roduction of biogas from agricultural biomass through anaerobic digestion is increasing rapidly, providing us with both environmental and economic benefits. However, when we compare the anaerobic digestion (AD) of a biogas reactor with that of a rumen system, the latter is more efficient and quicker to produce biogas due to its complex microbial consortia. Among those microbes, anaerobic fungi (AF) are important contributors to the fiber digestion process using a wide variety of extracellular hydrolytic enzymes. In this study, we investigated using AF as a pretreatment step for hydrolysis of lignocellulosic biomass with the aim of increasing biogas yield during AD. Recently, studies utilizing AF to improve biogas production and speed up substrate degradation have been reported. (Procházka et al., 2012; Nkemka et al., 2015). Our main objective of this work was to determine the difference in the rate of lignocellulose degradation between three different anaerobic fungal genera using two different plant species as biomass. Thus, we hypothesize that pretreating lignocellulosic substrates with AF will increase their degradability and thereby help improve methane production inside biogas reactors. In this research, hydrolysis was conducted in batch reactors using two different substrates i.e., corn (Zea mays L.) silage and reed (Phragmites australis (cav.) trin. ex Steud.). The substrates were bioaugmented separately with pure cultures of three different AF previously isolated from ruminant animals i.e., Neocallimastix frontalis 27, Piromyces rhizinflatus YM600 and Anaeromyces mucronatus YE505. Hydrolysis of substrates was characterized by measuring evolution of H2 during fermentation, pH and soluble COD of fermentation broth, and composition of cellulose/hemicellulose/lignin in substrates before and after hydrolysis

1. Effect of bioaugmentation of
anaerobic fungi on the hydrolysis of
reed and corn silage
Bhargavi Ravi1,2 (MSc. Candidate)
V. Nkemka1, X. Hao1, T.A. McAllister1, D. Vedres1, R. Gruninger1,
J. Yanke1, H. Lee2, B.H. Gilroyed3
1 Agriculture and Agri-Food Canada Lethbridge Research Station, Lethbridge, Canada
2 University of Guelph, School of Environmental Sciences, Guelph, Canada
3 University of Guelph Ridgetown Campus, Centre for Agricultural Renewable Energy and Sustainability, Ridgetown,
Canada

2. Project Background
Materials and Experimental Design
Results
Discussion and Future Directions

3. PROJECT
BACKGROUND

4. Potential Biogas Feedstocks in Canada
4
Canadian Biogas Study Summary, 2013

5. Bottleneck in Biogas Production
1. Only 60% of agricultural
substrates can be degraded
during anaerobic digestion
2. Results in Poor Bioenergy
Return
3. Reason: Lignocellulosic
structure
5
http://vunature.com/sun-sunset-hay-nature-road-straw-haystack-trees-field-photos-with-captions/

6. Current Pre-Treatment Strategies
1. Physical pretreatment
2. Chemical pretreatment
3. Physiochemical pretreatment
4. Biological pretreatment
6
https://www.slideshare.net/danialali18/green-conversion-of-oil-palm-
empty-fruit-bunch-into-fermentable-sugars-research-progress

7. Natural Vs Artificial Anaerobic Digesters
7
?
http://www.freechoiceminerals.com/The-Rumen http://www.host.nl/en/biogas-plants/farm-scale-biogas-plants/
http://www.icr.org/article/just-how-simple-are-bacteria/

8. Rumen fungi
1. Initial colonizers in lignocellulose
degradation
2. Enzymatically degrades plant cell
walls using a diverse suite of
extracellular hydrolyzing enzymes
3. Only known members of the
kingdom fungi to possess
‘Cellulosomes’
4. Produce enzymes feruloyl and p-
coumaroyl which help cleave
lignin away from cellulose and
hemicellulose
8
http://www.soi.wide.ad.jp/class/20070046/slides/02/27.html

9. Project Objectives
1. To perform hydrolysis as a
pretreatment step
2. To study the impact of three
different rumen fungal species
(Neocallimastix frontalis 27,
Piromyces rhizinflatus YM600 and
Anaeromyces mucronatus YE505)
on hydrolysis of two different
lignocellulosic substrates, corn (Zea
mays L.) silage and reed
(Phragmites australis (Cav.) Trin.
ex Steud.)
9

10. Materials and
Experimental Design

11. Anaerobic Digestion Substrates
1. Corn silage (Zea mays) 2. Reed (Phragmites australis)
11

12. Anaerobic Digestate Medium
1. Obtained from biogas facility
(Lethbridge Biogas LP)
2. Autoclaved
3. Used as a buffering medium
for hydrolysis experiments
I. pH 7.88
II. Total bicarbonate alkalinity
16.66 g/L
12

13. Neocallimastix frontalis 27
Bovine source
Anaeromyces mucronatus
YE505 Elk source
Piromyces rhizinflata YM600
Moose source
Fungal Strains
13

14. Experimental Set-up
14

15. N. frontalis A. mucronatus P. rhizinflata
Controls
N0
Treatments
N
Controls
A0
Treatments
A
Controls
P0
Treatments
P
Corn Silage
CS
CS + N0
X 3
CS + N
X 3
CS + A0
X 3
CS + A
X 3
CS + P0
X 3
CS + P
X 3
Reed
R
R + N0
X 3
R + N
X 3
R + A0
X 3
R + A
X 3
R + P0
X 3
R + P
X 3
Controls and Treatments
15

16. TS(g)
added
VS(g)
added
% TS in
reactors
Water in
substrate
Water
added
Autoclaved
sludge
Fungi
added
20% (mL)
Active
reactor
volume
(mL)
Corn
silage
31.12 30 7.9 61.71 100 200 78.56 471.40
Reed 31.59 30 7.9 26.07 140 200 79.53 477.20
Feeding characteristics (Mesophilic @ 40°C)
16

17. Experimental Set-up

18. Analytical methods conducted
1. Gas chromatography carried to
measure H2, Volatile fatty
acids(VFA)
2. Soluble Chemical Oxygen
demand (COD)
3. pH
4. Fiber analysis
18

19. Results

20. 20
Corn Silage and Reed Characteristics
Initial Substrate characteristics of corn silage and reed used for hydrolysis
experiments. Values shown are means (n=3) and standard error.
a Total solids b Volatile solids c Acid detergent lignin

21. Average Cumulative H2 yield
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12
AveragecummulativeH2yieldmL/g
VSadded
Corn silage average cumulative H2 yield
21
-10
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12
AveragecummulativeH2yield
mL/gVSadded
Time (Days)
Reed average cumulative H2 yield

22. Total Volatile Fatty Acids
0
2
4
6
8
10
12
14
16
18
20
0 2 4 6 8 10 12
Totalvolatilefattyacids(g/L)
Corn silage -Total volatile fatty acids
22
0
5
10
15
20
0 2 4 6 8 10 12
Totalvolatilefattyacids(g/L)
Time (days)
Reed- Total volatile fatty acids

23. 4
5
6
7
8
9
0 2 4 6 8 10 12
pH
pH of corn silage Corn silage + Neo control
Corn silage + Neo
Corn silage + Anaero control
Corn silage + Anaero
Corn silage + Piro control
Corn silage + Piro
23
4
5
6
7
8
9
0 2 4 6 8 10 12
pH
pH of Reed Reed + Neo control
Reed + Neo
Reed + Anaero control
Reed + Anaero
Reed + Piro control
Reed + Piro
pH and COD Changes
0
10
20
30
40
50
0 2 4 6 8 10 12
CODg/L
Time (Days)
COD of corn silage
0
10
20
30
40
50
0 2 4 6 8 10 12
CODg/L
Time (Days)
COD of Reed

24. Discussions and Future Directions
1. Bioaugmentation with three different rumen fungal species did not
significantly improve hydrolysis of corn silage or reed
• No significant improvement in production of H2, VFA, or COD or in the
degradation of either substrate
2. Survival of rumen fungi inside fermentation systems needs to be
explored further
• Nkemka et al. 2015. Bioresource Technology 185: 79-88
3. Focus on hydrolytic enzymes and genes from anaerobic fungi may
be alternative strategy for enhancing degradation of lignocellulosic
material
24

25. 25
Dr. Brandon
Gilroyed
Dr. Xiying Hao Dr. Tim McAllister Dr. Valentine Nkemka
Nkongndem
Research Committee

26. 26
Project Funding
Program of Energy
Research & Development
CENTRE FOR AGRICULTURAL RENEWABLE
ENERGY AND SUSTAINABILITY

27. Vielen Dank!
27