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Two-Stage Anaerobic Digestion of Energy Crops:Methane Production, Nitrogen Mineralisation andHeavy Metal MobilisationA. Lehtomäki & L. BjörnssonPublished online: 11 May 2010.

To cite this article: A. Lehtomäki & L. Björnsson (2006) Two-Stage Anaerobic Digestion of Energy Crops: MethaneProduction, Nitrogen Mineralisation and Heavy Metal Mobilisation, Environmental Technology, 27:2, 209-218, DOI:10.1080/09593332708618635

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Environmental Technology, Vol. 27. pp 209-218© Selper Ltd., 2006

TWO-STAGE ANAEROBIC DIGESTION OF ENERGYCROPS: METHANE PRODUCTION, NITROGEN

MINERALISATION AND HEAVY METAL MOBILISATION

A. LEHTOMÄKI1,2* AND L. BJÖRNSSON2

1Department of Biological and Environmental Sciences, University of Jyväskylä, P.O. Box 35,FI-40014 University of Jyväskylä, Finland

2Department of Biotechnology, Lund University, PO Box 124. SE-22100 Lund, Sweden

(Received 13 June 2005; Accepted 16 October 2005)

ABSTRACT

Energy crops (willow, sugar beet and grass silage) were digested in pilot scale two-stage anaerobic digesters. The specificmethane yields obtained were 0.16, 0.38 and 0.39 m3 kg-1 added volatile solids (VSadded) for willow, sugar beet and grass,respectively, corresponding to yearly gross energy yields of 15, 53 and 26 megawatt-hours (MWh) per hectare. With grassand sugar beets as substrate, 84-85 % of the harvestable methane was obtained within 30 days. In pilot scale two-stagedigestion of willow and sugar beet, 56 and 85 % of the laboratory scale methane yields were obtained, but digestion of grassin two-stage reactors yielded 5 % more methane than digestion in laboratory scale completely mixed low solids systems,possibly due to the pH conditions favourable to hydrolysis in the two-stage system. In digestion of grass and sugar beet theliquid at the end of digestion was rich in ammonium nitrogen, and the nitrogen in the substrate was efficiently mineralised.The results show that heavy metal concentrations are not likely to limit the utilisation of residues from digestion of non-metal accumulating crops. Efficient mobilisation of heavy metals during the acidic phase of digestion revealed the possibilityof removing metals from leachate generated in two-stage anaerobic digestion of phytoextracting crops.

Keywords: Biogas, methane, two-stage process, energy crops, nutrients

INTRODUCTION

Production of methane-rich biogas through anaerobicdigestion of organic materials provides a clean and versatileform of energy. The methane can be used for heat and powergeneration or as a vehicle fuel, in replacement for fossil fuels,thus cutting down the emissions of greenhouse gases andslowing down climate change [1]. The European Union (EU)has set a target of increasing the share of biofuels in vehiclesto 5.75 % by year 2010 in each member state [2]. In Swedentoday, methane and ethanol are the two main commerciallyavailable vehicle biofuels, and the domestic productionaccounted for 0.7 % of the total fuel consumption in 2003 [3].Utilisation of energy crops and crop residues will be aninteresting option for increasing the domestic biofuelproduction. Methane production through anaerobic digestionhas been evaluated as one of the most energy-efficient andenvironmentally benign ways of producing vehicle biofuel [4].It has been estimated that within agriculture in the EU, 1 500million tons of biomass could be anaerobically digested each

year, and half of this potential is accounted for by energycrops [5]. Most of the conventional agricultural crops aresuitable for anaerobic digestion if harvested beforelignification begins [6].

The predominant anaerobic digestion technology usedtoday is designed for waste fractions with high water content,such as sewage sludge or manure [7, 8]. Energy cropstypically have a rather high total solids (TS) content of 10-50%. In order to treat this kind of material in a one-stagecompletely mixed low solids system, the solids must usuallybe homogenised and diluted with external water, increasingthe volume to be treated and thus the energy required forheating and pumping. It is energetically and economicallywasteful to treat this kind of solid material in liquid phaselow solids digesters, and, thus, alternative approaches need tobe developed. Furthermore, the rate-limiting step in anaerobicdigestion of solid materials such as energy crops and cropresidues is hydrolysis of complex polymeric substances, suchas lignocellulose [9, 10, 11, 12]. The rate and extent oflignocellulose utilisation is limited due to the intense cross-

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linking of cellulose with hemicellulose and lignin. Moreover,the crystalline structure of cellulose prevents penetration bymicroorganisms or extracellular enzymes [13]. An alternativeprocess, which has previously been applied for digestion oforganic fraction of municipal solid waste [14, 15] andagroindustrial residues [16-20] is the two-stage process, wherethe first stage includes both hydrolysis/acidification andliquefaction of the substrate. In this process, hydrolysis andmethanogenesis can be optimised separately to suit the needsof each bacterial community [21]. The solids are hydrolysed ina first stage by circulating liquid over a bed of organic matter.The leachate is then pumped to the second stage, themethanogenic reactor, for further degradation. Since theleachate has low solids content, high-rate reactors such asupflow anaerobic sludge blanket reactors (UASBs) oranaerobic filters can be used in the second stage. A high solidretention time is achieved in these reactors throughattachment of biomass on carriers. The microbial growth in abiofilm also has the advantage of protecting the sensitivemethanogens from toxic shocks and overloads [21]. A two-stage approach is used in 10% of the anaerobic digesters formunicipal solid waste in Europe [22]. However, experiencesfrom digestion of energy crops using a two-stage approachare still limited. The use of various energy crops in biogasproduction has been suggested [23]. Perennial herbaceousgrasses are commonly cultivated as forage in northerncountries, and they are among the most efficient producers ofherbaceous biomass in boreal conditions [24]. Grass can easilybe stored as silage, therefore facilitating the continuousavailability of substrate for a biogas process all year round[25]. Cultivation of sugar beets is a widespread activity inSouthern Sweden. It gives high hectare yields of readilydegradable biomass, which can provide a suitable substratefor agricultural biogas production [6]. Willow is anestablished energy crop in Sweden, and it is routinelyharvested for combustion in the third or fourth year of growth[26]. If harvested at the end of the first growing season, theshoots will be less lignified and therefore more suitable forbiological degradation, yet giving high biomass yield perhectare with low cultivation input compared with manyherbaceous energy crops.

Production of inorganic fertilisers, especially thecapture of nitrogen from the atmosphere, is a very energy-

intensive process. For example, in cultivation of grass theproduction and application of inorganic fertilizers accountsfor 35 % of total energy input [27]. In order to maintain apositive energy balance in farm-scale biogas production, theuse of inorganic fertilisers in cultivation of energy cropsshould be minimised. The residues from anaerobic digestioncontain mineralised nitrogen, which is readily available forgrowing plants, as well as phosphorous and residual carbon,and they can thus be returned to the cultivation soil as afertiliser and a soil-improvement medium [28, 29, 30].However, very little information is available in the literatureon the distribution and mineralisation of nitrogen in a two-stage anaerobic digestion system. In a two-stage system,phosphorous is likely to remain in the solid residues, whereasthe mineralised nitrogen is more likely to end up in the liquidfraction. This could offer possibilities for more precisefertilisation, as well as for using the liquid as nitrogen-richirrigation water. When using organic residues as fertilisers, itis important to control the contents of unwanted compoundsin the residue. Metals like cadmium (Cd), lead (Pb), copper(Cu), nickel (Ni) and zinc (Zn) are abundant in Swedishagricultural soils, and could potentially be enriched by plantuptake [31]. The acidic conditions in the first stage of two-stage digestion would be expected to result in solubilisationof the metals, thus presenting a possibility of metal removalbefore recycling the digested residue back to the field.

The aim of this study was to evaluate the suitability oftwo-stage anaerobic digestion technology for methaneproduction from three very different energy crops, namelyfirst year shoots of energy willow, sugar beets and grasssilage. Furthermore, in order to study the possibilities ofusing residues from two-stage anaerobic digestion of energycrops as fertilisers and soil-improvement media, the fate ofnitrogen and heavy metals in different residual fractions ofthe process were investigated.

MATERIALS AND METHODS

Substrates

The substrates used were energy willow Salix viminalis(run A), sugar beet Beta vulgaris (run B) and grass silage(run C) (Table 1). The shoots of willow were harvested and

Table 1. Characteristics of substrates.

Substrate TS VS C TKN C/N Crudefibre

NDF ADF Lignin Higher heatcontent

(%) (%) (% TS) (% TS) (% TS) (% TS) (% TS) (% TS) (kWh kg-1 TS)Willow 49.5 48.7 49.3 0.9 55 53.1 71.0 63.5 13.8 5.5Sugar beet 20.2 17.9 41.8 0.9 47 8.7 17.1 11.3 1.5 4.6Grass 31.8 27.9 45.7 3.7 12 20.5 28.7 28.8 5.4 5.3

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chopped with an agricultural precision chopper at the end ofthe first growing season, and this fresh material was directlyloaded to the digesters. Whole sugar beet, i.e. both the beetsand tops were used as substrate, and before loading to thereactors, the beet tops stored as silage where mixed with thestored beets in ratio 1:3 on wet weight (WW) basis andchopped in an agricultural mixer. Grass silage was preparedfrom a mixture of English ryegrass Lolium perenne (50%) andwhite clover Trifolium repens (50%) by chopping at harvestwith an agricultural precision chopper and storing in bales.Before being loaded in the reactors the grass silage (laterreferred to as grass) was mixed in an agricultural mixer.

Pilot Scale Reactor Set-Up and Operation

Two parallel reactor set-ups (1 and 2) were used. Thehydrolytic reactor (1st stage, referred to as H1 in set-up 1 andH2 in set-up 2) of both set-ups consisted of a 10 m3 (7.6 m3

active volume) leaching bed hydrolytic reactor equipped withleachate circulation. The 2nd stage of both reactor set-ups wasa 2.6 m3 methanogenic reactor (referred to as MF1 in set-up 1and MF2 in set-up 2) built of stainless steel and equipped withleachate recirculation (Figure 1). The reactors were filled withdifferent carrier materials. MF1 was a downflowmethanogenic filter packed with pre-digested straw, whereasMF2 was an upflow methanogenic filter packed with plasticcarriers (HUFO 120 m2 m-3, Nordiska Plast AB). The effluentsfrom the methanogenic filters were recycled back to therespective hydrolytic reactor. In each set-up, the same pump

was used for circulation over both stages, and the flow wasswitched between the 1st and the 2nd stage by pneumaticvalves (Figure 1). At each flow change, a set volume of 8 l wasexchanged between the two reactors, and the number ofcirculation changes over a day determined the amount ofliquid exchanged between the two reactors. All reactors wereoperated under mesophilic temperature conditions. At eachstart-up, the liquid in the 1st stages was replaced with freshwater, whereas the liquid in the 2nd stages was reused andthus not replaced.

Substrate was added to the 1st stage in a removable cagein a batchwise manner, after which 2 m3 of fresh water wasadded. Initially, the 1st stage was operated with internalrecirculation of leachate, with the leachate collected from thebottom sprayed on top of the bed. Circulation over the 2nd

stage was initiated when the chemical oxygen demand (COD)of the leachate reached a level of 10 g l-1 and was continueduntil pH in the 1st stage was higher than 7 and methane (CH4)production had begun. Then the circulation over the 2nd stagewas terminated and the hydrolytic reactor was operated as aone-stage process until the gas production became negligibleand the runs were terminated.

The loading rates to the methanogenic filters variedbetween 3-19 kg COD m-3 d-1 (Table 2). If the pH in theeffluent from the methanogenic filters decreased to below 7,the feeding was interrupted and the methanogenic filterswere operated with internal circulation until the effluent pHwas stabilised above 7. Durations, amounts of substratesadded and temperature conditions in the different runs arepresented in Table 2.

Figure 1. Schematic drawing of the reactor set-up 1 (H = hydrolytic reactor, MF = methanogenic filter). Set-up 2 is identicalexcept for the reversed flow direction in MF2. Dashed lines represent the flow of process liquid.

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Table 2. The durations, amounts of substrates added, loading rates and temperature conditions in different runs (averages,where applicable, in parentheses).

R u n Substrate Dura-tions1

(d)

Amount ofsubstrate added

(kg WW)

Loading ratesto MFs

(kg COD m-3 d-1)

Temperature conditions(°C)

Set-up 1 Set-up 2 Set-up 1 Set-up 2 Set-up 1 Set-up 2

H1 MF1 H2 MF2

A Willow 82 1300 1300 5-10 (6) 5-10 (6) 35.9±0.1 36.4±1.9 36.0±0.1 34.1±4.7B Sugar beet 55 1970 1842 3-10 (6) 8-19 (11) 34.9±3.1 36.6±2.5 36.3±0.4 36.7±2.5C Grass 50 1940 1940 5-20 (11) 5-20 (11) 36.2±0.7 36.6±0.8 36.1±0.1 36.7±0.7

1 Total duration of the run in both set-ups.

Laboratory Scale Batch Digestions

Laboratory scale batch digestions were performed inorder to determine a reference methane yield for finelychopped substrates in completely mixed low solids digestionunder optimal conditions. Inoculum was obtained from amesophilic completely mixed low solids reactor treating cropresidues as substrate. Substrate/inoculum ratio of 1 onvolatile solid (VS) basis was used in all batch experiments,performed in 500 ml bottles (working volume 300 ml) intriplicate, incubated on a shaking water bath (70 roundsminute-1 (rpm)) at 37±1°C, and continued until methaneproduction became negligible (<5 ml CH4 d-1). Results oflaboratory scale batch digestions are presented as specificmethane yields, where methane yield of inoculum issubtracted.

Analyses and Calculations

The pilot scale reactors were equipped with on-line pHand temperature indicators (MiniCHEM-pH Process Monitor,TPS Pty Ltd.) and gas volume meters (Gallus 2000, ActarisTechnologies AB). The total solids (TS) and volatile solids (VS)were determined according to standard methods [32]. Liquidsamples were centrifuged at 3 000 rpm for 3 minutes and thesupernatant was used for COD, ammonium nitrogen (NH4-N)and volatile fatty acid (VFA) measurements. Dr Lange cuvettetests were used for measuring COD and NH4-N (LCK 114 andLCK 914 for COD and LCK 303 and LCK 302 for NH4-N, Dr.Bruno Lange GmbH). Volatile fatty acids (VFAs) weredetermined as previously described [33]. Samples for totalnitrogen (N-tot) were digested in a 2012 Digester Unit DS12(Foss Tecator AB) according to the manufacturer’sinstructions, and analysed colorimetrically using a FIAStar5000 analyser coupled with a 5027 sampler (Foss Tecator AB).Gas composition was analysed with a gas chromatograph(Agilent Technologies 6890 Network GC system) equippedwith Haysep (N 80/100, 9 ft, 1/8) and Molesieve (5 A 60/80, 6ft, 1/8) columns and a thermal conductivity detector. Helium

was used as the carrier gas at a flow rate of 30 ml min-1. Thecolumn temperature was 70°C and the injector and detectortemperatures were 110°C and 150°C, respectively. Heavymetals (Cd, Cu, Ni, Pb, Zn, arsenic (As), chrome (Cr) andmercury (Hg)) were extracted from the samples by theautoclave digestion method according to EN ISO 1483 [34]and analysed by inductively coupled plasma massspectrometry (ICP-MS) as previously described [35]. Samplesof substrates and solid residues were analysed at AnalyCenNordic AB (Lidköping, Sweden) for total carbon (C), crudefibre, neutral detergent fibre (NDF), acid detergent fibre(ADF), lignin, energy content and NH4-N. Organic nitrogen(org-N) was calculated as the difference between N-tot andNH4-N.

RESULTS AND DISCUSSION

Energy crops (willow in run A, sugar beet in run B andgrass in run C) were digested in pilot scale two-stageanaerobic digesters (Table 2, Figure 2). Hydrolysis beganrapidly in the 1st stage in all runs, as seen by the increase inleachate COD values (Figure 2). In run A (Figure 2), the CODin leachate from the 1st stage reached a level of 12 g l-1 on day3, and circulation over the methanogenic filters (MFs) couldbe initiated. The loading rate to the MFs was maintained at 10kg COD m-3 d-1 for the first 3 days, after which it decreasedwith decreasing COD concentration, until circulation overMFs was terminated on day 9. By then the methaneconcentrations in gas produced in the 1st stages were 47 and43 % in H1 and H2, respectively. The reactors wereterminated after 82 d of operation, after which 84 % of totalCH4 was produced in the 1st stages, and, due to lowsolubilisation of organic matter, only 16 % in the MFs.

In run B high COD values of 42-44 g l-1 were observedin the leachates from the 1st stages after 24 hours of internalcirculation (Figure 2), and circulation over the MFs wasinitiated at a loading rate of 10 kg COD m-3 d-1. However, thisloading led to a drop in pH in MFs, and on day 3 themethanogenic filters were put on internal circulation. MF2

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Figure 2. pH and COD profiles of effluents from all reactors and daily gas productions in 1st stage in different runs. Values ofcarbon dioxide (CO2) production in run 3 for day 6 (out of scale) are 21 517 (H1) and 12 479 (H2) l d-1.

recovered rapidly, and loading rates of 8-19 kg COD m-3 d-1

were applied between days 7-23. By day 23 the CH4

concentration in gas from H2 had increased to 56 %, pH was7.3, and circulation over MF2 was terminated. It took a longertime for MF1 to recover from the pH drop, and a low loadingrate of 3 kg COD m-3 d-1 was applied on day 14. Loading ratewas maintained at 5-10 kg COD m-3 d-1 until day 44, whenmethanogenic filter was considered redundant, pH in H1being 7.5 and CH4 concentration 68 %. Run B was terminatedon day 55. In this run, 17 % of total CH4 was produced in the1st stages, and 83 % in the MFs.

In run C, high COD values of 55-61 g l-1 were observedin the leachate from the 1st stage after 24 hours of internalcirculation (Figure 2), and circulation over the MFs wasstarted at an organic loading rate of 5 kg COD m-3 d-1. Loadingwas then gradually increased to 10 and 20 kg COD m-3 d-1.With these high loading rates MF2 showed reliableperformance with rather stable pH, whereas MF1 had lowCOD removal efficiency and fluctuations in pH. H2 becamemethanogenic slightly faster, and MF2 was closed on day 22,whereas circulation was continued over MF1 until day 28.Run C was terminated on day 50. In run C, 36 % of total CH4

was produced in the 1st stages and 64 % in the MFs.MF1 with digested straw as carrier material had been in

operation for about two years before initiation of theseexperiments, and the earlier results with this reactor showsimilar and even superior performance over MF2 [36]. It ispossible that the straw bed ageing could have caused acollapse in the structure or that channelling could haveoccurred. Biological materials such as straw are cheap to useas a carrier material, but the risk of unreliable performancedue to ageing and the need for periodic renewal andconsequent long start-up periods are disadvantages, makingplastic carriers more reliable. Furthermore, the loading ratesto the methanogenic filters in these set-ups were manuallycontrolled, and thus relatively difficult to maintain.Consequently, automatic control may be necessary in order tomake the systems more stable and easier to operate. Forexample, on-line monitoring of biological oxygen demand(BOD) or VFAs have been suggested as control strategies fortwo-stage processes [37, 38].

Reactor runs were continued for an excessively longtime to ensure all extractable methane was harvested. Despitethe problems in MF1 in runs B and C causing delays in reactoroperation, the total methane yields of the two reactor set-upswere very similar, and average values are presented in Table3. Assuming average hectare (ha) yields typical for SouthernSweden of 20 (after 1st growing season), 80 (both beets and

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Table 3. Methane production and reduction of different carbon fractions and of heat content in different runs.

Parameter Unit RUN A: Willow RUN B: Sugar beet RUN C: GrassMethane production m3 CH4 kg-1 VS 0.16 0.38 0.39

m3 CH4 t-1 WW 79 68 109

VS reduction % 46 96 59Heat content reduction % 35 94 51Crude fibre reduction % 32 87 43NDF reduction % 32 88 27ADF reduction % 49 83 41

leaves) and 25 tons (t) WW ha-1 per year (a) for willow, sugarbeet and grass, respectively, the methane yields from two-stage digestion correspond to 1 600, 5 400 and 2 700 m3 CH4

ha-1 a-1, respectively. These values correspond to gross energyyields per hectare of 15, 53 and 26 megawatt-hours (MWh)ha-1 for willow, sugar beet and grass, respectively. The VSreductions in runs A, B and C amounted to 46, 96 and 59 %,respectively (Table 3). Sugar beet was highly degradableunder anaerobic conditions, containing only 17 % NDF and 2% lignin (Table 1), whereas high concentrations of lignin (14%) and NDF (71 %) made willow quite resistant to anaerobicdegradation. NDF includes hemicellulose, cellulose and ligninas major components, whereas ADF is part of NDF andconsists mostly of cellulose and lignin [39]. ADF degradationwas in most cases higher than NDF degradation, and sincelignin is known to be refractory to anaerobic decomposition[40, 41, 42], this can be considered as an indication of the highextent of cellulose degradation. Thus, in the two-stage systemrelatively efficient degradation of these recalcitrant fibres was

obtained.In run A, 59 % of the total methane production was

attained on day 30, whereas the corresponding figures forruns B and C were higher, 84 and 85 %, respectively (Figure3). Thus, most of the harvestable methane was obtainedwithin 30 days, indicating that one month could be asufficient digestion time. The specific methane yields fromlaboratory scale batch digestions were 0.29, 0.45 and 0.37 m3

CH4 kg-1 VSadded for willow, sugar beet and grass, respectively(Figure 3). Thus, 56 and 85 % of the laboratory scale methaneyields could be obtained in pilot scale two-stage digestion ofwillow and sugar beet, respectively. However, digestion ofgrass in pilot scale two-stage reactors yielded 5 % moremethane than digestion in laboratory scale completely mixedlow solids systems. This could be due to the more efficienthydrolysis obtained in the two-stage reactors by physicallyseparating the rate limiting hydrolysis from methanogenesis.As indicated in Figure 2, higher pH prevailed in the 1st stagein run C during the hydrolytic phase, than in the other runs.

Figure 3. Total and short-term (30 d) specific methane yields of different substrates in laboratory and pilot scale experiments(H = hydrolytic reactor, MF = methanogenic filter). For pilot experiments with sugar beet, values from set-up 1 byday 30 are excluded due to operational problems. Values above bars represent the duration of the experiment indays.

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The alkalinity in the leachate from the 1st stage was also muchhigher in run C than in the other runs (data not shown). Thiswas probably due to the high amount of clover in the grassmixture. Legumes are known to have very high bufferingcapacities, caused by the high content of organic acids, saltsand proteins [43]. Many extracellular cellulolytic enzymesproduced by hydrolytic bacteria are known to have pHoptimum of around 6 [41]. In contrast, in laboratory scalebatch assays all reactions took place under neutral pHconditions, and conditions for hydrolysis were thus notoptimal. In two-stage digestion of sugar beet (run B) the pH inthe 1st stage remained below 5 for 14-20 days, which couldhave enhanced the inhibiting effect of undissociated VFAs[44]. However, the high nitrogen concentration together withthe high pH towards the end of run C may have increased therisk of higher ammonia volatilisation and loss of nitrogenthrough the gas phase.

Organically bound nitrogen (Org-N) and mineralisednitrogen (NH4-N) were analysed in all solid and liquidfractions at the start and end of the experiments (Figure 4).Grass was the only substrate to contain NH4-N initially. At theend of run A, the amount of mineralised nitrogen was low,and equally distributed between the liquid and solid phases.In run B, the majority of the nitrogen was converted toammonia, and 87% of the nitrogen was available as NH4-N inthe liquid. In run C, the amount of nitrogen in the substratewas the highest (23 kg), but the mineralisation rate was lower,and at the end of the run, 29% of the nitrogen was available asNH4-N in the liquid. NH4-N is the preferred form of nitrogenin the residue when considering the use of residue asfertiliser, since it is in a form readily available to plants [28].

The liquid fraction from digestion is relatively easy to spreadand to store, and it can also be used as nitrogen-rich irrigationwater. Thus, it would be preferable to have as high an amountof nitrogen as possible in the form of NH4-N in the liquidphase at the end of the digestion. Nitrogen concentrations asrelated to TS in the solid fractions increased 2 fold in runs Aand C and 4 fold in run B during digestion. In an earlierstudy, nitrogen concentrations in the solid fraction werereported to increase 4-5 fold during digestion of sorghumbiomass [29]. Solid residues from runs B and C had C/Nratios of 10-11 and 7-9, respectively, whereas that from run Awas 24-31. It has been reported that digestates with C/Nratios of around 10 are suitable for incorporation to soil assoil-improvement media [28]. Thus, the solid residues fromrun B and C were well suited for this purpose.

Concentrations of heavy metals in different fractionsduring and after digestion were determined. Metalconcentrations in the digested residues along with the limitvalues for use of digested residues as fertiliser in Sweden [31]are given in Table 4. In the solid residue from run A,cadmium concentration exceeded the Swedish limit value foruse of digested residues. In runs B and C, metals that areknown to be abundant in agricultural soils [31] weremonitored in the liquid in the 1st stage both during the acidicphase (day 3 in run B and day 2 in run C) and at the end ofeach run. With few exceptions the heavy metals were stronglymobilised during the acidic phase (Figure 5). This could offera possibility of precipitating metals from the leachate duringthe acidic phase and thus removing the metals from thefarming system. This option is especially interesting in areaslike Southern Sweden, where cadmium concentrations in

Figure 4. Initial distribution of organic and mineralised nitrogen in substrate and process liquid, and percentage ofmineralised nitrogen after digestion as total and in liquid phase.

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Table 4. Limit values for utilisation of digestate as soil fertiliser [31], and concentrations of heavy metals in the solid residuesin the different runs.

Metals (µg g-1 TS) As Cd Cr Cu Hg Ni Pb Zn

Limit value - 2 100 600 2.5 50 100 800

RunA 0.55 4.66 9.64 17.53 0.02 0.47 1.35 183.21B 2.84 1.52 54.76 96.54 0.08 40.35 6.62 194.12C 1.23 0.56 23.56 58.82 0.05 9.90 4.05 107.23

Figure 5. Metal concentrations in leachate from 1st stage under low pH (acidic phase) and neutral (end of run) conditions.Values for zinc / acidic phase are out of range with 8 600 and 3 400 µg l-1 in runs B and C, respectively. Please notethat concentrations of lead and cadmium are one order of magnitude lower.

arable land are high due to anthropogenic inflows (landapplication of inorganic fertilisers and atmosphericdeposition) [31]. Cultivation of crops with the ability toaccumulate more cadmium than most agricultural crops hasbeen suggested for phytoextraction of metals from arable land[45]. Consequent two-stage anaerobic digestion of these cropswith simultaneous removal of heavy metals from leachates,e.g. through biological precipitation by sulphate-reducingbacteria [46], could offer possibilities in remediating pollutedsoils.

CONCLUSIONS

High methane yields and efficient degradation oforganic matter was obtained in two-stage anaerobic digestionof grass and sugar beets, whereas first year shoots of willow

proved to be a too recalcitrant substrate for biologicalconversion through anaerobic digestion without pre-treatment. With grass and sugar beets as substrate, 84-85 % ofthe harvestable methane was obtained within 30 d, indicatingthat one month would be a sufficient digestion time.Digestion of grass in two-stage reactors yielded 5 % moremethane than digestion in laboratory scale completely mixedlow solids systems. This could be due to the more efficienthydrolysis obtained in the two-stage reactors by physicallyseparating the hydrolysis and methanogenesis to differentcontainers and allowing pH conditions favourable to thehydrolytic bacteria to prevail in the first stage. In digestion ofgrass and sugar beet the liquid at the end of digestion wasrich in NH4-N, and the nitrogen in the substrate wasefficiently mineralised. Heavy metal concentrations are notlikely to limit the utilisation of residues from digestion of non-

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metal accumulating crops. Efficient mobilisation of heavymetals during the acidic phase of digestion revealed thepossibility of removing (precipitating) metals from leachategenerated in two-stage anaerobic digestion of phytoextractingcrops, thus remediating contaminated soils and removingmetals from a farming system.

ACKNOWLEDGEMENTS

This study was supported by NorFA, Finnish GraduateSchool for Energy Technology and the Swedish EnergyAgency. We thank our colleagues in the biogas researchgroup at Department of Biotechnology for technical assistanceand advice.

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