1. Anaerobic Digestion: The Production of Bioenergy from Biomass
Faria Nusrata,*, Sunirat Rattanab, and Donna E. Fennellb,**
a Bloomfield College, 467 Franklin St. Bloomfield, NJ 07003
b Department of Environmental Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901
Conclusions
Thailand enrichments showed greater resistivity to
TAN stress
Thailand enrichments had more CH4 production; less
acetate and propionate accumulation
Decline in CH4 results from lack of carbon substrate
for 49 days
Thailand enrichments demonstrated propionate-
oxidizing bacteria and methanogens are able to
acclimate to high TAN
Ammonia stress selects for different microbial
communities.
The assay to characterize dnaK genes will be used to
compare the presence and diversity of these stress
genes in the NJ and Thailand enrichments.
Future Work
Functional Gene Analysis of trkA gene involved in
potassium transport that affects ammonia tolerance
References
Babson, David M., Bellman, Karen, Prakash, Shaurya, Fennell, Donna
E. (2013). Anaerobic digestion for methane generation and ammonia
reforming for hydrogen production: A thermodynamic energy balance
of a model system to demonstrate net energy feasibility. Elsevier, 56:
493-505.
Chen, Ye, Cheng, Jay J., Creamer, Kurt S. (2007). Inhibition of
anaerobic digestion process: A review. Elsevier, Bioresource
Technology 99: 4044-4064.
Rittmann, Bruce E. (2008). Opportunities for Renewable Bioenergy
Using Microorganisms. Biotechnology and Bioengineering. Vol. 100, No.
2.
Hofman-Bang, Jacob, Lange, Marianne, Conway de Macario, Everly,
Macario, Alberto J.L., Ahring, Birgitte K. (1999). The genes coding for
the hsp70(dnaK) molecular chaperone machine occur in the moderate
thermophilic archaeon Methanosarcina thermophila TM-1. Gene, 238:
387–395.
Throback, Ingela Noredal, Enwall, Karin, Jarvis, Asa, Hallin, Sara.
(2004). Reassessing PCR primers targeting nirS, nirK and nosZ genes for
community surveys of denitrifying bacteria with DGGE. FEMS
Microbiology Ecology, 49: 401–417
Acknowledgments
This work was funded by the National Science Foundation (NSF) under
grant number 1263250. We thank Miss Amanda Luther and Ms. Maria
Rivera of the Department of Environmental Sciences and Dr. Kimberly
Cook-Chennault of the Department of Mechanical and Aerospace
Engineering for technical support.
*Author: Faria_Nusrat@bloomfield.edu
**Corresponding Author: fennell@envsci.rutgers.edu (D.E. Fennell)
Results
0
20
40
60
80
0 50 100 150 200 250 300
MethaneVolume(mL)
High Ammonia Active Control
0
20
40
60
80
0 100 200 300 400
MethaneVolume(mL)
0
500
1000
1500
2000
2500
0 50 100 150 200 250 300
AcetateConcentration
(mg/L)
0
1000
2000
3000
4000
5000
0 100 200 300 400
PropionateConcentration
(mg/L)
Time (Days)
Abstract
Methods
EXPERIMENTAL
• Replicate (5) enrichments inoculated with
• Thai landfill leachate
• NJ landfill leachate
• Active controls and TAN stressed in each set
• 10 mM glutamate as carbon substrate
• Anaerobic media was provided
• NH4Cl (12.5g NH4
+-N/L) was added to TAN-stressed
enrichments
• Operated under a semi-continuous flow through a
fill and draw regime
ANALYTICAL
• Biogas volume: Water displacement
• CH4: Gas Chromatography- Flame Ionization
Detection (GC-FID)
• VFAs: High Performance Liquid Chromatography
(HPLC)
• TAN: Ion Chromatography (IC)
• Microbial communities identified by PCR coupled
with DGGE
Anaerobic digestion (AD) is a renewable energy
resource that produces bioenergy (i.e. methane) from
organic matter. For AD to be universally stable,
stresses in the environment must be overcome. A
prevalent stressor in digesters is ammonia (NH3),
which accumulates from the breakdown of organic
nitrogenous matter. Mesophilic enrichments derived
from two different landfill leachate were fed
glutamate and monitored for methane (CH4), volatile
fatty acids (VFAs), total ammonia nitrogen (TAN). The
dominant bacterial communities were identified using
polymerase chain reaction (PCR) of 16S rRNA genes
followed by denaturing gradient gel electrophoresis
(DGGE). A polymerase chain reaction assay was
developed to detect dnaK, general stress genes that
encode for the 70 kDa heat shock proteins (molecular
chaperones), and which may be involved in ammonia
tolerance. It was found that microorganisms in
leachate from Thailand showed greater resistivity to
high TAN and ammonia stress selects for different
microbial communities.
0
1000
2000
3000
4000
5000
0 50 100 150 200 250 300
PropionateConcentration
(mg/L)
0
1000
2000
3000
4000
5000
6000
0 100 200 300 400
AcetateConcentration
(mg/L)
Time (Days)
New JerseyThailand
Objectives
 Identify microbial communities tolerant to high
ammonia stress
 Detect stress genes of the dnaK locus present in the
digester microbial communities
Time (Days)
Time (Days)
Fig.2 Methane production in Thailand
leachate inoculated reactor
Time (Days)
Time (Days)
Fig.3 Methane production in NJ
leachate inoculated reactor
Fig.4 Acetate concentration in
Thailand leachate inoculated reactor
Fig.5 Acetate concentration in NJ
leachate inoculated reactor
Fig.6 Propionate concentration in
Thailand leachate inoculated reactor
Fig.7 Propionate concentration in NJ
leachate inoculated reactor
Fig.1
Top row: Thailand
leachate inoculated
reactors
Bottom row: NJ leachate
inoculated reactors
Fig.8 Gel
Electrophoresis of
PCR-amplified dnaK
gene fragment for
bacteria. From left to
right: DNA standard,
negative control,
positive control,
Thailand enrichment
2, Thailand leachate
active control 2, DNA
standard, Thailand
enrichment 2,
Thailand enrichment
3, NJ enrichment 2,
NJ enrichment 3, NJ
leachate active
control 2, and
negative control.
Peptostreptococcus
russellii DNA served as
the positive control.
Fig.9 DGGE of PCR-amplified 16S
rRNA genes in Thailand leachate
inoculated reactors.
Lanes 1, 2, and 3 contain TAN-
stressed samples. Lanes 4 and 5
contain active controls.
DNAStandard(LambdaHindIII)
NegativeControl
PositiveControl(P.russellii)
ThailandEnrichment
ThailandActiveControl
1 2 3 4 5
ThailandEnrichment
ThailandEnrichment
NJEnrichment
NJEnrichment
NJActiveControl
NegativeControl
DNAStandard(LambdaHindIII)