inhibition of total gas production, methane, hydrogen sulfide, and sulfate-reducing bacteria from in...

USDA - ARS - National Center for Agricultural Utilization Research

INHIBITION OF TOTAL GAS INHIBITION OF TOTAL GAS PRODUCTION, METHANE, HYDROGEN PRODUCTION, METHANE, HYDROGEN

SULFIDE, AND SULFATE-REDUCING SULFIDE, AND SULFATE-REDUCING BACTERIA FROM IN VITRO STORED BACTERIA FROM IN VITRO STORED

SWINE MANURE USING CONDENSED SWINE MANURE USING CONDENSED TANNINSTANNINS

Terence R. WhiteheadTerence R. Whitehead

Bioenergy Research UnitBioenergy Research Unit

USDA-ARS-NCAUR, Peoria, IllinoisUSDA-ARS-NCAUR, Peoria, Illinois

Pig House

Manure Storage

Underground Storage ofSwine Manure

Partial List of Identified Volatile Organic Compounds From

Swine Manure

1-Methylethanol Acetic AcidPropanol Propanoic acid2-Methoxyethanol 2-Methylpropanoic acid2-Methylpropanol Butanoic acidButanol Hexanoic acid3-Methylbutanol 2-PhenylethanolPentanol p-Cresol3-OH-butanone 3-Methylindole3-Octanol p-Ethylphenol2,3,4-Trithiapentane o-Methoxyphenol

Additional Emissions Also Which May Play Important Roles From Swine

Manure

• Ammonia

•Hydrogen Sulfide

•Methane

Identification of Bacteria Involved inManure 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.

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

Peoria Illinois USA World Manure Pit103

106

109

1012

1015

1018

1021

Comparison of Human Populations Versus Swine Manure Pit Bacteria

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.

• 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.

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

In Vitro Manure Assay Bottles.

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) 100.0Quebracho (0.5% w/v) 2.2C-KPN (0.5% w/v) 53.2C-KPS (0.5% w/v) 55.6NA-1 (0.5% w/v) 188.1TARA (0.5% w/v) 155.6WE-ME (0.5% w/v) 43.0_______________________________________________________1Quebracho tannin experiment terminated after 42 days. Othertannins ended after 58 days.

Inhibition of H2S Production by 0.5% Quebracho Tannins

Effects of 0.5% Quebracho Tannins on Total SRB Levels

1.0E+09

1.0E+10

1.0E+11

1.0E+12

0 1 3 7 14 21 28

Time (days)

cop

ies

16S

rD

NA

/ m

l sl

urr

ycontrol

0.5% tannins

**

**

***

Effects of 0.5% Quebracho Tannins on Bacterial Numbers

Inhibition of Methane Production by 0.5% Quebracho Tannins

Future Work: Known Knowns, Known Unknowns, and Unknown Unknowns

• Onsite Farm Testing with Quebracho Tannins

• Other Tannins Useful?

•Other Compounds for Testing? -Ionophores?

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.

Acknowledgments

NCAUR - Peoria

Dr. Michael CottaRhonda ZeltwangerDr. Cheryl Spence

ARS – Bowling Green, KY

Dr. Kim Cook