Semi-continuous anaerobic digestion of solid slaughterhouse waste

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Journal of Environmental Chemical Engineering 2 (2014) 819825Semi-continuous anaerobic digestion of solid slaughterhouse wasteAnia Escudero a,1,*, Arrate Lacalle a,1,**, Fernando Blanco a, Miriam Pinto a, Ignacio Daz b,Antonio Domnguez ba NEIKER-TECNALIA Basque Institute for Agricultural Research and Development, Berreaga 1, E-48160 Derio, Spainb BIOGAS FUEL CELL, S.A. ParqueTecnologico de Gijon, Avda Byron 107, 18 Izq. 33203, Gijon, SpainA R T I C L E I N F OArticle history:Received 21 November 2013Accepted 11 February 2014Keywords:Slaughterhouse wasteAnaerobic digestionWaste treatmentProduct inhibitionA B S T R A C TThe disposal of solid slaughterhouse waste can lead to contamination of the environment with organiccompounds and pathogens. The principal aim of this study was to evaluate a semi-continuous anaerobicdigestion procedure as a treatment for solid slaughterhouse by-products (low risk material) following aslow acclimatization stage and prior pasteurization of the substrate. The material was digested induplicate glass tank reactors (of volume 8 L), under mesophilic conditions (35 8C) and with continuousstirring. The procedure consisted of the gradual acclimatization of the inoculum (fed at a loading rate of0.31.6 gVS L1 d1 and with a hydraulic retention time [HRT] of 105160 days). The loading rate wasincreased and the HRT was decreased as the system reached stability. Gradual, slow acclimatization ofthe sludge and prior pasteurization of the substrate yielded biogas (methane content 6570%) at a rate of2 L L1 d1 and 1.07 L gVS1 added, for an organic loading rate of 1.3 gVS L1 d1, an HRT of 125 days anda waste:water dilution rate of 1:2.7. However, at the maximum loading rate of 1.6 gVS L1 d1 and anHRT of 105 days, the biogas production decreased, indicating microbial inhibition that was probablycaused by accumulation of volatile fatty acids (VFAs) and long chain fatty acids (LCFAs), due to the highlipid content of the substrate. In addition, the high protein content of the substrate led to theaccumulation of NH4-N in the reactors. An operational alternative must be developed to prevent systeminstability. 2014 Elsevier Ltd. All rights reserved.Contents lists available at ScienceDirectJournal of Environmental Chemical Engineeringjou r n al h o mep ag e: w ww .e lsev ier . co m / loc ate / jec eIntroductionAs a result of legal restrictions, rising treatment costs andpressure from environmentally aware consumers, the treatment ofsome solid residues and waste has emerged as a major concern inmany industries [18].Slaughterhouses generate substantial amounts of animal by-products, which are defined as parts of the animal not intended forconsumption, either because they are not fit for human consump-tion or because there is no market for them.This type of waste typically contains large amounts of organicmatter, mainly composed of proteins and lipids [4] and pathogens,and its disposal can lead to serious environmental problems[3,7,9,10].* Corresponding author. Tel.: +34 627973229.** Corresponding author. Tel.: +34 627973412.E-mail addresses:, (A. Escudero), (A. Lacalle).1 These authors contributed equally to the work. 2014 Elsevier Ltd. All rights reserved.European Commission Regulation (EC) [11] No. 1774/2002categorizes different animal by-products and indicates how theyshould be used, commercialized and eliminated. Only category 2(condemned meat, fallen stock, manure, digestive tract content)and category 3 (livers, lungs, spleens, fats, oesophagus) by-products (i.e. generally low risk by-products) can be treated byanaerobic digestion. All category 1 by-products, which includehigh-risk products (such as animals suspected or confirmed asbeing infected with Transmissible Spongiform Encephalopathy[TSE], animals killed in the context of TSE controls and specifiedrisk material) can only be eliminated by an incineration processand are excluded from this treatment.Anaerobic digestion has become established as a means ofmanaging solid organic waste. Besides generating biogas forenergy use, the process also destroys pathogens and producesstabilized material to be used as fertilizer in land applications.However, solid slaughterhouse waste is considered recalcitrant toanaerobic treatment, mainly because of its high lipid and proteincontents [4,12,13]. Protein degradation releases ammonium,which at high concentrations has inhibitory effects on anaerobicmicroorganisms [14,15]. During anaerobic digestion, lipids are Escudero et al. / Journal of Environmental Chemical Engineering 2 (2014) 819825820hydrolysed, and long-chain fatty acids (LCFAs) are then releasedinto the liquid phase. Under anaerobic conditions, biomassproduces volatile fatty acids (VFAs), which are then convertedinto biogas [7]. High concentrations of lipids can also causeproblems with the anaerobic digestion process as they tend togenerate floating foam and can also lead to the accumulation ofintermediate degradation compounds, such as LCFAs and VFAs,which can inhibit degradation [2,7,1618].However, Affes et al. [19] and Palatsi et al. [4] have stated thatslaughterhouse waste can yield large amounts of methane as aconsequence of the high theoretical methane potential ofefficiently degraded lipids. In a batch study, Cirne et al. [20]showed that although the inhibition caused by lipids was related tothe slow hydrolysis of these compounds, the process was able torecover from the inhibition. Furthermore, Marcos et al. [3] andSalminen and Rintala [1] also reported that anaerobic digestion issuitable for the management and valorization of slaughterhousewaste. If the process is well controlled, anaerobic digestion can becarried out as a result of the gradual acclimatization of the bacterialpopulation to an ammonia-rich medium [21,22] and to high fat andLCFA concentrations [8,17]. Rodrguez-Abalde et al. [13] andBattimelli et al. [7] reported that thermal pre-treatments can beapplied to increase the bioavailability of the protein and fat contentof the waste, thus producing a significant increase in the amount ofmethane produced from fatty waste.The main objective of this work was to study a semi-continuousanaerobic digestion procedure as a treatment for category 3 solidslaughterhouse waste following a slow acclimatization stage andprior pasteurization of the substrate.Materials and methodsInoculum and characterization of solid slaughterhouse wasteThe reactors were inoculated with methanogenically activebiomass from the Iurreta Wastewater Treatment Plant (Spain). Thetotal solid (TS) and volatile solid (VS) contents of the inoculumwere 4.2 and 1.9% respectively; the pH was 8.0 and the chemicaloxygen demand (COD) was 18.3 gO2 L1.In this study, the substrate used for the anaerobic digestionprocess was from beef and classified as category 3. The solidslaughterhouse waste was collected from the Durango slaughter-house (Spain). The heterogeneity of the substrate made samplingdifficult and led to a certain degree of analytical variability, andtherefore the values of the parameters measured in the substrateare shown as ranges (Table 1).The waste was mechanically chopped into pieces of size 23 cm, ground with water (kg waste:kg water, 1:5) in a glass grinder(Palson Bali 30525), and stored at 20 8C until required. Wheneverwaste was sampled from the slaughterhouse, the samples werechopped and analyzed. The average VS content in each collectedsample was used to manage the organic loadings in digesters.Table 1Characteristics of the solid slaughterhouse waste under studybefore mixing it with water. Values are expressed in fresh mattercontent. Average values are shown in quotations.Parameters Solid slaughterhouse wasteTS (%) 4060 (46)VS (%) 3959 (45)COD (gO2 kg1) 400520 (426)N Kjeldhal (%) 2.53.1 (2.7)Total fat (%) 1217 (14.5)pH 78 (7.7)According to Commission Regulation (EU) [23], category 3material, which is used as raw material in biogas plants, must betreated by pasteurization. Thermal pre-treatments increase thebioavailability of the protein and fat content of the waste andproduce a significant increase in methane production in fattywastes [7,13]. The substrate was therefore pasteurized for 2 h at70 8C in an oven (Selecta Digitheat 2001244) before being fed intothe digesters for anaerobic digestion.DigestersThe digestion experiments were performed in duplicate glasstank reactors, of volume 8 L (D1 and D2), which were equippedwith a mechanical shaker (IKA Eurostar power P4 model, operatingat 13 rpm), a thermostatic bath with recirculation (Hubber, CC-250B model, set at 35 8C), a pH meter (KNICK 911 model, with PoliliteLab electrode for a range of 014 pH), a gas flowmeter (RitterMilliGas counter MGC-1), a portable biogas detector (Drager X-AM7000) and gas sampling bags (RITTER 5, 10, 40 and 60 L) (Fig. 1).The substrate was stirred continuously during processing and theexperiments were carried out under mesophilic conditions (35 8C).Experimental procedureThe digesters were initially charged with 8 L of inoculum andthereafter fed once a day, except at weekends. The substrate wasintroduced by an in-feed located at the top of the digester. Prior tointroducing the feeding substrate, the inoculum was stirred tohomogenize the material and the corresponding amount ofdigestate was removed from the digester.Fig. 1. Digesters equipped with the items used in the experimental trial. (1) pHmeter,(2) mechanical shaker, (3) thermostatic bath with recirculation, (4) gas flowmeter and (5) portable biogas detector.Table 2Operational parameters in digesters D1 and D2 during the anaerobic digestion assay.Days Loading rate (gVS L1 d1) Hydraulic retention time (days) Waste:water ratio Influent daily volume rate (mL)113 0.3 No digestate extracted 1:5 341325 0.5 160 1:5 562541 0.7 160 1:4 534159 0.9 125 1:4 6859106 1.3 125 1:2.7 68106152 1.6 105 1:2.7 87A. Escudero et al. / Journal of Environmental Chemical Engineering 2 (2014) 819825 821A slow, gradual acclimatization strategy was used to enable thestability of the system to be assessed [8,17,21,22], and the loadingrate was increased and the hydraulic retention time (HRT)decreased as the system reached stability, as shown in Table 2.The stability was verified by periodic determination of thefollowing control parameters: alkalinity, biogas production,ammonium concentration, pH, redox potential and biogascomposition. The initial loading rate was 0.3 gVS L1 d1 andHRT was 160 days. The loading rate was increased and the HRTdecreased throughout the experiment, so that the final loading ratewas 1.6 gVS L1 d1 and the HRT was 105 days. To increase theloading rate without modifying the HRT, the ratio of waste to waterwas increased (Table 2).Chemical analysesThe pH, TS and VS were measured according to the standardmethods outlined by the APHA [24]. The amount of NH4-N in thesupernatant of the digestate was determined according to acolorimetric method based on the Berthelot reaction [25]. The CODwas determined colorimetrically at 605 nm after 2 h digestion at150 8C, with potassium dichromate in sulphuric acid medium. Thelipid content of the substrate was measured following theapplication note AN301 of FOSS TECATOR and using petroleumether as solvent [26].The alkalinity was measured by a method proposed by Hills andJenkins [27], in which a two stage titration is carried out with 0.1 NHCl, first to 5.75 and subsequently to 4.3. Considering these two pHendpoints, three alkalinity measurement parameters were de-fined: total alkalinity (TA), measured to pH 4.3, partial alkalinity(BA), associated with bicarbonate alkalinity and measured to pH5.75, and intermediate alkalinity (IA), associated with theconcentration of VFAs and estimated as the difference betweenTA and BA. Changes in the VFA content in the process can bemonitored by changes in alkalinity during the experiment [27].Fig. 2. Changes in the relative production of biogas in digesters D1 and D2 during the semioperational parameters. The operational parameters (loading rate, waste:water ratio aResults and discussionThe gradual increase in the loading rate led to an increase in therelative production of biogas, which reached approximately2 L L1 d1 (Fig. 2) with a organic loading rate of 1.3 gVS L1 d1,an HRT of 125 days and a waste:water dilution rate of 1:2.7, whichis equivalent to 1.07 L gVS1added. In comparison, Edstrom et al.[22] reported a biogas production rate of 1.14 L gVS1added with aprior substrate pasteurization step, similar to production achievedby Cuetos et al. [2] for pre-treated slaughterhouse waste (Table 3).However, with similar loading rates, Alvarez and Liden [28]reached lower productions rates (0.55 L gVS1added). On the otherhand, decreasing the loading rates (0.5 gVS L1 d1) Marcos et al.[3] reported production rates of around 3 L gVS1added.The difference in relative biogas production between bothdigesters, observed on days 7891, was caused by a leakage ofbiogas in D2 during this period (Fig. 2). The leakages were alsoreflected in the cumulative biogas production, as the cumulativebiogas production curves diverged during those days (Fig. 3). Oncethis problem was resolved, similar progress was observed in bothdigesters.The biogas composition in both reactors remained constantthroughout the trial (at about 6570% of CH4 and 3530% of CO2),except when acid was added, when the concentrations of CH4decreased to 60% as a result of the greater amount of CO2 in thebiogas. It was not possible to measure the biogas compositionwhen relative production was close to 0.1 L L1 d1. The methaneyield achieved with a organic loading rate of 1.3 gVS L1 d1, anHRT of 125 days and a waste:water dilution rate of 1:2.7 was0.64 LCH4 gVS1added. These results were higher than thosereported in the literature for category 2 animal by-products andco-digestion of organic fraction of municipal solid waste andslaughterhouse wastes (0.32 and 0.3 LCH4 gVS1added respectively)[29,30], and similar to those published by Bayr et al. [31,32](Table 3). Salminen and Rintala [1] also reported a methane yield of-continuous anaerobic digestion process. The vertical lines indicate modifications tond HRT) in each stage are shown in the figure.Table 3Previously reported data on anaerobic digestion of slaughterhouse wastes.Substrate Feeding T (8C) OLR(gSV L1 d1)HRT(days)Production ReferenceBiogas(L gVS1added)Methane(L gVS1added)Poultry slaughterhouse waste Semi-continuous 31 0.8 50100 * 0.520.55 [1]Pre-treated slaughterhouse waste Semi-continuous 34 1.2 36 0.9 0.5 [2]Slaughterhouse wastes (84% wastewater + 10% slurry + 6% solids) Semi-continuous 37 0.5 18 3.2 2.4 [3]Solid slaughterhouse wastes Batch 37.5 * * * 0.390.99 [5]Pasteurized animal by-products Batch 37 2 * 1.14 0.76 [22]Solid slaughterhouse waste + fruit and vegetable wastes + manure Semi-continuous 35 0.31.3 30 0.55 0.3 [28]Category 2 animal by-products Batch 35 0.64 * * 0.32 [29]Organic fraction of municipal solid waste + slaughterhouse wastes Semi-continuous 36 2 30 * 0.3 [30]Pig slaughterhouse waste Semi-continuous 35 1.5 30 * 0.65 [31]Rendering plant wastes + slaughterhouse by-products Semi-continuous 35 11.5 50 * 0.260.57 [32]Slaughterhouse solid wastes Semi-continuous 35 1.3 125 1.07 0.64 This study*data not availableFig. 3. Changes in the cumulative biogas production in digesters D1 and D2 duringthe semi-continuous anaerobic digestion process.A. Escudero et al. / Journal of Environmental Chemical Engineering 2 (2014) 8198258220.520.55 LCH4 gVS1added for poultry slaughterhouse waste.According to Pitk et al. [5], the methane potential of solidslaughterhouse waste rendering products is between 0.39 and0.99 LCH4 gVS1added.The release of ammonia from protein caused an increase inalkalinity, so that the pH values tended to increase constantly up topH 8 (Figs. 4 and 6), which is higher than the range of valuesreported as optimal for the development of methanogenicmicroorganisms (6.37.8) [8,33]. At high pH, the ammonia-nitrogen released during fermentation of proteins exists largelyas the unionized free ammonia (NH3), which is more toxic tomethane-forming microorganisms than the ionized NH4+ formas it diffuses more rapidly through the cell membrane of theFig. 4. Changes in pH in digesters D1 and D2 during tmicroorganisms [34]. Numerous studies have attempted to comeup with the solutions to prevail over the ammonia inhibitionduring the digestion process of substrates with high proteincontent. On one hand, acclimation of microflora can influence thedegree of ammonia inhibition, as adaptation of microbes to highammonia concentration could accelerate the ammonia tolerance[14,35]. Secondly, the temperature is also considered an importantfactor which can affects the threshold of ammonia inhibition. Anincrease in temperature of anaerobic digestion usually increasesthe metabolic rate of the microorganisms but also results in ahigher NH3 concentration [14,35]. On the other hand, as a propercontrol of pH during anaerobic digestion process may reduce theammonia toxicity of the microorganisms, the strategies reported toreduce the ammonia-induced inhibition of methane productionfrom livestock waste included reduction of the pH by addition ofacid [14,33,34,36,37]. Shanmugam and Horan [38] reported thatammonia toxicity only occurred at pH values greater than 7.6.Furthermore, in a recent study [39] it was shown that the additionof HCl to decrease pH from 8 to 7.6 improved methane yieldstreating slaughterhouse waste, manure and organic by-products.Therefore, to avoid a system inhibition, when the value approachedpH 8 (day 94), an appropriate amount of 10%HCl was added todecrease the pH to about 7.6. After 21 days, between day 115 andday 127, the HCl was replaced with 20% acetic acid, as this is anintermediate compound of anaerobic digestion and thus assimila-ble by microorganisms. The days on which acid was added arereflected in the abrupt decreases in pH in Fig. 4. No changes in NH4+concentration were observed after addition of acid (Fig. 5).Constant accumulation of ammonium was observed in bothsystems, up to a concentration of 6.9 g L1 (Fig. 5). Inhibition ofanaerobic digestion of slaughterhouse waste may be attributed tothe accumulation of high levels of ammonia resulting from thedegradation of the protein content of this type of waste [2,8,16,17].The threshold considered as inhibitory varies widely, between 1.7he semi-continuous anaerobic digestion process.Fig. 6. Changes in the total alkalinity (TA), partial alkalinity (BA), intermediate alkalincontinuous anaerobic digestion process.Fig. 5. Changes in NH4+ in digesters D1 and D2 during the semi-continuousanaerobic digestion process.A. Escudero et al. / Journal of Environmental Chemical Engineering 2 (2014) 819825 823and 14 g L1 [14,15]. These differences are attributed to variablefactors such as operational conditions (pH, temperature), type ofsubstrate, source of inoculum and microbial adaptation [34], andtherefore we cannot conclude whether the ammonium contribut-ed to inhibition of the system.Total and partial alkalinities (TA and BA) decreased withaddition of acid (Fig. 6), which modified the bicarbonate, carbonateand CO2 balance, leading to release of CO2. Furthermore, briefpeaks in relative biogas production coincided with the addition ofacid as a result of the release of CO2 (Fig. 2).On day 135 of the anaerobic digestion process and after 3 weeksof feeding the tanks at a loading rate of 1.6 gVS L1 d1, the biogasproduction decreased sharply to about 0.1 L L1 d1 (Fig. 2),indicating inhibition of the system.The TA and BA increased in parallel, as shown in Fig. 3a and b,while the alkalinity associated with the VFAs (IA) remainedconstant, ensuring equilibrium of the system. However, from day126, the IA increased consistently, thereby increasing the IA/TAratio up to a limit of 0.5, indicative of an imbalance in the system,probably due to accumulation of VFAs [27,40,41]. The increase inthe IA/TA ratio coincided with a relative decrease in biogasproduction (Fig. 2). Several authors also observed VFA accumula-tions in anaerobic digestion of slaughterhouse wastes[22,28,30,32].ity (IA) and IA/TA ratio (a) in digester D1 and (b) in digester D2 during the semi-A. Escudero et al. / Journal of Environmental Chemical Engineering 2 (2014) 819825824Inhibition of anaerobic digestion of slaughterhouse waste hasbeen attributed, among other things, to accumulation of LCFAs asconsequence of lipid degradation [2,22]. LCFAs constitute the mainintermediate by-product of the lipid degradation process anddegradation of these may be the limiting step in the anaerobicdegradation of solid slaughterhouse waste due to the slow growthof LCFA-consuming bacteria [17,42,43]. Furthermore, accumula-tion of LCFAs may cause problems in anaerobic digestion of solidslaughterhouse waste because these compounds are toxic toanaerobic microorganisms, particularly acetogens and methano-gens [16,17,4345]. It is therefore reasonable to assume that theaccumulation of LCFAs would also contribute to system inhibitionand a consequent decrease in biogas production due to the highlipid content of the substrate (Table 1) [14,16,46].ConclusionsAnaerobic digestion, including gradual slow acclimatization ofthe sludge and prior pasteurization of the substrate, appears to be asuitable treatment for solid slaughterhouse waste, yielding abiogas production rate of around 2 L L1 d1 and 1.07 L gVS1added,for a organic loading rate of 1.3 gSV L1 d1, a HRT of 125 days anda waste:water dilution rate of 1:2.7. However, with a loading rateof 1.6 gSV L1 d1 and a HRT of 105 days, biogas productiondecreased, indicating microbial inhibition, which was probablycaused by an accumulation of VFAs and LCFAs, due to the high lipidcontent of the substrate.The high protein content of the waste led to the accumulation oflarge amounts of ammoniacal nitrogen, which, in conjunction withchanges in pH, can produce of ammonia at inhibitory concentra-tions, which must be controlled. An operational alternative mustbe developed in order to prevent system instability.AcknowledgementsFunding for this study was provided by Biogas Fuel Cell S.A. andthe Centre for Industrial Technologic Development (CDTI) of theMinistry of Economy and Competitiveness of the SpanishGovernment.References[1] E.A. Salminen, J.A. Rintala, Semi-continuous anaerobic digestion of solid poultryslaughterhouse waste: effect of hydraulic retention time and loading, WaterResearch 36 (2002) 31753182.[2] M.J. Cuetos, X. Gomez, M. Otero, A. 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Angelidaki, X.Flotats, Long-chain fatty acids inhibition and adaptation process in anaerobicthermophilic digestion: batch tests, microbial community structure and mathe-matical modelling, Bioresource Technology 101 (2010) 22432251.[46] J.J. Kabara, R. Vrable, M.S.F. Liekenjie, Antimicrobial lipids natural and syntheticfatty-acids and monoglycerides, Lipids 12 (1977) 753759. anaerobic digestion of solid slaughterhouse wasteIntroductionMaterials and methodsInoculum and characterization of solid slaughterhouse wasteDigestersExperimental procedureChemical analysesResults and discussionConclusionsAcknowledgementsReferences


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