Waste Management 32 (2012) 404–409

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Waste Management

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Anaerobic digestion of chicken feather with swine manure or slaughterhousesludge for biogas production

Yun Xia a, Daniel I. Massé a,⇑, Tim A. McAllister b, Carole Beaulieu c, Emilio Ungerfeld b

a Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, 2000 College Street, Sherbrooke, Quebec, Canada J1M 0C8b Lethbridge Research Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, Alberta, Canada T1J 4B1c Département de Biologie, Université de Sherbrooke, 2500 De l’Université Boulevard, Sherbrooke, Quebec, Canada J1K 2R1

a r t i c l e i n f o

Article history:Received 29 March 2011Accepted 21 October 2011Available online 15 November 2011

Keywords:Anaerobic digestersFeather degradationSwine manureSlaughterhouse sludge

0956-053X/$ - see front matter Crown Copyright � 2doi:10.1016/j.wasman.2011.10.024

Abbreviations: COD, chemical oxygen demanSS, slaughterhouse sludge; SM, swine manure; VFA,volatile solids.⇑ Corresponding author. Tel.: +1 819 565 9171; fax

E-mail address: Daniel.Masse@agr.gc.ca (D.I. Mass

a b s t r a c t

Biogas production from anaerobic digestion of chicken feathers with swine manure or slaughterhousesludge was assessed in two separate experiments. Ground feathers without any pre-treatment wereadded to 42-L digesters inoculated with swine manure or slaughterhouse sludge, representing 37% and23% of total solids, respectively and incubated at 25 �C in batch mode. Compared to the control withoutfeather addition, total CH4 production increased by 130% (P < 0.001) and 110% (P = 0.09) in the swinemanure and the slaughterhouse sludge digesters, respectively. Mixed liquor NH4AN concentrationincreased (P < 0.001) from 4.8 and 3.1 g/L at the beginning of the digestion to 6.9 and 3.5 g/L at theend of digestion in the swine manure and the slaughterhouse sludge digesters, respectively. The fractionof proteolytic microorganisms increased (P < 0.001) during the digestion from 12.5% to 14.5% and 11.3%to 13.0% in the swine manure and the slaughterhouse sludge digesters with feather addition, respectively,but decreased in the controls. These results are reflective of feather digestion. Feather addition did notaffect CH4 yields of the swine manure digesters (P = 0.082) and the slaughterhouse sludge digesters(P = 0.21), indicating that feathers can be digested together with swine manure or slaughterhouse sludgewithout negatively affecting the digestion of swine manure and slaughterhouse sludge.

Crown Copyright � 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Four million tons of feathers are generated annually as a poultryby-product making disposal an important part of solid waste man-agement (Onifade et al., 1998; Gousterova et al., 2005). Feathersare composed of P90% keratin (Astbury and Beighton, 1961),mostly as b-keratin (Sawyer et al., 2000) that is hard to degradeby commonly known proteases due to the presence of extensivedisulfide bonds and cross-linkages (Papadopoulos et al., 1986).Traditionally, feathers are incinerated or disposed at waste dis-posal sites (Salminen and Rintala, 2002a). Disposal by such meth-ods is wasteful as feather keratin is rich in useful amino acids andgenerates green house gases (Salminen and Rintala, 2002a). Effortshave been made to develop more attractive alternatives. For exam-ple, feathers have been used in agriculture production as an animalprotein supplement after cooking under high pressure and temper-ature (Odetallah et al., 2003) or as slow-releasing nitrogen fertilizer

011 Published by Elsevier Ltd. All

d; TKN, Kjeldahl nitrogen;total volatile fatty acids; VS,

: +1 819 564 5507.é).

in agriculture (Choi and Nelson, 1996). However, use of by-products of animal production for animal feeding is becomingrestricted (Salminen and Rintala, 2002a).

Anaerobic digestion is a promising alternative for the treatmentof feathers (Bourne, 1993; De Baere, 2000) because it combinesmaterial recovery and energy production (Salminen and Rintala,2002a). During the anaerobic digestion, feather keratin is hydro-lyzed by keratinase into its constituent polypeptides and aminoacids. Amino acids are than fermented via different pathways to var-ious organic compounds (predominantly short-chain and branched-chain organic acids), ammonia, carbon dioxide and small amounts ofhydrogen and sulfur-containing compounds. Finally organic acidsand hydrogen can be used to produce methane through methano-genesis (Sawyer et al., 2000; Ramsay and Pullammanappallil, 2001).

Raw feathers without pre-treatment are poorly degraded underanaerobic conditions due to the complex, rigid and fibrous structureof feather keratin (Salminen and Rintala, 2002a). Pre-treatmentsincluding thermal, chemical and enzymatic methods have beenused in attempts to increase digestion rates (Onifade et al., 1998;Coward-Kelly et al., 2006). Feather degradation has been reportedto occur in anaerobic digesters treating poultry waste includingmanure and/or mixed fractions of bone, trimmings and offal underthermophilic (Williams and Shih, 1989) or mesophilic (Salminen

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Y. Xia et al. / Waste Management 32 (2012) 404–409 405

and Rintala, 2002b) conditions. However, apart from poultry waste,no attempts have been made to digest feathers in anaerobic digest-ers with other animal wastes. Whether the poor feather degrada-tion under anaerobic digestion is due to suboptimal ecologicalselection of keratin-degrading microorganisms is still not clear.

Recently, anaerobic digestion of feather keratin has attracted alot of attention because keratin-hydrolyzing bacteria and keratin-ase enzyme mixtures have been reported to digest prions respon-sible for transmissible spongiform encephalopathies or priondiseases including bovine spongiform encephalopathy (BSE), sheepscrapie, deer chronic waste disease and human Creutzfeldt–Jakobdisease (Gupta and Ramnani, 2006). This is due to the fact thatthe structures of prions and feather keratin share many character-istics, both being fibrous and insoluble proteins rich in b-sheet(Fraser et al., 1972; Gushterova et al., 2005). Keratinases secretedby Bacillus licheniformis strain PWD-1 (Langeveld et al., 2003) andBacillus MSK103 (Yoshioka et al., 2007) were reported to be ableto inactivate prions. BSE is the most notorious disease associatedwith prions, and has cost the Canadian cattle industry approxi-mately $6.3 billion since 2003, as a result of closed markets andconsumer concerns over food safety issues related to it (http://www.agric.gov.ab.ca). Therefore, the control and inactivation ofprions from cattle carcasses during processing have been majorpublic and animal health concerns (Hill et al., 1997).

In preliminary experiments, chicken feather was added in nylonbags to anaerobic digesters inoculated with swine manure (SM) orslaughterhouse sludge (SS) to investigate the feasibility of featherdigestion together with these substrates for biogas production.The aim of this study was to further evaluate the effect of featherdigestion on anaerobic digestion of SM and SS for biogas produc-tion. Ground feathers were directly added into the digesters.Chemical transformations with or without feather addition weremonitored and the relative abundances of proteolytic microorgan-isms were determined using BODIPY fluorescence casein (BODIPYFL casein) staining.

Table 2Physicochemical characteristics of the anaerobic digestion inocula.

2. Methods

2.1. Source and preparation of chicken feathers

Freshly plucked white chicken feathers (10 kg) were collectedfrom a slaughterhouse (Saint-Anselme, QC, Canada) and trans-ferred to the laboratory in a clean 15 L plastic barrel within 4 h.The chicken feathers were divided into 2 kg portions, placed inclean cotton bags and washed (delicate cycle) in a washing ma-chine (Frigidaire, Martinez, GA, USA) with tap water. The washedfeathers were divided into 100 g portions, placed individually intoUnithern dryer boxes (Construction CQLTD, UK) and then dried at45 �C. The weight of each dryer box was recorded daily until a con-stant weight was reached (about 8 weeks). The feather sampleswere ground through a 4-mm screen (Thomas-Wiley LaboratoryMill) before being added to the digesters. Physicochemical charac-

Table 1Physicochemical characteristics of chicken feather.

Variable/fractiona

Chemical oxygen demand (w/w, DM) 1.20 ± 0.02Dry matter (DM, %) 94.7 ± 0.44Organic matter (%, DM) 99.2 ± 0.69Fat (%, DM) 2.79 ± 0.032Crude protein (%, DM) 92.0 ± 0.48Keratin (%, DM)b 82.8 ± 0.51

a Average ± standard deviation based on at least triplicate measurements.b Keratin content was estimated according to Astbury and Beighton (1961).

teristics of the feather samples used in this study are listed inTable 1.

2.2. Digester operation

Eight 42-L Plexiglas anaerobic digesters as described previously(Masse et al., 2001) were used in this study. Adapted SM and SS,each representing 100% of the volume (35 L), were used as the inoc-ula. The physicochemical characteristics of the inocula are listed inTable 2. The SM inoculum was obtained after mixing from a com-mercial anaerobic digester of a pig farm (Sherbrooke, Quebec) byusing a submersible pump (Butt’s Pumps & Motors Ltd., ON, Can-ada). The SS inoculum was collected from a 7-m3 semi-industrial di-gester located at Dairy and Swine Research and DevelopmentCentre, Agriculture and Agri-Food Canada, Sherbrooke, Quebec,Canada treating slaughterhouse wastewater solids obtained froma commercial cattle slaughterhouse (Colbex, Levinoff, Quebec) witha retention time of 14 days.

Four 42-L digesters were inoculated with a same substrate (SMor SS) and were run in parallel. Ground feathers were added to twodigesters in duplicate. Four hundred and forty grams ground dryfeathers representing 37% (w/w) total solids (manure plus feath-ers) were added to a digester inoculated with 35 L adapted SM,and 380 g ground dry feathers representing 23% (w/w) total solids(manure plus feathers) were added to a digester inoculated with35 L adapted SS. The other two digesters containing only SM orSS were used as negative controls. All digesters were operated inbatch mode in a closed room at 25 �C for 146 days and were thor-oughly mixed 5 min daily via circulation by a pump.

2.3. Analytical procedures

Total solids, volatile solids (VS), total suspended solids and vol-atile suspended solids were determined according to standardmethods (APHA, 1998). Similarly, total chemical oxygen demand(COD); soluble COD and COD of the raw feathers were determinedaccording to the closed reflux colorimetric method also describedin the standard methods (APHA, 1998). Fat content of the rawfeathers was determined according to Schrooyen et al. (2000).Briefly, chicken feather samples (ca. 30 g) were extracted withpetroleum ether (40–60 �C) for 12 h using Soxhlet extraction andthe fat extracted was measured. Feather dry matter, organic mat-ter, ash content and protein content were determined accordingto the standard methods (APHA, 1998). Biogas composition (CH4,CO2, H2S and H2) pH, mixed liquor total Kjeldahl nitrogen (TKN),mixed liquor ammonium nitrogen (NH4AN) and soluble volatilefatty acids (VFAs) were analyzed following procedures describedby Masse et al. (1996). Biogas composition was analyzed weeklyusing a HachCarle 400 AGC gas chromatograph (Hach, Love-land

Variablesa (in g L�1 except pH) Swine manure Slaughterhouse sludge

Total chemical oxygen demand 20.6 ± 4.2 20.2 ± 3.3Soluble chemical oxygen demand 7.18 ± 1.2 2.2 ± 0.09Total solids 21.6 ± 5.7 36.0 ± 5.9Total volatile solids 11.5 ± 4.5 23.8 ± 3.6Volatile suspended solids 7.41 ± 3.4 12.0 ± 2.5Acetic acid 0.030 ± 0.01 0.04 ± 0.01Propionic acid 0.000 ± 0.000 0.005 ± 0.001Butyric acid 0.030 ± 0.01 0.000 ± 0.000Total nitrogen 5.7 ± 1.7 4.5 ± 0.02Ammonia nitrogen 4.8 ± 2.0 3.2 ± 0.02pH 7.8 ± 2.2 7.8 ± 1.8Alkalinity (CaCO3) 26.3 ± 5.8 8.7 ± 2.0

a Average ± standard deviation based on triplicate measurements.

406 Y. Xia et al. / Waste Management 32 (2012) 404–409

Co.). The value of pH was measured using a pH meter (PHM92 Lab,Radiometer Analytical, Bagsvaerd, Denmark). TKN and NH4ANwere analyzed using a Kjeltec auto-analyzer (Tecator AB. Box 70.S-26301 Hoganas, Sweden). VFA concentrations were measuredusing a Perkin–Elmer gas chromatograph (model 8310), with aDB-FFAP high resolution megabore column (Perkin–Elmer Corpo-ration; Norwalk, CT 06859, USA) connected to a flame ionizationdetector.

2.4. BODIPY fluorescence casein staining

In order to label proteolytic microorganisms, BODIPY FL casein(EnzChek Protease Assay Kit, E6638) was purchased from Molecu-lar Probe (Burlington, ON, Canada). The staining with BODIPY FLcasein was carried out according to the procedures described pre-viously (Xia et al., 2007, 2008) with a slight modification in samplepreparation. Fresh mixed liquor samples were collected directlyfrom a valve close to the bottom of the digesters after thoroughmixing. A volume of 400 lL mixed liquor was centrifuged at4500g for 10 min, and the supernatant was discarded. The biomassresidue was re-suspended in 1� Tris–HCl, and BODIPY FL caseinworking solution was added at a final concentration of 0.5 mg/gtotal suspended solids with a final reaction volume of 600 lL.The mixture was placed in a 10-mL serum bottle wrapped in alu-minum foil and incubated at 25 �C for 30 min on a rotating disk(220 rpm). Incubated samples were spread evenly on three-well(10 lL in each well) Teflon printed slides (Electron MicroscopySciences) and dried in a dark room before being mounted withantifade reagent CITI fluor (Electron Microscopy Sciences) andexamined microscopically.

2.5. Enumeration of microorganisms positively stained with BODIPY FLcasein

The numbers of proteolytic microorganisms were estimated bycounting the number of cells positively stained with BODIPY FL case-in against the total cell number stained with DAPI (40,6-diamidino-2-phenylindoledihydrochloride) in the same microscopic field. DAPIstaining was carried out using ProLong Gold antifade reagent(Molecular Probe, Burlington, ON, Canada) according to the manu-facturer’s instructions. Cells stained positively with BODIPY FL case-in were randomly captured and their positions on the gelatin-coatedslides recorded. Then, the CITI fluor was washed away by gently rins-ing slides (for 1 min) with 70% ethanol. Finally, DAPI staining wasperformed and fluorescent signals of the cells of interest werecaptured after their relocation. Images of bacterial samples stainedwith BODIPY FL casein and DAPI were examined and captured usingan epifluorescence microscope (Image-Pro Plus, Olympus) equippedwith a CCD digital camera SC500J (CoolSNAP-Procf, MediaCybernetic). Microbial cells on digital images were counted inImageJ (Abramoff et al., 2004). For each enumeration, at least 60microscopic images taken with the 100� objective lens from at leastthree slide wells (20 images from each well) were examined.

2.6. Statistical analysis

Different models were explored and chosen based on minimiz-ing the root mean square of the error to study the evolution of fer-mentation variables over time. The evolution of total gas, CH4

production and CH4 yield per unit VS over time were modeledusing a Michaelis–Menten kinetics-like equation:

Y ¼max� t=ðt1=2 þ tÞ ð1Þ

where Y = response variable, max = maximum production, t = timein days and t1/2 = time in days needed to obtain half of maximumproduction.

The evolution of CH4%, VS content, and acetate concentration inSM over time were modeled using inverted exponential first order-kinetics:

Y ¼ aþ b½1� expð�c � tÞ� ð2Þ

where Y = response variable, a + b = maximum production andc = fractional production rate.

Finally, NH4AN concentration in SM was modeled as a cubicpolynomial least square regressions as a function of time:

NH4—N concentration ¼ aþ b� t þ c � t2 þ d� t3 ð3Þ

The effect of feather addition was evaluated by comparingparameters obtained from 2-parameters Michaelis–Menten kinet-ics (i.e. time in days needed to obtain half of maximum production(t1/2) and maximum production) and first order-kinetics (i.e. initialand final concentration or percentage, and fractional rate ofchange) for each experiment (i.e. for each inoculum) using a 2-tailt statistic for unequal variances, and significance was declared atP < 0.05. For cubic polynomial models, the effect of feather additionwas evaluated as its main effect and through its interactions withthe first, second and third order polynomial coefficients for thetime effect.

Acetate concentration and NH4AN concentration in slaughter-house sludge, and number of proteolytic bacteria in both inoculahad different responses between treatments and sometimes be-tween reactors of the same treatment; for those response variables,mean values throughout the digestion period were compared forfeather addition and its absence using a 2-tail t statistic for unequalvariances and significance was declared at P < 0.05.

3. Results

3.1. Raw feather and inoculum characterization

Feather samples used in this study contained 94.7% dry matter(DW), of which 99.2% was organic matter (Table 1). Crude proteinand fat constituted 92.0% and 2.79% of feather DW, respectively.The COD value per g raw feathers was 1.2 g. The physiochemicalcharacteristics of the stabilized SM and SS inocula are listed inTable 2. The SM inoculum contained 7.41 g/L volatile suspendedsolids, lower than those measured in the SS (12.01 g/L) inoculum.The mixed liquor NH3AN level was higher in the SM inoculum(4.8 g/L) than in the SS inoculum (3.2 g/L). The alkalinity levels inSM inoculum were substantially higher in the SM inoculum(26.3 g/L) than in the SS inoculum (8.7 g/L).

3.2. Biogas production

The cumulative total gas and CH4 profiles from the digesters areshown in Fig. 1. In SM digesters, total gas (Fig. 1B) and total CH4

(Fig. 1A) were greater with feather addition than in the controls,increasing by 124% (633 vs 282 L; P < 0.001, SED = 3.36) and130% (418 vs 182 L; P < 0.001, SED = 4.57), respectively. The t1/2

for total gas and total CH4 were also greater in the SM digesterswith feather addition than the controls, increasing by 168%(30.26 vs 11.31 day; P = 0.022, SED = 1.36) and 215% (28.0 vs8.89 day; P = 0.025, SED = 1.00), respectively. Compared to thecontrols, feather addition had a tendency (496 vs 452 L/kg VSfed; P = 0.082, SED = 9.20) to increase CH4 yield per unit VS. In SSdigesters, total gas (Fig. 1B) was greater with feather addition thanin their controls, increasing by 183% (294 vs 104 L; P = 0.031,SED = 9.69). Feather addition had a tendency (196 vs 93.4 L;P = 0.090, SED = 14.6) to enhance total CH4 production (Fig. 1B).CH4 yield (162 vs 112 L/kg VS fed; P = 0.21, SED = 17.5) and thet1/2 for total gas (79.2 vs 154 day, P = 0.22, SED = 26.7) and total

Fig. 1. Cumulative methane (A) and total gas (B) production in anaerobic digesters inoculated with SM or SS and with or without feather addition.

Fig. 2. Acetic acid production (mg/L mixed liquor) in anaerobic digesters inoculated with SM (A) and SS (B) with or without feather addition.

Y. Xia et al. / Waste Management 32 (2012) 404–409 407

CH4 (75.6 vs 223 day; P = 0.22, SED = 54.5), and CH4 yield (75.6 vs224 day; P = 0.22, SED = 54.5) were not affected by feather additionin SS digesters. In both SM and SS digesters, final CH4 percentage(in total gas) with feather addition was similar to the controls[(65.5% vs 66.9%, P = 0.24, SED = 0.61) and (74.9% vs 68.7%;P = 0.14, SED = 1.38), respectively].

Fig. 3. NH4AN production (g/L mixed liquor) in anaerobic digesters inoculated withSM or SS and with or without feather addition.

3.3. VS degradation rate, VFA and NH4AN production and pH

At the end of the digestion, more VS was left in SM digesters (15.1vs 11.3 g/L; P = 0.05, SED = 0.311) and SS digesters (26.9 vs 15.4 g/L;P = 0.003, SED = 0.56) with feather addition than in their respectivecontrols. The VS degradation rates determined in SM and SS digest-ers with feather addition were similar to those determined in theircorresponding controls [(5.8 vs 3.3% per day; P = 0.42, SED = 2.2%)and (2.1 vs 2.0% per day; P = 0.94, SED = 1.0%), respectively].

Of all the VFAs tested only acetic acid was detected in SM (Fig. 2A)and SS (Fig. 2B) digesters in relatively abundant amount during thedigestion. Other VFAs including propionic acid and isobutyric acidwere only detected in trace concentrations (data not shown). InSM digesters, the maximum acetic acid concentration with featheraddition was 3.3 times higher than the controls (Fig. 2A) (806 vs245 mg/L; P = 0.023, SED = 55.6). The final acetic acid concentrationwith feather addition was 1.5 times higher than the control (Fig. 2A)(23.8 vs 16.2 mg/L; P = 0.011, SED = 0.8). Acetic acid production in SSdigesters was not affected (P = 0.10) by feather addition (Fig. 2B).The pH values remained relatively constant at 7.6–7.9 throughoutthe digestion in all SM or SS digesters examined.

NH4AN responded to feather addition in both SM and SS digest-ers. NH4AN increased (P < 0.001) from 4.8 and 3.1 g/L at the begin-ning of the digestion to 6.9 and 3.5 g/L at the end of digestion in SMand SS digesters with feather addition, respectively (Fig. 3). NH4ANwithout feather addition fluctuated between 4.8 and 5.3 g/L andbetween 3.2 and 3.4 g/L in SM digesters and SS digesters through-out the digestion, respectively (Fig. 3).

3.4. Abundance of proteolytic microorganisms

The proteolytic microorganisms in the mixed liquor of SM andSS digesters were visualized using BODIPY FL casein staining(Fig. 4). Their numbers increased (P < 0.001) with feather additionfrom 12.5% to 14.5% and 11.3% to 13.0% in SM and SS digesters,respectively (Fig. 5). In controls, the number of proteolytic micro-organisms decreased gradually throughout the digestion (Fig. 5).

4. Discussion

In both SM and SS digesters, feather addition enhanced total gasand total CH4 production, and increased mixed liquor NH4AN andthe number of proteolytic microorganisms in the mixed liquors.Estimation based on the difference in the amount of CH4 betweenthe digesters with feather addition and their control digesterswithout feather addition, 96% and 44% of feathers added in SMand SS digesters, respectively were used for methanogenesis dur-ing 146-day digestion. The digesters with a mixture of SM and

Fig. 4. Mixed liquor samples after BODIPY FL casein staining and DAPI staining. (A) a fluorescence BODIPY FL casein staining image and (B) a color-combined image from aBODIPY FL casein staining image (set as red color) and a DAPI staining image (set as green color) captured at the same microscopic field. The yellow-colored cells areproteolytic microorganisms. The ruler bars equal 10 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thisarticle.)

Fig. 5. The relative abundance (%) of proteolytic microorganisms in anaerobicdigesters inoculated with SM or SS and with or without feather addition.

408 Y. Xia et al. / Waste Management 32 (2012) 404–409

feathers were more efficient in biofuel production than the digest-ers with a mixture of SS and feathers. However, the feather diges-tion based on the amount of net CH4 generation might have beenoverestimated because the possibility that feather digestion stim-ulated digestion of SM and SS could not be ruled out althoughthe fact that feather addition did not affect the VS degradationrates in both SM and SS digesters seems to suggest that the possi-bility is small.

The increase in the t1/2 of total gas, total CH4, and CH4 yield perunit VS in SM digesters with feather addition reflects feather diges-tion. This is because feathers contain more VS than SM (as well asSS) and their addition increased total organic loading of a digester,thus a longer time was needed for the digester to reach the maxi-mum production of total gas, total CH4 and CH4 yield. In contrast, asimilar trend was not observed in SS digesters. The t1/2 of thesethree parameters in SS digesters was not affected by feather addi-tion. This is not caused by abnormal fermentation, pH level and/orNH4AN level (see following sections for more detail) because nosubstantial accumulation of VFAs and NH4AN inhibition werefound in SS digesters and pH level fluctuated within a narrowrange. Therefore, the lack of effect of feather addition on t1/2 of totalgas, total CH4 and CH4 yield may reflect a poor digestion of feathersin SS digesters, a result in line with the fact that only 44% of feath-ers added in SS digesters were used for methnogenesis, while 96%of feathers were used to do it in SM digesters.

Feather digestion rates detected in SM and SS digesters corre-lated to SM and SS digestion rates. SM used in this study is moredigestible than SS, with an average digestion rate of VS at 3.3%and 2.0% per day, respectively. Mixing with feathers did not changetheir digestion rates significantly suggesting that feathers aredigestible in these digesters but their digestion rates dependedon the digestion rates of SM and SS. The mechanism behind thishas to be further investigated. Moreover, feather addition did notchange CH4 yield per VS unit in SM and SS digesters. Therefore,more feathers were digested in SM digesters than in SS digesters,a result in line with that estimated using net CH4 generated. How-ever, this has to be further confirmed since SM and SS digesterswere run in separate experiments and the results obtained cannotbe compared together.

CH4 yield from the control SM digesters (0.42 L/g VS fed) wasnumerically similar to the value reported by Masse et al. (1996)using similar substrate and experimental setups. They observedCH4 yield of 0.48 L/g VS fed in their swine manure digesters. TotalCH4 and CH4 yield per unit VS (0.04 L/g VS fed) from the controltreatments of the SS digesters was numerically lower than that(0.09 L/g VS fed) reported by Masse and Masse (2000) using similarsubstrate and experimental setups. This difference could be attrib-uted to the fact that digesters in that study were regularly fed withfresh SS while digesters in this study were not.

The low level accumulation of soluble acetic acid, especially atthe beginning of the digestion, was observed in SM digesters withfeathers. A similar trend was also observed in SS digesters. Theiraverage acetic acid concentrations detected from day 0 to 60 arehigher in the digesters with feathers than those without feathers.The accumulation was mainly due to feather digestion instead ofinhibition of acetotrophic methanogens by e.g. NH4AN since an en-hanced CH4 generation and an increase in the number of proteo-lytic microorganisms were observed in both SM and SS digestersthroughout the digestion.

In both SM and SS digesters, NH4AN increased with featheraddition. Methanogens are the least tolerant and the most likelyto cease growth due to NH4AN inhibition (Kayhanian, 1994). Lossof activity (�56.5%) of methanogens has been reported at anNH4AN ranging from 4.1 to 5.7 g/L (Chen et al., 2008). However,in the current study, continuous CH4 production was detectedthroughout the digestion in SM and SS digesters. Therefore, itappears that high NH4AN levels found in these digesters, especiallyin SM digesters, did not markedly affect biogas production. This

Y. Xia et al. / Waste Management 32 (2012) 404–409 409

could be due to presence of the methanogens that can survive andfunction at high NH4AN (Jarrell et al., 1987).

As discussed earlier, both feather keratin and prions are fibrousand insoluble proteins rich in b-sheet conformation (Fraser et al.,1972; Gushterova et al., 2005). Feather keratin has been used asa model protein to investigate prion degradation (Suzuki et al.,2006; Tsiroulnikov et al., 2004). Therefore, in addition to resolvingthe problem of feather disposal, SM and SS digesters could perhapsbe applied to the treatment of specified risk materials potentiallycontaminated with prions. This remains to be further investigated.

5. Conclusion

Ground raw feathers without any other pretreatment can bedigested together with SM and SS at 25 �C for biofuel production.Mixing feathers with SM is more efficient in biofuel productionthan mixing feathers with SS. Feather digestion rates correlatedto digestion rates of SM and SS. Most of the feathers added to SMdigesters were digested and used for methanogenesis during146-day digestion. In contrast, less than half of the feathers addedin SS digesters were used for methanogenesis. Mixing featherswith SM in anaerobic digestion recovered more energy and gener-ated less waste sludge thus being a potentially better waste treat-ment process.

Acknowledgments

Financial support for Y. X was through a peer review project andthe specified risk material disposal project of Agriculture and Agri-Food Canada. We thank Denis Deslauriers and Gilles Grondin fortheir excellent technical support.

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