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Anaerobic digestibility of beef hooves with swine manure or slaughterhouse sludge
Waste Management 38 (2015) 443–448
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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. Massé 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 Cajón, Vilcún, Región de la Araucanía, 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 13 January 2015 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 significantly 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 significantly 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 identified 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 Ó 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 (Edström 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: [email protected] (D.I. Massé). http://dx.doi.org/10.1016/j.wasman.2014.12.017 0956-053X/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
slaughterhouses with additional valuable sources of income (Auvermann et al., 2004). However, outbreak of diseases like bovine spongiform encephalopathy in cattle and Creutzfeld–Jacob 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 effluents 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
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chains in a-helices and b-sheets respectively (Parry and North, 1998). Both are stabilized by high degrees of disulfide 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 fluorescence casein (BODIPY-FluoC) staining combined with fluorescence 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 3–5 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 O’Neil 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 (Massé 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 Massé 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 (22–25 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 Massé 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 modification. 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 3–5 min on a sieve and re-suspended in 50 ml bacteria-free filtrate (Xia et al., 2011). The filtrate was prepared from the mixed liquor of the same digester by centrifugation at 1600g for 30 min and filtration through a series of filters (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 filtered through 8-layer sterilized cheese clothes and the collected filtrate 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) Teflon-printed slides (Electron Microscopy Sciences, Hatfield, PA, USA) and dried in a dark room before being mounted with CITIfluor (Electron Microscopy Sciences, Hatfield, 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 field 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. 1000–3000 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 significant (P < 0.05), effects of one factor were evaluated across levels of the other factor. If the interaction was not significant (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-fluorescence. FISH combined with BODIPY-FluoC staining for identification of proteolytic microorganisms was carried out according to Xia et al. (2007). Briefly, cells staining positively with BODIPY-FluoC were detected microscopically and their positions on the three-well gelatine-coated slides were recorded. Then CITIfluor on the slides was washed away by gently rinsing (for ca. 1 min) with 70% ethanol before fixation 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 modified from O’Sullivan and Burrell (2007):
Methanogenesis% bovine hooves ¼ ðCODCH4 hoof CODCH4 ControlÞ=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 ¼ a þ b½1 expðctÞ 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).
¼ intercept þ inoculum þ beef hooves þ inoculum beef hooves þ residual
Significance was declared at P < 0.05 and tendencies at 0.05 < P < 0.10. If the interaction was significant, effects of one factor were evaluated across levels of the other factor. If the interaction was not significant (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 ¼ intercept þ inoculum þ beef hooves þ inoculum beef hooves þ residual where Yti is equal to response of Y at time i, i = 0, 45 or 90 d.
a
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 findings 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. Massé 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 fluorescence image showing BODIPY fluorescencelabelled 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.
Massé et al. (2001)). Furthermore, bovine hooves were more comprehensively digested in the SMDs than SSDs. This agrees with findings 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 reflect 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 first 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 first 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. Identification 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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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 efficiently 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). References Abramoff, M.D., Magalhaes, P.J., Ram, S.J., 2004. Image processing with image. J. Biophotonics Intern. 11, 36–41. Amann, R.I., 1995. In situ hybridization of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes, part 3.3.6. In: Akkermans, A.D.L., van Elsas, J.D., de Bruijn, F.J. (Eds.), Molecular Microbial Ecological Manual. Kluwer Academic Publications, London, pp. 1–15. APHA, AWWA, WPCF, 1998. In: Clesceri, L.S., Greenberg, A.E., Eaton, A.D., Franson, M.A.H. (Eds.), Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC. Auvermann, B., Kalbasi, A., Ahmed, A., 2004. In Carcass disposal: a comprehensive review. In: Proceedings of the August 2004 Carcass disposal working group, USDA APHIS Cooperative Agreement Project, National Agricultural Biosecurity Center Consortium, Kansas State University. Banks, C.J., Wang, Z., 1999. Development of a two phase anaerobic digester for the treatment of mixed abattoir wastes. Water Sci. Technol. 40, 69–76. Batstone, B.J., Tait, S., Starrenburg, D., 2009. Estimation of hydrolysis parameters in full-scale anaerobic digesters. Biotechnol. Bioeng. 102, 1513–1520. Cai, C.G., Lou, B.G., Zheng, X.D., 2008. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis. J. Zhejiang Univ.: Sci. B 9, 60–67. Chao, Y.P., Xie, F.h., Yang, J., Lu, J.h., Qian, S.j., 2007. Screening for a new Streptomyces strain capable of efficient keratin degradation. J. Environ. Sci. 19, 1125–1128.
Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: a review. Bioresour. Technol. 99, 4044–4064. Cho, J.K., Park, S.C., Chang, H.N., 1995. Biochemical methane potential and solid state anaerobic digestion of Korean food wastes. Bioresour. Technol. 52, 245– 253. Edström, M., Nordberg, A., Thyselius, L., 2003. Anaerobic treatment of animal byproducts from slaughterhouses at laboratory and pilot scale. Appl. Biochem. Biotechnol. – Part A Enzyme Eng. Biotechnol. 109, 127–138. Fuchs, E., 1995. Keratins and the skin. Annual Rev. Cell Development Biol. 199, 123– 154. Gallert, C., Winter, J., 1997. Mesophilic and thermophilic anaerobic digestion of source-sorted organic wastes: effect of ammonia on glucose degradation and methane production. Appl. Microbiol. Biotechnol. 48, 405–410. Hejnfelt, A., Angelidaki, I., 2009. Anaerobic digestion of slaughterhouse by-products. Biomass Bioenergy 33, 1046–1054. Jarrell, K.F., Saulnier, M., Ley, A., 1987. Inhibition of methanogenesis in pure cultures by ammonia, fatty acids, and heavy metals, and protection against heavy metal toxicity by sewage sludge. Can. J. Microbiol. 33, 551–555. Kirchmayr, R., Resch, C., Mayer, M., Prechtl, S., Faulstich, M., Braun, R., Wimmer, J., 2007. Anaerobic degradation of animal by-products. In: Oreopoulou, V., Russ, W. (Eds.), Utilization of by-Products and Treatment of Waste in the Food Industry. Springer, New York, pp. 159–191. Massé, D.I., Patni, N.K., Droste, R.L., Kennedy, K.J., 1996. Operation strategies for psychrophilic anaerobic digestion of swine manure slurry in sequencing batch reactors. Can. J. Civil. Eng. 23, 1285–1294. Massé, D.I., Massé, L., Verville, A., Bilodeau, S., 2001. The start-up of anaerobic sequencing batch reactors at 20 °C and 25 °C for the treatment of slaughterhouse wastewater. J. Chem. Technol. Biotechnol. 76, 393–400. O’Sullivan, C.A., Burrell, P.C., 2007. The effect of media changes on the rate of cellulose solubilisation by rumen and digester derived microbial communities. Waste Manage. 27, 1808–1814. Parry, D.A.D., North, A.C.T., 1998. Hard a-keratin intermediate filament chains: Substructure of the N-and C-terminal domains and the predicted structure and function of the C-terminal domains of type I and type II chains. J. Struct. Biol. 122, 67–75. Resch, C., Grasmug, M., Smeets, W., Braun, R., Kirchmayr, R., 2006. Optimised anaerobic treatment of house-sorted biodegradable waste and slaughterhouse waste in a high loaded half technical scale digester. Water Sci. Technol. 53 (8), 213–221. Salminen, E., Rintala, J., 2002. Anaerobic digestion of organic solid poultry slaughterhouse waste – a review. Bioresour. Technol. 83, 13–26. Tritt, W.P., 1992. The anaerobic treatment of slaughterhouse wastewater in fixedbed reactors. Bioresour. Technol. 41, 201–207. Tritt, W.P., Schuchardt, F., 1992. Materials flow and possibilities of treating liquid and solid wastes from slaughterhouses in Germany. A review. Bioresour. Technol. 41, 235–245. Wallner, G., Amann, R., Beisker, W., 1993. Optimizing fluorescent in sit hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14, 136–143. Xia, Y., Kong, Y., Nielsen, P.H., 2007. In situ detection of protein-hydrolysing microorganisms in activated sludge. FEMS Microbiol. Ecol. 60, 156–165. Xia, Y., Massé, D.I., McAllister, T.A., Beaulieu, C., Talbot, G., Kong, Y., Seviour, R., 2011. In situ identification of keratin-hydrolyzing organisms in swine manure inoculated anaerobic digesters. FEMS Microbiol. Ecol. 78, 451–462. Xia, Y., Massé, D.I., McAllister, T.A., Beaulieu, C., Ungerfeld, E., 2012a. Anaerobic digestion of chicken feather with swine manure or slaughterhouse sludge for biogas production. Waste Manage. 32, 404–409. Xia, Y., Massé, D.I., McAllister, T.A., Kong, Y., Seviour, R., Beaulieu, C., 2012b. Identity and diversity of archaeal communities during anaerobic co-digestion of chicken feathers and other animal wastes. Bioresour. Technol. 110, 111–119.
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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. Massé 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 Cajón, Vilcún, Región de la Araucanía, 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 13 January 2015 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 significantly 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 significantly 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 identified 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 Ó 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 (Edström 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: [email protected] (D.I. Massé). http://dx.doi.org/10.1016/j.wasman.2014.12.017 0956-053X/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
slaughterhouses with additional valuable sources of income (Auvermann et al., 2004). However, outbreak of diseases like bovine spongiform encephalopathy in cattle and Creutzfeld–Jacob 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 effluents 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
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chains in a-helices and b-sheets respectively (Parry and North, 1998). Both are stabilized by high degrees of disulfide 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 fluorescence casein (BODIPY-FluoC) staining combined with fluorescence 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 3–5 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 O’Neil 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 (Massé 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 Massé 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 (22–25 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 Massé 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 modification. 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 3–5 min on a sieve and re-suspended in 50 ml bacteria-free filtrate (Xia et al., 2011). The filtrate was prepared from the mixed liquor of the same digester by centrifugation at 1600g for 30 min and filtration through a series of filters (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 filtered through 8-layer sterilized cheese clothes and the collected filtrate 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) Teflon-printed slides (Electron Microscopy Sciences, Hatfield, PA, USA) and dried in a dark room before being mounted with CITIfluor (Electron Microscopy Sciences, Hatfield, 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 field 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. 1000–3000 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 significant (P < 0.05), effects of one factor were evaluated across levels of the other factor. If the interaction was not significant (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-fluorescence. FISH combined with BODIPY-FluoC staining for identification of proteolytic microorganisms was carried out according to Xia et al. (2007). Briefly, cells staining positively with BODIPY-FluoC were detected microscopically and their positions on the three-well gelatine-coated slides were recorded. Then CITIfluor on the slides was washed away by gently rinsing (for ca. 1 min) with 70% ethanol before fixation 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 modified from O’Sullivan and Burrell (2007):
Methanogenesis% bovine hooves ¼ ðCODCH4 hoof CODCH4 ControlÞ=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 ¼ a þ b½1 expðctÞ 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).
¼ intercept þ inoculum þ beef hooves þ inoculum beef hooves þ residual
Significance was declared at P < 0.05 and tendencies at 0.05 < P < 0.10. If the interaction was significant, effects of one factor were evaluated across levels of the other factor. If the interaction was not significant (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 ¼ intercept þ inoculum þ beef hooves þ inoculum beef hooves þ residual where Yti is equal to response of Y at time i, i = 0, 45 or 90 d.
a
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 findings 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. Massé 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 fluorescence image showing BODIPY fluorescencelabelled 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.
Massé et al. (2001)). Furthermore, bovine hooves were more comprehensively digested in the SMDs than SSDs. This agrees with findings 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 reflect 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 first 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 first 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. Identification 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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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 efficiently 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). References Abramoff, M.D., Magalhaes, P.J., Ram, S.J., 2004. Image processing with image. J. Biophotonics Intern. 11, 36–41. Amann, R.I., 1995. In situ hybridization of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes, part 3.3.6. In: Akkermans, A.D.L., van Elsas, J.D., de Bruijn, F.J. (Eds.), Molecular Microbial Ecological Manual. Kluwer Academic Publications, London, pp. 1–15. APHA, AWWA, WPCF, 1998. In: Clesceri, L.S., Greenberg, A.E., Eaton, A.D., Franson, M.A.H. (Eds.), Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC. Auvermann, B., Kalbasi, A., Ahmed, A., 2004. In Carcass disposal: a comprehensive review. In: Proceedings of the August 2004 Carcass disposal working group, USDA APHIS Cooperative Agreement Project, National Agricultural Biosecurity Center Consortium, Kansas State University. Banks, C.J., Wang, Z., 1999. Development of a two phase anaerobic digester for the treatment of mixed abattoir wastes. Water Sci. Technol. 40, 69–76. Batstone, B.J., Tait, S., Starrenburg, D., 2009. Estimation of hydrolysis parameters in full-scale anaerobic digesters. Biotechnol. Bioeng. 102, 1513–1520. Cai, C.G., Lou, B.G., Zheng, X.D., 2008. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis. J. Zhejiang Univ.: Sci. B 9, 60–67. Chao, Y.P., Xie, F.h., Yang, J., Lu, J.h., Qian, S.j., 2007. Screening for a new Streptomyces strain capable of efficient keratin degradation. J. Environ. Sci. 19, 1125–1128.
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