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Journal of Environmental Management 136 (2014) 9e15

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Journal of Environmental Management

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

Semi-continuous anaerobic co-digestion of cow manure andsteam-exploded Salix with recirculation of liquid digestate

Maria M. Estevez a,*, Zehra Sapci a,b, Roar Linjordet c, Anna Schnürer d,e, John Morken a

aDepartment of Mathematical Sciences and Technology, Norwegian University of Life Sciences, P.O. Box 5003, 1432 Ås, NorwaybDepartment of Environmental Engineering, Bitlis Eren University, 13000 Bitlis, TurkeycBioforsk, Norwegian Institute for Agricultural and Environmental Research, Frederik A. Dahls vei 20, 1432 Ås, NorwaydDepartment of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, 1432 Ås, NorwayeDepartment of Microbiology, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7025, SE 750 07 Uppsala, Sweden

a r t i c l e i n f o

Article history:Received 20 September 2013Received in revised form20 January 2014Accepted 22 January 2014Available online 15 February 2014

Keywords:Anaerobic co-digestionSalixSteam explosionManureLiquid digestate recirculation

* Corresponding author. Tel.: þ 47 64 96 54 93; faxE-mail address: maria.magdalena.estevez@nmbu.n

0301-4797/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.jenvman.2014.01.028

a b s t r a c t

The effects of recirculating the liquid fraction of the digestate during mesophilic anaerobic co-digestionof steam-exploded Salix and cow manure were investigated in laboratory-scale continuously stirred tankreactors. An average organic loading rate of 2.6 g VS L�1 d�1 and a hydraulic retention time (HRT) of 30days were employed. Co-digestion of Salix and manure gave better methane yields than digestion ofmanure alone. Also, a 16% increase in the methane yield was achieved when digestate was recirculatedand used instead of water to dilute the feedstock (1:1 dilution ratio). The reactor in which the largerfraction of digestate was recirculated (1:3 dilution ratio) gave the highest methane yields. Ammonia andvolatile fatty acids did not reach inhibitory levels, and some potentially inhibitory compounds releasedduring steam explosion (i.e., furfural and 5-hydroxy methyl furfural) were only detected at trace levelsthroughout the entire study period. However, accumulation of solids, which was more pronounced in therecycling reactors, led to decreased methane yields in those systems after three HRTs. Refraining from theuse of fresh water to dilute biomass with a high-solids content and obtaining a final digestate withincreased dry matter content might offer important economic benefits in full-scale processes. To ensurelong-term stability in such an approach, it would be necessary to optimize separation of the fraction ofdigestate to be recirculated and also perform proper monitoring to avoid accumulation of solids.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years, increased attention has been focused on solu-tions aimed at mitigating nutrient losses and emissions of green-house gases from agricultural production in Norway, and one ofthese strategies entails anaerobic digestion of the organic wastesthat are generated within this sector. In such digestion, organiccompounds are degraded by microorganisms under anaerobicconditions, which yields various compounds, mainly methane andcarbon dioxide (i.e., biogas), that can be used to produce heat orelectricity, or as vehicle fuel. Mineral nutrients are also releasedduring the degradation process, which gives rise to a residue that isvaluable as fertilizer (Angelidaki and Ellegaard, 2003; Deublein andSteinhauser, 2008; Massé et al., 2011).

: þ47 64 96 54 01.o (M.M. Estevez).

All rights reserved.

Fast growing (short rotation) energy crops such as Salix are goodalternatives for bioenergy production. Comparing to softwoods, Salixcan sequester more carbonwithin a growing season (Kuzovkina andQuigley, 2005) and can easily adapt to extreme soil conditions, andhence it is a very viable source of biomass from an economicperspective (Sassner et al., 2008). Salix viminalis (basquet willow),which is widely cultivated in the Nordic countries, can produce up to35 � 103 kg of stems per hectare per year (Kuzovkina and Quigley,2005). However, this species has a high lignocellulosic content andis therefore less susceptible to degradation as a substrate duringanaerobic digestion. The degradability of Salix can be increased byuse of various mechanical, chemical, biological and thermal pre-treatment techniques (Sassner et al., 2008; Hendriks and Zeeman,2009; Bruni et al., 2010a). One of the most effective methods ishigh-pressure-steam treatment (Brownell and Saddler,1987; Ramos,2003; Bruni et al., 2010a), which combines high temperatures and arapid pressure reduction to achieve physical disruption of thelignocellulosic structures. Previous batch-scale studies have shownthat steam explosion of Salix as a pre-treatment can increase the

Table 1Characteristics of the substrates.

Material pH TSa VSb TOCa Total-Na NHþ4 eN

a

Treated Salix(210 �Ce10 min)

3.8 17.8 98.0 12.20 0.19 n.d.

Manure 6.8 11.3 85.8 4.49 0.20 0.17Inoculum 7.2 2.6 81.0 0.85 0.15 0.07

n.d. ¼ not determined.a Amounts expressed as percentage of wet weight.b Amount expressed as percentage of dry weight.

M.M. Estevez et al. / Journal of Environmental Management 136 (2014) 9e1510

yield of methane by up to 50% compared to digestion of untreatedmaterial (Horn et al., 2011a; Estevez et al., 2012).

Lignocellulosic biomass is rich in carbon, and thus it is essentialthat such material be co-digested together with nitrogen-richmaterials in order to achieve good nutrient balance, good yields,and stability in an anaerobic reactor (Deublein and Steinhauser,2008; Jagadabhi et al., 2008; Seppälä et al., 2008). In an earlierstudy (Estevez et al., 2012), we performed batch assays and foundthat co-digestion of steam-exploded Salix and manure led to fasterand more stable methane production compared to separatedigestion of these substrates (Estevez et al., 2012). The highestmethane yields were achieved at C/N ratios of 35e40, whichcorrespond to about 30e40% volatile solids (VS) from Salix in thedigestion mixture (Estevez et al., 2012).

In general, about 25% of the methane potential of substrateswith a high fiber content remains unexploited after anaerobicdigestion (Hartmann et al., 2000; Jagadabhi et al., 2008) and theresidual potential can be as high as 30% for anaerobic digesters atbiogas plants (Angelidaki et al., 2005; Seppälä et al., 2008). Failureto make effective use of this potential would entail not only eco-nomic, but also environmental implications, due to the subsequentloss of methane from storage tanks (Seppälä et al., 2008). Theanaerobic digestion process can be made more efficient and sus-tainable in the following ways: (i) by using gas-tight post-storagetanks (Jagadabhi et al., 2008; Massé et al., 2011); (ii) by applying apre-treatment and/or longer hydraulic retention times (Angelidakiet al., 2005; Massé et al., 2011); (iii) by recirculating the digestateand allowing a longer solid retention time in the digester and thusprolonging degradation. The results of previous studies of the ef-fects of recycling the digestate have varied. For example, Jagadabhiet al. (2008) found that attempts to recycle the solid digestatefraction separated from the liquid fraction did not enhance butrather decreased the production of methane. When digestate fibersare separated from the liquid phase, most of the organic materialremains in the liquid fraction, which thus has a much higher biogaspotential. Recirculation of the liquid fraction of the digestate hasproven successful, as indicated by increased methane production(Jarvis et al., 1995; Nordberg et al., 2007). Moreover, dilution to anappropriate total solids (TS) content is necessary to achieve satis-factory mixing during digestion of high-solids biomass (Angelidakiet al., 2006; Nordberg et al., 2007), and consumption of water inthat context can be decreased by substituting liquid digestate forwater. Such an approach will also lower the generation of effluents,and the one produced will contain a higher solids content, whichwill decrease both transport costs and storage capacityrequirements.

Thus far, few studies have focused on systems using continu-ously stirred tank reactors (CSTRs) for co-digestion of crops andmanure.

In this study we aim to investigate the stability and methaneproduction from a mixture of 40% VS from steam-exploded Salixand 60% manure in mesophilic small-scale CSTRs, as well asascertain the effects of recirculating the liquid digestate whileminimizing the consumption of water and manure. For compari-son, a control reactor degradingmanure only was also evaluated. Toour knowledge, no previous studies have explored the concept ofrecycling digestate from reactors processing Salix.

2. Materials and methods

2.1. Steam-exploded Salix

The samples used in this study consisted of Salix viminalis“Christina” that was harvested in November 2009 after the secondgrowing season in a 7-year-old short-rotation coppice plantation

located near Grimstad, Norway (58�200 N 8�310 E). The shoots werecollected manually and chopped to a nominal length of 7 mm in astandard disk chipper. The material was stored at �20 �C untilpretreated by steam explosion at the Norwegian University of LifeSciences in Ås, Norway, as described by Horn et al. (2011b). Atemperature of 210 �C and a residence time of 10 min were used.The pretreatedmaterial was packed in vacuum-sealed polyethylenebags and kept refrigerated at 4 �C until used. Characteristics oforganic components of untreated and steam-exploded Salix mate-rial are described elsewhere (Estevez et al., 2012), and Table 1 givesinformation on parameters such as pH, solids, total organic carbon(TOC) and total nitrogen (total-N) for the material subjected tosteam-explosion at 210 �C for 10 min.

2.2. Cow manure

Fresh cow manure was obtained from the farm run by theNorwegian University of Life Sciences (Ås, Norway) and was kept in20 kg containers at 4 �C until fed to the reactors. Some character-istics of the manure are listed in Table 1.

2.3. Inoculum

The inoculum used in the current reactors originated from aprevious experiment in which the gas potential of different mix-tures of pre-treated Salix and cattle manure was evaluated (Estevezet al., 2011). The digested material from the earlier experiment waspooled in a container and stored anaerobically for oneweek at 37 �Cbefore being used as inoculum in the CSTRs. Some characteristics ofthe inoculum are presented in Table 1.

2.4. Semi-continuous anaerobic digestion process

Four CSTRs (BELACH BIOTEKNIK AB, Sweden) with a nominalworking volume of 6 L were run at a temperature of 37 �C and astirrer speed of 18.85 rad s�1. The reactors were coupled to BluesensKombi-CO2/CH4 infrared dual-wave-length gas sensors to deter-mine the methane composition of the biogas produced. The soft-ware employed (BIOPHANTOM�) allowed continuous real-timemonitoring of pH, stirrer speed, temperature, gas flow, gas vol-ume and gas composition.

During the start-up period, the reactors were filled with 3 L ofinoculum. Feeding of the reactors up to their full working volumewas begun at a low organic loading rate (OLR) of 1 g VS L�1 d�1 ofthe respective substratemixtures andwas successively increased toa maximum of 3 g VS L�1 d�1. The start-up period lasted 3 weeks.

After the start-up period, normal operation of the reactorsconsisted on one daily feeding, 6 days a week, at an OLR of 3 g VSL�1 d�1 (one whole week with a daily load of 2.6 g VS L) and ahydraulic retention time (HRT) of 30 days. Thus 200 mL of freshlyprepared substrate mixture was added to each reactor on eachoccasion and an equivalent volume was removed before eachaddition so to maintain the volume inside the reactor constant.

Fresh substrates

Liquid digestate

solid digestateSieve

Fig. 1. Diagram of the recirculation system (reactors B and C).

M.M. Estevez et al. / Journal of Environmental Management 136 (2014) 9e15 11

To evaluate the effects of co-digestion reactor A was fed with amixture of 40% steam exploded Salix and 60% cow manure (VSbasis), and the substrate mixture was diluted to 200 mL with water.Reactor B was fed with the samemixture as reactor A, but in reactorB recirculation of the sieved digestate took place, using it as dilutingagent instead of water (dilution ratio substrate-liquid 1:1). ReactorC was fed with less manure than reactors A and B and had moredigestate recirculated (dilution ratio 1:3). These configurationsaimed to test the effects that dilution of the feedstock with liquiddigestate would have on the methane yield and the stability of theprocess, while saving resources as water and manure. Finally,reactor D acted as a control and was fed solely with cow manure,being diluted to 200 mL with water. Details of the feeding schemeare presented in Table 2. For the reactors B and C in which recir-culationwas applied, the output fraction that was removed prior toeach feeding (200 mL) was filtered through a sieve (2.5-mm mesh)and the resulting liquid fraction was returned to the digester(Fig. 1). Considering the VS content of the recirculated sieveddigestate, the average OLRs of B and C were 3.1 and 2.9 g VS L�1 d�1

respectively, and the HRT differed from the solids retention time(SRT), the latter being approximately 43 and 51 days for B and C,respectively. Only the daily additions of fresh VS were taken intoaccount in calculations of the specific methane yields. The anaer-obic digestion process was followed for a period of 100 days.

2.5. Analytical tools

Elemental analysis for determination of TOC and total-N in thefresh substrates, and subsequently also the C/N ratio, were per-formed as described by Nelson and Sommers (1982) and Bremmerand Mulvaney (1982), respectively. Samples were evaluated in aLECO CHN 1000 analyzer equipped with infrared (IR) cells todetermine TOC, and thermal conductivity (TC) cells to determinetotal-N. TS and VS content were analyzed by standard methods(APHA, 1995), and pH values were measured using a WTW Multi�350i meter equipped with a WTW pH electrode (Sen Tix 41), asreported by Allen (1989).

Volume of biogas produced, pH in the digester and methanecontent were continuously monitored by the reactor control pro-gram throughout the entire experiment. NH4

þ-N, TS and VS content,total-N, and chemical oxygen demand (COD)were determined oncea week. Samples for assessment of volatile fatty acids (VFAs) werecollected weekly and were analyzed by EUROFINS (Moss, Norway)according to the method described by Jonsson and Borén (2002).Furfural, 5-hydroxy methyl furfural (HMF), and phenolic com-pounds were analyzed at every HRT by ultra-high performanceliquid chromatography with diode array detection (UHPLC-DAD)performed on an Agilent Infinity 1290 system equipped with aZorbax Eclipse Plus C18 column (2.1�150mm,1.8 mm) fittedwith a0.5-mm frit inline filter. Samples were prepared by centrifugation(1466 rad s�1, 10 min) and acidification of the supernatant withH2SO4 72% to pH below 2.5.

The concentration of NH4þ-N in the digestates was monitored

using an Ion Selective Electrode (Orion-Thermo Scientific�). Total-N

Table 2Feeding schemes for the four reactors.

Reactor Feedstock mixture

Fresh substrates Dilution ratio(substrate mix.: liquid)

Salix (% VS) Manure (% VS)

A 40 60 1:1 with waterB 40 60 1:1 with digestateC 47 53 1:3 with digestateD e 100 1:1 with water

and COD were analyzed spectrophotometrically by use of MerckSpectroquant� Kits.

2.6. Statistical analysis

All standard deviations reported here were calculated usingstatistical functions of Microsoft Excel 2007. The statistical softwareMinitab� 16.1.1 was employed to evaluate the relationship betweenpaired experimental data. The results were assessed with p-valuesto reflect the statistical significance between paired groups (con-fidence level 95%). For each reactor, methane yield and volumetricgas production were evaluated by one way analysis of variance(ANOVA) followed by Fisher’s least significant difference method. Apaired t-test was used to compare variations in the methane yieldbetween weeks 4e8 and weeks 9e13.

3. Results and discussion

3.1. Effects of employing steam exploded Salix as a co-substrate

The specific methane yield of reactor A, having steam explodedSalix added as co-substrate, was 18% higher than the one of reactorD, digesting only manure (Table 3, Fig. 2), even though the co-digestion reactor showed a lower methane content (48% vrs.54%). Co-digestion with 40% VS content of steam exploded Salixproved to be beneficial to increase the yield, although the semi-continuous yields obtained were slightly lower than the valuesrecorded after 30 days of batch digestion of the same mixture ofSalix-manure (193 mL CH4 g VS�1) and manure alone (186 mL CH4g VS�1) (Estevez et al., 2012).

3.2. Effects of performing digestate recirculation

Performing recirculation and thus increasing the retentiontimes resulted in a comparably higher yield from the indicatedmixture of Salix-manure. In reactor B, when liquid digestate wasused for dilution of the fresh substrate mixture rather than water,

Digestate VS (%) in 200 mLof feedstock mixture

C/N of feedstock(incl. digestate VS)

OLR g VS L�1 d�1

(incl. digestate VS)

e 39 2.615 34 3.125 32 2.9e 23 2.6

Table 3Operational conditions for the four reactors.

Reactor AveragepH

Averagemethanecontent (%)

Max. specificmethaneyield (mL g�1)a

Max. volumetricproduction(L m�3 d�1)a

A 7.7 � 0.2 48.4 � 1.8 185.3 � 10.5 556.0 � 31.5B 7.3 � 0.1 55.9 � 1.8 215.2 � 15.7 645.5 � 47.2C 7.3 � 0.1 59.5 � 1.2 235.0 � 17.2 626.6 � 45.7D 7.6 � 0.1 54.3 � 1.2 156.5 � 11.7 469.4 � 33.4

a Based on weekly average production.

M.M. Estevez et al. / Journal of Environmental Management 136 (2014) 9e1512

there was a 16% increase in themethane production (Table 3). Thus,substituting liquid digestate for water resulted in significantlygreater generation of methane. Furthermore, the highest methaneyield among all the reactors was noted for C, in which part of thefresh manure was also replaced with filtered digestate and so thedilution ratio was higher (1:3) (Table 3). Applying a one-wayANOVA test based on Fisher’s method, we found that themethane yields differed significantly between all of the systems.The methane content of the biogas produced was higher in thereactors subjected to digestate recycling than in the reactorswithout recycling (Table 3). Notably, Jarvis et al. (1995) observedsimilar effects in a two-phase anaerobic digestion process fed withsilage: the methane yield of their whole system rose by 22% wheneffluent from the methanogenic reactor was recirculated back tothe acidogenic reactor. Moreover, Nordberg et al. (2007) found thata 25% increase in the OLR could be achieved by initiating recircu-lation of the liquid fraction in one-phase anaerobic digestion ofalfalfa silage, and the recycling in that system led to increased pH,alkalinity, and stability. Such influence on stability was also re-ported by Hartmann and Ahring (2005), who found that processliquid recirculation had an immediate buffering effect.

0

50

100

150

200

250

300

0 20 40

Met

hane

yie

ld (

mL

g - 1

VS)

A B

0

150

300

450

600

750

0 20 40

Vol.

met

hane

pro

d. (L

m-3

.d-1

)

Fig. 2. Specific methane yield (mL CH4 g VS�1) and volumetric methane production (L CH4

reactors. Reactors designations: A, run on Salix 40% VS and manure 60% VS, dilution with waand manure 53% VS, dilution with digestate; D, run on 100% VS manure, dilution with wat

There are several possible explanations for the positive effectsobserved in connection with recirculation of liquid digestate. Oneexplanation could be that suspended material, i.e. microbialbiomass, is being reintroduced in the reactor during recycling,enriching the degradation process (Nordberg et al., 2007). Also, theincrease in microbial biomass improved the contact between mi-croorganisms and substrate, making this process configurationmore robust to fluctuations (Hartmann and Ahring, 2005).Furthermore, the difference in yields between A and the reactorswith recirculation in our study might be partially explained bymore extensive degradation of VS in the liquid digestate as a resultof the prolonged retention time.

3.3. Accumulation of substances in the semi-continuous processwith recirculation

The maximum levels of volumetric methane production in therecycling reactors were achieved during the second HRT period andwere higher than those observed in the non-recycling digester A(Table 3, Fig. 2). However, during the last HRT, therewas a tendencytoward a decrease in the yields from B and C (Fig. 2). Degradation ofVS (considering both fresh and recirculated VS), was similar in re-actors A and B (average values 34 � 3% and 37 � 1%, respectively),higher in C (average 42� 4%), and lower in D (average 28� 5%). Dueto recycling, the SRT differed from the HRT, and both were longerthan 30 days. It is possible that the extended retention of solids in Band C enhanced the production of methane from the fraction of VSthat was still degradable in those reactors (Angelidaki et al., 2005).Likewise, part of the recalcitrant VS was also recycled and thusaccumulated, and hence probably gave rise to the observed increasein the VS content over time in the recirculating reactors (Fig. 3).Accumulation of organic and inorganic compounds during process

60 80 100

C D

60 80 100Days

m�3 d�1) calculated as weekly averages with standard deviations for each of the fourter; B, fed Salix 40% VS and manure 60% VS, dilution with digestate; C, fed Salix 47% VSer.

M.M. Estevez et al. / Journal of Environmental Management 136 (2014) 9e15 13

liquid recirculation accompanied by a gradual decrease in methaneyield has also been noted in a study of two-step digestion of alfalfasilage (Nordberg et al., 2007). The buildup of organic material inthat investigation led to an increase in the VFAs levels and even-tually to process inhibition. By comparison, in our study, VFAs werehardly detected, and thus therewas no organic overloading present,which implies that most of the accumulated solids were recalci-trant. Furthermore, Hartmann and Ahring (2005) observed loweraverage levels of VS degradation when liquid recirculation wasperformed in a co-digestion system that included organic fractionof municipal solid waste and manure. This finding suggests thatrecirculation of liquid digestate can eventually lead to inhibitioncaused by accumulation of solids. However, with proper control ofthe system and an optimum recirculation ratio, accumulation ofsolids may not represent a problem (Nordberg et al., 2007).

Showing a similar tendency to the VS content profile, CODvalues increased throughout the period for all the reactors (Fig. 3),while COD degradation ranged from 60% in the non-recirculatedreactors, to 64% in the recirculated ones.

As mentioned above, VFA contents were very low in all the re-actors throughout the entire experimental period, and themaximum total concentrations did not surpass 320mg L�1, which isfar below the level at which process imbalance can occur (i.e.,approx. 3000 mg L�1)(Ahring et al., 1995; Holm-Nielsen et al.,2007). Nordberg et al. (2007) noted that recycling 100% of liquiddigestate during digestion of alfalfa silage led to accumulation oforganic compounds, including VFAs, and subsequently to inhibitionof the system, whereas recycling of a mixture of 50% digestate and50%water caused onlymoderate accumulation of VFAs. Similarly, ina previous study conducted by our group (Estevez et al., 2011) 100%of liquid digestate was recirculated and mixed with steam-exploded Salix without the addition of manure or water, whichresulted in acidification of the process and collapse of the reactor.Thus, the mentioned results also support the conclusion that aproper recirculation ratio is necessary to balance the levels of VFAs,ammonium and solids in anaerobic digestion.

0

20

40

60

80

0 20 40 60 80 100

CO

D (

g L-1

)

A B C D

0

2

4

6

8

10

0 20 40 60 80 100

VS (%

)

Days

Fig. 3. Total chemical oxygen demand (COD) and volatile solids (VS) content (withstandard deviations) for the four reactors. Reactors designations: A, run on Salix 40% VSand manure 60% VS, dilution with water; B, fed Salix 40% VS and manure 60% VS,dilution with digestate; C, fed Salix 47% VS and manure 53% VS, dilution with digestate;D, run on 100% VS manure, dilution with water.

A possible drawback of using steam explosion as a pre-treatment to disrupt lignocellulosic materials is the accompa-nying release of phenolic compounds and furfurals, which mayinhibit the biomethanation process (Castro et al., 1994; Ramos,2003; Bruni et al., 2010b). However, in our reactors, only tracelevels of HMF and furfural were detected over the entire experi-mental period. Minor accumulation of HMF was detected in therecirculating reactors B and C seen as a 2.5-fold increase by the endof the period (100 days), whereas no HMF was found in reactor Ddigesting only manure. Furfural was present in all the reactorsand after three HRT increased twofold in A and B and threefold inC, although all of the recorded values were at trace levels. Thus,it seems unlikely that the decrease in gas production observedin our reactors was caused by inhibitory components. Moreover,this decrease was not statistically significant (p < 0.05) when thegas production profiles of the different reactors for weeks 4e8(second HRT) were compared with the profiles for weeks 9e13(third HRT).

Total-N increased slowly throughout the study period in all thereactors (Fig. 4). Average levels of NHþ

4eN were stable over theentire period andwere below inhibitory concentrations, even in therecycling reactors (Fig. 4), which is interesting considering thatrises in NHþ

4eN levels have been observed in other investigations inwhich silages were digested at mesophilic temperatures (Jarviset al., 1995; Nordberg et al., 2007). It seems that the C/N ratio ofthe substrates in our reactors was well balanced, and NHþ

4eN didnot reach levels that could inhibit the bacteria. Further researchshould include applying higher levels of nitrogen to investigateprocess stability during recirculation of digestate. Mesophilic pro-cesses with high concentrations of ammonia have been foundto exhibit a shift from acetoclastic methanogenesis to syntrophicacetate oxidation in the methane-producing pathway, and this al-lows stable operation of reactors at high ammonia levels (Schnüreret al., 1999). The doubling time of a syntrophic acetate oxidizingco-culture is longer than that of acetoclastic methanogens, so

0

1

2

3

4

0 20 40 60 80 100

Tota

l-N (g

L-1

)

A B C D

0

1

2

3

0 20 40 60 80 100

NH4+ -

N (g

L-1

)

Days

Fig. 4. Total nitrogen (total-N) and ammonium nitrogen (NHþ4 eN) content (with

standard deviations) for the four reactors. Reactors designations: A, run on Salix 40% VSand manure 60% VS, dilution with water; B, fed Salix 40% VS and manure 60% VS,dilution with digestate; C, fed Salix 47% VS and manure 53% VS, dilution with digestate;D, run on 100% VS manure, dilution with water.

M.M. Estevez et al. / Journal of Environmental Management 136 (2014) 9e1514

operation at high ammonia levels may require a comparatively longHRT to avoid washing out the syntrophic microorganisms, and thiscan be achieved by recirculating the digestate.

3.4. Benefits of co-digestion of steam exploded Salix and manureand digestate recirculation

Co-digestion of steam exploded Salix and manure gave an in-crease of themethane production of 18%. The ratio of Salix in the co-digestion mixture (40% VS basis) shows that if available, animportant amount of lignocellulosic biomass can be used as a co-substrate giving a stable process and better energy output thanthe use of only manure. This can be beneficial for large-scale andfarm-scale processes aiming to use lignocellulosic biomass as a co-substrate. The steam explosion pre-treatment was proven tosignificantly increase the specific methane production from alignin-rich biomass as Salix viminalis (Estevez et al., 2012), and suchpre-treatment can be applied to high solids-biomass types withoutthe need of adding chemicals and with minimal sample handling(Horn et al., 2011a,b). However, it requires high temperatures andthus energy consumption. In full scale plants using steam explosionas a pre-treatment for anaerobic digestion, continuous steam-explosion batches are run and so volatile materials that might becontained in the flash steam are also accounted for methane pro-duction, while the flash steam’s heat is recovered and used forheating the anaerobic digestion feedstock, making the whole pro-cess energetically self-sufficient and economically profitable(Pérez-Elvira and Fernández-Polanco, 2012).

Using filtered digestate to dilute high-solids feedstockreduces water consumption and the need to pre-heat the feedstockmixtures, which means lower process costs for full-scale plants(Jarvis et al., 1995; Angelidaki et al., 2006). Compared to A, reactor Bsaved roughly 3 L of water and reactor C 4 L of water per HRT,respectively.

Considering manure as a feedstock resource, dosifying its con-sumption may benefit farm-scale biogas plants that rely on sea-sonal animal manure for their biogas production. Approximatelyhalf the amount of fresh manure fed to reactor D was fed to C (3 kgless per HRT), that being in C co-digested and diluted with diges-tate, ended up giving better methane yields. Thus, in our study,performing digestate recirculation (16e27% yield increase) inaddition to co-digestion (18% yield increase) caused an increase ofthe methane production of 45%.

Furthermore, an average of 35% dry matter reduction was ach-ieved with the 2.5-mm e mesh sieve, so the final solid fraction inthe process digestate had a dry matter content of about 10%. In-creases on this level can lead to economic savings in relation totransport and handling of such material for subsequent use asfertilizer.

4. Conclusion

Co-digestion of Salix and manure in CSTRs gave better yieldsthan digestion ofmanure alone. Recirculation of the liquid digestateincreased methane productions, and inhibitions due to ammonia,VFA or furfural accumulation did not occurred. However, in the longterm recirculation caused a decrease in process efficiency, probablydue to accumulation of solids. Nevertheless, recirculation might bea good option to avoid the use of water for dilution of substrateswith high solids content, and to reduce manure consumption. Theincrease in degradation time for the not easily digestible materialsmay enhance methane yields. Clearly, it will be necessary to opti-mize the recycling ratio and solids content of the recirculateddigestate in order to avoid problems related to accumulated solidsin the long run.

Acknowledgments

All experimental work was carried out at the Norwegian Uni-versity of Life Sciences (NMBU) facilities. We are grateful to Svein J.Horn, Jane Agger and Elisabeth F. Olsen at the department ofChemistry, Biotechnology and Food Science (IKBM-NMBU) for theirhelp with steam-explosion and analytical techniques, and SusanneEich-Greatorex and Trine A. Sogn at the department of Plant andEnvironmental Sciences (IPM-NMBU) for the analysis of the sub-strates. This study was funded by the Norwegian Research Council(project n� 190877) in collaboration with the company CAMBI.

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