Bioresource Technology 102 (2011) 1005–1011

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Bioresource Technology

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Anaerobic co-digestion of desugared molasses with cow manure; focusingon sodium and potassium inhibition

Cheng Fang, Kanokwan Boe, Irini Angelidaki ⇑Department of Environmental Engineering, Technical University of Denmark, Building 113, DK-2800 Kgs. Lyngby, Denmark

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 July 2010Received in revised form 17 September2010Accepted 20 September 2010Available online 27 September 2010

Keywords:Anaerobic digestionCo-digestionManureDesugared molassesInhibition

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.09.077

⇑ Corresponding author. Tel.: +45 4525 1429; fax: +E-mail address: ria@env.dtu.dk (I. Angelidaki).

Desugared molasses (DM), a syrup residue from beet-molasses, was investigated for biogas production inboth batch and in continuously-stirred tank reactor (CSTR) experiments. DM contained 2–3 times higherconcentration of ions than normal molasses, which could inhibit the biogas process. The effect of sodiumand potassium concentration on biogas production from manure was also investigated. Fifty percent inhi-bition occurred at sodium and potassium concentration of 11 and 28 g/L, respectively. The reactor exper-iments were carried out to investigate the biogas production from DM under different dilutions withwater and co-digestion with manure. Stable operation at maximum methane yield of 300 mL-CH4/gVS-added was obtained at a mixture of 5% DM in cow manure. The biogas process was inhibited at DM con-centrations higher than 15%. Manure was a good base substrate for co-digestion, and a stable anaerobicdigestion could be achieved by co-digesting DM with manure at the concentration below 15% DM.

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1. Introduction

Concerns about climate changes, and the need for energy supplysecurity, have led to increased interest for sustainable technologiesof renewable energy production. Anaerobic digestion (AD) has be-come an important technology for stabilization of waste streams,along with production of methane (Angelidaki et al., 2003). ADhas a number of advantages compared to other biomass conversiontechnologies, for example, it reserves nutrients and structure ofbiomass, opposite to incineration. AD requires less energy and ishighly efficient in utilizing all types of organic materials. Moreover,AD is relatively simple and straight forward to implement.

In year 2009, global sugar production was around 160 milliontons, which was 4.5% higher than in year 2008 (World sugar mar-ket review, 2009). Beet sugar production generates several streamsof organic by-products. The three main streams are molasses, beetpulp and grass cut-offs. Molasses is a by-product from sugarextraction process, which can contain up to 48% sugar (Satyawaliand Balakrishnan, 2007). Technological advances in sugar industryhave made it possible to extract more sugar from the normalmolasses. Desugared molasses (DM) is the syrup residue fromthe desugaring process of normal molasses (Olbrich, 1963). Fromthe factory data (DANISCO, Denmark), every ton of beet sugar pro-duced generates 0.24 ton of DM, 0.33 tons of beet pulp, and0.53 tons of grass cut-offs (Sugar production, 2001). Normal

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45 4593 2850.

molasses has several commercial applications, for example, as car-bon source for fermentation industry, as fertilizer, and as animalfeed, and it has also been reported to be used for biogas production(Lo et al., 1991). DM has less economic value than normal molasses(Olbrich, 1963), but application as animal feed has also been re-ported (Shellito et al., 2006). In general, DM contains lower concen-tration of sugar and higher concentration of ions such as sodiumand potassium, than normal molasses due to the desugaring pro-cess. Due to its relatively high organic loading, DM would consti-tute an attractive substrate for AD for production of biogas.However, its high ions concentration might cause a problem forthe AD process. The high ion concentration could cause inhibitionin the biogas process (McCarthy and McKinney, 1961; Mosey andHughes, 1975).

For solving inhibition problems from toxic compounds, dilutionor co-digestion with animal manure or other types of organicwastes, can be applied. It is proved that co-digestion offers eco-nomic and environmental benefits due to cost-sharing by process-ing multiple waste streams in a single facility (Margarita et al.,2009). There are two advantages using animal manure forco-digestion. First, it is a source for nutrients, trace metals, vita-mins and other compounds necessary for microbial growth. Sec-ond, it plays a role in neutralizing pH and improving bufferingcapacity (Angelidaki and Ellegaard, 2005). Although sodium andpotassium cations are found in several organic wastes used asfeedstocks in biogas reactors, there is relatively limited knowledgeabout their effect on the AD process. McCarthy and McKinney(1961) reported the effect of these ions on the biogas process using

Table 2Characteristics of desugared molasses, normal molasses and cow manure.

Component Units Desugaredmolasses

Normalmolassesa

Cowmanure

Total solids % w/w

49.8 77.0 5.1

Volatile solids % w/w

32.6 n/a 3.7

Lipid g/L 2.84 0 2.1–5b

Total sugar g/L 167 480 17–28.1b

Ammonium-N g-N/L 0.74 n/a 2.21Total-N g-N/L 6.72 n/a 3.33Protein g/L 37.4 60.0 7.0Bromide g/L 0.06 n/a 0Nitrate g/L 1.12 n/a 0Sulfate g/L 5.5 5.0 0Chloride g/L 19.8 9.0 19.8Calcium g/L 4.6 2.0 1.03Potassium g/L 159.6 47.0 4.4Sodium g/L 36.3 10.0 1.1Magnesium g/L 0.03 n/a 0.55

n/a, not available.a Data from Satyawali and Balakrishnan (2007).b Data from Boe et al. (2009) and Boe and Angelidaki (2009).

1006 C. Fang et al. / Bioresource Technology 102 (2011) 1005–1011

acetate as pure substrate. They found that the inhibiting concen-tration of sodium and potassium was at 10 g/L. Similar inhibitinglevel for sodium was obtained by Shin et al. (1994). However, inhi-bition of these cations on the AD process of mixed substrates stillneeds to be investigated.

In this study, possibility of utilizing DM for biogas productionwas investigated in batch and semi-continuously fed reactors. AsDM contained very high concentration of sodium and potassiumwhich could inhibit the biogas process, batch experiments werecarried out to investigate the effect of these ions on biogas produc-tion. Moreover, different dilutions and co-digestions of DM werealso tested for improving biogas production in both batch and reac-tor experiments.

2. Methods

Desugared molasses (DM) was obtained from a beet-sugarprocessing factory, (DANISCO, Nakskov, Denmark). Cow manurewas obtained from a Danish full-scale biogas plant (Hashøj Biogas,Denmark). All substrates were obtained in one batch and stored at�18 �C. The frozen substrates were thawed at 4 �C for 2–3 days be-fore use.

2.1. Methane potential of DM and the effect of dilution

A batch assay was set up to determine methane productionfrom DM at different dilutions with water. The batch assay wascarried out in 540 mL glass bottles in triplicates. Each bottle wasadded with 160 mL of inoculum and 40 mL of substrate. The sub-strate used was a mixture of DM diluted with distilled water atfour different dilutions as shown in Table 2. The inoculum wasfrom full-scale biogas plant co-digesting manure with industrialorganic wastes. After inoculation, the bottle headspace was flushedwith a mixture of N2:CO2 gas (80:20) and then closed with rubberstopper, sealed with aluminium cap and placed in a 55 �C incuba-tor. The methane content in the headspace was regularly measuredto follow the methane production until the maximum methaneproduction has reached.

2.2. Inhibition effect of sodium and potassium

Another batch experiment was set up to investigate the effect ofsodium and potassium on biogas production from cow manure.The batch experiment followed the same setup as for methanepotential of DM. The substrate used in this experiment was40 mL of cow manure supplemented with a mixture of differentsalts to provide the desired ion concentrations. Each cation wastested at six different concentrations. The maximum concentrationof each cation was chosen based on the actual concentration ofions found in DM. A mixture of NaCl, Na2CO3, NaHSO4�H2O andNa2HPO4�7H2O with the ratio of 2:2:5:5 by weight, was used to

Table 1Summarized data of substrate fed into the reactors.

Experiment Operatingtemperature (�C)

Day Substrate composition (%w/w)

Desugared molasses Cow manure

1 37 0–25 50 02 55 0–24 0 100

25–50 27 33 55 0–14 0 100

15–69 5 9570–163 15 85164–206 50 50

50% Inhibition level from batch results

provide sodium cation to the manure. The concentrations testedfor sodium were 1, 6, 10, 14, 19 and 36 g Na+/L. Likewise a mixtureof KNO3, K2CO3, KHCO3 and K2HPO4 with the ratio of 6:4:6:5 byweight was used to provide potassium cation to the manure. Theconcentrations tested for potassium were 5, 10, 15, 20, 82 and160 g K+/L. The ratio of different Na-salts was chosen so that eachsalt provides equal amount of Na+ ion to the mixture. Differentanions were used with Na+ or K+ in order to minimize the effectof anion concentration. The pH of Na- and K-salt mixture was 6.8and 6.99, respectively.

The inhibition effect was expressed as% inhibition, which wasdefined by equation:

% Inhibition ¼ A� BA� 100%

where A and B were the methane yield from the batch bottles with-out and with inhibition, respectively. Uninhibited methane produc-tion was obtained from the control where no extra ions were added.

2.3. Reactor experiments

Three reactor experiments were carried out for the biogas pro-duction of DM under different dilutions and co-digestions withmanure. A 4.5 L continuously-stirred tank reactor (CSTR) with 3 Lworking volume was used in each experiment at hydraulic reten-tion time approx. 20 days. In the first experiment, the reactorwas inoculated with mesophilic inocula and operated at 37 �C;while in the next two experiments the reactors were inoculated

Ion concentrations(g/L)

Average methane yield(mL-CH4/gVS-added)

Organic loading rate(gVS/(L-reactor�d))

Water Na+ K+

50 18.2 79.8 160 7.90 1.1 4.4 300 1.7

70 9.8 43.2 250 3.60 1.1 4.4 200 2.00 2.9 12.2 260 2.10 6.4 27.7 190 2.10 18.7 82.0 70 4.5

11 28

Table 3Methane yield of desugared molasses from batch experiment at different dilutions.

Desugared molassesconcentrationin water (%w/w)

Initial concentrationof desugared molasses(gVS/L)

Methane yield(mL-CH4/gVS-added)

1 3.26 300 ± 0.35 16.3 260 ± 0.6

10 32.6 190 ± 1.315 48.9 120 ± 1.3

Fig. 1. Inhibition effect of sodium and potassium.

C. Fang et al. / Bioresource Technology 102 (2011) 1005–1011 1007

with thermophilic inocula and operated at 55 �C. The substrate wasautomatically fed by peristaltic pump four times per day. The sub-strate compositions in each experiment, ion concentrations and or-ganic loading rates to the reactors are shown in Table 1. Biogasproduction was measured by an automated displacement gasmetering system with 100 mL cycle (Angelidaki et al., 1992). Bio-gas production was recorded daily, while pH and volatile fattyacids concentration were measured twice a week.

The reactor was built from double glass cylinder fitted withstainless steel plates as top and bottom. The top plate supportedthe mixer, mixer motor, feed tube, and effluent tube, temperaturemeasuring port and sampling port. The bottom plate had one sam-pling port. Stable reactor temperature was maintained by circulat-ing hot water in the space between the reactor glass walls. Reactormixer was controlled by timer and relay. It operated on a cycle of20 min mixing followed by 20 min stop. Pressure from the newfeed and biogas production inside the reactors pushed out theeffluent from liquid surface through the effluent tube on top ofreactor. The effluents were collected in effluent bottles.

2.4. Analytical methods

Total solids (TS), volatile solids (VS), total carbon, inorganic car-bon, pH, total Kjeldahl nitrogen (TKN), and ammonium nitrogenwere determined according to the Standard Methods (APHA,1995). Lipid content was determined by Soxhlet extraction method(APHA, 1995). Biogas composition (CH4 and CO2) was measuredusing a gas chromatograph (Mikrolab, Aarhus A/S, Denmark),equipped with a thermal conductivity detector (TCD). Volatile fattyacids (VFA) concentrations were measured using a gas chromato-graph (Shimadzu GC-2010AF, Kyoto, Japan), equipped with a flameionization detector (FID) (Angelidaki et al., 2009).

Sugar concentration was measured using a high performance li-quid chromatograph (HP series1100, Germany) equipped with acolumn BioRad Aminex HPX-87 H at 63 �C and a refractive indexdetector (RID1362A), using 0.6 mL/min of 4 mM H2SO4 as eluent.The lower detection limits for glucose, xylose and arabinose were0.011, 0.002 and 0.014 g/L, respectively. Chloride, bromide, nitrateand sulfate were measured using the method for determination of

inorganic anions by ion chromatography (Pfaff, 1993). For calcium,potassium and sodium determination, the samples were digestedusing HNO3 and HCl, followed by elemental analysis using atomicabsorption spectrometry with graphite atomizer (Alpha 4 AAS,Chem Tech Analytical, UK). All analyses were done in triplicates.Total organic carbon was calculated from the difference of totalcarbon and inorganic carbon. The protein content (g-protein/L)was calculated from an organic nitrogen (g-N/L) times a factor of6.25 (AOAC, 2000).

3. Results and discussion

3.1. Waste characteristics

Chemical composition of DM, normal molasses, and manure arepresented in Table 2. The anions and cations contained in DM wereevaluated in respect to their potential toxicity on the AD process.Concentration of several cations and anions in DM (Na+, K+, Ca2+

and Cl�) were found at concentrations more than twice highercompared to normal molasses. Although the concentration ofCa2+ in DM was higher than normal molasses, it was below the re-ported inhibiting level of 5 g/L Ca2+ (Ahn et al., 2006). Moreover,when comparing anions, SO4

2� concentration in DM was similarto normal molasses, while Cl� concentration in DM was similar le-vel as in manure. Both manure and normal molasses can be suc-cessfully used as pure substrates for anaerobic digestion (Parket al., 2010) and we could therefore, conclude that both SO4

2�

and Cl� concentrations in DM were below inhibitory levels. Like-wise, NO3

� concentration in DM was far below the reported inhib-iting level (Percheron et al., 1999). Due to very high concentrationof sodium and potassium in DM, it could be suspected that substi-tution of normal molasses with DM could be problematic foranaerobic digestion. On the other hand, manure contained highcontent of water, which could help diluting the potential inhibitorswhen applying for co-digestion. The organic matter in manure con-sists of carbohydrates, mainly as biofibers (almost 75% of the totalorganic content), proteins, volatile fatty acids and smaller concen-trations of other organics (Hartmann et al., 2000). Thus, it has goodproperties for neutralizing the potential inhibiting organic wastes.

3.2. Methane potential of DM and the effect of dilution

In order to investigate the inhibitory effect of DM on the biogasprocess, different dilutions of DM with water were tested for bio-gas production in batch experiment. The results showed that theinhibition decreased with increased dilution as seen from the in-crease of methane production (Table 3). In all bottles, the maxi-mum methane production was reached after 25 days. Themethane production rates in all bottles were relatively slow butno lag phase was observed (data not shown). This implies thatthe microorganisms did not adapt to the DM, but continued theirfunction at lower rates, i.e. growing at sub-optimal conditions.From the lipid, sugar and protein content in DM, the theoreticalmethane yield could be estimated to approx. 430 mL-CH4/gVS-added. However, from the batch experiment, the maximum meth-ane yield was only 300 mL-CH4/gVS-added at a DM concentrationof 1% in water, which corresponded to 70% of the theoretical yield.The incomplete conversion of organic matter to biogas could bedue to several reasons such as consumption of substrate for bio-synthesis, recalcitrance of some of the organic content, and/or byinhibition occurring at even this very low DM concentration.

1008 C. Fang et al. / Bioresource Technology 102 (2011) 1005–1011

3.3. Inhibition effect of sodium and potassium

Based on the chemical characteristics of DM, the inhibitionfound in the first batch experiment could potentially be due to highconcentration of ions in DM. Focusing on sodium and potassium,which were found at high concentrations in DM, another batch as-say was setup to investigate the effect these cations on the biogasprocess. The results showed that high concentrations of sodiumand potassium could inhibit the biogas process (Fig. 1). Sodiumshowed stronger inhibition than potassium, under the same cationconcentrations. This could be due to difference in the biologicalfunction of these cations in the microbial cells. Sodium affects bothoxidation mechanisms and is involved in transportation of sub-strate and ions through the cell membrane, while potassium isneeded particularly for oxidative activity (Payne, 1960). From thecorrelation in Fig. 1, 50% inhibition occurred at a cation concentra-tion of approx. 11 and 28 g/L for sodium and potassium, respec-tively. 50% inhibition was the cation concentration, resulting inhalf methane yield compared to the methane yield without the cat-ion. In general, the activity of the methanogenic archaea is influ-enced by several parameters such as temperature, pH, volatileacids, salts and other toxic compounds. The results from thisexperiment showed that high concentration of sodium and potas-sium can also affect the anaerobic digestion process.

He et al. (2006) investigated the effect of sodium and potassiumat the concentration of 25 and 50 g/L on anaerobic hydrolysis andacidogenesis of vegetable wastes. They observed that acidogenesiswas more sensitive than hydrolysis and it was necessary to controlpH when the cation concentration was high in order to ensure suc-cessful acidogenesis. Sodium cation has been reported to causemoderate inhibition at 3.5–5.5 g/L and strong inhibition at 8 g/L(Kugelman and McCarty, 1965). De Baere et al. (1984) reported ini-

Fig. 2. Reactor performance of the anaerobic digestion process of desugared molasses dilumethane yield and organic loading rate; (b) VFA and pH.

tial inhibition by Na+ at 30 g/L in a biofilm reactor and suggestedthat the high tolerance of Na+ in their study was due to the protec-tion of the microbial communities in biofilm, which mediated con-centration gradient, resulting in lower concentrations in thevicinity of the microorganisms. More recent studies also suggestedthat immobilized reactor systems are in general more tolerant todifferent inhibitors (Kolmert et al., 1997).

All organisms with a semi-permeable membrane are subjectedto osmotic pressure, resulting in water moving in and out of thecells according to the salt concentrations in the medium. High con-centrations of salts in the medium can, as result to the osmoticpressure, cause dehydration of microbial cells (Brock, 1970). In thiscase, the osmotic stress could have been the reason for the toxicitywe observed.

3.4. The effect of dilution and co-digestion in reactor experiments

The performance from the mesophilic digestion of 50% DM di-luted in water, in the CSTR reactor is shown in Fig. 2. The AD pro-cess was slowly inhibited as seen from VFA concentrationgradually increased. The pH was in the range of 7.2–8. Methaneyield was clearly dropped when the total VFA concentration wasover 300 mM. Although VFA increased to very high levels, pHwas relatively stable and remained above 7. The ammonia concen-tration in the reactor was in the range of 2.3–2.8 g-N/L (data notshown). The ammonium level in the reactor was not high enoughto explain the inhibition observed. Ammonia inhibition is occur-ring at ammonia concentrations higher than 4 g-N/L (Angelidakiand Ahring, 1993; Hansen et al., 1998). The inhibition in this pro-cess could possibly be due to the combination of high organicloading rate and high salt concentration in DM. Moreover, DM

ted with water (50%w/w) at mesophilic temperature 37 �C; (a) methane production,

C. Fang et al. / Bioresource Technology 102 (2011) 1005–1011 1009

could possibly lack of some important nutrients/micronutrient formicrobial growth.

In the second reactor experiment, a mixture of DM and manurediluted in water (27% DM, 3% manure and 70% water) was tested inat thermophilic temperature 55 �C because only thermophilic inoc-ulum was available at that time. The results are shown in Fig. 3.The methane yield when feeding with 100% manure ranged around200–400 mL-CH4/gVS-added and the VFA concentration was below50 mM. The pH was in the range of 7.5–8. During the first 5 daysafter introducing the new substrate mixture, the methane produc-tion increased as the results of increase organic loading. However,the VFA started accumulating slowly, indicating that the processwas inhibited. The inhibition was not obvious during the first5 days probably due to the accumulated salt concentration in thereactor was still low. Similar to the first reactor experiment, themethane production clearly dropped when the total VFA concen-tration was over 300 mM. The pH did not show significant re-sponse despite the increase of VFA. The high VFA concentrationsin the reactor clearly showed that the process became unstableafter introducing the new substrate, and therefore could be con-cluded that 27% DM dilution in 3% manure and 70% water was

Fig. 3. Reactor performance of co-digestion of 27% desugared molasses, 3% manure and 7and organic loading rate; (b) VFA and pH.

not enough to counteract the inhibition exhibited by DM. It isworth noticing that the highest methane production rate of1000 mL-CH4/(L-reactor�d) was obtained at 3 days after introduc-ing the DM mixture, indicating that the DM had good potentialfor methane production. However, the later decrease in methaneproduction and increase of VFA concentration was probably dueto accumulation of salt in the reactor.

In the third reactor experiment, co-digestion of DM and manureat different mixture ratios was tested. The results are shown inFig. 4. The average methane yield when feeding with 100% manuresubstrate was 200 mL-CH4/gVS-added, which was similar to theperiod feeding with a mixture of 5% DM and 95% manure. Duringboth periods, the VFA concentration was stable below 100 mMand pH around 8. The similar reactor performance during thesetwo periods was probably due to the organic loading rate was sim-ilar at approx. 8 gVS/(L-reactor�d), and the amount of cations in 5%DM was very low. When increasing the DM concentration to 15%under the same organic loading rate as the previous period, themethane yield decreased to approx. 100 mL-CH4/gVS-added, pHremained in the range of 7.5–8.1, while the VFA concentration in-creased up to 400 mM. This clearly indicated that the process was

0% water at thermophilic temperature 55 �C; (a) methane production, methane yield

Fig. 4. Reactor performance of co-digesting desugared molasses with manure at different desugared molasses concentrations at thermophilic temperature 55 �C; (a) methaneproduction, methane yield and organic loading rate; (b) VFA and pH.

1010 C. Fang et al. / Bioresource Technology 102 (2011) 1005–1011

stressed due to higher%DM which led to higher cations concentra-tion, and not from OLR. The average methane yield of 15% DM inmanure was 190 mL-CH4/gVS-added in continuous reactor experi-ment (Table 1), while the methane yield of 15% DM in water was120 mL-CH4/gVS-added from the batch experiment (Table 3). Thisproves that co-digestion with manure could increase the methaneyield. When the DM concentration was increased to 50%, corre-sponding to the organic loading rate of 4.5 gVS/(L-reactor�d). Afluctuation in methane production was observed, while pHdropped from 8 to 6.4, meanwhile VFA sharply increased up to1200 mM. The process broke down after 10 days, as seen from al-most no methane production. These results indicated that the con-centration of DM in manure should be kept lower than 15% in orderto avoid process instability.

In the present study, both mesophilic and thermophilic temper-ature were tested in the reactor experiments. In the first experi-ment, the reactor was operated at mesophilic temperature, andin the second and third experiment at thermophilic temperature.However, there was not any apparent difference was seen in per-formance, which indicates that the inhibition occurs at bothtemperatures.

Several studies have reported that the biogas process could beimproved and stabilized by application of co-digestion strategy(Totzke, 2009). Gelegenis et al. (2007) reported 10% improvementin biogas yield from applying co-digestion of olive-oil mill waste-water with diluted poultry manure compared to digestion of poul-try manure alone. In the present study, co-digestion of 5% DM withmanure did not show significant difference in methane yield fromthe digestion of 100% manure (approx. 300 mL-CH4/gVS-added)which could imply that this was the maximum practical potential

of DM. McCarthy and McKinney (1961) suggested that processimbalance due to high cation concentration i.e. sodium in the sub-strate was difficult to solve by neutralization alone, and dilutionwas a more proper solution. In this study, the co-digestion withcow manure also helped dilute the cations. Moreover, manurecontains various nutrients important of microbial growths. Thiscould be the reason why co-digestion with manure showed betterresults than dilution with water. The methane yield of 300 mL-CH4/gVS-added could be obtained at 1% DM when diluted withwater, while the same methane yield could be obtained at higherDM concentration up to 5% when mixed with manure. This indi-cated that higher organic load from DM could be applied whenco-digested with manure. In addition, it would not be sustainableto add water, which would result in larger wastewater volumes,while co-digestion with manure would not cause this problem asmanure already exists.

4. Conclusions

Desugared molasses (DM) contained more than 2–3 times high-er concentration of ions, especially sodium and potassium, thannormal molasses, which could strongly inhibit the biogas process.50% inhibition of manure digestion occurred at sodium and potas-sium concentration of 11 and 28 g/L, respectively. In order to min-imize the inhibition, co-digestion with manure can be a goodstrategy. The maximum methane yield of DM was 300 mL-CH4/gVS-added. Successful anaerobic digestion of a mixture of 5% DMwith cow manure was achieved and a stable methane productioncould be obtained at the concentration lower than 15% DM.

C. Fang et al. / Bioresource Technology 102 (2011) 1005–1011 1011

Acknowledgement

This work was supported by the Ph.D. scholarship from theDepartment of Environmental Engineering, Technical Universityof Denmark.

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