Articles
CHANGES IN BACTERIAL COMMUNITIES IN DAIRY MANURE DURING NINE MONTHS OF COMPOSTING AS DETERMINED BY DENATURING GRADIENT GEL ELECTROPHORESIS
Article number
1034_49
Pages
399 – 407
Language
English
Abstract
Composted dairy manure has strong potential for use as a potting mix amend-ment, but prospective users are concerned that harmful microorganisms may be present in the compost.
The objective of this study was to determine the changing bacterial communities in dairy manure during 9 months of composting by using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Dairy manure was collected 10 days (considered month 0), 3, 6, and 9 months after the composting process was started.
Collected manure was homogenized and then extracted using the Mobio Powersoil® DNA Isolation Kit.
Polymerase chain reaction (PCR) was used to amplify bacterial DNA. Three universal 16S rRNA bacterial primer sets, 63F/518R, 341F/907R and 968F/1401R, were used to amplify bacterial DNA and mitigate the effects of primer bias.
PCR products were loaded onto denaturing gradient gels that contained a urea and formamide gradient from 40 to 80% (month 0) or 55 to 80% (months 3, 6 and 9). After electrophoresis, distinct DNA bands were excised, and reclaimed DNA was amplified again by PCR and submitted to a commercial sequencing company.
Identifications to the species level were made if DNA sequences matched > 97%; genus level designations were made for matches > 95%; order level identifications were assigned if similarity was ≥ 90%; and any matches less than 90% similarity were designated unclassified bacteria.
Five bacterial phyla, the Firmicutes, Proteobacteria, Actinobacteria, Deinococcus-Thermus, and Bacteroidetes were identified as the dominant populations during the 9 months of composting.
Relative abundances of the phyla changed over time with the most changes in the abundance of Firmicutes and Bacteroidetes between the 0 and 3 month samples.
The pH and salt concentration of the compost strongly impacted bacterial populations, promoting halotolerant/halophilic and alkaliphilic bacteria.
Although taxonomic resolution was insufficient to state pathogenic microorganisms were absent, DNA sequences with strong homology to fecal bacteria, such as E. coli or Salmonella, were not present in any samples collected during the study.
Results from this study indicated the composting process successfully reduced the levels of fecal coliforms to below the PCR-DGGE detection limit.
The objective of this study was to determine the changing bacterial communities in dairy manure during 9 months of composting by using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Dairy manure was collected 10 days (considered month 0), 3, 6, and 9 months after the composting process was started.
Collected manure was homogenized and then extracted using the Mobio Powersoil® DNA Isolation Kit.
Polymerase chain reaction (PCR) was used to amplify bacterial DNA. Three universal 16S rRNA bacterial primer sets, 63F/518R, 341F/907R and 968F/1401R, were used to amplify bacterial DNA and mitigate the effects of primer bias.
PCR products were loaded onto denaturing gradient gels that contained a urea and formamide gradient from 40 to 80% (month 0) or 55 to 80% (months 3, 6 and 9). After electrophoresis, distinct DNA bands were excised, and reclaimed DNA was amplified again by PCR and submitted to a commercial sequencing company.
Identifications to the species level were made if DNA sequences matched > 97%; genus level designations were made for matches > 95%; order level identifications were assigned if similarity was ≥ 90%; and any matches less than 90% similarity were designated unclassified bacteria.
Five bacterial phyla, the Firmicutes, Proteobacteria, Actinobacteria, Deinococcus-Thermus, and Bacteroidetes were identified as the dominant populations during the 9 months of composting.
Relative abundances of the phyla changed over time with the most changes in the abundance of Firmicutes and Bacteroidetes between the 0 and 3 month samples.
The pH and salt concentration of the compost strongly impacted bacterial populations, promoting halotolerant/halophilic and alkaliphilic bacteria.
Although taxonomic resolution was insufficient to state pathogenic microorganisms were absent, DNA sequences with strong homology to fecal bacteria, such as E. coli or Salmonella, were not present in any samples collected during the study.
Results from this study indicated the composting process successfully reduced the levels of fecal coliforms to below the PCR-DGGE detection limit.
Authors
A. Knerr, R.R. Tripepi
Keywords
compost, cattle manure, polymerase chain reaction, plant growth media, bacteria, denaturing gradient gel electrophoresis
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