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Renewable Energy 66 (2014) 550e558

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Renewable Energy

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Incorporation of fish by-product into the semi-continuous anaerobicco-digestion of pre-treated lignocellulose and cow manure, withrecovery of digestate’s nutrients

Maria M. Estevez a,*, Zehra Sapci a,b, Roar Linjordet c, 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, Norway

a r t i c l e i n f o

Article history:Received 16 May 2013Accepted 2 January 2014Available online 28 January 2014

Keywords:Anaerobic digestionFish by-productLiquid digestate recirculationAmmoniumeNStruviteBentonite adsorption

* Corresponding author. Tel.: þ47 64965493; fax: þE-mail address: maria.magdalena.estevez@nmbu.n

0960-1481/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.renene.2014.01.001

a b s t r a c t

Norway’s fish processing industry generates large amounts of fish waste every year. The high-risk wastefraction with most of its oil removed has not yet been tested for energy production. The stability of ananaerobic digestion process that incorporates this material with steam exploded Salix and cow manurewas tested using mesophilic, semi-continuous laboratory-scale digesters. The effects of recycling theliquid digestate fraction were also investigated. The removal of ammonium (NH4

þ) and phosphate (PO43�)

from the rejected digestate using struvite precipitation and bentonite adsorption were tested to generatea nutrient-enriched, final solid fertiliser. Adding 20% fish by-product (volatile solids basis) increasedmethane yields by 35%, while recycling the digestate caused a slight increase. The NH4

þeN levels reached4e5 g l�1 in the reactors with recirculation and fish feed. Although these levels may threaten meth-anogenesis, the stability of the process was maintained during the entire period due to the good balancebetween the lignocellulose, proteins and fats provided by the co-digestion mixture and the positiveeffects of recirculation. The NH4

þ and PO43� were successfully removed from the rejected liquid digestate.

The reductions using struvite reached 87% and 60% (pH 9.5 and Mg2þ:NH4þ:PO4

3� ratio of 1.2:1:1), whilebentonite achieved 82% and 52%, respectively.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic digestion (AD) under Norwegian conditions requiresoptimisation before becoming an applicable process for energyproduction. In a country with limited cultivation and farming land,the biomass used for biogas productionmust in addition come fromother areas, such as the industrial, municipal and forestry sectors.Fish processing is a vast industry in Norway, and Norwegian fish-eries produce more than 815,000 tons of by-products annually,accounting for more than 30% of all the fish caught and farmed inNorway [1].

The amount of “category 2” fish by-product was estimated to be40e50 thousand tons in 2009, and this amount should increase byca. 5% annually to match the general increases in aquaculture [1].According to the Norwegian Food and Safety Authority [2], thisconsists of “material with risks of animal and fish diseases andmaterial with residues of drugs content over the limit”. Examples

47 64 96 54 01.o (M.M. Estevez).

All rights reserved.

would be dead fish, disease infected fish and material with drugsresidues. Consequently, this material cannot be used to producefeed; it must be hygienised and subsequently treated using alter-native methods, such as composting or biogas production. Thehygienisation treatment performed in Norway is often called the“Fish Silage Processing Method” (FSPM) [3,4] and is adapted fromEU regulations EC 1069/2009 and EU 142/2011 [5]. This processconsists of ensiling thematerial with formic acid at a pH below 4 formore than 24 h, followed by a heat treatment above 85 �C for25 min. After hygienisation, the material is processed to separatethe oil fraction. Fish wastes normally contain higher lipid andprotein contents than cattlemanure [6], and the processed category2 material contains mostly the protein fraction. This compositionmay correspond to a greater potential for producing ammoniaduring AD, possibly inhibiting the process [6,7]. According to theliterature, inhibition can occur at various concentrations of totalammonium nitrogen (TAN) (1.4e17 g l�1) [7,8]. However, meth-anogenic bacteria can adapt to high ammonia levels duringanaerobic digestion. Schnürer and Nordberg [9] reported that highconcentrations of ammonia may shift the methane-producingpathway during mesophilic digestion, changing it from

M.M. Estevez et al. / Renewable Energy 66 (2014) 550e558 551

aceticlastic methanogenesis to syntrophic acetate oxidation (SAO),because the co-culture is less affected by the ammonia [10].

Co-digestion with carbon rich material, such as lignocellulosicbiomass, can help balance the high nitrogen input. Salix is a shortrotation energy crop commonly found in the Nordic countries; thismaterial was studied as substrate for biogas production after beingpre-treated using steam explosion [11]. The results indicated thatthe methane yield can be increased up to 50% at temperaturesabove 210 �C while using short retention times [11,12]. The co-digestion of steam-exploded Salix (up to 40% based on volatilesolids (VS)) with cow manure provided good yields [11].

The study’s first aim was to investigate the feasibility ofemploying a nutrient rich material, processed fish waste, for biogasproduction. The effects that higher ammoniumeN (NH4

þeN) levelswould have in the stability of the AD process can be demonstratedusing semi-continuous systems. The co-digestion of processed fishwaste, steam-exploded Salix and manure was evaluated as a mea-sure to avoid inhibition. Recirculation of the digestate was assessedfor water savings and increased methane yield; this process maylead to ammonium accumulation and subsequent process inhibi-tion. Therefore, alternatives to reduce the NH4

þeN concentration inthe digestate were studied.

One of the most promising techniques to remove NH4þeN from

wastewater and recover it as a slow release fertiliser [13] is toprecipitate it as magnesium ammonium phosphate hexahydrate,i.e., struvite:

Mg2þ þ NHþ4 þ PO3�

4 þ 6H2O4MgNH4PO4$6H2OðsÞ (1)

For this reactiontooccur, the three componentsmustbepresent instoichiometric ratios and the pH should be above 7.5 [14,15]. Inaddition, the accumulation of struvite is a frequent problem atwastewater treatment plants; this material clogs pumps and pipesdownstream from the sludge dewatering system for AD and post-digestion processes [14]. Therefore, recovery after AD would alsohelp to mitigate these problems. While most studies on struvite for-mation for recovery of nutrients focus onmunicipal wastewater [14],some address recovery from AD effluents [13]. When substrates ofvery different characteristic are being co-digested, the formation ofstruvitemay be hampered by the presence of other components, andthe applicability needs to be investigated inpristine feedstock blends.

Removing nutrients using adsorption on clay minerals isanother viable method because this process is inexpensive andcompetitive compared to biological and chemical treatments[16,17]. Zeolites and bentonites should be promising materials forremoving NH4

þ ions fromwastewater because they can act either asstrong acidic adsorbents or ion exchangematerials [16,17]. The highnegative charge on the bentonite surfaces is usually balanced byalkali metals and cations (typically sodium (Naþ) and calcium(Ca2þ)). According to Saltali et al. [16], clay minerals can be used assoil conditioners, improving the physicochemical properties of thesoil, reducing nutrient and water losses, and minimising NH3volatilisation.

Table 1Characteristics of the materials employed.

Material pH COD (g l�1) Total-N (g l�1) NH4þeN (g l�1)

Salix steam exploded 3.8 n.a. n.a. n.a.Manure 7.6 52.8 2.2 1.3Fish by-product 3.9 377.5 13.5 1.8Inoculum Ånaa 7.8 35.4 4.9 4.4

n.a. ¼ not available.a The inoculum was diluted for the startup period to levels of VS: 2% and NH4

þeN: 3.2b Water content determination by Karl Fisher.c Total carbon and nitrogen values from dried samples.

Phosphate (PO43�) is another important fertilising nutrient found

in agro-industrial effluents; in addition, the natural viable reservesof this compound will be depleted within measurable time [18,19].Therefore, alternatives for its recovery must be investigated further.Struvite precipitation would also remove PO4

3� from the rejecteddigestate, improving the nutrient composition of the final soliddigestate. During the sorption-removal of PO4

3� with bentonite, thebentonite cations can be replaced by inorganic hydroxyl-metalpolycations, such as aluminium and iron [20].

The second aim of this study was then, to investigate theremoval of NH4

þ and PO43� from the anaerobic digestion liquid

digestate by using struvite precipitation and bentonite adsorption.These techniques might prevent the accumulation of ammonium toinhibitory levels when recirculating digestate while enabling therecovery of NH4

þ and PO43� in solid fractions, increasing the value of

the final biofertiliser.

2. Materials and methods

2.1. Steam-exploded Salix

Samples of 4-year-old Salix “Tora” (Salix Orm x S. viminalis)harvested in Uppsala, Sweden, with 41.9% Total Solids (TS) werereceived chopped to a 0.5 cm particle size and frozen. The materialwas pre-treated using steam explosion at 210 �C for 10 min, at thepilot unit located at the Norwegian University of Life Sciences’campus (Ås, Norway) as described by Horn et al. [21]. The pre-treated material was stored in vacuum-sealed polyethylene bagsat 4 �C and later added to the reactor’s substrates mixtures. Thecharacteristics of the steam-exploded material are shown inTable 1.

2.2. Cow manure

Fresh cowmanurewas obtained from the farm of the NorwegianUniversity of Life Sciences and stored in a 20 kg container at 4 �Cbefore being fed to the reactors. The details are presented in Table 1.

2.3. Fish by-product

Category 2 fish by-product that was pre-treated according theFSPM and had its oil fraction removed to its greatest extent, wasprovided by BIOKRAFT MARIN AS (Trondheim, Norway). It wasstored at 4 �C before being used. The characteristics of the materialare given in Table 1.

2.4. Inoculum

The inoculumwas collected from amesophilic (37� 1 �C) biogasdigester at the farm Åna, in Rogaland, Norway, that process cowmanure with approx. 7e10% fish silage by weight as co-substrate.This inoculum was chosen because it was already adapted to afeeding composition similar to the co-digestion mixture in our

TS (%) VS (% dry basis) Cc (% dry basis) Nc (% dry basis) C/Nc

24.1b 97.0 51.2 0.6 8511.3 85.8 45.8 2.0 22.925.6b 96 53.6 9.8 5.56.9 79.2 40.6 3.9 10.4

g l�1.

M.M. Estevez et al. / Renewable Energy 66 (2014) 550e558552

study. It had a high nitrogen and VS content (Table 1), so it wasdiluted with water to a VS level of 2% and an NH4

þeN content of3200 mg l�1.

2.5. Methodology

2.5.1. Semi-continuous co-digestionFour 10 l CSTR (Belach Bioteknik AB, Sweden) with a nominal

working volume of 6 l were run at 37 � 1 �C and stirred 180 rpm.The reactors software (BIOPHANTOM�) allowed continuous, real-time monitoring of the pH, stirrer speed, temperature, gas flowand gas volume produced. The produced biogas was measuredusing volume displacement in glass columns provided with sen-sors. On the first day, 3 l of the diluted inoculum were fed to thereactors and maintained for a week to reduce the endogenousmethane production. The feeding of the respective substrate mix-tures began with a low organic loading rate (OLR: 1 g [VS] l�1 d�1)and was successively increased until an OLR of 3 g [VS] l�1 d�1 wasreached. The start-up period lasted 5 weeks.

During the experimental period, the reactors were fed once aday, 6 days a week, with 200 ml of freshly prepared feedstockmixtures, an OLR of 3 g [VS] l�1 d�1 and a hydraulic retention time(HRT) of 30 days, according to the feeding scheme detailed inTable 2. The liquid digestate was recirculated in three of the fourreactors (GA2, GB1 and GB2); the volume was removed before thedaily feeding and was filtered through 2.5 and 1 mm mesh sizesieves, and the liquid fractionwas used to dilute the fresh feedstockand so added back to the digester (approx. 100 ml). The remainingliquid fraction (i.e., rejected digestate) was collected and usedduring the nutrient removal trials. A diagram of the experimentalsetup for the process with recirculation is presented in Fig. 1. Forreactor GA1, the feedstock was diluted to 200ml with tap water. Forthe specific methane yield calculations, only the fresh daily VSadditions were considered. The anaerobic digestion process wasfollowed for 132 days.

2.5.2. Struvite precipitation trials on the digestateThe struvite trials were performed in duplicate at

Mg2þ:NH4þ:PO4

3� ratios of 1:1:1 and 1.2:1:1. MgCl2$6H2O andKH2PO4 were added to 300 ml aliquots of the rejected sieved (2.5and 1 mm mesh) digestate from reactor GA2 according to thestoichiometric calculations (Table 3). The content was agitated at300 rpm for 15 min and allowed to settle for 15 min before beingdivided into four 45-ml aliquots. These aliquots were adjusted topH levels of 7, 8, 9, and 9.5 with 1 N NaOH. The NH4

þeN, PO43� and

soluble chemical oxygen demand (COD) contents in the superna-tants were determined spectrophotometrically after a 24 h settlingperiod for the samples with visible sedimentation.

2.5.3. Bentonite adsorption trials on the digestateThe adsorption trials employing commercial Naþ bentonite

were performed in triplicate by adding 1.5, 3, 4, 6, and 9 g to 50 mlaliquots of the rejected sieved digestate from reactor GA2. Themixtures were agitated at 600 rpm for 5 min and allowed to settle

Table 2Feeding scheme.

Reactor Feedstock mixture

Fresh substrates

Salix (% VS) Manure (% VS) Fish (% VS)

GA1 40 40 20GA2 40 40 20GB1 40 50 10GB2 40 60 e

for 24 h before the spectrophotometric analyses for NH4þeN, PO4

3�

and soluble COD were performed.

2.5.4. Analytical toolsElemental analyses were used to determine the total carbon (C)

and nitrogen (N) on dried samples of each substrate, revealing theirC/N ratio, as stated by Estevez et al. [11]. These analyses were alsoperformed for the final digestate fractions, as well as the totalphosphorous (P), potassium (K) and heavy metals content, deter-mined by inductively coupled plasma-mass spectrometry (ICP-MS)after an ultra-clave digestion at 260 �C in concentrated, double-distilled HNO3 (0.25 ge0.3 g sample in 5 ml) and subsequentdilution to 50 ml. The equipment used was a triple quad ICP-MSAgilent 8800 QQQ Agilent technologies (California, USA) with thefollowing instrumental parameters: 1.05 l/min nebulizer gas flow,1550 W power on plasma, loop injection with 0.4 ml/min flow assample uptake, 100e250 ms dwell time, and three instrumentalreplicates. The sensitivity of the equipment is high; 500000 cps/ppb with 0e0.05 cps in background.

The biogas produced in the CSTRs were collected in gas-tightpolyethylene bags to measure the biogas composition with an SRIgas chromatograph (Model 8610 C) equipped with a thermal con-ductivity detector (TCD) and a 2 m. Haysep-D column. The injector,detector, and column were operated at 41, 153 and 81 �C, respec-tively. Helium was used as a carrier gas at 20 ml min�1. A standardgas mixture (CH4/CO2) at 65/35% was used for calibration. Thechromatogram was analysed using the PeakSimple 3.67 program.

The NH4þeN, TS and VS contents in the reactors were deter-

mined once a week, while the total N and total COD were deter-mined every 10 days. The concentration of NH4

þeN in the reactorswas followed with an Ion Selective Electrode (Orion-Thermo Sci-entific�), while the total N and COD were analysed spectrophoto-metrically with Merck Spectroquant� Kits.

The TS and VS contents were determined according to standardmethods [22] except for the fish by-product and pre-treated Salix.For those materials, a Karl Fisher titration of the water content wasused to determine their TS with a Metrohm automated volumetricsystem (Tampa, USA). The Karl Fisher method was chosen forsamples containing volatiles, such as alcohols and acids that are lostduring oven drying at 105 �C. The titrant was CombiTitrant 5 fromMerck (Darmstadt, Germany), containing iodine, sulphur dioxide,base and an alcohol; while dry methanol was the working mediumin the titration cell.

Determination of the volatile fatty acid (VFA) content was per-formed in the inoculum, fish by-product, and in samples collectedweekly from the reactors, by HPLC on a Dionex Ultimate 3000chromatographic system with UV detection. The VFAs weremeasured at 210 nm with a Zorbax Eclipse Plus C18 column fromAgilent Technologies (150 � 2.1 mm (3.5 mm particles)) equippedwith guard column by the same brand (12.5 � 2.1 mm (5 mmparticles)). Operating conditions were as follows: 40 �C columntemperature, 0.3 ml min�1

flow; the eluents were 100% methanoland 2.5 mM H2SO4; 1 ml sample injection volume. The sampleswere acidified before the analyses to pH <2.5 with concentrated

C/N of feedstock mixture(incl. digestate’s VS)

Dilution ratio (substrate mix.:liquid)

1:1 with water 441:1 with digestate 391:1 with digestate 411:1 with digestate 43

C

A

B

struvite precipitation bentonite adsorption

Solid digestate fraction (10-12% TS)

Fresh substrate mixture

Liquid digestate fraction not being recycled (rejected digestate, ca. 50%)

RecoveredNH4

+ & PO43-Final digestate

Liquid digestate fraction recycled (3.5-4.5% TS)

D

Fig. 1. Schematic diagram of the experimental set-up for the process with digestate’s recirculation; A: anaerobic co-digestion, B: filtration with sieves, C: struvite-nutrient removaltrials, D: bentonite adsorption-nutrient removal trials.

Table 3Struvite trials dosage of chemicals.

Mg2þ NH4þ PO4

3� MgCl2$6H2O added (g)to 300 ml digestate

KH2PO4 added (g)to 300 ml digestate

Initial concs. in filtrated digestate (mg l�1) 110 2300 56Initial molar ratio 1 28 0.13

Stoichiometric ratio for precipitation 1 1 1 7.5 5.2Stoichiometric ratio for precipitation 1.2 1 1 9 5.2

M.M. Estevez et al. / Renewable Energy 66 (2014) 550e558 553

H2SO4 (72%) and centrifuged twice for 10 min at 14,000 rpm toremove any particulate matter. The long chain fatty acids (LCFA) inthe fish by-product and in the inoculumwere analysed by Eurofins(Moss, Norway), according to the AOCS method [23] based onmethyl esterification and gas chromatography.

The magnesium (Mg), P, PO43�, NH4

þeN and the soluble CODcontents were determined in the accumulated digestate, struviteand bentonite trials with spectrophotometric Merck Spectroquant�

Kits. When necessary, the samples were diluted and/or centrifuged(6000 rpm, 5 min) before filtration through a 0.45 mm glass fibrefilter. All analyses were performed in triplicate.

2.5.5. Statistical analysesThe reported standard deviations were calculated using the

statistical functions in Microsoft Excel 2007. To evaluate the rela-tionship between the paired experimental data, the Minitab� 16.1.1statistical software was used. A two sample t-test was used tocompare the methane production profiles. The results wereassessed using p-values to reflect the statistical significance (con-fidence level 95%).

3. Results and discussion

3.1. Process stability and methane production

Average specific daily methane yields (ml [CH4] g [VS]�1) overall 132 days were: 180.5 � 43.0 for GA1, 191.3 � 39.9 for GA2,159.3 � 34.7 for GB1 and 140.9 � 26.6 for GB2. The average yieldwas 6% higher for GA2 than for GA1, although the maximum dailyyield was 279e292 ml [CH4] g [VS]�1 for GA1 versus 270 ml [CH4] g[VS]�1 for GA2 (Fig. 2). Both reactors were fed with the same sub-stratemixture including steam-exploded Salix, manure and fish by-

product, but in GA2, the digestate was recirculated. Statistically, therecirculation effect was significant when comparing the two pro-files (two sample t-test; p ¼ 0.034). The increased yield may becaused by positive effects of recirculating the digestate, such aspreserving trace-elements, enhancing adaptation to changes for anenriched microbial community and longer biomass retention [24e26].

When comparing the yields from the reactors fed with the fishby-product, GA1 and GA2, with GB2 which was fed with onlysteam-exploded Salix and manure, both reactors were significantlydifferent from GB2 (p ¼ 0.000 for both reactors). The yields for GA1and GA2 averaged 28 and 35% higher, respectively; adding the fishby-product in a proportion of 20% VS (7.2% in weight) caused aconsiderable increase. When this VS proportion was 10% in GB1(3.7% inweight), the average increase in yield compared to GB2 wasonly 13%. The potential maximum experimental yields for themixtures were calculatedwhile accounting for themaximumyieldsachieved for each substrate during previous biochemical methanepotential trials (BMP) and the fraction of VS applied to the feedstockmixture. For GA1 and GA2, the potential calculated maximum yieldwould be 291 ml [CH4] g [VS]�1, while in GB1, it would be 269 ml[CH4] g [VS]�1. These values were very similar to our experimentalresults: 292 ml [CH4] g [VS]�1 for GA2 and 264ml [CH4] g [VS]�1 forGB1 (Fig. 2).

Compared to studies of the co-digestion of fish waste with othersubstrates, an increase of 8% in the yield in sequential batch reactorprocess was achieved when the fish waste was co-digested withfruit and vegetable waste (1.4% by weight of fish waste in themixture), yielding 320ml [CH4] g [VS]�1 added [27]. Callaghan et al.[6] found that increasing the fraction of solid fish waste duringcontinuous co-digestion with cattle manure at high OLR (5.1e6.3 g[VS] l�1 d�1) deteriorated the process due to long chain fatty acid

0

50

100

150

200

250

300

350

0 20 40 60 80 100 120 140

mL

CH

4g -

1 VS

Days

GA1 GA2

0

50

100

150

200

250

300

350

0 20 40 60 80 100 120 140

mL

CH

4g-

1 VS

Days

GB1 GB2

Fig. 2. Specific methane yields (ml [CH4] g [VS]�1 d�1) for each reactor during 132 days. GA1 fed with Salix (40% VS), manure (40% VS) and fish (20% VS); GA2 fed with Salix (40%VS), manure (40% VS) and fish (20% VS) with recirculation of digestate; GB1 fed with Salix (40% VS), manure (50% VS) and fish (10% VS) with recirculation of digestate; GB2 fed withSalix (40% VS) and manure (60% VS) with recirculation of digestate.

M.M. Estevez et al. / Renewable Energy 66 (2014) 550e558554

(LCFA) inhibition; themaximumyield was between 270 and 300ml[CH4] g [VS]�1 added when the fish fraction was 4% by weight.Similarly, Eiroa et al. [28] also reported inhibition due to LCFA andVFA when performing the BMP assays for different solid fishwastes; for the mackerel type with a higher fat content, a higheraccumulation of LCFAwas observed. Table 4 shows the compositionof different types of solid fish waste. Category 2 underwent pre-treatment and homogenisation, and even though the lipid con-tent is reduced in our material, the amount is comparable to that offish offal and exceeds that of tuna processing waste. The VFAanalysis for the inoculum and fish by-product samples showed thatonly small quantities of acetic acid were present on the former(50 mg l�1), while the fish by-product contained approximately1200 mg l�1 of acetic and 480 mg l�1 of propionic acid. During theexperimental period, VFA content showed initial levels of propionic

Table 4Characteristics of different types of fish wastes.

Type of fishwaste

TS (%) Fat (%) Protein (%) Maximum methaneyield (ml g [VS]�1)

Literaturesource

Tuna solid waste 37 3.74 22.6 280 [28]Mackerel solid

waste32 11.8 17.82 350 [28]

Fish offala 29.6 8.2 20.3 [6]Fish silage 50.2 30 e 450e500 [1]Fish category 2b 25.6 7.4 10.2e14.8 500

a Fish offal accounts for the macerated mix of heads, tails and viscera produced bygutting, from a rainbow trout fish farm [6].

b Fish category 2 is the substrate employed and its results from itscharacterisation.

acid (maximum 1600 mg l�1) for the reactors containing the fishwaste and no signs of inhibition (Fig. 3). The buildup in propionicacid during the first HRT was higher for the reactor having a higherproportion of fish in its feeding (20% of total VS) and it may thus bea consequence of the higher load of easily degradable compounds,having a faster hydrolysis process than methanogenesis. Wheneasily degradable nitrogen-rich compounds increase in the feedingthis will affect methanogenesis, causing an accumulation of acids[29,30]. Acid accumulation would be normally accompanied by adrop in pH if the system’s buffer capacity is not robust enough[29,30] but this was not seen in our system, where lower concen-trations of VFA after the first HRT indicated that the process runstable.

The pH values in all the reactors increased 0.3 points after thefirst HRT, corresponding to the decrease in propionic acid levels,and the values for the reactors remained constant throughout theentire period: 7.42 � 0.15, 7.51 � 0.2, 7.43 � 0.18 and 7.37 � 0.13 forGA1, GA2, GB1 and GB2, respectively. The average methane con-tents ranged from 58 to 63% for the reactors containing fish andfrom 56 to 59% for GB2; the production yields remained stable forthe entire period (Fig. 2). Therefore, inhibition due to acids was notdetected in our systems. However, the LCFA were analysed for theinitial samples of inoculum and fish by-product, and theywere onlydetected in the latter. Unsaturated fatty acids, such as oleic (3.9%fish weight, 35% of total fatty acids) and linoleic (1.2% fish weight,11% of total fatty acids) were predominant. Although the LCFA areconverted into acetate, hydrogen, and eventually methane by themethanogens consortia [28,31,32], their accumulation can inhibitbacterial growth and biogas production [31,33]. Adsorption onto

0

400

800

1200

1600

2000

0 20 40 60 80 100 120 140

Prop

ioni

c ac

id (

mg

L-1 )

Days

GA1 GA2 GB1 GB2

0

400

800

1200

1600

2000

0 20 40 60 80 100 120 140

Acet

ic a

cid

(mg

L-1 )

Days

Fig. 3. Concentrations (mg l�1) of propionic and acetic acids. GA1 fed with Salix (40% VS), manure (40% VS) and fish (20% VS); GA2 fed with Salix (40% VS), manure (40% VS) and fish(20% VS) with recirculation of digestate; GB1 fed with Salix (40% VS), manure (50% VS) and fish (10% VS) with recirculation of digestate; GB2 fed with Salix (40% VS) and manure(60% VS) with recirculation of digestate.

M.M. Estevez et al. / Renewable Energy 66 (2014) 550e558 555

cell membranes and interference in the transport or protectionmechanisms are proposed to be the inhibitory effects [28,33]. Thesetoxic effects were observed in AD studies using fish wastes with acomposition similar to category 2 and without lignocellulosicbiomass as co-substrate [6,28]. However, some studies demon-strated that this type of inhibition is reversible, with processes thatrecover when LCFA concentrations decrease [34] or with processesthat can adapt to high lipid levels if fat is added in continuous cycles[32]. Co-digestion with lignocellulosic biomass and the longerretention times provided by the recirculation may have helped toavoid inhibition during our process.

The NH4þeN content increased during the period mostly for the

recirculating reactors containing fish by-products (GA2 and GB1)while the reactor with recirculation and without fish retainedstable levels (GB2) (Fig. 4). In GA1 with fish and without recircu-lation, the increasewas less pronounced compared to GA2 and GB1.The maximum NH4

þeN levels reached 5 g l�1 for GA2 during astable process run, a level that, according to literature, can inhibitmethanogenesis [7]. The co-digestionwith lignocellulose may haveaided our process by achieving stability and overcoming this inhi-bition. Furthermore, our process may have benefited fromemploying an inoculum possibly acclimated to high NH4

þeN levelsdue to the digestion of fish waste. When using digested swinemanure acclimated to 2.4 g l�1 NH4

þeN as the inoculum, themethane production occurred, even at 5 g/l of NH4

þeN [35]. Fornon-adapted inoculums, the methanogenesis ceases when theNH4

þeN levels reach 1.7e2 g l�1 [30].While recirculating the digestate may have promoted the

increased NH4þeN levels, it also may have helped acclimate the

microbial consortia by providing longer biomass retention times.Most studies have shown that acetoclastic methanogens are more

susceptible to free ammonia inhibition than hydrogenotrophicmethanogens [7,8,35]. When methane production from acetatefollows the SAO pathway, another group of non-methane-producing bacteria that is less affected by ammonia converts ace-tate into H2 and CO2; these components are used by the hydro-genotrophs to produce methane [10]. The co-culture responsiblefor SAO has a longer doubling time than methanogens [10], andtherefore, recirculating the digestate may have promoted a changeto the SAO pathway and the acclimation to high ammonia levels.

An estimation of the total COD degradation was calculated asapprox. 43e50% for the reactor without recirculation (GA1).Recirculating the digestate in GA2 produced higher total CODvalues during the process, with a total COD degradation of approx.43%. Solid accumulation is an important issue when performingdigestate recirculation. The VS content profiles showed anincreasing tendency overall for the entire experimental period(Fig. 4), although the incremental increases were less pronouncedthan in a previous study with steam-exploded Salix co-digestedwith manure and recycled digestate (unpublished results). At theend of the entire period, the VS content inside the reactors withrecirculation reached 6e7%, reaching a 1.5-fold increase, while inthe previous study, a 2.6-fold increase relative to the initial VScontent was obtained. Incorporating a 1 mm mesh in addition tothe 2.5 mm screen provided a better separation of the recalcitrantmaterial, allowing the process to run stable. The reactor withoutrecirculation exhibited the lowest VS content. The recalcitrantsolidsmay also accumulate due to recycling [26,36], translating intothe loss of process capacity and less degradation. Our VS degrada-tions during the entire period (accounting the VS added with therecycled digestate), ranged from 36 to 43%; the lower values cor-responded to GB2. Although the degradations in GA1 and GA2were

0

1

2

3

4

5

6

0 20 40 60 80 100 120

NH

4+ -N

(g L

-1)

Days

GA1 GA2 GB1 GB2

0

2

4

6

8

10

0 20 40 60 80 100 120

VS (%

)

Days

Fig. 4. AmmoniumeN (g l�1) and volatile solids levels (%) inside each reactor. GA1 fed with Salix (40% VS), manure (40% VS) and fish (20% VS); GA2 fed with Salix (40% VS), manure(40% VS) and fish (20% VS) with recirculation of digestate; GB1 fed with Salix (40% VS), manure (50% VS) and fish (10% VS) with recirculation of digestate; GB2 fed with Salix (40%VS) and manure (60% VS) with recirculation of digestate.

0

20

40

60

80

100

pH 9/ 1:1:1 pH 9.5/ 1:1:1 pH 9/ 1.2:1:1 pH 9.5/ 1.2.1:1

%

Ammonium removal Phosphate removal sCOD removal

Fig. 5. Removal efficiency (%) of ammoniumeN, phosphate and soluble COD from thefiltrated digestate after struvite precipitation for the 1:1:1 and 1.2:1:1 molar ratios, andpH 9 and 9.5.

M.M. Estevez et al. / Renewable Energy 66 (2014) 550e558556

similar (42 and 43%, respectively), the reactor with recirculation(GA2) produced slightly more methane possibly due to the longerhydraulic (60 days) and solid retention times (43e51 days). Theseconditions were expected to provide a significantly highermethaneyield for the recirculating reactors. Therefore, the main benefit ofrecirculationwas water savings, accounting for approximately 2.6e3 l per HRT when comparing GA1 and GA2. The lower VS degra-dation for the recirculated reactors were also reported by Hart-mann and Ahring [25] and Nordberg et al. [26], who stated thatproper control over such systems must be undertaken to avoid theaccumulation of recalcitrant substances.

3.2. Recovery of digestate nutrients

After analysing the amount of NH4þeN in the feedstock mixture

withfishbeforeanaerobicdigestion, this compoundaccounted for ca.36% of the total-N;whilst in thedigestateNH4

þeN represented94%ofthe total-N. This NH4

þeN remainedmostly (70%) in the liquid fractionafter separationwith themesh. Process inhibitiondidnotoccur inourreactors, and the removal of NH4

þeN was performed in the rejectedliquid digestate that was accumulated throughout the period.

Compared to nitrogen, the phosphorous content was low in alldigestates, ranging from 60 to 100 mg l�1. PO4

3� accounted for ca.92% of the total-P in the digestate, with proportions of 64% and 36%in the liquid and solid digestate fractions, respectively. The diges-tates with higher values of both NH4

þeN and PO43� were those from

the co-digestion of fish by-product and recirculation (GA2 andGB1), with 70% and 40% higher NH4

þeN levels than GB2,

respectively. The PO43� content was 18e28% higher for the reactors

co-digesting fish. Therefore, the rejected digestate from GA2 waschosen for NH4

þ and PO43� removal trials (Table 3).

3.2.1. Struvite trialsThe results of the NH4

þeN, PO43� and soluble COD reductions

achieved for the liquid phase of GA2 digestate after struvite pre-cipitation are presented in Fig. 5. The best sedimentation resultswere obtained at pH 9 and 9.5 for both ratios. The samples at pH 7and 8 did not always achieve a clear phase separation after 24 h, butlonger settling times allowed a clearer sedimentation; whenever

0

20

40

60

80

100

0 2 4 6 8 10

%

bentonite (g) added to 50 mL digestate

Ammonium removal Phosphate removal sCOD removal

Fig. 6. Adsorption efficiency (%) of ammoniumeN, phosphate and soluble COD againstmass of added bentonite (g) to 50 ml digestate aliquots.

M.M. Estevez et al. / Renewable Energy 66 (2014) 550e558 557

the supernatant was analysed, the NH4þeN reductionwas similar to

that for pH 9 and 9.5. The NH4þeN reductions levels were compa-

rable to those obtained by Altinbas et al. [13] and Yetilmezsoy andSapci-Zengin [37] for the pH levels between 9 and 9.2 and stoi-chiometric ratios of 1:1:1 and 1.5:1:1.

Reducing the original PO43� content was not as apparent as for

NH4þeN because the levels were not as high in the liquid fraction

(Table 3) and KH2PO4 was added to reach the appropriate stoichi-ometry. Positive reduction was only possible for the 1.2.1:1 ratio,and the best overall results were achieved at pH 9.5.

3.2.2. Bentonite adsorption trialsThe adsorption results are presented in Fig. 6. The removal ef-

ficiency for the NH4þ ions with bentonite increasedwith the amount

of bentonite added to the 50 ml digestate aliquots up to 76e82%. Aplateau occurred for doses above 4 g of bentonite in the 50 ml al-iquots, indicating that the removal efficiency is negligible at higherbentonite concentrations, most likely due to particle interactionsand aggregate formation.

When removing PO43� and soluble COD, the reductions

regarding the original levels did not exceed 40e50% (Fig. 6). TheCOD removal followed a trend similar to NH4

þeN, while PO43� was

removed better when lower doses of bentonite were applied.Therefore, when only removing NH4

þeN, bentonite use is a goodstrategy.

The elemental analysis of the dried digestate samples showedthe following N:P:K ratios: the accumulated digestates beforesieving yielded approximately 4:1:7; the solid fractions with fishyielded 3:1:4, and the fractions without fish yielded 2:1:5. A lowN:P ratio would translate to a good soil amendment for fruit trees,carrots or other root vegetables [38], but the recovered NH4

þ fromthe liquid fraction, when incorporated with the solid fraction, canimprove this ratio.

The limits for heavymetal and pathogen contents established bythe Norwegian Ministry of Agriculture and Food [39] must be ful-filled when employing the final digestate as organic fertiliser inNorway. Results showed cadmium (Cd) as the only heavy metallimiting the solid digestate’s class as organic fertiliser. Levels of0.6 mg Cd/kg TS would position it as organic fertiliser class 1 [39],able to be used in limited amounts in agricultural areas and parks.Overall, solid digestate coming from the digestion of Salix andmanure showed lower levels of zinc, cadmium and chromium thanthe digestate including fish.

According to the Norwegian Scientific Committee for Food andSafety [4], the FSPM applied to the category 2 fish by-product willinactivate Salmonella sp., non-spore-forming bacteria, Clostridiumperfringens, viruses and parasites. Manure that has been digestedfor several months at high ammonium concentrations is thought to

have pathogens inactivated [40]. Thus, regulations on organic fer-tiliser’s hygienic quality that prohibit the content of Salmonella sp.and restrict the content of thermo-tolerant coliformswould bemet.

4. Conclusions

The co-digestion of the organic- and nitrogen-rich category 2fish by-product together with steam-exploded Salix and manurewas successful. Adding this material increased the yields up to 35%,and the process was stable, even at high and potentially inhibitorylevels of NH4

þeN. The mixture with lignocelluloses, as well as thelonger retention times and enriched nutrients, micronutrients andmicrobial biomass content provided by recirculating the digestatemay have enhanced the stability of the process regarding the po-tential inhibitory problems caused by ammonia and free fatty acids.In countries where fish industry is extensive, the positive use ofunexploited value-added products, such as category 2 fish by-product, in stable anaerobic digestion processes, is of greatimportance.

The solid digestate fraction complied with the Norwegian reg-ulations regarding its use as organic fertiliser class 1, while itsnutrient content could be improved by recovering NH4

þ and PO43�

from the rejected liquid fraction. The cost-effective methods ofstruvite precipitation and bentonite adsorption were investigatedon the rejected digestate from the co-digestion of fish by-product,Salix and manure, containing high levels of NH4

þeN (2300 mg/L).Struvite was superior for NH4

þeN removal (87%); concurrently, 60%of the PO4

3� was removed.

Acknowledgements

We would like to express our gratitude to Anna Schnürer fromthe Swedish University of Agricultural Sciences (SLU), Elisabeth F.Olsen and Jane Agger from IKBM-NMBU, and Susanne Eich-Greatorex from IPM-NMBU. This study was financed by the Nor-wegian Research Council (project n� 190877 UB) in collaborationwith CAMBI.

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