1.
USDA - ARS - National Center for Agricultural Utilization Research
INHIBITION OF TOTAL GASINHIBITION OF TOTAL GAS
PRODUCTION, METHANE, HYDROGENPRODUCTION, METHANE, HYDROGEN
SULFIDE, AND SULFATE-REDUCINGSULFIDE, AND SULFATE-REDUCING
BACTERIA FROM IN VITRO STOREDBACTERIA FROM IN VITRO STORED
SWINE MANURE USING CONDENSEDSWINE MANURE USING CONDENSED
TANNINSTANNINS
Terence R. WhiteheadTerence R. Whitehead
Bioenergy Research UnitBioenergy Research Unit
USDA-ARS-NCAUR, Peoria, IllinoisUSDA-ARS-NCAUR, Peoria, Illinois

2.
Pig House
Manure Storage
Underground Storage of
Swine Manure

3.
Partial List of Identified
Volatile Organic Compounds From
Swine Manure
1-Methylethanol Acetic Acid
Propanol Propanoic acid
2-Methoxyethanol 2-Methylpropanoic acid
2-Methylpropanol Butanoic acid
Butanol Hexanoic acid
3-Methylbutanol 2-Phenylethanol
Pentanol p-Cresol
3-OH-butanone 3-Methylindole
3-Octanol p-Ethylphenol
2,3,4-Trithiapentane o-Methoxyphenol

4.
Additional Emissions Also Which May
Play Important Roles From Swine
Manure
• Ammonia
•Hydrogen Sulfide
•Methane

5.
Identification of Bacteria Involved in
Manure Breakdown and Odor Production
I. Classical Microbiology
• Pure culture isolation and identification using
habitat simulating media, biochemical techniques
and 16S rDNA gene sequence analyses.
II. Molecular Ecology (Culture Independent)
• Isolate total DNA from fecal and manure samples,
determine DNA sequence of isolated 16S rDNA
genes, and identify bacteria by comparison with
known sequences in GenBank database.

6.
Miscellaneous Parameters for
Swine Manure Pits
•Temperature: 24°C
• pH: 6.85
•3-Ft. Pit: 1 x 1010
Cells/ml
• Approx.Volume: 500,000 Gallons
•Approx. Bacterial Number: 2 x 1019

7.
Peoria Illinois USA World Manure Pit
103
106
109
1012
1015
1018
1021
Comparison of Human Populations Versus
Swine Manure Pit Bacteria

8.
Summary
•Microbial populations of stored swine manure
are dominated by Gram-positive, low %G+C,
anaerobic bacteria.
•The vast majority of the bacterial isolates (and
DNA clones) could only be identified to the
genera level, and many strains represent new
genera and species.
•Swine manure represents a rich ecosystem for
isolation of new and novel bacteria.

9.
• Using 16S rDNA sequence analyses and biochemical
studies we have been able to identify new bacterial
genera and species.
• Characterized populations of sulfate-reducing bacteria
(SRB), which produce hydrogen sulfide, using PCR and
qRT-PCR. Archeal 16S rDNA gene and dissimilatory
sulfite reductase (dsrA) gene primer sets were used for
analyses.
• Identified 3 new groups of SRB.
• Groups fell within known lineages of SRBs; however,
the clones represented novel species within these genera.

10.
Schematic representation of a condensed tannin
molecule. Condensed tannins can be linear (with 4→8
bounds) or branched (with 4→6 bounds - dotted line).

11.
In Vitro Manure Assay Bottles.

12.
Table 1. Effects of Addition of Various Condensed Tannins on
Total Gas Production by In Vitro Swine Manure Slurries
Tannin % Control (Cumulative Gas
Production)1
Control (No Tannin) 100.0
Quebracho (0.5% w/v) 2.2
C-KPN (0.5% w/v) 53.2
C-KPS (0.5% w/v) 55.6
NA-1 (0.5% w/v) 188.1
TARA (0.5% w/v) 155.6
WE-ME (0.5% w/v) 43.0
_______________________________________________________
1
Quebracho tannin experiment terminated after 42 days. Other
tannins ended after 58 days.

13.
Inhibition of H2S Production by 0.5% Quebracho Tannins

14.
Effects of 0.5% Quebracho Tannins on Total SRB Levels

15.
1.0E+09
1.0E+10
1.0E+11
1.0E+12
0 1 3 7 14 21 28
Time (days)
copies16SrDNA/mlslurry control
0.5% tannins
**
**
**
*
Effects of 0.5% Quebracho Tannins on Bacterial Numbers

16.
Inhibition of Methane Production by 0.5% Quebracho Tannins

17.
Future Work: Known Knowns, Known
Unknowns, and Unknown Unknowns
• Onsite Farm Testing with Quebracho
Tannins
• Other Tannins Useful?
•Other Compounds for Testing?
-Ionophores?

18.
POSTER SESSION:
Combination of Borax and Quebracho Condensed
Tannins Treatment to Reduce Hydrogen Sulfide,
Ammonia and Greenhouse Gas Emissions from
Stored Swine Manure. Yokoyama et al.

19.
Acknowledgments
NCAUR - Peoria
Dr. Michael Cotta
Rhonda Zeltwanger
Dr. Cheryl Spence
ARS – Bowling Green, KY
Dr. Kim Cook