Project: MLMMI 02-01-11
Objective
Identify unculturable bacteria responsible for odourous compounds in swine manure. To identify these bacteria the Polymerase Chain Reaction, denaturing gradient gel electrophoresis and DNA sequencing will be used on fresh feces, stored manure and composted manure. When unculturable bacteria are identified their contribution to odour generation can be determined or inferred. Changes to manure storage conditions can be rationally planned to reduce odour production.
Performer
Paul Holloway
Details
Status: Completed
Started: 2002-12-01
Completed: 2003-06-11
Funding Partners: who have contributed to MLMMI in support of this project:
Manitoba Pork Council and industry groups - $6,676
Amount Funded: $6,676.00
Performer Funded: $5,450.00
Total Cost: $12,226.00
Activity
First Progress report received Mar 5/03
Final report due June 1, 2003; received June 11/03.
Summary
One of the most important objections to the expansion of the Manitoba livestock industry is the problem of odour from animal wastes. Bacteria ferment manure under anaerobic conditions to produce most of the objectionable odour compounds. Little is known about the microbiology of anaerobic bacteria in manure. Microbiological research has relied on conventional techniques such as growing the bacteria and measuring biochemical properties. Techniques that rely on growing bacteria underestimate the population and the diversity of species of microorganisms present in manure. Most bacterial species present in an environment such as manure cannot be grown. If we cannot culture these bacteria, we cannot measure their contributions to odour compound generation.
To overcome this problem, new techniques from the field of molecular genetics have been applied in this study. For each sample, the DNA from all of the bacteria was isolated. The polymerase chain reaction (PCR) technique was applied to produce enough of a specific gene which every bacterium carries, the 16S rRNA gene. While every bacterium carries this gene, the precise sequence of nucleotides of the gene varies amongst different species and can be used to identify a particular bacterium. Nucleotides are the individual building blocks of all genes. Determining the nucleotide sequences is expensive so, in this study, a technique was first used to determine how many different 16S rRNA genes were present. Once this was accomplished, then the most abundant genes actually had their individual nucleotide sequences determined. The technique to indicate the number of different 16S rRNA genes present is called denaturing gradient gel electrophoresis. This technique is based on the principle that each different type of 16S rRNA gene melts or denatures at a different temperature and can be separated from all the others.
In this project, bacterial DNA was isolated from fresh swine manure and from composted solids manure. The quantity of the 16S rRNA gene was increased for both types of samples. Using denaturing gradient gel electrophoresis, seven types of bacteria were detected in the fresh manure sample and five types of bacteria were detected in the composted manure sample. Many other faint bands were detected in both samples, revealing that many other types of bacteria are also present at lower levels. Bacteria in the fresh manure sample were identified by sequencing as Lactobacillus plantarum, Streptococcus alactolyticus, Clostridium butyricum, and a Eubacterium species. Sequencing is a technique which establishes the identity and order of the nucleotides making up the 16S rRNA gene. The other two types of bacteria did not resemble any known bacteria very well. In the composted manure samples bacteria were identified as Bacillus sylvestris, Clostridium butyricum, Clostridium celatum and probably a Lactobacillus species. The other bacterium did not resemble any known bacteria very well.
This project has increased understanding of the microbiology occurring in swine manure by identifying many species of bacteria which are common in manure. The techniques used to identify these bacteria have just begun to be used in this type of microbiology and should provide much information in the future. With a full and complete knowledge of the types of bacteria in swine manure and what each species does metabolically, a better understanding of the production of odour compounds will be realized. This will lead to better management strategies for the collection and treatment of swine manure in the future.