Waste Management 33 (2013) 1362–1371

Contents lists available at SciVerse ScienceDirect

Waste Management

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

The influence of slaughterhouse waste on fermentative H2 production fromfood waste: Preliminary results

Maria Rosaria Boni, Silvia Sbaffoni, Letizia Tuccinardi ⇑Department of Civil and Environmental Engineering, SAPIENZA University of Rome, via Eudossiana 18, 00184 Rome, Italy

a r t i c l e i n f o

Article history:Received 30 August 2012Accepted 28 February 2013Available online 30 March 2013

Keywords:BiohydrogenCo-digestion processFood wasteSlaughterhouse waste

0956-053X/$ - see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.wasman.2013.02.024

⇑ Corresponding author. Tel./fax: +39 06 44585015E-mail address: letizia.tuccinardi@uniroma1.it (L.

a b s t r a c t

The aim of this study was to evaluate the influence of slaughterhouse waste (SHW; essentially the skin,fats, and meat waste of pork, poultry, and beef) in a fermentative co-digestion process for H2 productionfrom pre-selected organic waste taken from a refectory (food waste [FW]). Batch tests under mesophilicconditions were conducted in stirred reactors filled with different proportions of FW and SHW. The addi-tion of 60% and 70% SHW to a mixture of SHW and FW improved H2 production compared to that in FWonly, reaching H2-production yields of 145 and 109 ml g VS�1

0 , respectively, which are 1.5–2 times higherthan that obtained with FW alone. Although the SHW ensured a more stable fermentative process due toits high buffering capacity, a depletion of H2 production occurred when SHW fraction was higher than70%. Above this percentage, the formation of foam and aggregated material created non-homogenousconditions of digestion. Additionally, the increasing amount of SHW in the reactors may lead to an accu-mulation of long chain fatty acids (LCFAs), which are potentially toxic for anaerobic microorganisms andmay inhibit the normal evolution of the fermentative process.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

In the anaerobic digestion process, the use of suitable co-sub-strates usually improves the biogas yields due to positive syner-gisms established in the digestion medium and to the supply ofmissing nutrients by the co-substrates. In addition, the diverse bac-terial communities of different types of waste can contribute to theoptimization of the digestion process (Marcias-Corral et al., 2008).For example, Hartmann and Ahring (2005) conducted a laboratory-scale experiment using a completely mixed thermophilic reactorfor co-digestion of the organic fraction of municipal solid waste(OFMSW) and cow manure. Their co-digestion experimentsshowed higher biogas production and a more stable process. Mar-cias-Corral et al. (2008) used the same substrate (OFMSW) with theaddition of agricultural waste and demonstrated that intrinsic cel-lulose-degrading bacteria and nutrients improved the digestion ofthe fiber present in the agricultural waste and in the paper fractionof the OFMSW. The authors concluded that OFMSW and cow man-ure have a synergistic effect on the digestion process that over-comes the imbalance in nutrients and improves biodegradation.Carucci et al. (2005) performed small-scale laboratory experimentson the co-digestion of food waste (FW) and aerobic sludge fromindustrial wastewater treatments. These experiments showed thatthe co-digestion of the two wastes can reduce the inhibition of

All rights reserved.

.Tuccinardi).

methanogenesis and increase methane yield. Murto et al. (2004)used a mixture of organic wastes (i.e., slaughterhouse waste[SHW], pig manure, vegetable waste, and industrial waste) toobtain a more buffered system and a well-functioning process toproduce methane. Edstrom et al. (2003) co-digested animalby-products with SHW, FW and liquid manure at a pilot scale toimprove methane yields.

In the field of anaerobic digestion, researchers have recentlybeen focused on dark fermentation for H2 production from organicwaste (e.g., FW, corn straw, kitchen waste, different types of man-ure; Lay et al., 2005; Fan et al., 2006; Li et al., 2007; Kim and Shin,2008; Wang and Zhao, 2009; Xing et al., 2010). The aim of thesestudies was to investigate energetic processes generated via envi-ronmentally sustainable methods and to significantly reduce thereliance on landfill disposal (Li and Fang, 2007; Wang and Wan,2008a,b; Zhang and Jahng, 2012).

Among the various possible substrates, FW might be suitable forfermentative hydrogen production because it is a carbohydrate-rich and easily hydrolysable waste (Gómez et al., 2006; Lee andChung, 2010; Tuccinardi et al., 2011; Redondas et al., 2012). How-ever, during the rapid hydrolysis/acidogenesis of substrates, theaccumulation of volatile fatty acids (VFAs) in the reactor could leadto the inhibition of the anaerobic digestion process. For this reason,the co-digestion of different substrates is a strategy used as analternative to more expensive supplementation with bufferingagents and minerals (Zhu et al., 2008; Lateef et al., 2012) to betterregulate the pH and other operative conditions (e.g., C/N ratio,

Table 1Main parameters of the physical–chemical characterization of the waste and theinoculum used.

Parameters Unitsa FW SHW Inoculum

pH – 5.84 ± 0.03 6.45 ± 0.01 6.77 ± 0.2TS % 23.76 ± 0.4 56.06 ± 0.2 1.09 ± 0.02VS % 94.86 ± 0.2 99.74 ± 0.1 60.74 ± 0.9TOCb % 42.83 ± 0.2 22.82 ± 0.7 23.62 ± 0.7TKNc % 1.91 ± 0.03 5.93 ± 0.1 4.22 ± 0.2C/N – 22.43 ± 0.4 3.85 ± 0.1 5.59 ± 0.4

a The percentages are expressed as grams per dry weight of the material (g/g ofdry weight of the sample).

b TOC = total organic carbon.c TKN = total Kjeldahl nitrogen.

M.R. Boni et al. / Waste Management 33 (2013) 1362–1371 1363

water content, VFAs production, micronutrient levels) within thereactor. In fact, the nitrogen and phosphorus content in fruit andvegetable waste is often low, and for this reason, these wastes havebeen used in co-digestions with wastes that have higher N and Pcontents (Alvarez and Lidén, 2008; Zhu et al., 2008). This combina-tion leads to an increase in biogas production, and different sub-strates can thus be stabilized.

The co-digestions of animal manure/sewage sludge and carbo-hydrate-rich feed (Kim et al., 2004, 2012; Zhu et al., 2009) and ofanimal manure and other wastes/feedstock (Yokoyama et al.,2010; Gilroyed et al., 2010; Lateef et al., 2012) for hydrogen pro-duction have been suggested. Nevertheless, the existing scientificliterature does not provide sufficient data on co-digestion pro-cesses dedicated to bio-H2 production. Thus, taking into accountthe above considerations regarding FW and SHW, the aim of thisexperimental activity was to evaluate the addition of different per-centages of SHW and FW in a fermentative co-digestion process.The potential enhancement of hydrogen production from FW wasalso investigated. The present authors have already tested FWregarding fermentative H2 generation (Boni et al., 2009). At pres-ent, the use of SHW as a co-substrate was studied only for methanegeneration (Alvarez and Lidén, 2008; Cuetos et al., 2008; Ek et al.,2011).

SHW are generally regarded as difficult substrates for anaerobicdigestion, mainly because of their typically high lipid and proteincontents (Banks and Wang, 1999). The degradation of proteins re-leases ammonia, which at high concentrations is suggested to beinhibitory for anaerobic microorganisms (Angelidaki and Ahring,1992; Hansen et al., 1998). Lipids may also cause problems inanaerobic digestion because of their tendency to promote floatingscum and due to possible accumulation of degradation-inhibitingintermediates, such as long-chain fatty acids (LCFAs) (Angelidakiet al., 1990; Angelidaki and Ahring, 1992; Broughton et al.,1998). Due to these properties, the use of SHW in its original undi-luted form (Hejnfelt and Angelidaki, 2009) for biogas production isgenerally precluded. Therefore, co-digestion with complementarywaste, such as municipal solid waste or FW, could be a moreattractive option to reduce problems associated with the accumu-lation of intermediate volatile compounds and high ammonia con-centrations (Cuetos et al., 2008).

2. Materials and methods

2.1. Materials

Co-digestion tests were carried out using two materials:

– Organic FW derived from a refectory in Rome (Lazio, Italy). TheFW consisted primarily of kitchen waste (i.e., pasta, bread, fruit,vegetables, meat, and fish).

– SHW consisting of skin, fat, and meat waste of pork, poultry,and beef, taken from a butcher’s shop in Rome.

The two substrates were separately shredded in a blender toguarantee better homogenization. The substrates were divided intoaliquots stored in a laboratory freezer (T = �4 �C) and used withinthree weeks.

The physical–chemical parameters, such as the pH and the con-centrations of total solids (TSs), volatile solids (VSs), total organiccarbon (TOC) and total Kjeldahl nitrogen (TKN), of the FW, SHWand inoculum were measured using standard methods (DIVAPRA,1998 and APHA, 2005). Duplicate tests were conducted, and thedata shown in Table 1 represent the average values of the mainparameters.

The FW was characterized by a high moisture content (Mc)(72%) and level of volatile solids (VSs) (96%). The SHW presentedhigher percentages of volatile solids (99%) and TKN (5.93%), anda lower moisture content (44%).

The inoculum was an activated aerobic sludge sampled from adomestic wastewater-treatment plant in Rome (Lazio, Italy). To in-crease the hydrogen production, the sludge was subjected to aheat-shock treatment (100 �C for 30 min). This treatment is com-monly used to inactivate hydrogen consumers, whereas hydro-gen-producing bacteria are capable of surviving due to theirability to produce spores (Li and Fang, 2007; Boni et al., 2009).

2.2. Experimental apparatus

Batch tests were conducted in 1.5-L glass reactors (Fig. 1a) thatwere hermetically sealed with gas-tight septa on the top to enablecollection of gas samples. The reactors were equipped with amechanical stirring system to guarantee better homogenizationas well as with a system on the bottom to sample the digestingmaterials for VFAs and pH analyses to investigate the evolutionof the fermentative process.

The total volume of the produced biogas was measured basedon the displacement of a saturated saline solution into a graduatecylinder system connected to the reactors.

Before the test started, each of the reactors was flushed withnitrogen to establish an anaerobic environment and was placedon a heating plate to ensure the establishment of mesophilic con-ditions (36 ± 2 �C). The experimental apparatus is shown in Fig. 1b.

2.3. Batch tests

Batch tests on FW conducted in previous experimental studiesby the authors (Boni et al., 2009) allowed for the selection of wetanaerobic digestion as the optimal operating condition for H2 pro-duction. This condition was also adopted in the present researchactivity with the aim of enhancing H2 production from the FWthrough co-digestion. Both semi-dry and dry conditions have beenshown to be inappropriate for the digestion of this material. Fur-thermore, the high moisture content of the FW made it difficultto achieve the TS content that is typical of semi-dry or dryconditions.

Nine co-digestion batch tests were conducted with differentproportions of the FW and SHW to test the influence of the SHWon the fermentative process. In addition, two control tests on FWand SHW separately were performed. Each test had the same initialconditions:

– Working volume: 800 ml.– Liquid–solid ratio (L/S): 8 l kg�1.– 100 g of substrate composed of different FW and SHW contents

(Table 2), adequately mixed and homogenized.

Fig. 1. (a) The reactor and (b) the complete experimental apparatus.

Table 2Identification of the different batches.

Batch label Composition of themixture (%w/w)

FW 100% FW90F-10S 90% FW-10% SHW80F-20S 80% FW-20% SHW70F-30S 70% FW -30% SHW60F-40S 60% FW-40% SHW50F-50S 50% FW-50% SHW40F-60S 40% FW-60% SHW30F-70S 30% FW-70% SHW20F-80S 20% FW-80% SHW10F-90S 10% FW-90% SHWSHW 100% SHW

1364 M.R. Boni et al. / Waste Management 33 (2013) 1362–1371

– Thermally pre-treated inoculum (30% by weight of the workingvolume).

– 6.5 g l�1 of CaCO3.– Distilled water.

The desired value of the L/S ratio was established to maintainwet conditions by adding demineralized distilled water in quanti-ties dependent upon the water content of the waste. All of the testswere performed in duplicate, and the results shown represent theaverage values.

2.4. Analytical Methods

The biogas composition (H2, CH4, and CO2) was analyzed dailyusing a gas chromatograph (Varian 3600 CX; Varian, Inc. AgilentTechnologies 2700 Mitchell Drive, Walnut Creek, CA 94598-1675/USA) equipped with a thermal conductivity detector (TCD) and a2 m stainless-steel packed column (ShinCarbon ST) with an innerdiameter of 1 mm. The operating temperatures at the injectionport and at the detector were 250 �C and 300 �C, respectively.The method used an initial column temperature of 80 �C held for2 min which was subsequently increased to 100 �C at a rate of2.5 �C min�1 and then held for 5 min.

The H2 yield was calculated as the total quantity of H2 producedper gram of initial total volatile solids (VS0) within the reactors.

Liquid sampling for the pH and VFAs analyses was performeddaily. The pH values were determined on the liquid uncentrifugedsamples. The concentration of VFAs (acetate, propionate, butyrate,isobutyrate, valerate, and isovalerate) was measured in centrifuged(4000 rpm for 15 min) and filtered samples (1.2 lm fiberglass fil-ter) using a GC equipped with a flame ionization detector (FID)

and a 30 m packed capillary column (TRB-WAX) with an innerdiameter of 0.53 mm. The operating temperatures of the injectionport and detector were 250 �C and 300 �C, respectively. The initialtemperature of the oven was 140 �C, which was held for 8 min,subsequently increased to 165 �C at a rate of 3 �C min�1, and finallyincreased to 230 �C at a rate of 30 �C min�1 and held for 3 min. He-lium was used as the carrier gas at a flow rate of 22 ml min�1.

3. Results and discussion

3.1. H2-production yields

In all of the batches, the H2 production ended within five days.Fig. 2 shows the final cumulative H2 yield measured during theexperimental tests for each mixture and for FW and SHWindividually.

The results demonstrated that the performance in terms of theH2-production yields of each of the batch tests was quite differentfrom that calculated based on a linear combination of the yieldsobtained using FW and SHW alone. In fact, a non-linear effect inthe measured H2 yields was observed with increasing SHW inthe mixture (Fig. 2), most likely due to the above-mentioned prop-erties of SHW in terms of composition and buffering capacity asdiscussed below. In particular, the batch tests with a percentageof SHW lower than 50% presented similar H2-production yields:the test control FW reached a final yield production of70.34 ml g VS�1

0 . Increasing the percentage of SHW up to 50% (i.e.,90F-10S, 80F-20S, 70F-30S, 60F-40S, and 50F-50S) produced onlya slight variability (less than 20 ml g VS�1

0 ) in the final yield, as alsoshown in Fig. 3 for H2 yields over time. Thus, it can be concludedthat when added in a percentage 650%, SHW did not exert any sig-nificant effect on the H2 production.

As the SHW percentage increased up to 70%, a change in the H2

yield was observed. In particular, the mixtures 40F-60S and 30F-70S reached a cumulative H2 production (145.09 ml g VS�1

0 and to109.40 ml g VS�1

0 , respectively) approximately 1.5–2 times higherthan that obtained with FW alone. These tests also showed thehighest H2-production rate (Fig. 4).

With an SHW level of 80% (i.e., 20F-80S), the hydrogen yield de-creased to a value similar to that reached using FW alone (approx-imately 74 ml g VS�1

0 ), and also, the experimental time profile of H2

production was quite similar to the FW experimental curve (Fig. 5).When the SHW level was higher than 80%, a decreased productionrate occurred, with a strong depletion of the final cumulative H2

yield. In 10F-90S, the yield was 44.43 ml g VS�10 , approximately

37% lower than that obtained with FW alone.

Fig. 3. Time profile of cumulative H2-production yields corresponding to SHW 6 50%.

Fig. 2. Cumulative H2 yields measured and calculated based on the linear combination of H2 production from the pure materials in the different batches.

M.R. Boni et al. / Waste Management 33 (2013) 1362–1371 1365

Although the mixture 10F-90S showed the lowest H2 yield, thisvalue was substantially higher than that reached using SHW alone(approximately 9 ml g VS�1

0 ) but much lower than that producedusing FW alone. This pattern was due to the high percentage of lesseasily hydrolyzed matter supplied in addition to FW, which led to adecrease in H2 production (Karlsson et al., 2008). It is worth noting

that in both the 80F-20S and 90F-10S treatments, mechanical stir-ring was not sufficient to prevent the formation of foam and aggre-gated material on the top of the reactors, demonstrating the hightendency of SHW to generate a non-uniform distribution in thedigesters. This pattern was also reported by Cuetos et al. (2008)and Salminen et al. (2001), who found that lipids have a tendency

Table 3Grams of VS0 and VSF together with the SR percentage.

Batch VS0 (g)a VSF (g)b SR (%)c

FW 24.68 15.78 36.0690F-10S 25.87 12.35 52.2480F-20S 27.07 6.63 75.5270F-30S 30.14 8.96 70.2860F-40S 18.59 7.43 60.0250F-50S 12.15 5.26 56.7240F-60S 11.59 4.98 57.0630F-70S 11.10 5.40 51.3820F-80S 9.96 4.68 53.0210F-90S 8.44 2.94 65.14SHW 18.73 11.96 36.15

a VS0: grams of initial volatile solids in the batches.b VSF: grams of final volatile solids in the batches.c SR: percentage of the efficiency of solids removal (VS0 � VSf)�100/VS0.

Fig. 4. Time profile of cumulative H2-production yields for the tests 40F-60S and 30F-70S (SHW = 60% and SHW = 70%, respectively).

Fig. 5. Time profile of cumulative H2-production yields in treatments with SHW P 80% (20F-80S and 10F-90S).

1366 M.R. Boni et al. / Waste Management 33 (2013) 1362–1371

to form floating aggregates and foam that may cause problems ofstratification. Because the floating of LCFAs may reduce theirbioavailability and simultaneously increase their potential toxicityto microorganisms due to LCFA accumulation in the reactor,H2-production inhibition can occur.

VS0 was considered the most reliable parameter to compare theresults obtained from the different mixtures because volatile solidsconsumption is in general related not only to H2 production butalso to fermentative generation of CO2 and soluble metabolites.Consequently, it was not possible to find a perfect correlation be-tween the H2 yields and solids removal (SR). In fact, the mixtureshad different digestibilities, as shown by the levels of the initialand the final volatile solids (VSF) measured before and after eachfermentation test (Table 3). In addition, the highest values of SRwere measured for the 80S-20F and 70F-30S treatments, whichwere not the best performing mixtures. The efficiency of solidremoval ranged between 51% and 75%, except for FW and SHW,in which SR was equal to approximately 36%.

During these tests, methane was not detected in the producedbiogas.

3.2. VFAs

The daily analysis of the digesting materials showed that acetic(HAc) and butyric (HBu) acids were the predominant solublemetabolites, with lower production of propionic acid (80–800 mg kg�1 of digestate). Thus, it can be suggested that H2 wasproduced by an acetic–butyric fermentation. In Fig. 6, the HAcand HBu concentrations are shown along with the H2 production.

In general, the maximum HAc and HBu values were observed tocorrespond to the H2-production peak. The highest values of bothof the acids (over 3000 mg kg�1 of digestate) were detected inthe FW-only treatment (Fig. 6j) and in those with significant FW

percentages (>50%), which also presented the highest H2-produc-tion peaks (Fig. 6a–c). As the SHW content increased to 80% and90% (Fig. 6h and i), the maximum concentrations of acetic and bu-tyric acid reached quite similar values but lower than thosereached in other tests, with an H2-production depletion. For SHW(Fig. 6k), the lowest cumulative and peak H2 production (156 ml)and the most delayed start of the acidogenesis were observed, de-spite the relatively high butyric and acetic acid concentrations(maximum values of 3100 and 1480 mg kg�1 of digestate, respec-tively). This pattern was most likely due to the high viscosity ofthe material, which made it difficult to maintain homogenous con-ditions within the reactor, affecting the fermentation process. Inaddition, as found by other authors (Salminen and Rintala, 2002;Broughton et al., 1998; Salminen et al., 2001), a sort of inhibitionof the anaerobic systems occurred in the case of the digestion ofsolid SHW with a high content of LCFAs, derived from the

Fig. 6. HAc and HBu concentrations and H2 production in the batch tests: (a) 90F-10S, (b) 80F-20S, (c) 70F-30S, (d) 60F-40S, (e) 50F-50S, (f) 40F-60S, (g) 30F-70S, (h) 20F-80S,(i) 10F-90S, (j) FW and (k) SHW.

M.R. Boni et al. / Waste Management 33 (2013) 1362–1371 1367

hydrolysis of lipids. LCFAs can accumulate, leading to toxic effectsto anaerobic microorganisms. In our study, this phenomenon oc-curred in the tests in which SHW was the main component, thuscontributing to the depletion of H2 production.

3.3. pH

Although SHW is generally considered a difficult substrate foranaerobic digestion, primarily due to its high protein and lipid con-

Fig. 6. (continued)

1368 M.R. Boni et al. / Waste Management 33 (2013) 1362–1371

tent (Hejnfelt and Angelidaki, 2009), it can, in co-digestion sys-tems, improve the C/N ratio (Cuetos et al., 2008), thus limitingthe potential inhibition of microorganisms. Moreover, becauseSHW is able to influence the buffering capacity of the reaction mix-ture and the resulting pH values (through balancing ammonia pro-duction, dissolved CO2, and VFAs production), this substrate can

avoid the excessive acidification of the system and the potentialinhibition of the anaerobic digestion process, thus improving fer-mentative H2 production. Generally, as reported by Kim et al.(2012), organic waste characterized by a high nitrogen content(such as SHW) combined with a material characterized by a highcarbon content (such as FW) can lead to a versatile mixture for

Fig. 7. Minimum and maximum pH values in the different batch tests.

Table 4CaCO3 added to the different reactors during the test.

Batches FW 90F-10S 80F-20S 70F-30S 60F-40S 50F-50S 40F-60S 30F-70S 20F-80S 10F-90S SHW

CaCO3 (g) 6.9 1.5 4.5 1.5 – – – – – – –pHF 5.22 5.47 5.20 5.15 5.13 5.15 5.21 5.11 5.18 5.53 5.98

M.R. Boni et al. / Waste Management 33 (2013) 1362–1371 1369

anaerobic processes that could be optimized in terms of the per-centages of each waste to maximize the desired product. Duringthe tests, the pH was adjusted with CaCO3 to avoid an excessivepH decrease, which is linked to the natural acidification of the sys-tem as an unavoidable consequence of the fermentation. CaCO3 (inquantities ranging between 0.5 and 1 g) was added when pH 6 5.0and when two successive temporal measurements showed a pHdecrease (especially during the first few hours of the fermenta-tion). Although some studies have reported that the presence ofCa2+ has a controversial effect on the anaerobic wastewater treat-ment in a bioreactor (Jackson-Moss et al., 1989; van Langeraket al., 1998; Yu et al., 2001; Liu et al., 2011; Ahn et al., 2006; Ferná-ndez-Nava et al., 2008), excessive amounts of calcium may reducespecific methanogenic activity (Chen et al., 2008). Experimentaldata regarding the effect of Ca2+ on fermentative hydrogen produc-tion are scarce in the literature; however, some studies reported apositive effect connected to the presence of calcium ions in fer-mentative systems. In fact, even if abundant calcium can suppressH2 production, Ca2+ additions have been proven to enhance the fer-mentative H2 production from sucrose (Chang and Lin, 2006; Wuet al., 2012) and glucose (Liu et al., 2012) due to the combinationwith CO2�

3 , which is able to eliminate the potential toxicity ofexcessive calcium content (Liu et al., 2012).

In this study, the presence of SHW in the mixture appears to ex-ert a buffering effect in the reactor. In fact, when the proportion ofSHW was higher than 30%, the pH showed an increasing trend inboth the minimum and maximum values (Fig. 7) without the needto adjust the pH by adding CaCO3 during the process (Table 4). Onthe contrary, in the mixtures with a high percentage of FW, the ini-tial pH adjustment used was apparently not sufficient to buffer thepH of the system, which tended to decrease as an unavoidable re-sult of the acidification occurring during the first phase of thefermentation process. This acidification led to a pH lower than 5in the FW, 90F-10S, 80F-20S, 70F-30S treatments. Consequently,additional CaCO3 was provided to adjust the pH until a stable valuehigher than 5 was achieved.

The pH adjustment was essential because biohydrogen ismainly produced at a pH range of 5–7, which most likely favorsthe activity of the hydrogenases and is suitable for microbial devel-opment in dark fermentation (Li and Fang, 2007; Li et al., 2007;Guo et al., 2010). In the case of FW, the optimal pH values for H2

production range between 5/5.5 and 6, as reported by severalauthors (van Ginkel et al., 2001; Kim et al., 2004; Shin and Youn,2005; Karlsson et al., 2008). In the best-performing mixtures(40F-60S and 30F-70S), the pH range was consistent with such val-ues and, therefore, was suitable for hydrogen production: theranges were 5.18–5.57 and 5.11–5.70 for the 30F-70S and 40F-60S treatments, respectively. Additionally, the pH stability, withlimited variation of the pH values during the fermentation of thetwo mixtures, may have contributed to an improvement in theH2 production.

The buffering capacity of a system combining FW + SHW mightalso be determined by the potential ammonia release from SHW. Infact, because SHW is a protein-rich substrate, it is a well-knownsource of ammonia during anaerobic degradation, with consequentpH increases in the digesters (Ek et al., 2011).

4. Conclusions

The preliminary results obtained for biohydrogen productionfrom the co-digestion of FW with SHW demonstrated that thepresence of SHW is able to affect the H2 production from FW.

The improvement in the production of H2 cannot be explainedby the simple effect resulting from the addition of SHW to FWbut rather by a sort of synergism resulting from the simultaneouspresence of the two substrates in certain percentages.

In particular, when the proportion of the SHW was lower than50%, the H2 yields were similar to that of FW alone (ranging be-tween 70 and 90 ml g VS�1

0 ). Higher SHW percentages (60% and70%) led to an improvement in H2 production, with cumulativeH2 yields of 145 ml g VS�1

0 and to 109 ml g VS�10 , respectively

(approximately 1.5–2 times higher than that obtained with FW

1370 M.R. Boni et al. / Waste Management 33 (2013) 1362–1371

alone). This behavior can be ascribed to the more stable fermenta-tive process ensured by the presence of SHW, which guaranteed acertain pH stability (no buffering agent was added during thesetests in the ranges suitable for hydrogen production).

Nevertheless, a further increase of the proportion of SHW(above 70%) led to a depletion in H2 production, most likely dueto the formation of foam and aggregated material resulting fromnon-homogenous conditions of digestion, despite mechanical stir-ring of the systems. These preliminary results suggested that theco-digestion of SHW and FW can improve H2 production, com-pared to the FW alone, only when SHW was added in percentagesranging between 60% and 70%. Future research is recommended tobetter understand the behavior of the mixture and to optimize theco-digestion system through additional investigations of the opti-mal pH, ammonia level, lipid content, and C/N during the test,which can influence the fermentation process. In particular, theprotein content of SHW and ammonia release during fermentativetests should be known step by step during each test to betterunderstand the evolution of the buffering capacity of the system.In addition, to better explain the unfavorable results obtainedwhen the SHW content was high in the batches, the effective sol-uble TOC could be analyzed. Because different parameters influ-enced the evolution of the fermentative process of the complexmixtures, a statistical analysis of the results may allow for a betterunderstanding of the influence of different factors affecting theprocess.

Acknowledgments

The authors thank Chiara Iobbi, who carried out the experimen-tal tests for her degree thesis. The authors are grateful to nativespeaker Dr. Maurice Shindler for assistance in improving the Eng-lish language of this manuscript.

References

Ahn, J.H., Do, T.H., Kim, S.D., Hwang, S., 2006. The effect of calcium on the anaerobicdigestion treating swine wastewater. Biochemical Engineering Journal 30 (1),33–38.

Alvarez, R., Lidén, G., 2008. Semi-continuous co-digestion of solid slaughterhousewaste, manure, and fruit and vegetable waste. Renewable Energy 33, 726–734.

Angelidaki, I., Ahring, B.K., 1992. Effect of free long-chain fatty acids onthermophilic anaerobic digestion. Applied Microbiology and Biotechnology37, 808–812.

Angelidaki, I., Petersen, S.P., Ahring, B.K., 1990. Effects of lipids on thermophilicanaerobic digestion and reduction of lipid inhibition upon addition of bentonite.Applied Microbiology and Biotechnology 33, 469–472.

APHA, AWWA, WEF. 2005. Standard Method for the examination of water andwastewater. 21st ed. Washington, DC.

Banks, C.J., Wang, Z., 1999. Development of a two phase anaerobic digester for thetreatment of mixed abattoir wastes. Water Science and Technology 40, 69–76.

Boni, M.R., Sbaffoni, S., Tuccinardi L., 2009. Lab-scale tests in different operatingconditions for the hydrogen and methane production from organic waste. In:Proceedings of Sardinia 2009. Twelfth International Waste Management andLandfill Symposium. S. Margherita di Pula, Cagliari, Italy.

Broughton, M.J., Thiele, J.H., Birch, E.J., Cohen, A., 1998. Anaerobic batch digestion ofsheep tallow. Water Research 32, 1423–1428.

Carucci, G., Carrasco, F., Trifoni, K., Majone, M., Beccari, M., 2005. Anaerobicdigestion of food industry wastes: effect of co-digestion on methane yield.Journal of Environmental Engineering 131 (7), 1037–1045.

Chang, F.Y., Lin, C.Y., 2006. Calcium effect on fermentative hydrogen production inan anaerobic up-flow sludge blanket system. Water Science and Technology 54(9), 105–112.

Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: areview. Bioresource Technology 99, 4044–4064.

Cuetos, M.J., Gómez, X., Otero, M., Moràn, A., 2008. Anaerobic digestion of solidslaughterhouse waste (SHW) at laboratory scale: influence of co-digestion withthe organic fraction of municipal solid waste (OFMSW). BiochemicalEngineering Journal 40, 99–106.

DIVAPRA, IPLA, ARPA. 1998. Metodi di analisi del compost: determinazionichimiche, fisiche, biologiche e microbiologiche; analisi merceologica deirifiuti. Collana Ambiente, Regione Piemonte, Assessorato all’Ambiente.

Edstrom, M., Norberg, A., Thyselius, L., 2003. Anaerobic treatment of animal by-products from slaughterhouses at laboratory and pilot scale. AppliedBiochemical and Biotechnology 109, 127–138.

Ek, A.E.W., Hallin, L., Vallin, L., Schnurer, A., Karlsson, M., 2011. Slaughterhousewaste co-digestion-Experences from 15 years of full-scale operation. In: WorldRenewable Energy Congress 2011-Sweden, 8-13 May 2011, Linkoping, Sweden.

Fan, Y., Zhang, Y., Zhang, S., Hou, H., Ren, B., 2006. Efficient conversion of wheatstraw wastes into biohydrogen gas by cow dung compost. BioresourceTechnology 97 (3), 500–505.

Fernández-Nava, Y., Marañón, E., Soons, J., Castrillón, L., 2008. Denitrification ofwastewater containing high nitrate and calcium concentrations. BioresourceTechnology 99 (17), 7976–7981.

Gilroyed, B.H., Li, C., Hao, X., Chu, A., McAllister, T.M., 2010. Biohydrogen productionfrom specified risk materials co-digested with cattle manure. InternationalJournal of Hydrogen Energy 35, 1099–1105.

Gómez, X., Morán, A., Cuetos, M.J., Sánchez, M.E., 2006. The production of hydrogenby dark fermentation of municipal solid wastes and slaughterhouse waste: atwo phase process. Journal of Power Sources 157, 727–732.

Guo, X.M., Trably, E., Latrille, E., Carrère, H., Steyer, J.P., 2010. Hydrogen productionfrom agricultural waste by dark fermentation: a review. International Journal ofHydrogen Energy 35, 10660–10673.

Hansen, K.H., Angelidaki, I., Ahring, B.K., 1998. Anaerobic digestion of swinemanure: inhibition by ammonia. Water Research 38, 5–12.

Hartmann, H., Ahring, B.K., 2005. Anaerobic digestion of the organic fraction ofmunicipal solid waste: influence of co-digestion with manure. Water Research3, 1543–1552.

Hejnfelt, A., Angelidaki, I., 2009. Anaerobic digestion of slaughterhouse by-products.Biomass and Bioenergy, 1046–1054.

Jackson-Moss, C.A., Duncan, J.R., Cooper, D.R., 1989. The effect of calcium onanaerobic digestion. Biotechnology Letters 11, 219–224.

Karlsson, A., Vallin, L., Ejlertsson, J., 2008. Effects of temperature, hydraulicretention time and hydrogen extraction rate on hydrogen production fromthe fermentation of food industry residues and manure. International Journal ofHydrogen Energy 33 (3), 953–962.

Kim, S.H., Shin, H.S., 2008. Effects of base-pretreatment on continuous enrichedculture for hydrogen production from food waste. International Journal ofHydrogen Energy 33 (19), 5266–5274.

Kim, S.-H., Han, S.K., Shin, H.S., 2004. Feasibility of biohydrogen production byanaerobic co-digestion of food waste and sewage sludge. International Journalof Hydrogen Energy 29 (15), 1607–1616.

Kim, M., Yang, Y., Morikawa-Sakura, M.S., Wang, Q., Lee, M.V., Lee, D.Y., Feng, C.,Zhou, Y., Zhang, Z., 2012. Hydrogen production by anaerobic co-digestion of ricestraw and sewage sludge. International Journal of Hydrogen Energy, 3142–3149.

Lateef, S.A., Beneragama, N., Yamashiro, T., Iwasaki, M., Ying, C., Umets, K.,2012. Biohydrogen production from co-digestion of cow manure and wastemilk under thermophilic temperature. Bioresource Technology 112, 251–257.

Lay, J., Fan, K., Hwang, J., Chang, J., Hsu, P., 2005. Factors affecting hydrogenproduction from food wastes by clostridium-rich composts. Journal ofEnvironmental Engineering 131 (4), 595–602.

Lee, Y.W., Chung, J., 2010. Bioproduction of hydrogen from food waste by pilot-scalecombined hydrogen/methane fermentation. International Journal of HydrogenEnergy 35, 11746–11755.

Li, C., Fang, H.H.P., 2007. Fermentative hydrogen production from wastewaterand solid wastes by mixed cultures. Environmental Science Technology 37,1–39.

Li, Y.F., Ren, N.Q., Chen, Y., Zheng, G.X., 2007. Ecological mechanism of fermentativehydrogen production by bacteria. International Journal of Hydrogen Energy 32,755–760.

Liu, J., Hu, J., Zhong, J., Luo, J., Zhao, A., Liu, F., Hong, R., Qian, G., Xu, Z.P., 2011. Theeffect of calcium on the treatment of fresh leachate in an expanded granularsludge bed bioreactor. Bioresource Technology 102, 5466–5472.

Liu, Q., Zhang, X.L., Jun, Z., Zhao, A.H., Chen, S.P., Liu, F., Tai, J., 2012. Effect ofcarbonate on anaerobic acidogenesis and fermentative hydrogen productionfrom glucose using leachate as supplementary culture under alkalineconditions. Bioresource Technology 113, 37–43.

Marcias-Corral, M., Samani, Z., Hanson, A., Smith, G., Funk, P., Yu, H., Longworth, J.,2008. Anaerobic digestion of municipal solid waste and agricultural waste andthe effect of co-digestion with dairy cow manure. Bioresource Technology 99,8288–8293.

Murto, M., Bijornsson, L., Mattiasson, B., 2004. Impact of food industrial waste onanaerobic co-digestion of sewage sludge and pig manure. Journal ofEnvironmental Management 70, 101–107.

Redondas, V., Gómez, X., García, S., Pevida, C., Rubiera, F., Morán, A., Pis, J.J., 2012.Hydrogen production from food wastes and gas post-treatment by CO2

adsorption. Waste Management 32, 60–66.Salminen, E., Rintala, J., 2002. Anaerobic digestion of organic solid poultry

slaughterhouse waste – a review. Bioresource Technology 83, 13–26.Salminen, E., Einola, J., Rintala, J., 2001. Characterisation and anaerobic batch

degradation of materials accumulating in anaerobic digesters treating poultryslaughterhouse waste. Environmental Technology 22, 577–585.

Shin, H., Youn, J., 2005. Conversion of food waste into hydrogen by thermophilicacidogenesis. Biodegradation 16 (1), 33–44.

Tuccinardi, L., Sbaffoni, S., Boni, M.R., 2011. Hydrogen production from householdwaste in semicontinuous reactor: selection of the optimal operatingconditions. In: Proceedings of Sardinia 2011. Thirteenth International WasteManagement and Landfill Symposium. 3–7 October 2011, S. Margherita diPula, Cagliari, Italy.

M.R. Boni et al. / Waste Management 33 (2013) 1362–1371 1371

van Ginkel, S., Sung, S., Lay, J.J., 2001. Biohydrogen production as a function of pHand substrate concentration. Environmental Science Technology 35 (24), 4726–4730.

van Langerak, E.P.A., Gonzalez-Gil, G., Van Aelst, A., Van Lier, J.B., Hamelers, H.V.M.,Lettinga, G., 1998. Effects of high calcium concentrations on the development ofmethanogenic sludge in up-flow anaerobic sludge bed (UASB) reactors. WaterResearch 32 (4), 1255–1263.

Wang, J.L., Wan, W., 2008a. Comparison of different pretreatment methods forenriching hydrogen-producing cultures from digested sludge. InternationalJournal of Hydrogen Energy 33, 2934–2941.

Wang, J.L., Wan, W., 2008b. Effect of Fe2+ concentrations on fermentative hydrogenproduction by mixed cultures. International Journal of Hydrogen Energy 33,1215–1220.

Wang, X., Zhao, Y., 2009. A bench scale study of fermentative hydrogen andmethane production from food waste in integrated two-stage process.International Journal of Hydrogen Energy 34 (1), 245–254.

Wu, S.Y., Chu, C.Y., Shen, Y.C., 2012. Effect of calcium ions on biohydrogenproduction performance in a fluidized bed bioreactor with activated carbon-

immobilized cells. International Journal of Hydrogen Energy 37, 15496–15502.

Xing, Y., Li, Z., Fan, Y.T., Hou, H.W., 2010. Biohydrogen production from dairymanures with acidification pretreatment by anaerobic fermentation.Environmental Science and Pollution Research 17, 392–399.

Yokoyama, H., Yamashita, T., Ogino, A., Ishida, M., Tanaka, Y., 2010. Hydrogenfermentation of cow manure mixed with food waste. Japan AgriculturalResearch Quarterly 44 (4), 399–404.

Yu, H.Q., Tay, J.H., Fang, H.H.P., 2001. The roles of calcium in sludge granulationduring UASB reactor start-up. Water Research 35 (4), 1052–1060.

Zhang, L., Jahng, D., 2012. Long-term anaerobic digestion of food waste stabilized bytrace elements. Waste Management 32, 1509–1515.

Zhu, H., Parker, W., Basnar, R., Proracki, A., Falleta, P., Beland, M., Seto, P., 2008.Biohydrogen production by anaerobic co-digestion of municipal food wasteand sewage sludges. International Journal of Hydrogen Energy 33, 3651–3659.

Zhu, J., Li, Y., Wu, X., Miller, C., Chen, P., Ruan, R., 2009. Swine manure fermentationfor hydrogen production. Bioresource Technology 100, 5472–5477.