Report


1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Storage of swine manure is associated with the production of a variety of odorous chemicals and emissions including ammonia, methane, organic acids and alcohols, and sulfides. Much of this problem is due to the incomplete digestion process associated with anaerobic systems. Although production of these chemicals is the result of microbiological activity, little is known about the types of microorganisms responsible for their production. Traditional microbiological analysis involves the isolation, identification, and enumeration of microorganisms present in environmental samples. This approach limits the examination to those organisms which can be easily isolated and cultivated using conventional selective and non-selective media. This greatly reduces the scope of the potential environmental samples and experimental treatments that can be evaluated. To overcome these problems, newly developed molecular biological approaches are being applied to rapidly screen and identify microorganisms in environmental samples. In addition, these findings are directing the assembly of a pure culture collection of swine fecal and manure bacteria needed for physiological and biochemical studies of odor production. The information gained from this research will be critical to the evaluation of methods to control odor by manipulation of animal diet or by the use of additives to manure storage.

This project has three specific goals: .
1)Identification of predominant bacterial populations present in swine feces and manure storage pits by direct isolation and culture-independent molecular methods;.
2)evaluation of compounds to inhibit bacterial activity in stored swine manure and reduce emissions; and.
3)development of molecular methods for monitoring changes in bacterial populations in response to diet manipulation and management (e.g., handling procedures, addition of metabolic inhibitors) and their impact on emissions from swine facilities.

This research falls under National Program 206 – Manure and Byproduct Utilization and addresses goals as described under the Emissions Component of the National Program Action Plan.

The major product of our research is new information and techniques that can be shared with collaborators. We provide technical guidance to other members of the Emissions Team of National Program 206 on molecular diagnostics. We serve as a member of an advisory panel to a national producer group on Manure Safety which meets periodically to evaluate research proposals, collect information on manure safety and potential for spread of zoonotic diseases, and make recommendations regarding the possible dangers associated with manure handling (National Pork Board). Members of the research group participate in information sharing activities including ARS workshops on Air Quality and Pathogens. The work unit maintains an anaerobic microbial culture collection, acquiring new strains, and providing these strains and advice to researchers both within and outside of ARS.


2.List by year the currently approved milestones (indicators of research progress)
FY 2005 1.1 Primary bacterial populations identified. 1.2 Producers of odorous compounds identified. 1.3 Real-time PCR techniques developed. 1.4 Real-time PCR techniques for sulfate-reducing bacteria populations developed. 2.1 Complete testing of monensin for emissions inhibition in vitro. 2.2 New inhibitory compounds identified.

FY 2006 1.1 Primary bacterial populations identified. 1.2 Producers of odorous compounds identified. 1.3 Real-time PCR techniques developed. 1.4 Real-time PCR techniques for sulfate-reducing bacteria populations developed. 2.1 Complete testing of monensin for emissions inhibition in vitro. 2.2 New inhibitory compounds identified. 3.1 Complete diet manipulation studies. 3.2 Complete analyses of manure samples from diet studies.

FY 2007 1.1 Primary bacterial populations identified. 1.2 Producers of odorous compounds identified. 1.3 Real-time PCR techniques developed. 2.2 New inhibitory compounds identified. 2.3 In vitro effects of inhibitory compounds on bacterial populations completed. 3.2 Complete analyses of manure samples from diet studies.

FY 2008 1.1 Primary bacterial populations identified. 1.3 Real-time PCR techniques developed. 2.2 New inhibitory compounds identified. 2.3 In vitro effects of inhibitory compounds on bacterial populations completed. 2.4 Studies testing selected inhibitory compounds in stored swine manure completed. 2.5 Development of inhibition technology continued. 3.3 Manure sampling/analyses from swine facilities continued.

FY 2009 1.1 Primary bacterial populations identified. 2.3 In vitro effects of inhibitory compounds on bacterial populations completed. 2.4 Studies testing selected inhibitory compounds in stored swine manure completed. 2.5 Development of inhibition technology continued. 3.3 Manure sampling/analyses from swine facilities continued.


4a.List the single most significant research accomplishment during FY 2006.
DEVELOPMENT OF QUANTITATIVE REAL-TIME PCR METHODS FOR DETECTION OF SULFATE-REDUCING BACTERIA IN STORED SWINE MANURE. This research contributes to solving problems outlined in the Atmospheric Emissions Research Component of National Program 206. A variety of odorous chemicals are produced by the anaerobic bacterial consortium that constitutes stored swine manure. One of the more odorous and potentially health-threatening of these compounds is hydrogen sulfide. The primary producers of this compound are sulfate-reducing bacteria, but little is known about the presence of these bacteria in stored swine manure. This research resulted in the isolation and identification of sulfate-reducing bacteria from swine manure and development of PCR methods for the detection and quantitation of different groups of these bacteria. The information gained from this and additional studies will be useful in isolating, identifying, and quantitating other microbial groups involved in the anaerobic production of volatile emissions from stored manure.


4b.List other significant research accomplishment(s), if any.
DETERMINATION OF THE BREAKDOWN PATHWAY OF TRYPTOPHAN AND INDOLE ACETIC ACID TO SKATOLE BY CLOSTRIDIUM DRAKEI, CLOSTRIDIUM SCATOLOGENES, AND SWINE MANURE SLURRY. This research contributes to solving problems outlined in the Atmospheric Emissions Research Component of National Program 206. One of the more odorous compounds produced by anaerobic bacteria present in stored swine manure is 3-methyl-indole, also known as skatole. Identification of the biochemical pathway for skatole production from the amino acid tryptophan, a crucial constituent of swine feed, is important for determining potential methods for intervening in this breakdown. Collaborative research was carried out at the National Center for Agricultural Utilization Research, Peoria, IL, between our research group and members of the Bioproducts & Biocatalysis Research Unit. This research resulted in the confirmation of the pathway of skatole production from tryptophan and indole acetic acid by two bacterial species and slurry from stored swine manure, and provided evidence for the presence of bacteria in stored swine manure that can produce skatole. The information gained from this and additional studies will be useful in isolating and identifying microorganisms involved in the anaerobic production of skatole and other volatile emissions in stored manure.


4c.List significant activities that support special target populations.
None.


5.Describe the major accomplishments to date and their predicted or actual impact.
This research program has focused on the identification of the bacteria present in swine manure and storage facilities (primarily pits) and contributes to solving problems outlined in the Atmospheric Emissions Research Component of National Program 206. This identification employs state-of-the-art molecular biology techniques as well as conventional microbiological methods. Using these approaches, we have established that the primary bacterial populations present in both the swine feces and the manure storage to be Gram (+) anaerobic bacteria. The information gathered from these studies enabled us to develop diagnostic techniques (i.e., quantitative real-time PCR) that use specific genes to identify different groups of microorganisms. These approaches enable us to detect differences in the microorganisms present in feces, stored manure, and with animals at various stages of production.

Many new bacterial groups and species have also been discovered during the course of this research. Studies with pure cultures of manure bacteria have identified species capable of producing known odorous compounds, including ammonia, volatile fatty acids, and sulfides. Monensin, an ionophore normally fed to ruminants to reduce ammonia production by Gram (+) bacteria in the rumen, was tested in in vitro swine manure slurries prepared in the laboratory. Gas production (methane, carbon dioxide, hydrogen) was reduced significantly by addition of this compound. Production of volatile fatty acids was also altered. The techniques developed and information gained from this research demonstrates the potential for controlling microbial activity as a means towards selectively inhibiting odor producing microbes. Decreasing odorous emissions from swine production facilities will improve environmental quality surrounding these farms and foster better relations between producers and rural neighbors.


6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Scientific papers were presented in the literature and scientific meetings to other scientists that conveyed results of our work on bacterial populations of swine feces and stored manure.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
None.


Review Publications
Whitehead, T.R., Cotta, M.A., Collins, M.D., Falsen, E., Lawson, P.A. 2005. Bacteroides coprosuis sp. nov., isolated from swine-manure storage pits. International Journal of Systematic and Evolutionary Microbiology. 55:2515-2518.

Price, N.P., Whitehead, T.R., Cote, G.L. 2006. Global metabolic flux analysis of the bifidobacterial bifido shunt pathway. Biocatalysis and Biotransformation. 24(1-2):95-98.

Wang, Y., Wang, G., Shoemaker, N.B., Whitehead, T.R., Salyers, A.A. 2005. Distribution of the ermG gene among bacterial isolates from porcine intestinal contents. Applied and Environmental Microbiology. 71(8):4930-4934.

Whitehead, T.R., Price, N.P., Cotta, M.A. 2006. Determination of breakdown pathway of tryptophan and indole acetic acid to skatole by Clostridium drakei and swine manure slurry [abstract]. Reproduction Nutrition Development. 46(S60):113.

Adeuya, A., Whitehead, T.R., Price, N.P. 2006. Stable isotopes and mass spectrometry. Flux analysis of the fructose-6-p-phosphoketolase shunt pathway in bifidobacterium [abstract]. American Society for Mass Spectrometry. 17(5S):118S.

Hamady, Z.Z., Farrar, M.D., Whitehead, T.R., Lodge, J.P., Carding, S.R. 2006. Engineering of the human commensal Bacteroides ovatus for the in situ delivery of immunomodulatory proteins [abstract]. Reproduction Nutrition Development. 46(S64):0-18.

Last Modified: 4/20/2014
Footer Content Back to Top of Page