i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 9 9 – 1 1 0 5

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Biohydrogen production from specified risk materialsco-digested with cattle manure

Brandon H. Gilroyed a,b, Chunli Li a, Xiying Hao a, Angus Chu b, Tim A. McAllister a,*a Agriculture and Agri-Food Canada, Lethbridge Research Centre, P.O. Box 3000, Lethbridge, Alberta T1J 4B1, Canadab Department of Civil Engineering, Schulich School of Engineering, University of Calgary, Calgary, Alberta T2N 1N4, Canada

a r t i c l e i n f o

Article history:

Received 15 September 2009

Received in revised form

18 November 2009

Accepted 19 November 2009

Available online 6 December 2009

Keywords:

SRM

Hydrogen

Anaerobic digestion

Fermentation

* Corresponding author. Tel.: þ1 403 381 515E-mail address: tim.mcallister@agr.gc.ca

0360-3199/$ – see front matter Crown Copyrigdoi:10.1016/j.ijhydene.2009.11.072

a b s t r a c t

Biohydrogen production from the anaerobic digestion of specified risk materials (SRM) co-

digestedwith cattle manure wasassessedin a 3� 5 factorial design.Totalorganic loading rates

(OLR) of 10, 20, and 40 g L�1 volatile solids (VS) were tested using manure:SRM (wt/wt) mixtures

of 100:0 (control), 90:10, 80:20, 60:40, and 50:50 using five 2 L continuously stirred biodigesters

operating at 55 �C.Gas sampleswere taken daily todetermine hydrogenproduction, andslurry

samples were analyzed daily for volatile fatty acid (VFA) concentration, total ammonia

nitrogen (TAN), and VS degradation. Hydrogen production (mL g�1 VS fed) varied quadratically

according to OLR (P< 0.01), with maximum production at OLR20, while production decreased

linearly (P< 0.0001) asSRMconcentration increased. Reducedhydrogenproduction associated

with SRM inclusion at>10% VS may be attributed to a rapid increase in TAN (r¼�0.55) or other

inhibitors such as long chain fatty acids. Reduced hydrogen production (P< 0.01) at OLR40

versus OLR20 may be related to increased rate of VFA accumulation and final VFA concen-

tration (P< 0.001), as well as inhibition due to hydrogen accumulation (P< 0.001). Biohydrogen

production from SRMco-digestedwith cattlemanure may not befeasible onanindustrialscale

due to reduced hydrogen production with increasing levels of SRM.

Crown Copyright ª 2009 Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu.

All rights reserved.

1. Introduction In order to collect hydrogen as an end product of fermen-

Hydrogen, which is a clean and renewable fuel with high

energy density (122 kJ g�1), has the potential to become a green

source of energy in the future if renewable production levels

can be increased in tandem with the development of efficient

storage technology. Hydrogen can be produced biologically

through the anaerobic digestion (AD) of organic waste, coupling

energy production with waste treatment in a similar fashion to

methane producing AD systems [1]. In contrast to methane

production via AD, which results principally in the conversion

of biodegradable substrates to CH4 and CO2, hydrogen is an

intermediate product of acidogenesis and acetogenesis in the

anaerobic fermentation pathway.

0; fax: þ1 403 352 4526.(T.A. McAllister).ht ª 2009 Published by Else

tation, consumption of hydrogen by methanogenic bacteria

must be prevented [2]. This can be accomplished by reducing

the fermentation pH to �6.0 [1], increasing dilution rates or

shortening retention time [3]. Treatment of the inoculum with

heat can also be used to inactivate methanogenic bacteria

within the overall bacterial community [4]. Removal of meth-

anogenic bacteria from the AD system alters the overall efficacy

of degradation, resulting in elevated concentrations of volatile

fatty acids (VFA) which in turn inhibit continued substrate

degradation [5]. As such, removal of chemical oxygen demand,

which is used as an indicator of the efficiency of biodegrada-

tion, is typically less than 20% in fermentative biohydrogen

systems [6]. The conversion of substrate to VFA under hydrogen

vier Ltd on behalf of Professor T. Nejat Veziroglu. All rights reserved.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 9 9 – 1 1 0 51100

producing conditions has been adapted for use in two-stage AD

systems, in which acid production occurs during the first step

and subsequent methanogenesis takes place in a separate

reactor in the second step [7]. Depending on the substrate, two-

stage processes enable a higher conversion of biomass to

biogas than single stage digestion, making the process more

efficient and economical [8].

The microbial community present during the production of

hydrogen differs from that present during methane produc-

tion, particularly if fermentation is occurring under conditions

of low pH or if the inoculum is heat-treated [9]. This may result

in altered degradation efficiency and could influence the

characteristics of the effluent generated during AD. The effi-

cacy of pathogen inactivation during AD is typically consid-

ered to be based on a time–temperature relationship [10].

However, the efficacy of pathogen inactivation during AD

under hydrogen producing conditions has not been well

studied [11].

Specified risk materials (SRM) are a waste byproduct of the

agricultural slaughter industry, consisting of tissues which

are deemed most likely to contain prions. Prions are the

infectious protein particles (e.g., PrPBSE, PrPSc, PrPCWD)

thought to cause transmissible spongiform encephalopathies

such as bovine spongiform encephalopathy in cattle, scrapie

in sheep, and chronic wasting disease in cervids, respec-

tively. Bovine SRM include the brain, spinal cord, dorsal

ganglia, distal ileum, eyes, tongue, and tonsils of slaughtered

cattle �30 months of age, and the distal ileum of cattle <30

months of age. These tissues are collected and disposed of in

an effort to prevent the infective agent from entering the

food chain [12]. Cattle with BSE have been identified in

Canada, the United States, Europe, and Japan, the disease

believed to have arisen mainly from consumption of meat

and bone meal (MBM) contaminated with PrPBSE. To prevent

future outbreaks of BSE in Canada, a feed ban was instituted

in 1997 that prohibits the feeding of ruminant MBM to cattle.

This ban was enhanced in 2003 to restrict the feeding of SRM

to all livestock, as well as to exclude SRM as a fertilizer.

Consequently, the z250,000 tonnes of SRM produced annu-

ally in Canada has become an economic liability for the beef

industry as much of it is currently rendered and landfilled,

a practice that is environmentally questionable [13,14]. As

a result, alternative methods of SRM disposal which will be

both economically sound and environmentally responsible

are being investigated. We have previously investigated the

biodigestion of SRM as a means of methane production [15],

as well as the composting of SRM and the value of the

finished compost as a fertilizer [16,17].

The purpose of this study was to evaluate the hydrogen

production potential of SRM when co-digested with cattle

manure at different ratios and with different organic loading

rates (OLR) during AD.

2. Materials and methods

2.1. Materials

A 5� 3 factorial experiment evaluating the hydrogen produc-

tion potential of five ratios (wt:wt dry organic matter) of

manure:SRM (100:0, 90:10, 80:20, 60:40, 50:50) at three different

organic loading rates (OLR, 10, 20, 40 g volatile solids L�1) was

performed using five 2-L bench scale batch fermenters (Mini-

fors, Infors AG, Switzerland). Each of the 15 treatment types

was repeated in quadruplicate, for a total of 60 individual runs

(12 batches using 5 fermenters). Digesters were mixed

continuously at 600 rpm and maintained at 55 �C for a total of 4

days for each replicate.

Specified risk materials were obtained from a slaughter-

house and homogenized using a Waring blender. The homog-

enate was divided into aliquots (250 mL) which were frozen at

�21 �C until subsequent use. A subsample was evaluated for

volatile solid (VS) content according to standard methods [18].

Fresh cattle manure was collected from the concrete pad of

a feedlot to exclude bedding material prior to each batch run.

Manure was evaluated for VS content, and stored at 4 �C until

use (<72 h). Immediately prior to fermentation, cattle manure

was added to an aluminum tray and placed in an oven set to

90 �C for 3 h in order to inactivate methanogens as described by

Oh et al. [19]. Aliquots of manure, SRM, and distilled water were

combined and mixed in a Waring blender to produce 1.6 L of

slurry with the desired manure:SRM ratios and OLR prior to

addition to the fermenters. Helium was used to flush the

headspace of each fermenter prior to sealing. Slurry samples

were taken daily, analyzed for pH, and then frozen at�21 �C for

subsequent analysis.

2.2. Analyses

Total gas production was monitored daily using a water

displacement apparatus. Gas samples were taken daily and

stored in evacuated 6.8 mL Exetainers� (Labco Limited, UK)

until analyzed by gas chromatography (GC). The concentra-

tions of H2, N2, CO2, and CH4 were determined using a 2-

channel GC (Varian 4900 micro GC, Varian Inc., Palo Alto, CA)

equipped with a thermal conductivity detector and

a 2 m� 2 mm (inside diameter) stainless steel column packed

with Porapak N (80–100 mesh). Injector and column tempera-

tures were kept at 110 �C and 40 �C, respectively, for H2, and N2

analysis on Channel A, and 110 �C and 60 �C, respectively, for

CH4 and CO2 analysis on Channel B. Channel A used argon as

a carrier gas and helium was the carrier gas for Channel B, with

the carrier gasses for both channels maintained at a static

pressure of 150 kPa.

Slurry aliquots were analyzed for VS according to standard

methods [18]. Samples for VFA analysis were centrifuged at

13,000 g for 10 min, followed by acidification of the superna-

tant (1000 mL) with 25% m-HPO3 (200 mL). Samples were allowed

to stand for 30 min and then centrifuged again at 13,000 g for

10 min. A 1000 mL sample was used for VFA determination

using crotonic acid as an internal standard. VFA (acetate,

propionate, butyrate, isobutyrate, valerate, isovalerate, and

caproate) were analyzed using a Varian 3800 GC (Varian Inc.,

Palo Alto, CA) equipped with a flame ionization detector at

250 �C and a fused-silica capillary column (25 m� 0.32 mm

WCOT FFAP-CB). The oven temperature program was 80 �C for

5 min, ramp 20 �C min�1 to 225 �C for 8 min. The injector was

held at 280 �C and helium was used as the carrier gas with

a flow rate of 5 mL min�1. Total VFA concentration reported

was the sum of the concentrations of those VFA analyzed.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 9 9 – 1 1 0 5 1101

Slurry samples for NH4þ analysis were first centrifuged (10 min

at 13,000 g), and the supernatant was diluted either 50 or 100

times in deionized water to a final volume of 5 mL. Concen-

tration of NH4þ was determined colorimetrically at 650 nm

using an Auto Analyzer 3 (Branþ Luebbe, Germany).

Statistical analyses were performed using the general

linear models module of StatSoft Statistica version 8.0.

3. Results and discussion

3.1. Hydrogen concentration and production

Hydrogen concentration (%) in produced biogas decreased

linearly with both OLR (P< 0.001) and manure:SRM (P< 0.01)

over time (Fig. 1), with no interaction between the two (P> 0.05).

No methane was detected from the biogas in this study, which

suggests that the heat treatment employed had effectively

inactivated methanogens. In all treatments, maximum

hydrogen concentrations (H2max) were observed after 1 d, after

which point the concentration decreased (Fig. 1a and b).

Differences in hydrogen concentration among OLR treatments

were evident at 1 (P< 0.01) and 2 d (P< 0.001), but not for

Time (d)

0 1 2 3 4 5

H2 C

once

ntra

tion

(%)

0

10

20

30

40

50

OLR10OLR20OLR40

a

Time (d)

0 1 2 3 4 5

H2 C

once

ntra

tion

(%)

0

10

20

30

40

50

100:0 90:10 80:20 60:40 50:50

b

Fig. 1 – Variations in hydrogen concentration (% of total

biogas) over time in response to OLR (g LL1 VS fed) (a) and

manure:SRM (b). Values are means with standard error bars.

subsequent time intervals (P> 0.05). Hydrogen concentration

decreased most rapidly for OLR40 (slope¼�11.2) followed by

OLR20 (slope¼�8.9) and OLR10 (slope¼�5.7), respectively

(Fig. 1a). Based on manure:SRM, hydrogen concentration

decreased most rapidly for 90:10 (slope¼�10.4) followed by

100:0 (slope¼�8.9), 60:40 (slope¼�8.4), 80:20 (slope¼�8.3),

and 50:50 (slope¼�7.0) (Fig. 1b).

Maximum hydrogen concentration (%, denoted H2max)

showed 2-factor interactions between OLR and manure:SRM

(P< 0.001) (Table 1). H2max increased both linearly and

quadratically (P< 0.001) with increasing OLR. Conversely,

a linear decrease in H2max (P< 0.001) was observed as SRM

concentration increased. The greatest H2max (49.9%) was ach-

ieved with the 100:0 treatment at OLR40. The 60:40 treatment at

OLR10 had the lowest H2max (27.7%).

The total volume of hydrogen produced increased with

increasing OLR for all treatments tested (data not shown).

However, hydrogen production (mL H2 g�1 VS fed) was influ-

enced by both OLR and manure:SRM, with no interaction

between these two variables observed (Table 1). Hydrogen

production from the perspective of OLR was quadratic

(P< 0.001) with the greatest production found at OLR20. Inclu-

sion of SRM resulted in a linear (P< 0.001) decrease in hydrogen

production, although the difference in production between

100:0 and 90:10 was not significant (P> 0.05). At OLR10, differ-

ences in hydrogen production between manure:SRM treat-

ments were not significant (P> 0.05). However, differences

between manure:SRM treatments were significant at OLR20

(P< 0.01) and OLR40 (P< 0.001), with maximum hydrogen

production obtained when manure:SRM was 90:10 at both

OLR20 (33.1 mL g�1 VS fed) and OLR40 (27.3 mL g�1 VS fed)

(Table 1).

3.2. VS degradation

The percentage of VS degraded was dependent on OLR, dis-

playing a quadratic effect (P< 0.01) with maximum degra-

dation (41%) observed for the 90:10 treatment at OLR10

(Table 1).

3.3. VFA production

Total VFA production (Table 1) was dependent upon OLR

(P< 0.001) and manure:SRM (P< 0.01), with no interactions

between the two main effects (P> 0.05). Total VFA production

increased linearly (P< 0.001) with increasing OLR and

decreased linearly with the inclusion of SRM as substrate

(P< 0.01). The accumulation of VFA over time varied signifi-

cantly from the perspective of OLR (P< 0.001), with the rate of

increase greatest at OLR40 (slope¼ 6.2) and relatively less at

OLR20 (slope¼ 3.6) and OLR10 (slope¼ 1.4) (Fig. 2a). Variations

in total VFA accumulation over time were not different

(P> 0.05) among manure:SRM treatments (Fig. 2b). The overall

increase in total VFA as a percentage of the initial concen-

tration was not affected (P> 0.05) by OLR or manure:SRM.

Acetic acid was the predominant VFA present at 0 h for all

treatments, but declined after 1 d of AD (Fig. 2c). At 1 d, butyric

acid level increased for all treatments (Fig. 2d), followed by

a stabilization of the ratio of acetic acid to butyric acid (Fig. 2c

and d).

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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 9 9 – 1 1 0 51102

3.4. Total ammonia nitrogen production

Variation in TAN production was dependent upon OLR

(P< 0.001) and manure:SRM (P< 0.001), with no interaction

(P> 0.05) observed (Fig. 3a,b). TAN concentration increased

with increasing OLR, displaying a linear (P< 0.001) and

quadratic (P< 0.05) relationship. TAN concentration increased

linearly (P< 0.001) with increasing inclusion of SRM as

substrate. The overall increase in TAN concentration as

a percentage of the initial concentration was dependent on

manure:SRM (P< 0.001), linearly increasing with SRM concen-

tration, but was not dependent on OLR (P> 0.05).

3.5. Hydrogen production potential of SRM

The greatest hydrogen production (mL g�1 VS fed) was observed

for the 100:0 and 90:10 treatments, with increasing concentra-

tions of SRM resulting in reduced production (Table 1). It is

possible that inclusion of SRM reduced the diversity and

number of bacteria that were introduced into the biodigesters.

Lower microbial density could reduce the degradation of

substrate, and thus the amount of hydrogen produced.

However, our data show that there was no significant differ-

ence in VS degradation based on manure:SRM (Table 1). This

suggests that the microbial communities in each treatment

were capable of degrading VS to a similar extent despite

possible differences in the density and diversity of microbes

present in the initial inocula. This could be a function of the

degradability of the substrate, given that protein – and lipid-

rich SRM is more readily degradable than is cellulosic manure

[15]. This inherent dissimilarity could mask any potential

differences in overall degradation as a result of differences in

composition or microbial density. The fact that manure:SRM

did not influence VFA accumulation also suggests that

microbial populations had similar capacities to degrade the

substrates provided (Fig. 2b).

Free NH3 at concentrations exceeding 80 mg L�1 is known

to be toxic to the members of the microbial community

involved in methane production during AD, whereas NH4þ is

not inhibitory until concentrations exceed 1500 mg L�1 [20].

The effect of NH3 on hydrogen production via AD has been less

well studied. Zhu et al. [21] found that NH4þ concentrations of

170 mg L�1 did not inhibit hydrogen production. In contrast,

Wang et al. [22] showed that hydrogen production was

reduced when initial NH4þ concentrations exceeded

100 mg L�1. Salerno et al. [23] reported that total hydrogen

production was not inhibited by even higher (10 g L�1)

concentrations of ammonia, but that the lag time for

hydrogen production was substantially increased. Because

NH3 is produced during the deamination of amino acids, and

given the protein-rich nature of SRM [24], the observed

increase in TAN concentration based on the level of SRM

inclusion was anticipated and agrees with our previous find-

ings [15]. Given the acidic conditions present in this study

(Table 1) and ammonia’s pKa of 9.25, >99% of the ammonia

produced during AD would have been present in the ionized

form [25], indicating that inhibitory levels of free NH3, as

described above, were not reached. However, a negative

correlation (r¼�.55; P< 0.05) was observed between the rate

of hydrogen production (mL g�1 VS fed) and the increase in

Time (d)

0 1 2 3 4 5

Tota

l VFA

Con

cent

ratio

n (m

M)

0

10

20

30

40

50

OLR10 OLR20 OLR40

a

Time (d)

0 1 2 3 4 5

Tota

l VFA

Con

cent

ratio

n (m

M)

0

5

10

15

20

25

30

35

100:0 90:10 80:20 60:40 50:50

b

Time (d)

0 1 2 3 4 5

Acet

ic A

cid

(% T

otal

VFA

)

50

60

70

80

90

100

100:0 90:10 80:20 60:40 50:50

c

Time (d)

0 1 2 3 4 5

Buty

ric A

cid

(% T

otal

VFA

)

0

10

20

30

40

50

100:0 90:10 80:20 60:40 50:50

d

Fig. 2 – Total VFA (mM) accumulation over time in response to OLR (g LL1 VS fed) (a) and manure:SRM (b). Total VFA

concentration was initially composed almost entirely of acetic acid (c), but butyric acid levels rapidly increased in the first

24 h, then stabilized (d). Values are means with standard error bars.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 9 9 – 1 1 0 5 1103

TAN within the biodigesters. This suggests that the rapid

increase in TAN may have altered the microbial community in

some manner that was nonetheless inhibitory to hydrogen

production. Others have shown that an adequate period of

acclimation to increasing ammonia levels is necessary to

optimize hydrogen production [23], although the ammonia

concentrations observed in our study would still be consid-

ered to be relatively low. It is also possible that the negative

correlation between hydrogen production and TAN increase is

not due to a causative relationship. Rather, it may reflect an

inhibition attributable to a related metabolic process. For

example, as the lipid fraction of SRM was hydrolyzed, it is

possible that long chain fatty acids (LCFA) were released that

inhibited hydrogen production [26,27].

In the present study, hydrogen production was expected to

rank OLR40>OLR20>OLR10 as a result of the increasing

concentration of the substrate and inoculum with increasing

OLR. However, the hydrogen production of OLR40 was less

than that of OLR20, suggesting that other factors must have

contributed to the lower than expected production from

OLR40. VS degradation was actually lower for OLR20 than

OLR40, which suggests that the disparity in hydrogen

production was not a result of increased VS degradation in

OLR20. Rather, product (VFA) accumulation during degrada-

tion may have been responsible for the reduced hydrogen

production at OLR40. Total VFA concentration increased with

OLR, and the rate of increase was more rapid for OLR40 than

other treatments. Hydrogen production is a byproduct of

acidogenesis and the degradation of VFA to acetate, but the

thermodynamics of hydrogen-generating reactions are

unfavourable except under conditions of product (i.e., acetate

and hydrogen) removal [28]. Typically, methanogenic bacteria

fulfill this role by consuming acetate and hydrogen while

producing methane. However, in the absence of methano-

genic bacteria, as was the case in this study, an accumulation

of hydrogen and VFA can occur, causing inhibition of the

anaerobic degradation process [29]. The rapid increase and

higher concentration of total VFA likely accounts for the lower

hydrogen production observed with OLR40 as compared to

OLR10 and OLR20 treatments.

Further evidence of enhanced inhibition occurring at OLR40

is provided by the data obtained for H2max, which indicate that

the greatest hydrogen concentrations were observed for OLR40

(Fig. 3a). Given that high hydrogen concentrations are

Time (d)

0 1 2 3 4 5

TAN

(mg

L-1)

0

50

100

150

200

250

300

OLR10 OLR20 OLR40

a

Time (d)

0 1 2 3 4 5

TAN

(mg

L-1)

0

20

40

60

80

100

120

140

160

180

200

100:0 90:10 80:20 60:40 50:50

b

Fig. 3 – Changes in total ammonia nitrogen concentration

over time in response to OLR (g LL1 VS fed) (a) and

manure:SRM (b). Values are means with standard error bars.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 9 9 – 1 1 0 51104

inhibitory to further hydrogen production [30], the OLR40

treatment would be expected to be the most inhibited based on

H2max values. This is supported by the fact that after H2max was

reached, the rate of decrease in hydrogen concentration was

greatest for OLR40 (Fig. 3). Of the treatments examined, OLR10

also exhibited the lowest H2max and rate of decline in hydrogen

concentration, suggesting that hydrogen accumulation did not

inhibit hydrogen production. The level of substrate dilution

plays a key role in determining hydrogen production from

anaerobic systems [29]. The optimal OLR for hydrogen

production lies between 10 g VS L�1 and 40 g VS L�1, with

20 g VS L�1 demonstrating the greatest production in the

conditions tested here. This agrees with the findings of Argun

et al. [29], who found that the optimal substrate concentration

using powdered wheat starch was 20 g L�1.

Our findings suggest that SRM may not be a good candidate

for biohydrogen production using thermophilic AD, and

conversely, AD under hydrogen-producing conditions may not

be an ideal candidate for SRM treatment and disposal. Given

the reduced hydrogen production obtained at SRM levels

exceeding 10% of the substrate, industrial scale applications

may be unfeasible unless large quantities of manure are

available for co-digestion with SRM. It is possible that

enhanced hydrogen production from SRM could be achieved

using a continuous flow system in which acclimation of the

microbial community to both the substrate and end-product

accumulation (i.e., VFA, NH4þ) could occur. However, if LCFA

inhibition was responsible for reduced hydrogen production,

acclimation would not be expected to occur and hydrogen

production would remain poor [26]. Our previous findings

regarding production of methane from SRM co-digested with

manure indicated that biogas was enhanced more by the

inclusion of SRM at 37 �C than 55 �C [15]. It is possible that

mesophilic biohydrogen production would produce greater

yields than were observed in this study. However, we have

observed previously that hydrogen production was greater

under thermophilic conditions in AD that contained only heat-

treated manure [5]. Future studies could look at optimizing AD

temperature for biohydrogen production, or assessing the

feasibility of employing a two-stage system for SRM treatment

if hydrogen is a desired product of SRM treatment and

disposal.

4. Conclusions

The results of this study indicate that under thermophilic

conditions, including SRM as substrate at concentrations

>10% of total VS during manure-based AD reduced hydrogen

production. Product accumulation (i.e., VFA, NH4þ, LCFA) may

be responsible for the reduced hydrogen production associ-

ated with greater concentrations of SRM. SRM treatment via

AD may be more suitable to single stage methanogenic

digestion or to a two-stage hydrogen–methane process to

maximize total biogas yield.

Acknowledgements

Funding for this project was provided by the Specified Risk

Material Program of Agriculture and Agri-Food Canada, and the

Program of Energy Research and Development through Natural

Resources Canada and Agriculture and Agri-Food Canada. The

authors thank B. Hill and W. Smart for their technical

assistance.

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