Waste Management xxx (2015) xxxxxx

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

Anaerobic digestibility of beef hooves with swine manure or

slaughterhouse sludge
Yun Xia a,d, Ding-Kang Wang a, Yunhong Kong a, Emilio M. Ungerfeld b, Robert Seviour c, Daniel I. Mass d,
a

Key Laboratory of Special Biological Resource Development and Utilization of Universities of Yunnan Province, Kunming University, Kunming, China
Instituto de Investigaciones Agropecuarias, INIA Carillanca, km 10 Camino Cajn, Vilcn, Regin de la Araucana, Chile
c
Microbiology Dept., La Trobe University, Bundoora, Victoria, Australia
d
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, Quebec, Canada
b

a r t i c l e

i n f o

Article history:
Received 12 July 2014
Accepted 16 December 2014
Available online xxxx
Keywords:
Anaerobic digestion
a-Keratin
Beef hooves degradation
Swine manure
Slaughterhouse sludge
Fluorescence in situ hybridization (FISH)

a b s t r a c t
Anaerobic digestion is an effective method for treating animal by-products, generating at the same time
green energy as methane (CH4). However, the methods and mechanisms involved in anaerobic digestion
of a-keratin wastes like hair, nails, horns and hooves are still not clear. In this study we investigated the
feasibility of anaerobically co-digesting ground beef hooves in the presence of swine manure or slaughterhouse sludge at 25 C using eight 42-L Plexiglas lab-scale digesters. Our results showed addition of
beef hooves statistically signicantly increased the rate of CH4 production with swine manure, but only
increased it slightly with slaughterhouse sludge. After 90-day digestion, 73% of beef hoof material added
to the swine manure-inoculated digesters had been converted into CH4, which was signicantly higher
than the 45% level achieved in the slaughterhouse sludge inoculated digesters. BODIPY-Fluorescent
casein staining detected proteolytic bacteria in all digesters with and without added beef hooves, and
their relative abundances corresponded to the rate of methanogenesis of the digesters with the different
inocula. Fluorescence in situ hybridization in combination with BODIPY-Fluorescent casein staining identied most proteolytic bacteria as members of genus Alkaliphilus in the subfamily Clostridiaceae 2 of family Clostridiaceae. They thus appear to be the bacteria mainly responsible for digestion of beef hooves.
Crown Copyright 2014 Published by Elsevier Ltd. All rights reserved.

1. Introduction
Large amounts of organic by-products are produced from
slaughterhouse and meat-processing industries because of the high
demand for meat resulting from increased global populations and
their associated economic growth (Tritt, 1992; Tritt and
Schuchardt; 1992; Cho et al., 1995). Canada produced 3.5 billion
pounds of beef (1.6 billion kilograms carcass weight) in 2006, which
contributed $26 billion to its economy (Canfax, Statistics Canada
2006). Consequently, large quantities of animal by-products (ABPs)
including carcasses and products of animal origin rich in proteins
and lipids (Edstrm et al., 2003; Resch et al., 2006), which constitute approx. 25% of total animal weight not intended for human
consumption, are generated [Reg. (EC) No. 1069/2009 replacing
the ABP directive 1774/2002]. Traditionally ABPs have been treated
by rendering processes and used as animal fodder, thus providing
Corresponding author at: Dairy and Swine Research and Development Centre,
Agriculture and Agri-Food Canada, 2000 College Street, Sherbrooke J1M 0C8,
Quebec, Canada. Tel.: +1 819 565 9171; fax: +1 819 564 5507.
E-mail address: Daniel.Masse@agr.gc.ca (D.I. Mass).

slaughterhouses with additional valuable sources of income

(Auvermann et al., 2004). However, outbreak of diseases like bovine
spongiform encephalopathy in cattle and CreutzfeldJacob in
humans has prohibited their utilization as animal fodder. Consequently disposal of ABPs has emerged as a major concern in the
meat industry from an increasing awareness of the need for stringent hygiene regulations and tighter process control (Hejnfelt and
Angelidaki, 2009).
Anaerobic digestion is an effective method of treating ABPs, at the
same time generating energy in the form of CH4, and the resulting
nutrient rich digestion efuents can be used as agricultural fertilizers (Salminen and Rintala, 2002). Successful methanogenesis from
anaerobic digestion of poultry by-products like blood, meat and
bones (Salminen and Rintala, 2002), rumen digesta and cattle blood
(Banks and Wang, 1999), blood and category 3 materials (http://
europa.eu/legislation_summaries/food_safety/animal_nutrition/
f81001_en.htm) from pigs have been reported (Kirchmayr et al.,
2007). However, little information exists for the degradation of
keratin-rich materials like feathers, hairs, hooves, horns or toenails.
Keratins are categorized as a-keratin (e.g. bovine hooves) and
b-keratin (e.g. chicken feathers) consisting of tightly packed protein

http://dx.doi.org/10.1016/j.wasman.2014.12.017
0956-053X/Crown Copyright 2014 Published by Elsevier Ltd. All rights reserved.

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chains in a-helices and b-sheets respectively (Parry and North,

1998). Both are stabilized by high degrees of disulde and hydrogen
bond cross-linking, as well as hydrophobic interactions, which render them insoluble and resistant to biodegradation (Fuchs, 1995).
Few studies have looked at the anaerobic digestion of keratinrich wastes for biogas production. Hejnfelt and Angelidaki (2009)
reported that hair and skin from Danish piggeries supported high
CH4 yields under thermophilic conditions (55 C) compared to
other piggery by-products. We have used untreated chicken feathers (b-keratin wastes) as a model protein for prions to investigate
the feasibility of their anaerobic co-digestion with swine manure
or slaughterhouse sludge, and found that they could be digested
effectively (Xia et al., 2011, 2012a,b), where Alkaliphilus spp. were
the main feather-hydrolysers. However, similar information for the
anaerobic digestion of a-keratin wastes such as hairs, nails, horns
and hooves is lacking.
In this study, we have investigated the feasibility of co-digesting
ground beef hooves with no pre-treatment, and with addition of
swine manure or slaughterhouse sludge at 25 C, and attempted
to identify the keratin-hydrolyzing organisms involving in their
degradation using BODIPY uorescence casein (BODIPY-FluoC)
staining combined with uorescence in situ hybridization (FISH)
(Xia et al., 2007).
2. Methods
2.1. Preparation of bovine hoof samples
Fresh bovine hooves (10 kg) were collected from a local slaughterhouse (Colbex, Levinoff, QC, Canada) and transferred to the laboratory within 4 h. The hooves were then washed with deionized
water and dried at 45 C in a Unitherm dryer (Construction CQLTD,
England). The weight of each dryer box was recorded daily until a
constant weight for each was reached, which took about one week.
Subsequently, the hooves were cut manually into smaller pieces of
35 cm with a drill and then ground through a 2-mm screen (Thosmas-Wiley, Laboratory Mill). Samples were then ground in a blender (Vita-Mix5200, Vitamix Corporation) and passed through a
sieve with a pore-size of 500 lm. Homogenized hooves were
divided into aliquots of 33 g and placed in nitrogen-free polyester
forage bags with a pore size of 50 lm (ANKOM Technology, 2052
ONeil Road Macedon, NY14502, USA), sealed with plastic tie
wraps, and washed in a washing machine (Frigidaire, Martinez,
GA, USA) using the delicate cycle setting to remove as much
remaining residual material as possible. Washed hoof bags were
then dried at 45 C until their weights were constant (31 g each),
and were labelled and tied onto a steel stick and placed into the
digester (see below).
2.2. Digestion experiments
Two 7-m3 semi-industrial scale digesters (Mass et al., 1996)
were used to stabilize fresh swine manure collected from a commercial pig farm (Sherbrooke, Quebec), and slaughterhouse sludge
obtained from a commercial cattle slaughterhouse (Colbex, Levinoff, Quebec). These digesters had been operating at 25 C for more
than 2 years with a mean retention time of 14 d before the inocula
were used. Eight 42-L Plexiglas lab-scale digesters described by
Mass et al. (2001) were used for hoof incubation experiments.
Four 42-L digesters were fed 35-L of the swine manure. Two of
these were fed bovine hooves. The other two did not receive beef
hooves and served as controls. Four 42-L digesters were fed with
35-L of the slaughterhouse sludge and set up with or without beef
hooves as described for the swine manure fed digesters. Addition
of the beef hooves was achieved by adding 14 beef hoof bags per

digester, representing 28.9%, and 26.7% of the total chemical oxygen demand (COD) loading ratio for the swine manure digesters
(SMDs) and slaughterhouse sludge digesters (SSDs) respectively.
Digesters were operated in batch mode in a closed room maintained at 25 C for 90 d. Twelve hoof bags were removed at day
90 from each digester to measure beef keratin degradation. Also,
two bags were taken from each digester at different time intervals
(2225 d), sampled for BODIPY-FluoC staining, and returned
immediately. Digesters were mixed thoroughly daily and before
each sampling for 5 min using a circulation pump.
2.3. Analyses
Total solids (TS), volatile solids (VS), total suspended solids
(TSS), volatile suspended solids (VSS), total chemical oxygen
demand (COD), soluble COD of manure, and COD, dry matter,
organic matter, ash content, protein content of the raw beef hooves
were determined according to the standard methods (APHA, 1998)
as described previously (Xia et al., 2012a). Biogas composition (CH4,
CO2, H2S and H2), mixed liquor total Kjeldahl nitrogen (TKN), mixed
liquor ammonium nitrogen (NH+4-N) and soluble volatile fatty acids
(VFAs) were analyzed following procedures described by Mass et al.
(1996) as detailed previously (Xia et al., 2012a).
2.4. Bacterial sampling for BODIPY-FluoC staining
The sampling and staining of proteolytic microorganisms were
carried out according to the procedure described by Xia et al.
(2011) with a slight modication. To stain for organisms attached
to the hoof particles, fresh hoof samples (ca. 5 g) were collected
from a hoof bag after mixing carefully with a sterile glass rod at
each sampling point, drained for 35 min on a sieve and re-suspended in 50 ml bacteria-free ltrate (Xia et al., 2011). The ltrate
was prepared from the mixed liquor of the same digester by centrifugation at 1600g for 30 min and ltration through a series of lters (Whatman Nuclepore track-etched membranes) with pore
sizes of 3, 1, 0.45, and 0.2 lm, used sequentially. The mixture
was then transferred into a thick-walled polyethylene bag
(180  300 mm) and subjected to vigorous mechanical pummelling for 2 min using a Colworth Stomacher 400 (A.J. Seward &
Co., Ltd., London). The mixed liquid was subsequently ltered
through 8-layer sterilized cheese clothes and the collected ltrate
was centrifuged at 800g for 15 min to remove any large particles.
Then 200 ll of supernatant, which contained bacterial cells
washed from the beef particles, was mixed with the same volume
of freshly prepared 2  Tris-HCl (10 mM, pH 7.8) buffer before
addition of 200 ll BODIPY-FluoC working solution. The mixture
was incubated in a 10 ml serum bottle wrapped in aluminum
paper at 25 C for 30 min on a rotating platform (220 rpm). Incubated samples were evenly spread on 3-well (10 ll in each well)
Teon-printed slides (Electron Microscopy Sciences, Hateld, PA,
USA) and dried in a dark room before being mounted with CITIuor
(Electron Microscopy Sciences, Hateld, PA, USA) and examined
microscopically.
2.5. Enumeration of microorganisms positively stained with BODIPYFluoC
Numbers of proteolytic microorganisms were estimated by
counting the number of cells positively stained with BODIPY-FluoC
as a percentage of the total cell number determined after staining
with DAPI (40 , 6-diamidino-2-phenylindoledihydrochloride) in the
same microscopic eld according to the procedures described
previously (Xia et al., 2011). Cells on digital images were counted
in ImageJ (Abramoff, et al., 2004). Each image contained ca.
10003000 bacterial cells. For each enumeration, at least 60

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microscopic images taken with the 100  objective lens from at

least three slide wells, with 20 images from each well, were
examined.

If the interaction was signicant (P < 0.05), effects of one factor

were evaluated across levels of the other factor. If the interaction
was not signicant (P > 0.10), it was removed from the model.
JMP 10.0.1 was used in all statistical analyses.

2.6. FISH combined with BODIPY-FluoC staining

FISH was carried out according to Amann (1995). The oligonucleotide probe FIMs1029 (50 -GTCTCCTCTGTCCCCGAA-30 ) (Xia
et al., 2011) was labelled with Cy3 and purchased from Opron
(Huntsville, AL, USA). Probe NONEUB (Wallner et al., 1993) was used
as a negative control for detecting any general auto-uorescence.
FISH combined with BODIPY-FluoC staining for identication of
proteolytic microorganisms was carried out according to Xia et al.
(2007). Briey, cells staining positively with BODIPY-FluoC were
detected microscopically and their positions on the three-well gelatine-coated slides were recorded. Then CITIuor on the slides was
washed away by gently rinsing (for ca. 1 min) with 70% ethanol
before xation in either 4% paraformaldehyde or in 50% ethanol
for 2 h. FISH signals of the cells of interest were examined after
relocation.
2.7. Estimation of bovine hoof methanogenesis and statistical analysis
The CH4 data and initial bovine hoof masses were converted to
units (grams) of COD equivalents. Methanogenesis from bovine
hoof digestion was estimated according to the following equation
modied from OSullivan and Burrell (2007):

Methanogenesis% bovine hooves

CODCH4 hoof  CODCH4 Control=CODinitial  100%
where CODinitial is the bovine hoof COD initially added to a digester,
CODCH4hoof is the amount of CH4 COD produced from the bovine
hoof digesters and CODCH4control is the amount of CH4 COD produced from their corresponding control digesters.
For statistical analyses, the initial step was to model each
response variable for each individual digester. Negative exponential and quadratic and cubic polynomial regression models were
compared. Models that produced the lowest root mean square of
the error were selected for each response variable. As a result,
CH4 production was modelled as a negative exponential function:

3. Results and discussion

3.1. Physicochemical characterization of raw bovine hooves and
digester inocula
The physicochemical attributes of bovine hoof samples used in
this study are listed in Table 1. They contained 99% organic matter,
of which 95% was proteinaceous, and 1% fat. The COD value per g
raw hooves was 1.2 g. The physiochemical characteristics of the
stabilized swine manure and slaughterhouse sludge inocula are
listed in Table 2. Swine manure inoculum contained 16.1 g VSS
L1 mixed liquor, higher than those measured in the slaughterhouse sludge inoculum (14.09 g L1). The NH+4-N level was also
higher in the swine manure inoculum (5.58 g L1 mixed liquor)
than in the slaughterhouse sludge (3.09 g L1). Alkalinity levels followed the same pattern: 28.3 g L1 vs. 14.0 g L1 mixed liquor for
swine manure and slaughterhouse sludge, respectively.
3.2. Digesters fed with swine manure behaved better than digesters fed
with slaughterhouse sludge in methanogenesis of bovine hooves
Both the inoculum used and the addition of beef hooves affected
CH4 production at day 90. Thus, methane production (Fig. 1A and B)
at day 90 was greater with swine manure than with slaughterhouse
sludge as inoculum (average of incubations with and without beef
hooves: 162 vs. 62.2 L; SD = 1.8 L; P < 0.001; Fig. 1A and B). Methane
production at day 90 was greater with addition of beef hooves (average of incubations with swine manure and slaughterhouse sludge:
150 vs. 74.1 L; SD = 1.8 L; P < 0.001; Fig. 1A and B). There was an
interaction between the type of inoculum and the addition of beef
hooves on CH4 fractional production rate (P = 0.012). Addition of

Table 1
Physicochemical characteristic of beef hooves used in this study.

CH4 production : CH4 a b1  expct

where CH4 volume is expressed in litre, and t = time in days, and c is
the fractional rate of CH4 production. Subsequently, the fractional
rate of CH4 production, and the predicted CH4 production at 90 d
(CH4 90), estimated for each fermenter using the above model, were
modelled as a function of the inoculum (swine manure or slaughterhouse sludge), the addition of beef hooves, and their interaction:

c or CH4

90

a
b

Variable/fractiona

Beef hooves

Chemical oxygen demand (w/w DM)

Dry matter (DM %)
Organic matter (% DM)
Fat (% DM)
Crude protein (% DM)
Keratin (% DM)

1.20 0.03
91.4 0.38
99.1 0.73
1.11 0.026
94.6 0.69
87.98 0.48b

Average standard deviation based on triplicate measurements.

Keratin content was estimated according to Leach, D.H. (1980).

intercept inoculum beef hooves inoculum

 beef hooves residual

Signicance was declared at P < 0.05 and tendencies at

0.05 < P < 0.10. If the interaction was signicant, effects of one factor were evaluated across levels of the other factor. If the interaction was not signicant (P > 0.10), it was removed from the model.
pH was modelled as a quadratic polynomial, and beef hoof
methanogenesis rates, NH4-N concentrations and alkalinities were
modelled as cubic polynomials. Once polynomial responses were
parameterised, effects of the inoculum, of the addition of beef
hooves, and their interaction, on each response variable at 0, 45
and 90 d incubation, were analyzed similarly:

Table 2
Physicochemical characteristics of the anaerobic digestion inocula.

Yt i intercept inoculum beef hooves inoculum

 beef hooves residual
where Yti is equal to response of Y at time i, i = 0, 45 or 90 d.

Variablesa (in g L1 except pH)

Swine manure

Slaughterhouse sludge

Total chemical oxygen demand

Soluble chemical oxygen demand
Total solids
Total volatile solids
Volatile suspended solids
Acetic acid
Propionic acid
Isobutyric acid
Total nitrogen
Ammonia nitrogen
pH
Alkalinity (CaCO3)

36.67 6.2
9.55 2.2
38.35 7.7
22.48 4.5
16.1 4.4
0.26 0.01
0.83 0.02
0.02 0.001
6.7 1.7
5.58 2.0
8.05 2.2
28.3 5.8

40.95 7.3
2.29 0.09
37.38 5.9
24.72 3.6
14.09 2.5
0.04 0.01
0.002 0.001
0.000 0.000
4.48 0.02
3.09 0.02
7.75 1.8
14.03 2.0

Average standard deviation based on triplicate measurements.

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beef hooves increased the rate of CH4 production with swine manure
inoculum (0.026 vs. 0.013 d1; SD = 0.0015 d1; P = 0.001), but only
tended to increase it with slaughterhouse sludge (0.0053 vs.
0.0016 d1; SD = 0.0015 d1; P = 0.071). On the other hand swine
manure inoculum supported a higher beef hoof methanogenesis
rate at both day 45 (0.36% vs. 0.20%; SD = 0.0066%; P = 0.003) and
day 90 (0.73% vs. 0.45%; SD = 0.020%; P = 0.010) than that achieved
with the slaughterhouse sludge inoculum (initial beef hooves methanogenesis rate was zero for both treatments, so intercepts were not
compared). After 90-day incubation 73% and 45% of the ground
bovine hooves added to the SMDs and the SSDs respectively (each
containing 12.4 g bovine hooves L1 mixed liquor) were converted
to CH4. All these ndings would indicate that swine manure is a
more suitable inoculum than slaughterhouse sludge for co-digesting
beef hooves.
Swine manure as inoculum resulted in greater NH+4-N concentration in the mixed liquor (Fig. 1C and D) at day 0 (5559 vs. 3073
mg L1; SD = 15.1 mg L1; P < 0.001), day 45 (6010 vs. 3536 mg L1;
SD = 32 mg L1; P < 0.001) and day 90 (6241 vs. 3847 mg L1;
SD = 48.2 mg L1; P < 0.001) than with slaughterhouse sludge inoculum. Ammonia N concentration at day 0 was not affected by the
addition of beef hooves (average 4316 mg L1; SD = 15.1 mg L1;
P = 0.39), yet beef hoof addition regardless of the inoculum used
increased NH+4-N concentration at both day 45 (4942 vs.
4605 mg L1; SD = 32.0 mg L1; P < 0.001) and day 90 (5410 vs.
4678 mg L1; SD = 48.2 mg L1; P < 0.001).

Acetic acid (Fig. 1E and F) and propionic acid (Fig. 1G and H)

concentrations increased with bovine hoof addition with both
the inocula. Isobutyric acid was only detected in trace amounts
(data not shown). The pH was initially greater (7.99 vs. 7.79;
SD = 0.010; P < 0.001) than at day 45 (7.86 vs. 7.61; SD = 0.020;
P < 0.001) and day 90 (7.82 vs. 7.67; SD = 0.0042; P < 0.001) using
swine manure as inoculant. While adding beef hooves to the
digesters did not affect the initial pH (average = 7.89; SD = 0.010;
P = 0.16), their presence increased the pH at day 45 in the SMDs
(7.95 vs. 7.77; SD = 0.040; P = 0.011). The pH of the SSDs did not
vary much (average = 7.61; SD = 0.040; P = 0.20; interaction
P = 0.013). Addition of beef hooves increased the pH of the digesters with both inocula at day 90 (7.78 vs. 7.72; SD = 0.0042;
P < 0.001). At the end of digestion, more TS was left in the digesters
with hoof addition than in their respective controls [31.4(0.3) vs.
29.2(0.3) g L1 in the SMDs, 37.7(0.4) vs. 31.4(0.2) g L1 in the
SSDs].
In summary, we have demonstrated that ground bovine hooves
can be effectively digested in anaerobic digesters fed with swine
manure at 25 C. This is important because psychrotrophic anaerobic digestion (operating at 20 or 25 C) is more economic than
mesophilic (35 C) or thermophilic (55 C) digestion, especially in
Canada where the temperature falls below 20 C in many areas
most of the year. Moreover, it will also avoid the occurrence
of lowered digestion rates which are less stable in anaerobic
digesters operating at lower than 25 C (e.g. Mass et al., 1996;

Fig. 1. Chemical characterization of anaerobic digesters inoculated with swine manure or slaughterhouse sludge and with (square) or without (triangle) adding beef hooves.
(A) and (B) Cumulative methane production yields; (C) and (D) NH+4-N levels; (E) and (F) acetic acid concentrations; (G) and (H) propionic acid concentrations.

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reported that pig hair (mostly a-keratin) could be co-digested with

stabilized manure, and moreover produced more CH4 than any
other individual component of animal by-products under both
thermophilic (55 C) and mesophilic (37 C) conditions. However,
substrate digestion rate was not reported in their study so comparisons between the two studies are not appropriate.
3.3. Abundance of proteolytic organisms corresponds to the
methanogenesis rate

Fig. 2. A color-combined uorescence image showing BODIPY uorescencelabelled casein staining cells (red-colored), cells hybridizing with probe FIMs1029
(green-colored), and DAPI stained cells (blue-colored) showing that the pinkcolored rod-shaped keratin-hydrolyzing organisms (labelled by arrows) stained
positively with all three stains. The bar represents 10 lm.

Mass et al. (2001)). Furthermore, bovine hooves were more comprehensively digested in the SMDs than SSDs. This agrees with
ndings from our earlier study (Xia et al., 2012a), where co-digesting
chicken feathers (mostly b-keratin) with swine manure had a higher
methanogenesis potential than with slaughterhouse sludge.
It was clear that with both the SMDs and the SSDs, methanogenesis of the beef hooves slowed down gradually toward the
end of incubations. This may arise from an inhibition of the methanogenic communities by corresponding increases in NH+4-N levels
(Chen et al., 2008). Anaerobic protein digestion remains a challenge in processes of this kind since the NH+4-N generated is a toxic
substance commonly produced under anaerobic conditions
(Gallert and Winter, 1997). The methanogens are highly sensitive
to NH+4-N and the most likely to cease growth in its presence at
high levels. Loss of activity (57%) of methanogens has been
reported at NH+4-N concentrations ranging from 4.1 to 5.7 g L1
mixed liquor (Chen et al., 2008).
Although the NH+4-N concentrations in the SMDs were higher
than those in the SSDs, the methanogenesis rates of bovine hooves
were higher in the former than the latter. This may reect differences in physicochemical characteristics of the two inocula, and
in the composition and function of the microbiota associated with
each. We found earlier (Xia et al., 2012b) that methanogenic populations in swine manure inoculated digesters were substantially
different to those in slaughterhouse sludge inoculated digesters.
It was thought that the methanogenic populations in the SMDs
were more adapted than those in SSDs to high NH+4-N concentration (Jarrell et al., 1987). Hejnfelt and Angelidaki (2009) also

Proteolytic bacteria (PRBs) were detected using BODIPY-FluoC

staining inside the hoof bags, and also in the mixed liquors of each
digester with or without adding hooves. They were predominantly
small rods and in fewer cases, cocci (Fig. 2). The percentage abundances of the PRBs inside the hoof bags corresponded to the methanogenesis rates of the bovine hooves added into the SMDs and
SSDs (R2 = 0.91 and 0.92, respectively). Their relative abundances
inside the hoof bags in the SMDs (Fig. 3A) remained unchanged
over the rst 22 d but then increased from 6.3(0.6)% to
7.3(1.3)% of total DAPI stained cells at day 45, and then to
15.1(2.8)% at day 68, before decreasing to 12.0(3.9)% at day 90
(Fig. 3A). Similarly, the PRBs in the SSDs (Fig. 3B) did not change
in their relative abundance after the rst 22 d but they too
increased from 2.0(1.8)% to 5.6(1.7)% at day 45 and to
7.1(2.2)% at day 68, before decreasing to 5.0(1.6)% at day 90.
These data would indicate that these PRBs were the major organisms responsible for the hydrolysis of the hoof keratin. Hydrolysis
of organic substrates is considered to be the bottleneck of the
anaerobic digestion process (Batstone et al., 2009). More PRBs
meant generation of more protein hydrolysates whose further degradation would lead to production of more H2 and short chain fatty
acids. These then could be used by the methanogens as energy
source and/or carbon sources to generate CH4. That the PRBs were
more abundant in the SMDs than the SSDs also supports the view
that the physicochemical characteristics of the inoculum played a
major part in enrichment of the PRBs, as discussed above.
3.4. Identication of the proteolytic microbes responsible for digestion
of bovine hooves
Applying BODIPY-FluoC staining combined with FISH probing
revealed that the dominant (>90% of all the PRBs) rod-shaped hoof
a-keratin hydrolysers hybridized with the probe FIMs1029
designed for Alkaliphilus spp. in the subfamily Clostridiaceae 2 in
the family Clostridiaceae. Earlier (Xia et al., 2011), we found that
this group of bacteria were also b-keratin hydrolysers, thus suggesting that these uncultured Alkaliphilus proteolytic bacteria can
hydrolyze both a- and b-keratin. However, it is still unknown
whether the same Alkaliphilus populations are involved in the
hydrolysis of a- and b-keratin, or whether different populations

Fig. 3. Relationship of the abundance of proteolytic bacteria (square with black line) inside the hoof bags to hoof digestion rates (circle with broken line) in anaerobic
digesters inoculated with swine manure (A) or slaughterhouse sludge (B). Each standard deviation of the percentage values of the proteolytic bacteria was calculated based on
at 3 countings. Each was calculated from 20 enumerations (see Materials and methods for more details).

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(2015), http://dx.doi.org/10.1016/j.wasman.2014.12.017

Y. Xia et al. / Waste Management xxx (2015) xxxxxx

are responsible. Cai et al. (2008) reported that a mutant strain of

Bacillus subtilis utilized both a-keratin (from hair and wool) and
b-keratin (from feather and silk) as substrates. Chao et al. (2007)
also reported that Streptomyces sp. strain No. 16 efciently could
degrade a range of keratins including keratin azure, human hair,
and cock feather, and collagen, without strict limitations of pH,
temperature and ions. The identity of the coccus-shaped PRBs is
still to be investigated.
4. Conclusion
Ground bovine hooves with no physicochemical or enzymatic
pretreatments can be co-digested together with swine manure or
slaughterhouse sludge in an anaerobic digester at 25 C to generate
considerable amounts of methane. Swine manure is more suitable
than slaughterhouse sludge as the co-digesting inoculum, supporting higher methanogenesis rates. Bacteria belonging to the genus
Alkaliphilus in the subfamily Clostridiaceae 2 of family Clostridiaceae
were the dominant proteolytic populations observed by FISH in the
digesters with added beef hooves and with both inocula, and
should be considered as the major populations responsible for
hydrolysis of beef hooves.
Acknowledgement
This work was supported by National Natural Science Foundation of China (31360026 and 31260073).
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Please cite this article in press as: Xia, Y., et al. Anaerobic digestibility of beef hooves with swine manure or slaughterhouse sludge. Waste Management
(2015), http://dx.doi.org/10.1016/j.wasman.2014.12.017