Bioresource Technology 120 (2012) 106113

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Bioresource Technology
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Effect of additives on process stability of mesophilic anaerobic monodigestion of pig slaughterhouse waste
Suvi Bayr , Outi Pakarinen 1, Aki Korppoo 2, Saku Liuksia 2, Ari Visnen 3, Prasad Kaparaju, Jukka Rintala 4
University of Jyvskyl, Department of Biological and Environmental Science, P.O. Box 35, FI-40014 University of Jyvskyl, Finland

h i g h l i g h t s
" Anaerobic monodigestion of pig slaughterhouse waste was studied in CSTRs at 35 C. " Effect of two additives on process stability was studied as well. " Additive of trace elements, Fe and HCl improved process stability. " Additive of trace elements, Fe and HCl allowed higher OLRs to be used. " Additive of Fe and HCl reduced H2S from biogas, but did not facilate higher OLR.

a r t i c l e

i n f o

a b s t r a c t
The effect of two additives on anaerobic monodigestion of pig slaughterhouse waste was studied in three laboratory scale continuously stirred tank reactors (CSTRs) operated for 242284 days at 35 C. A higher organic loading rate (OLR), namely 2.25 kg volatile solids (VS)/m3 d with a hydraulic retention time (HRT) of 30 d was feasible with an additive containing Fe, HCl and trace elements while an OLR of 1.5 kg VS/m3 d (HRT 30 d) led to instabilities in CSTR with an additive consisting of Fe and HCl and in CSTR without additives. Methane yields of ca. 700 dm3 CH4/kg VSfed were obtained in all reactors under stable conditions. Both additives decreased H2S concentration of the biogas below detection limit 0.5 ppm while in a control reactor H2S concentration was up to 1900 ppm. In conclusion, the results suggest the possibility that additives may facilitate higher OLRs in monodigestion of pig slaughterhouse waste. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 27 March 2012 Received in revised form 6 June 2012 Accepted 10 June 2012 Available online 16 June 2012 Keywords: Additives Anaerobic digestion Mesophilic Process stability Slaughterhouse waste

1. Introduction Slaughterhouse wastes are potentially good substrates for biogas production because of high concentrations of proteins and lipids. However, in laboratory reactor studies only relatively low OLRs (0.81.7 kg VS/m3 d) with HRTs of 25100 d have been noticed to be feasible in anaerobic digestion of slaughterhouse waste (Bayr et al., 2012; Cuetos et al., 2008; Salminen and Rintala, 2002) because high protein and lipid contents of the material causes insta Corresponding author. Tel.: +358 40 8053904.
E-mail address: suvi.bayr@jyu. (S. Bayr). Present address: Jyvskyl Innovation Ltd. P.O. Box 27, FI-40101 Jyvskyl, Finland. 2 Kemira Oyj, Luoteisrinne 2, P.O. Box 44, FI-02271 Espoo, Finland. 3 University of Jyvskyl, Department of Chemistry, P.O. Box 35, FI-40014 University of Jyvskyl, Finland. 4 Present address: Tampere University of Technology, Department of Chemistry and Bioengineering, P.O. Box 541, FI-33101 Tampere.
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bility through intermediate products of the digestion process, viz. ammonia nitrogen (NH4-N), volatile fatty acids (VFAs) and longchain fatty acids (LCFAs). Ammonia is formed in anaerobic digestion of proteins and long chain fatty acids are formed in digestion of lipids. Ammonia nitrogen exists in two forms, ammonia ions (NH4+) and free ammonia nitrogen (NH3), the latter has been considered to be more inhibitory for the anaerobic digestion process than the former (Koster and Koomen, 1988). The concentration of each form depends on temperature and pH, the higher the temperature the higher the concentration of free ammonia. In a recent study (Karlsson and Ejlertsson, 2012) it was shown that the addition of HCl to decrease pH from 8 to 7.6 and 7.8 improved methane yields of laboratory CSTR reactors treating slaughterhouse waste, manure and organic by-products from 400 to 600 dm3 CH4/kg VSadded/d by decreasing NH3 levels of the reactors which increased degradation of VFAs. Anaerobic digestion of pig slaughterhouse waste has been studied in a few previous reactor experiments as well as in batch

0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.06.009

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experiments. In batch experiments methane productions of 430 dm3 CH4/kg VSadded (Bayr et al., 2012) and 580 dm3 CH4/kg VS (Rodrguez-Abalde et al., 2011) have been reported for pig slaughterhouse waste at 35 C. As far as we know, there are no results reported about anaerobic monodigestion of pig slaughterhouse waste in CSTR experiments. However, in mesophilic codigestion of ve fractions of rendering wastes together with poultry, cattle and pig slaughterhouse waste (25% of the substrate fresh matter (FM), Bayr et al., 2012) and of pig slaughterhouse waste (5% waste) with manure (Hejnfelt and Angelidaki, 2009), methane yields of 720 dm3 CH4/kg VSfed (Bayr et al., 2012) and 489 dm3 CH4/kg VS were obtained (Hejnfelt and Angelidaki, 2009). On the other hand, in monodigestion of poultry slaughterhouse waste in CSTRs, methane yields of 520550 dm3 CH4/kg VSfed (Salminen and Rintala, 2002) and 600700 dm3 CH4/kg VSfeed (Cuetos et al., 2008) were obtained at 35 C. For the anaerobic digestion process, macro- and micronutrients (referred to here as trace elements) are needed. The required macronutrients include e.g. carbon, nitrogen, phosphorus and sulphur, and the required trace elements include e.g. iron, nickel, cobalt, selenium and tungsten (Takashima and Speece, 1990). Microorganisms need trace elements as building blocks for growth, as well as to various other purposes, e.g. for enzymatic activities (Takashima and Speece, 1990). In a study of 10 biogas plants around Europe digesting manure and energy crops as well as industrial by-products and organic wastes, it was found that concentrations of trace elements in digestates varied greatly between individual biogas plants (Schattauer et al., 2011). No specic concentrations of needed elements can be determined, because the need depends on many factors such as substrate and the form in which the elements are in the process (Schattauer et al., 2011; Takashima and Speece, 1990). However, some suggestions have been made for concentrations of iron from 1000 to 5000 ppm (dry basis, Kayhanian and Rich, 1995), for cobalt from 0.148 to 0.580 mg/l (Lo et al., 2012) and from <1 to 3 ppm (dry basis, Kayhanian and Rich, 1995), for nickel from 5 to 25 ppm (dry basis, Kayhanian and Rich, 1995) and from 0.801 to 5.362 mg/l (Lo et al., 2012), for selenium <0.1 ppm (dry basis, Kayhanian and Rich, 1995) and for tungsten <0.1 ppm (dry basis, Kayhanian and Rich, 1995) and from 0.658 to 40.39 mg/l (Lo et al., 2012). Bioavailability of the trace elements can be different than the concentration of trace element in the process while the required minerals tend to co-precipitate (Takashima and Speece, 1990). Trace elements essential for microbes in anaerobic digestion process may have other impacts. At high concentrations trace elements can inhibit the microbiological process. They can also react chemically in the process e.g. iron reacts with H2S to form FeS. Therefore, the addition of iron can be used to control H2S concentration in the biogas, which is needed as H2S in biogas can cause corrosion in compressors, gas storage tanks and engines and it is toxic in concentrations of >5 ppm (Ryckebosch et al., 2011). Lack of trace elements has been considered in anaerobic digestion of e.g. energy crops, animal excreta, crop residues and organic fraction of municipal solid waste (OFMSW) as recently reviewed by Demirel and Scherer (2011). The effect of trace nutrients on anaerobic digestion of slaughterhouse waste has been studied (Ek et al., 2011), but to our knowledge no detailed results on process performance has been published before the present study. The effect of trace element addition on anaerobic monodigestion of pig slaughterhouse waste is interesting because trace elements have been suggested to be useful in digestion of such diversied substrate as OFMSW (Feng et al., 2010). Traditionally, the effects of addition of individual trace elements have been studied. In practice, commercial mixtures of trace elements and other additional substances are used in biogas plants. The objective of the present study is to evaluate the effects of two additives on OLRs and process stability

of semi-continuously fed mesophilic CSTRs digesting pig slaughterhouse waste. Both additives contained Fe and HCl while the other additive contained also several trace elements. Three parallel CSTRs were operated both with and without additives for 242 284 days. The impacts were evaluated by analysis of the digestates and methane yield as well as the biogas H2S concentration. 2. Methods 2.1. Substrate and inoculum The substrate of the experiments was pig slaughterhouse waste from Saarioinen Oyj, Finland, containing the contents of stomach and intestines. At the laboratory, the slaughterhouse waste was macerated (5 mm) by using a meat mincer (Talsa W 22) and the well homogenized material was stored at 20 C until further use. Inoculum was digestate from mesophilic farm biogas plant treating cow manure and confectionary by-products (Kalmari, Finland). Two additives delivered by Kemira Oyj were used in the study. Additive 1 contained Fe and HCl and additive 2 contained Fe, HCl and several trace elements. The characteristics of the substrate and inoculum are presented in Table 1 and consistency of the additives in Table 2. 2.2. Experimental set-up Experiments were carried out in three parallel semi-continuously fed CSTRs with total volume of 13 l each and liquid volume of 10 l each at 35 C. The temperature in the reactors was maintained by a heating coil wrapped around the insulated reactor and controlled thermostatically. Reactor contents were mixed mechanically with a timer (13 min on/16 min off). First, reactors were lled with 10 l of inoculum (day 0). Reactor contents were ushed with nitrogen for 10 min to remove oxygen. On the rst day, 5 g of pig waste was added to each reactor and daily feeding was started on day 4. Feeding was done through waterlocked pipelines. Biogas was collected in aluminium gas bags. Reactors were fed manually once per day from Monday to Friday. Feed was prepared daily by diluting pig waste with liquid (tap water + recirculated liquid fraction of the digestate) to get the HRT of 30 d and desired OLR (total solids (TS) of the substrate was 32%, TS of the reactor feed was 38%). From day 18 onwards, 3238% (95 ml/d) of the water was replaced with a liquid fraction of the reactor digestate. Recirculation of liquid fraction of the digestate was used to better simulate full scale plant conditions and to reduce the amount of pure water used in the feed. The liquid fraction was separated from the digestate by centrifuging (3000 rpm, 10 min). On day 82 reactor contents were mixed to get identical digestate in each reactor before additive additions were started

Table 1 Characteristics of the substrate and inoculum of the semi-continuously fed CSTRs digesting pig slaughterhouse waste. Pig waste TS (%/ww) VS (%/ww) VS/TS (%) SCOD (mg/gTS) NH4-N (g/kg) TKN (g/kg) CH4 (m3/tVS) CH4 (m3/tFM) Co (mg/kgTS) Fe (mg/kgTS) Ni (mg/kgTS) W (mg/kgTS) 32 31 97 94 1 15 630 120 200 40 1.7 180 2.2 4.5 Inoculum 5 4 76 8.6 1 4 2.6 621 4.8 3.1

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Table 2 Consistency of the additives and amounts added to reactors in semi-continuously fed CSTRs digesting pig slaughterhouse waste. Additive 1 Fe-tot (%) Fe3+ (%) Fe2+ (%) Free HCl (%) Trace elements (Co, Ni, Se, W) Addition to reactor g/reactor l/d S1 (218) S1 (224284) S2 (98125) S2 (126187) S2 (188242) S3 (98125) S3 (126187) S3 (188210) S3 (211223) S3 (224242) 10 2 6.7 1.4 3.3 0.7 2 0.5 No 0.054 0.076 0.016 0.021 0.024 Additive 2 91 6.0 1.4 3.0 0.3 2 0.5 Yes 0.875 0.067 0.078 0.067 0.067 0.089 0.100 0.110

on day 98. When additives were added, they were added to tap water before mixing with pig waste. Process parameters of the reactor experiments are presented in Table 3 and amounts of additives added to reactors are presented in Table 2. Reactor experiments were performed for 242284 days. Batch experiment to determine the methane potential of the substrate was performed as previously reported (Bayr et al., 2012), except for VS:VS ratio of inoculum to substrate was 1 in the present experiment. 2.3. Analyses and calculations TS, VS, pH, soluble chemical oxygen demand (SCOD), NH4-N, total Kjeldahl nitrogen (TKN) and VFAs were determined as described earlier (Bayr et al., 2012). VFA concentrations were converted to SCOD equivalents with the following coefcients: acetic acid

1.066, propionic acid 1.512, iso-butyric and butyric acid 1.816, iso-pentanoic (iso-valeric) and pentanoic (valeric) acid 2.036 and hexanoic (caproic) acid 2.204 (Ince, 1998). TVFA was calculated by summing individual acid concentrations that were converted to SCOD, together. In liquid fraction separation centrifuge Jouan (3000 rpm, 10 min) was used. For SCOD analysis of the substrate, pig slaughterhouse waste was extracted in a shaker for 1 h at 22 C. Gas composition (CH4, CO2 and H2) was measured by using a gas chromatograph (GC, Perkin-Elmer Arnel Clarus 500, Alumina column 30 m 0.53 mm) equipped with a thermal conductivity detector (TCD). Argon was used as a carrier gas and operation conditions were: oven 100 C, detector and injection port 225 C. The biogas volume was measured by water displacement method. Biogas results were converted to standard temperature and pressure conditions (STP, T = 273 K, p = 1 bar). Hydrogen sulphide, methylmercaptan and dimethylsulphide (DMS) were measured with a portable gas chromatograph with a detection limit of <0.5 ppm for all the gases (Photovac GC/ photoionization detector (PID)). Concentrations of trace elements and Fe were determined with a Perkin Elmer Optima 4300 DV inductively coupled plasma optical emission spectrometer (ICP-OES). The device was equipped with a Scott type double-pass spray chamber and a cross-ow nebulizer, RF power was 1400 W, plasma gas ow 15 l/min, nebulizer gas ow 0.6 l/min and auxiliary gas ow 0.2 l/min. Before analysis, samples were pretreated using the ultrasound-assisted digestion method. A sample volume of 20 ml was used throughout. The digestion solution of 6 ml of aqua regia and 4 droplets of HF was introduced into the sample vessel (50 ml centrifuge tube supplied by Sarstedt) and placed into the ultrasonic water bath. The sonication procedure was performed at 40 C and contains three steps: 3 min sonication, 15 min standing and 23 min sonication. After the sonication procedure the sample was centrifuged for 15 min at 3500 rpm and ltered using the (VWR GF/A Grade 691, 90 mm, 1.6 lm) lter. After the ltration, the addition of internal standard Yttrium was performed resulting in a concentration of 1 ppm in the nal sample volume of 50 ml. The detection limits

Table 3 Process parameters and characteristics of the digestates in semi-continuously fed CSTRs digesting pig slaughterhouse waste. HRT 30 d. Reactor S1 Days 417 1862 6370 7182 83125 126157 158169 170217 218284 417 1862 6370 7182 83155 156187 188195 196242 417 1862 6370 7182 83164 165187 188206 207224 225242 OLR (kgVS/m3 d) 1 1.5 2 N.f. 1.5 N.f. 1 1.5 1.5 1 1.5 2 N.f. 1.5 N.f. 1 1.5 1 1.5 2 N.f. 1.5 1.75 2 2.25 2.5 CH4 yield (m3/tVSfed) 690 150 680 60 540 720 40 750 10 630 80 760 140 600 10 680 80 290 670 110 580 290 740 50 620 250 700 50 560 720 20 720 20 670 10 710 80 410 160 pH 7.67.8 7.67.8 7.67.8 7.77.8 7.57.9 7.58.2 7.77.9 7.27.8 7.27.8 7.77.8 7.67.8 7.57.8 7.37.5 7.47.9 7.57.9 7.77.8 7.67.8 7.67.7 7.67.8 7.77.9 7.67.8 7.68.0 7.7 7.77.8 7.77.8 7.57.9 SCOD (g/l) 8.9 7.7 1.0 5.5 5.5 0.8 4.9 0.2 6.6 0.1 4.7 5.6 2.0 6.1 2.8 9 7.6 1.1 5.8 8.9 0.6 4.7 0.9 8.2 5.9 4.4 0.8 8.7 7.2 1.1 5.1 5.4 1.3 4.4 1.2 2.7 2.5 0.6 2.2 0 6.8 3.6 TVFAa (g/l) N.d. 0 (0) N.d. 0.4 0 0.8 1.3 2.1 2.4 00 1.8 2.1 N.d. N.d. 00 N.d. 4.6 1.2 0.8 1.2 2.0 2.5 0.9 0.1 0.1 N.d. 00 N.d. 00 00 00 00 00 2.6 3.4 NH4 (g/l) 1.5 0.1 1.7 0.1 1.8 2.3 2.2 0.1 2.5 0.3 2.2 2.2 0.1 2.4 0.1 1.6 0.1 1.6 0.1 1.7 2.5 2.2 0.1 2.7 2.7 2.5 0.1 1.6 0.1 1.7 0.1 1.8 2.4 2.5 0.2 2.3 2.6 0.1 2.7 3.2 0.1 TS (%) 4.4 0.2 2.4 0.6 1.2 1.0 1.2 0.4 0.9 0.0 2.2 0.1 1.0 0.4 1.2 0.7 4.4 0.3 2.4 0.6 1.2 1.1 1.2 0.4 3.0 3.0 2.4 0.4 4.4 0.4 2.6 0.5 1.7 1.7 2.2 0.3 1.7 0.2 1.6 0.2 2.0 0.1 1.8 0.5 VS (%) 3.3 0.2 1.8 0.5 0.8 0.7 0.8 0.3 0.6 0.0 1.8 0.0 0.7 0.3 0.9 0.6 3.3 0.2 1.8 0.5 0.8 0.8 0.8 0.4 2.4 2.4 1.9 0.3 3.3 0.3 1.9 0.4 1.2 1.3 1.7 0.3 1.4 0.2 1.3 0.2 1.6 0.1 1.4 0.4 VSremoval (%) -10 60 87 82 40 84 80 -10 60 87 82 20 58 -10 58 80 62 73 78 76 81

S2

S3

N.f. = Not fed. N.d. = Not determined. a Converted to SCOD.

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OLR (kgVS/m3d) Volumetric biogas production (m3/m3/d) Methane production (m3/tVSd) H2S of the biogas (ppm)

0 2

0 900

600

300

0 2100

1400

700

0 0 50 100 150 Time (d) 200 250 300

Fig. 1. Amount of biogas and methane produced, methane and H2S contents of the biogas in semi-continuously fed CSTRs digesting pig slaughterhouse waste; (e or ) S1, (h or ) S2, (D or ) S3. Additives were used as follows: S1 additive 2 on days 218284, S2 additive 1 on days 98187 and additive 2 on days 188242 and S3 additive 1 on days 126223 and additive 2 on days 98242.

of Co, Fe, Ni, and W resulted in 1.2, 4.9, 0.5, and 3.3 mg/kg, respectively. HRTs and OLRs were calculated by ve days per week feeding basis. VS removals were calculated by the equation 100 ((VSin-VSout)/VSin). NH3 concentrations were calculated as described earlier (Bayr et al., 2012). The standard deviation has been marked as when used.

3.2. Reactor experiments Anaerobic monodigestion of pig slaughterhouse waste and the effect of additives on the digestion process were studied in three parallel CSTRs with the same substrate. Process parameters and reactor performances are shown in Figs. 13 and summarized in Tables 2 and 3. At rst, all three reactors were operated similarly with OLR of 1 kg VS/m3 d from day 4 on and increased to OLR of 1.5 kg VS/m3 d on day 18 and to OLR of 2 kg VS/m3 d on day 70. On days 7177, 81 and 82 the reactors were not fed due to instabilities and from day 83 onward the reactors were fed with OLR of 1.5 kg VS/m3 d. After that the process parameters of the reactors differed due to different operational stability. Additives were fed to reactors S2 and S3 starting on day 98 whilst S1 was kept as a control reactor without additions, until additions to reactor S1 were started on day 218. During the period in which all the reactors were operated similarly (days 497), methane production of all the reactors uctuated between 600 and 800 dm3 CH4/kg VSfed. An increase of OLR

3. Results and Discussion 3.1. Material characterisation Substrate of the experiment, pig slaughterhouse waste, had TS concentration of 32% and part of VS from TS of 97% (Table 1) indicating high biodegradability. In batch experiments methane potential of the substrate was 630 120 dm3 CH4/kg VSadded. The substrate contained 15 g/kg of total nitrogen of which 1 g/kg was NH4-N.

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OLR (kgVS/m3d) pH SCOD (g/l) TVFA(g/l)

2 1 0 8.5

8.0

7.5

7.0 12 8 4 0 9 6 3 0 6 0.6 0.4 0.2 0.0 0 50 100 150 Time (d) 200 250 300

NH4-N (open, g/l)

4 2 0

Fig. 2. pH, SCOD, TVFA (converted to SCOD), NH4-N and NH3 of the digestates in semi-continuously fed CSTRs digesting pig slaughterhouse waste; (e or ) S1, (h or ) S2, (D or ) S3. Additives were used as follows: S1 additive 2 on days 218284, S2 additive 1 on days 98187 and additive 2 on days 188242 and S3 additive 1 on days 126223 and additive 2 on days 98242.

from 1.5 to 2 kg VS/m3 d (day 63) resulted in a decrease of the methane content and methane production in reactor S2 and thus all the reactors were kept unfed on days 7177, 81 and 82. On day 82 reactor contents were mixed to insure identical sludge content in each reactor (day 84 unfed) before starting additive feeding on day 98. After starting additive feeding, methane production of the reactors continued at the level of 700 dm3 CH4/kg VSfed. However, around day 123 with OLR of 1.5 kg VS/m3 d methane production of control reactor S1 started to decrease indicating instability and the reactor was not fed on days 126157. However, in other reactors methane production continued at the same level until it decreased in reactor S2 on day 152 (OLR 1.5 kg VS/m3 d) and on reactor S3 on day 235 (OLR 2.5 kg VS/m3 d). Methane yield of the reactors was at the same level as previously reported in co-digestion of rendering plant and slaughterhouse waste (720 dm3 CH4/kg VSfed, Bayr et al., 2012) and poultry slaughterhouse waste (600

700 dm3 CH4/kg VSfeed, Cuetos et al., 2008) at 35 C. According to the methane production results of the present study (Fig. 1), the addition feeding of additive 2 (reactor S3) improved process stability and thus enabled higher OLR. Methane content of the biogas uctuated between ca. 65 and 75% in all three reactors, which is typical for substrates containing high concentrations of proteins and lipids as also reported by 6572% methane content of the biogas in the digestion of poultry slaughterhouse wastewater in hybrid upow anaerobic sludge blanket reactor (Rajakumar et al., 2012). During the rst 70 days in all the reactors SCOD decreased from ca. 9 g/l to 46 g/l and TS and VS from 4.5 and 3.5% to 1.2 and 0.7% respectively (Fig. 2), indicating good degradation. However, concentration of SCOD and TVFA increased around day 70 in reactor S2, and in other reactors concentrations increased somewhat on day 81 after mixing and redistribution of the reactor contents.

NH3 (black, g/l)

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6000

3.0

S1
Acid (mg/l)
4000 2.0

2000

1.0

0.0

6000

3.0

S2
Acid (mg/l)
4000 2.0

2000

1.0

0.0

6000

3.0

S3
Acid (mg/l)
4000 2.0

2000

1.0

0 0 50 100 150 Time (d) 200 250 300

0.0

Fig. 3. Acetic (e) and propionic (h) acid concentrations of the digestates in semi-continuously fed CSTRs digesting pig slaughterhouse waste, () OLR; S1 in the highest and S3 in the lowest gure. Concentrations of other VFAs were less than 320 mg/l in all the reactors throughout the experiments. Additives were used as follows: S1 additive 2 on days 218284, S2 additive 1 on days 98187 and additive 2 on days 188242 and S3 additive 1 on days 126223 and additive 2 on days 98242.

After mixing, concentrations of SCOD and TVFA decreased in all the reactors, but increased again in reactor S1 around day 120, and in reactor S2 (additive 1 addition from day 98 on) around day 135. Thus, reactors S1 and S2 were kept unfed on days 126157 and 156187, respectively. In reactor S3 where the introduction of additive 2 was started on day 98, SCOD and TVFA did not increase before d 235 as explained more specically later on. SCOD of the reactor S2 stabilized to 4 g/l after feeding was restarted with additive 2 instead of additive 1. Similarly, SCOD of reactor S1 rst decreased, but increased again around day 210 onward from which it can be concluded that OLR 1.5 kg VS/m3 d was too high for anaerobic digestion of pig slaughterhouse waste without additives. On day 218, reactor S1 began receiving additive 2 by a higher oneday dose and from day 219 onward daily addition was fed. This led to decrease in SCOD and TVFA concentrations around day 240. In reactor S3 concentrations of SCOD and TVFA increased rapidly around day 235 with OLR of 2.5 kg VS/m3 d. From these results we may conclude, that without additions and with addition of additive 1 (Fe and HCl), process became unstable with OLR of

1.5 kg VS/m3 d and HRT of 30 d, but with additive 2 (trace elements, Fe and HCl) process did not become unstable before OLR 2.5 kg VS/m3 d. However, because in S3 operation with OLRs of 1.752.5 lasted for less than 1 HRT each, it cannot be ensured that OLR as high as 2.25 would be stable in the long run. However, the stabilization of reactors S1 and S2 after additive 2 was introduced may prove that the process is more stable with additive 2 at least with OLR of 1.5 kg VS/m3 d than without additive. Among VFAs, acetic acid was the main acid in all the reactors (Fig. 3) as also noticed in the anaerobic batch digestion of chicken feathers with cattle slaughterhouse sludge or swine manure at 25 C (Xia et al., 2012). In addition to VFAs, also LCFAs (not analysed in the present study) can accumulate to the digestion process of the lipid-rich substrates and inhibit it as noticed in the anaerobic batch digestion e.g. by Palatsi et al. (2011) with pig and cattle slaughterhouse waste and Vallado et al. (2011) with poultry slaughterhouse efuent. However, even high concentrations of LCFAs can be tolerated as the process can adapt to the higher fatty acid concentrations due to changes in microbial populations (Palatsi et al., 2011;

OLR (kgVS/m3d)

OLR (kgVS/m3d)

OLR (kgVS/m3d)

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Vallado et al., 2011). However, while the previous ndings on adaptation were based on batch studies, in the present study, in semi-continuous reactors process failure occurred with relatively low OLRs in the absence of additives and thus the role of adaptation could not be proven. In the present study, the NH4-N concentrations of all the reactors were around 1.52.5 g/l until it increased in reactor S3 around day 200 on, to 3.5 g/l on day 242 (Fig. 2). It needs to be kept in mind that feeds of the reactors were diluted and ca. one third of the dilution liquid was circulated from the reactor digestate also circulating NH4-N back to the reactor. The TKN of all the reactors was between 3 and 4 g/l, except for the last measurement of reactors S1 and S3, which were 4.5 g/l (day 284) and 5.7 g/l (day 242), respectively. It depends on e.g. substrate and inoculum, which NH4-N concentration inhibits the digestion process. Some inhibitory limits have been suggested as 4 g/l was found to be inhibitory in the digestion of mashed barley at 40 C (Prochzka et al., 2012), 4.9 g/l was found to decrease methane production by 39% and 5.8 g/l by 64% in the digestion of synthetic wastewater at 55 C (Sung and Liu, 2003) and 3.9 and 5.6 g/l were found to decrease methane production by 50% in the digestion of synthetic substrate (pet food) to simulate OFMSW waste at 35 and 55 C, respectively (Benabdallah El Hajd et al., 2009). On the other hand, anaerobic digestion of slaughterhouse waste with manure and industrial by-products was shown to be feasible with NH4-N concentrations of more than 6 g/l (Karlsson and Ejlertsson, 2012). Compared to these it could be speculated, that NH4-N concentrations in the present study were below inhibitory. Calculated NH3 concentrations uctuated between 0.05 and 0.22 g/l in all the reactors during the operational period (Fig. 2). These concentrations were low compared to 1.1 g NH3/l suggested to be inhibitory in a previous study digesting cattle and swine manure (Hansen et al., 1998) and less than or equal to the 0.215 g/l suggested to inhibit methane production of synthetic substrate (pet food) simulating OFMSW, by 50% (Benabdallah El Hajd et al., 2009). The H2S content of the biogas in all three reactors was between 1400 and 1800 ppm on day 67 (Fig. 1). After additive 1 was introduced to reactor S2 and additive 2 to reactor S3 on day 98, the content of the H2S fell below the detection limit of 0.5 ppm. In reactor S1, the H2S content uctuated between 600 and 1900 ppm until it decreased to less than 100 ppm after additive 2 feeding was started on day 218. The decrease of the H2S content of the biogas in all the reactors after the feeding of the additives was due to the added Fe reacting with H2S to form FeS (Ryckebosch et al., 2011). The Fe concentrations of the digestates were about fourfold in reactors with additives, (S2 250 mg/kg FM and for S3 243 mg/kg FM) than in the control reactor (S1 64 mg/kg FM) at the end of the experimental period. The present results show that the studied additive 2 enabled higher OLR with stable performance than without additive and thus could improve the capacity of the biogas plant. The use of additive 2 (Fe, HCl and trace elements) prevented the build-up of VFAs and thus enabled acetogenesis and further improved the acetoclastic methanogenesis, as also noticed in anaerobic digestion of grass silage supplemented by cobalt addition (Jarvis et al., 1997). From this it can be concluded, that Fe and HCl together with trace elements, Co, Ni, Se and W were essential to have the impact while the additive without trace elements reduced the H2S content of the biogas but did not facilitate better performance of the reactor than obtained without additives. The nding that HCl and Fe addition did not improve process performance is opposite to results reported by Karlsson and Ejlertsson (2012), who found that HCl addition improved methane yield by decreasing the level of NH3 which decreased pH and concentration of VFAs in the digestion of slaughterhouse waste, manure and organic by-products. At the end of the experiment, trace element concentrations of 0.03 mg/kg FM of Co,

1.10 mg/kg FM of Ni and 0.49 mg/kg FM of W were analysed in the reactor S1 while the respective concentrations were 0.07, 0.68 and 0.84 mg/kg FM in reactor S2 and 0.15, 0.49 and 1.13 mg/kg FM in reactor S3. The possible effects of feeding additives and subsequent increasing concentrations of trace elements, iron and sulphur in the digestate on the further digestate utilization e.g. as a fertilizer is a topic needing further study. 4. Conclusions For the rst time, anaerobic monodigestion of pig slaughterhouse waste as well as the effect of additives on process performance was studied in laboratory scale CSTRs at 35 C. OLR of 2.25 kgVS/m3 d was found feasible with additive containing trace elements, Fe and HCl. On the other hand, OLR of 1.5 kgVS/m3 d was found to cause instabilities without additives and with additive containing Fe and HCl. Methane yields of ca 700 dm3 CH4/kg VSfed were obtained in all the reactors. Both additives were seen to decrease H2S concentration of the biogas below the detection limit 0.5 ppm. Acknowledgements The authors greatly acknowledge Mervi Koistinen, Hanna Koponen, Ossi Tonteri, Ida Harlin and Markku Rahikainen for their excellent laboratory work. References
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