anaerobic digestion of grass silage in batch leach bed processes for methane production

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Bioresource Technology 99 (2008) 3267–3278

Anaerobic digestion of grass silage in batch leach bed processesfor methane production

A. Lehtomaki *,1, S. Huttunen, T.M. Lehtinen, J.A. Rintala

University of Jyvaskyla, Department of Biological and Environmental Science, P.O. Box 35, FI-40014 Jyvaskyla, Finland

Received 6 April 2007; received in revised form 6 April 2007; accepted 6 April 2007Available online 15 August 2007

Abstract

Anaerobic digestion of grass silage in batch leach bed reactors, with and without a second stage upflow anaerobic sludge blanket(UASB) reactor, was evaluated. Sixty six percent of the methane potential in grass was obtained within the 55 days solids retention timein the leach bed–UASB process without pH adjustment, whereas in the one-stage leach bed process 20% of the methane potential in grasswas extracted. In two-stage operation, adjustment of the pH of influent to the leach bed reactor to 6 with HCl led to inhibition of bothhydrolysis/acidogenesis and methanogenesis. In the leach bed–UASB process 39% of the carbohydrates and 58% of the acid soluble lig-nin were solubilised within the 49 days of operation, whereas Klason lignin was most recalcitrant. The methane potential of the digestatesvaried from 0.141 to 0.204 m3 CH4 kg�1 added volatile solids.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Anaerobic digestion; Energy crop; Methane production; Leach bed; UASB

1. Introduction

Methane-rich biogas produced through anaerobic diges-tion of organic materials provides a clean and versatile car-rier of renewable energy, as methane can be used inreplacement for fossil fuels in both heat and power gener-ation and as a vehicle fuel. Methane production throughanaerobic digestion has been evaluated as one of the mostenergy-efficient and environmentally benign ways of pro-ducing vehicle biofuel (LBS, 2002). The European Union(EU) has set a target of increasing the share of biofuelsin vehicles to 5.75% by year 2010 in each member state(European Parliament, 2003). The utilisation of energycrops and crop residues for methane production is an inter-esting option for increasing domestic biofuel production, asit has been estimated that within the agricultural sector in

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.04.072

* Corresponding author. Tel.: +358 14 4451 160; fax: +358 14 4451 199.E-mail address: [email protected] (A. Lehtomaki).

1 Presently at Jyvaskyla Innovation Ltd., P.O. Box 27, FI-40101Jyvaskyla, Finland.

the EU, 1500 million tons of biomass could be anaerobi-cally digested each year, half of this potential accountedfor by energy crops (Amon et al., 2001). In Finland, forexample, the Ministry of Agriculture has estimated thatby 2012 up to 500000 hectares (ha), an area correspondingto about one fourth of all arable land in Finland, could bededicated to energy crop production (Vainio-Mattila et al.,2005).

Energy crops and crop residues can be digested eitheralone or in co-digestion with other materials, employingeither wet or dry processes. Energy crops typically have ahigh total solids (TS) content of 10–50%, and in order totreat this kind of material in wet processes, the solids mustusually be homogenised and diluted with other materialshigh in water content. In the agricultural sector, the mostwidely applied solution is to co-digest crop biomass withanimal manures in wet processes (Lehtomaki et al., inpress). Dilution increases the volume to be treated and thusthe energy required for heating and pumping (Ghosh et al.,2000). Furthermore, floating of the crop materials alongwith crust or scum formation has been reported in

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3268 A. Lehtomaki et al. / Bioresource Technology 99 (2008) 3267–3278

digestion of crop materials in wet processes (Nordberg andEdstrom, 1997). Moreover, suitable materials for co-diges-tion may not always be available, and water would thenhave to be used for dilution.

The gas production per digester volume (volumetric gasproduction) can potentially be increased by operating thedigesters at a higher solids concentration. Batch high solidsreactors, characterised by lower investment costs than thoseof continuously fed processes, but with comparable opera-tional costs, are currently applied in the agricultural sectorto a limited extent (Kottner, 2002; Weiland, 2003). In thesesystems, digesters are filled with fresh substrate, with orwithout addition of inoculum, and allowed to go throughall the degradation steps sequentially. Batch reactors areoften leach bed processes where solids are hydrolysed by cir-culating leachate over a bed of organic matter. Recircula-tion of leachate stimulates the overall degradation owingto more efficient dispersion of inoculum, nutrients and deg-radation products (Chanakya et al., 1993; Lissens et al.,2001). Digestion of plant biomass in one-stage leach bedprocesses has been seldom reported in the literature (Table1), but in batch leach bed processes digesting barley straw,reductions in volatile solids (VS) of 45–60% and methaneyields of 0.159–0.226 m3 CH4 kg�1 VSadded were obtained(Torres-Castillo et al., 1995), and in one-stage leach bedprocesses fed on a weekly basis with various lignocellulosicsubstrates (such as water hyacinth, straw, bagasse, canetrash etc.) and vegetable wastes, VS removals and biogasyields ranging from 37% to 78% and from 0.26 to0.95 m3 biogas kg�1 VSadded, respectively, were reported(Chanakya et al., 1993, 1997; Ramasamy and Abbasi,2000) (Table 1).

Batch leach bed processes can also be operated in con-junction with a second stage methanogenic reactor, withthe leachate generated in the first stage pumped to themethanogenic reactor for further degradation (Ghosh,1984). Since the leachate has a low solids content, high-rate reactors such as upflow anaerobic sludge blanket

Table 1Examples of anaerobic digestion of plant material in one-stage leach bed proc

Feedstock Mode offeeding

Reactorvolume (l)

T(�C)

Feed TS(% ww)

VS(%

Barley straw Batch 220 35 35–36a 45Water

hyacinthWeekly 2 21–27 9.4 n.

Paddy straw Weekly 6 26 n.r. 56Bagasse Weekly 6 26 n.r. 37Cane trash Weekly 6 26 n.r. 49Synedrella Weekly 6 26 n.r. 68Parthenium Weekly 6 26 n.r. 78Vegetable

wasteWeekly 11 35 n.r. n.

n.r. = not reported.1: Torres-Castillo et al. (1995), 2: Chanakya et al. (1993), 3: Chanakya et al.a Values calculated from the data reported.b m3 CH4 kg�1 VSadded.c m3 biogas kg�1 VSadded.

reactors (UASBs) or anaerobic filters can be used in thesecond stage, and a high solid retention time is achievedin these reactors through the formation of granules orattachment of biomass to carriers (Henze and Harremoes,1983; Lettinga, 1995). Digestion of plant material in pro-cesses of this kind has been reported (Table 2), but exper-iments on digestion of energy crops in these processes arefew. Methane yields and VS removals of 0.27 to0.39 m3 CH4 kg�1 VSadded and 59–60%, respectively, wereobtained in two-stage anaerobic digestion of grass silagein batch leach bed processes connected to anaerobic fil-ters, in both laboratory (Cirne et al., in press) and pilottrials (Lehtomaki and Bjornsson, 2006) (Table 2).

In practice, not all of the methane potential in substratescan be extracted in anaerobic digestion within the reactorresidence time, and if the digestates are stored in uncoveredstorage tanks without gas collection, part of this methanecan be lost to the atmosphere through spontaneous degra-dation, contributing to climate change. Post-methanationof digestates in covered storage tanks offers the possibilityof both minimizing the potential methane emissions, as wellas contributing to an increase in the methane yields (Kap-araju and Rintala, 2003), as up to 15% more biogas hasbeen be obtained in post-methanation of digestates fromliquid phase low solids digesters (Weiland, 2003). However,to our knowledge the methane potential of digestates fromleach bed processes has not been determined previously.

The aim of this study was to evaluate the suitability ofleach bed reactors for methane production from grasssilage. Operation of a one-stage process consisting of abatch leach bed reactor and a two-stage process with leachbed reactor in connection with an UASB were compared.Furthermore, the effect of adjusting the pH of influent inthe first stage of the two-stage process was evaluated, andthe extent of degradation of different fractions of VS in var-ious stages of digestion was evaluated both by chemicalcharacterisation and by determining the methane potentialof the digestates.

esses, as reported in the literature

removal)

Gas production (m3 CH4 kg�1 VSadded orm3 biogas kg�1 VSadded)

References

–60 0.159–.226b 1r. 0.348c 2

.5 0.48a,c 3

.1 0.83a,c 3

.8 0.26a,c 3

.1 0.95a,c 3

.1 0.71a,c 3r. 0.513–0.869b 4

(1997), 4: Ramasamy and Abbasi (2000).

Table 2Examples of anaerobic digestion of plant material in two-stage processes consisting of a leach bed reactor and a methanogenic reactor, as reported in theliterature

Feedstock Mode of feedingin first stage

Type of reactor assecond stage

Reactor volume firststage/second stage (l)

T (�C) Feed TS(% ww)

VSremoval(%)

Spec. CH4 yield(m3 CH4 kg�1 VSadded)

Refs.

Fruit andvegetablewaste

Batch UASB-AF 1.3/0.5 35 5 83 0.345 1


Batch UASB-AF 1.3/0.5 35 5 82 0.355 1


Batch UASB-AF 1.3/0.5 35 5 87 0.368 1


Batch UASB-AF 1.3/0.5 35 5 90 0.383 1


Daily UASB-AF 1.3/0.5 35 6.4 72 0.405a 2


Daily UASB-AF 1.3/0.5 35 6.4 53 0.294a 2


Daily UASB-AF 1.3/0.5 35 6.4 38 0.187a 2


Daily UASB-AF 1.3/0.5 35 6.4 27 0.098a 2

Potato waste Batch UASB 2.0/0.84 37 19 n.r. 0.39 3Potato waste Batch AF 2.0/1.0 37 19 n.r. 0.39 3Sugar beet leaves Batch AF 7.6/2.6 35–37 n.r. n.r. 0.216a 4Unpeeled

potatoesBatch AF 7.6/2.6 35–37 n.r. n.r. 0.258a 4

Peeled potatoes Batch AF 7.6/2.6 35–37 n.r. n.r. 0.351a 4Sugar beet leaves,

potatoes 1:2Batch AF 7.6/2.6 35–37 n.r. n.r. 0.402a 4

Sugar beet leaves,potatoes 1:3

Batch AF 7.6/2.6 35–37 n.r. n.r. 0.402a 4

Grass waste Batch AF 8000/190 Ambient 92 67 0.165a 5Grass silage Batch AF 7.6/2.6 37 31.8 59 0.39 6Sugar beet Batch AF 7.6/2.6 37 20.2 96 0.38 6Willow Batch AF 7.6/2.6 37 49.5 46 0.16 6Grass silage Batch AF 0.75/0.9 37 27 60 0.27 7Sugar beet Batch AF 0.75/0.9 37 24 89 0.44 7Rice straw Batch ASBR 4.0/4.0 35 92 44 0.19a 8Rice straw Batch ASBR 4.0/4.0 35 92 45 0.19a 8Rice straw Batch ASBR 4.0/4.0 35 92 48 0.21a 8Water hyacinth Weekly AF 2.0/0.5 n.r. 9.6 n.r. 0.181b 9

UASB = upflow anaerobic sludge blanket reactor, AF = anaerobic filter, ASBR = anaerobic sequencing batch reactor.n.r. = not reported. References: 1: Martinez-Viturtia and Mata-Alvarez (1989), 2: Martinez-Viturtia et al. (1995), 3: Parawira et al. (2005), 4: Parawiraet al. (submitted for publication), 5: Yu et al. (2002), 6: Lehtomaki and Bjornsson (2006), 7: Cirne et al. (in press), 8: Zhang and Zhang (1999), 9: Chanakyaet al. (1992).

a Values calculated from the data reported.b m3 biogas kg�1 TSadded.

A. Lehtomaki et al. / Bioresource Technology 99 (2008) 3267–3278 3269

2. Methods

2.1. Origin of materials

Grass silage was obtained from a farm in central Fin-land (Kalmari farm, Laukaa) (Table 3). It was preparedat the farm from grass (75% timothy Phleum pratense,25% meadow fescue Festuca pratensis) harvested at early

flowering stage, chopped with an agricultural precisionchopper after 24 h of pre-wilting and ensiled in a bunkersilo with the addition of a commercial silage additive (lacticacid bacteria inoculant AIV Bioprofit, containing 60% Lac-

tobacillus rhamsonus and 40% Propionibacterium freud-

enreichii spp. shermanii (Kemira Growhow Ltd.) with atotal count of 5.8 · 1011 colony-forming units (CFU) g�1,diluted to 0.7 g l�1 in tap water and applied to the plant

Table 3Characteristics of grass silage and inoculum

Parameter Unit Grass silage Inoculum

pH 4.1 7.7TS % ww 25.9 6.6VS % ww 24.0 5.0SCOD mg g�1 TS 228 189Ntot mg g�1 TS 16.9 48.9NH4-N mg g�1 TS 1.4 17.2Klason lignin % TS 13.0 n.d.LigninAS % TS 4.1 n.d.Carbohydrates % TS 45.0 n.d.Extractives % TS 8.4 n.d.Proteins % TS 10.4 n.d.Higher heat content MJ kg�1 TS 19.8 n.d.

n.d. = not determined.


material in a ratio of 0.5% volume/weight, v/w). In the lab-oratory, the material was further chopped with a gardenchopper (Wolf Garten SD 180E) to a particle size ofapproximately 3 cm and then immediately frozen andstored at �20 �C. Before analysis and feeding to the reac-tors, the samples were allowed to thaw overnight at 4 �C.

The inoculum used to inoculate the one-stage leach bedreactors and methane potential assays was from a meso-philic farm digester (Laukaa, Finland) treating cow man-ure and industrial confectionary by-products as substrate

Run 1 Run 2

Ru

LB2

R1

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LB LB LB LB LB

R1

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R1

Fig. 1. Reactor set-ups in runs 1–4 (U = UASB, pH adj. = pH adjustment). Da

(Table 3). The UASB was inoculated with granular sludgeobtained from an internal circulation (IC) reactor treatingwastewater from sugar beet and vegetable processing(Sakyla, Finland).

2.2. Experimental set-up

In this study, a one-stage leach bed reactor (LB1) (run 1)and two two-stage processes combining LB (LB2 and LB3)and UASB reactors (UASB2, UASB3) (runs 2 and 3) wereused. Furthermore, in order to characterise the changes inLB material, six LBs in conjunction with a common UASB(UASB4) were operated (run 4) (Fig. 1).

All the LBs (plastic columns) and UASBs (glass col-umns) had a liquid volume of 1000 ml and they were oper-ated at 35 (±1) �C. Leachate from LBs was collected at thebottom of the reactors in a liquid reservoir (R1) and eithercirculated to an UASB or recycled back to the top of thereactor when internal recirculation was applied. The UASBeffluent was collected in another reservoir (R2), from whichit was recirculated to LB (Fig. 1). The biogas produced wascollected from the top of the reactors and the liquid reser-voirs into aluminium gas bags. Before starting the presentexperiments the UASBs had been inoculated with granularsludge (see Section 2.1) and operated for two months with

Run 3

n 4

U LB3

R1

U

R2pH adj.

U LB3

R1

U

R2pH adj.

U LB3

R1

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R2pH adj.

UU LB3

R1

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R2pH adj.

LB3

R1

U

R2pH adj.

LB U

R2

LB U

R2

shed lines represent the flow of process liquid during internal recirculation.


artificial wastewater at an organic loading rate (OLR) of5 kg chemical oxygen demand (COD) m�3 d�1.

The LBs (LB1–LB3) were filled with 50 g VS (208 g wetweight, ww) of grass silage mixed with 3.2 g VS (64 g ww)of inoculum (LB1) or without inoculum (LB2, LB3), afterwhich 750 ml of tap water was added to fill the reactor (aninitial liquid/solid (L/S) ratio of 17). After each sampling ofleachate an equivalent amount of tap water was added tothe liquid reservoir. When internal recirculation wasapplied, the recirculation rate of the leachate was750 ml d�1. In two-stage operation the OLR to the UASBwas maintained at 5 kg COD m�3 d�1, which determinedthe flow rate to both UASB and LBs. The pH adjustmentsof the influents to LBs in runs 1 (pH 7) and 3 (pH 6) wereperformed automatically with 1 M NaOH and 1 M HCl.

In run 4 six parallel LBs were filled with 50 g VS (208 gwet weight) of grass silage without inoculum, after which250 ml of tap water was added per reactor (1500 ml intotal) (initial L/S ratio 8). The leachate from all six LBswas collected in a common reservoir (R1) and circulatedfrom there to the common UASB at 5 kg COD m�3 d�1.The effluent from the UASB was collected in a reservoir(R2) and circulated back to the top of the LBs so that eachLB received the same liquid at the same flow rate. The LBswere terminated sequentially during the run to characterisethe residual materials.

The methane potentials of grass silage and digestateswere determined in triplicate batch experiments in 2 l glassbottles (liquid volume 1.5 l) and in 118 ml glass bottles,respectively, at 35 ± 1 �C. In assays with silage, inoculum(500 ml) and substrate in a VSsubstrate/VSinoculum-ratio of1 were added into the bottles, distilled water was addedto achieve a liquid volume of 1.5 l, and NaHCO3 (3 g l�1)was added as buffer. In assays with digestates, the dige-states (1 g VS) and inoculum corresponding to 1 g VS wereadded into the bottles. The contents of the bottles wereflushed with N2/CO2-gas (70%:30%, Aga Ltd.) for 5 minand the bottles were then sealed with butyl rubber stoppers.Bottles were manually mixed before each gas measurement.Assays with inoculum only were incubated to subtract themethane yield of the inoculum from those of substrates.The assays were terminated when CH4 production becamenegligible after 94–100 days.

The methane potentials of digestates were expressed asm3 CH4 kg�1 VSadded and m3 CH4 kg�1 VSoriginal. The lat-ter was calculated per VS of substrate originally added tothe reactor, taking into account the VS removal duringreactor operation.

2.3. Analyses and calculations

Methane and volatile fatty acids (VFAs) were measuredby gas chromatograph (GC) (methane: Perkin Elmer Cla-rus 500 GC with thermal conductivity detector and SupelcoCarboxenTM 1010 PLOT fused silica capillary column,30 m · 0.53 mm, and VFA: PE Autosystem XL GC withflame-ionisation detector and PE FFAP column,

30 m · 0.32 mm). Operating conditions were for methane:oven 200 �C, injection port 225 �C, detector 230 �C, andfor VFA: injection port and detector 225 �C, oven 100–160 �C (20 �C/min). Argon (methane) and helium (VFA)were used as carrier gases.

Metrohm 774 pH-meter was used in all pH measure-ments. TS and VS were determined according to the Stan-dard Methods (APHA, 1998) and COD according to theSFS 5504 (Finnish Standards Association, 1988). Total(Ntot), ammonium nitrogen (NH4-N) and proteins weredetermined according to the Tecator application note (Per-storp Analytical/Tecator AB, 1995) with a Kjeltec system1002 distilling unit (Tecator AB), protein content calcu-lated as 6.25 · Ntot. NH4-N and soluble COD (SCOD)from crop samples were analysed after extraction accord-ing to SFS-EN 12457-4 (Finnish Standards Association,2002) and the samples for NH4-N and SCOD determina-tion were filtered with GF50 glass fibre filter papers (Schlei-cher and Schuell). Extractives were determined by acetoneextraction according to the TAPPI Test Method T 280 pm-99 (TAPPI, 2000). For lignin and carbohydrate analyses,the acetone-extracted samples were hydrolysed accordingto the TAPPI Test Method T 249 cm-00 (TAPPI, 2000).Klason lignin content was measured according to theTAPPI Test Method T 222 om-98 (TAPPI, 2000). Acid sol-uble lignin (ligninAS) in hydrolysis filtrate was quantifiedspectroscopically (Beckman DU640 Spectrofotometer) onthe basis of ultra-violet absorption at 205 nm using anabsorptivity value of 110 l g�1 cm�1, and total lignin (lig-nintot) content was calculated as the sum of Klason ligninand ligninAS. The monosaccharides obtained (arabinose,galactose, mannose and xylose from the hemicellulose com-ponents and glucose from cellulose) were per(trimethylsi-lyl)ated and analysed with GC (HP 5890 Series II GCwith flame-ionisation detector and a DB-1701 column,60 m · 0.32 mm, Agilent Technologies, J&W Scientific).Operating conditions were injection port 290 �C and detec-tor 300 �C. Oven temperature was programmed to begin at100 �C (held for 2 min), rise 2 �C/min to 185 �C (22 min)and rise 39 �C/min to a final temperature of 280 �C(15 min). Nitrogen was used as carrier gas. Heat contentwas analysed as higher heat content with a bomb calorim-eter (IKA-Kalorimeter C400, Janke and Kunkel GmbH).

3. Results

3.1. One-stage leach bed reactors (run 1)

In the one-stage leach bed reactor (LB1) with internalrecirculation and pH adjustment of the influent (pH 7),the pH of the LB effluent decreased to 4.8 on day 1, butincreased to 6.3 by day 3 and from day 9 onwards variedbetween 6.9 and 7.8 for the rest of the 55 days run(Fig. 2). SCOD in effluent reached 15 g l�1 after 1 day, afterwhich it started to decrease, falling below 2 g l�1 by day 55(Fig. 2). VFA concentrations peaked at 5.2 g l�1 (totalVFA, TVFA) and accounted to 52% of the SCOD on

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(gl

-1)

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Fig. 2. SCOD and VFA concentrations and pH in effluent from the leach bed reactors in the one-stage leach bed process (run 1) and in the leach bed–UASB processes, without (runs 2 and 4) and with (run 3) pH adjustment. Dashed lines mark the time when the leach bed reactors were disconnected fromthe UASB.


day 13, and decreased steadily from then to <1 g l�1

(Fig. 2).Methane production and concentration remained low

(less than 5 ml d�1 and 2%, respectively) until day 9, thenstarted to increase. Methane concentration varied between34% and 53% from day 16 onwards, while methane pro-duction ranged mainly from ca. 30 to 90 ml d�1 (peakingat 120 ml d�1) and was ca. 30 ml d�1 when the run wasterminated (Fig. 3). During the 55 days run, VS removalin the LB1 amounted to 34% and the specific methaneyield was 0.060 m3 CH4 kg�1 VSadded and 15 m3 CH4 t�1

ww (tons of wet weight), corresponding to 20% of themethane potential in grass silage (Table 4). The methanepotential of the LB1 digestate was 0.204 m3 CH4 kg�1 dig-estate VSadded, and the sum of methane yields from LB1and from digestate amounted to 71% of the methanepotential in grass (Table 4).

3.2. Two-stage processes: leach bed reactor and UASB

3.2.1. Effect of pH adjustment (runs 2 and 3)

In runs 2 and 3, two leach bed reactors (LB2 and LB3)were operated initially with internal recirculation and thenin conjunction with UASB reactors. In run 3, the pH ofthe influent to the leach bed reactor (LB3) was adjusted to6. After 1 day of leachate recirculation, SCOD in both LBeffluents had increased to 11–12 g l�1, while pH 4.0–4.2,and circulation to the UASB was initiated. Circulationwas continued until days 9–10, when the SCOD in bothLB effluents had dropped to below 1 g l�1. After the UASBwas disconnected, the pH in the LB2 effluent initiallydecreased from 7.3 (day 9) to 6.1, and remained at 6.6 to6.8 for the rest of the run while in LB3 effluent pH variedbetween 5.5 and 5.8. Correspondingly, after UASB discon-nection SCOD increased in the LB2 effluent, peaking at

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Fig. 3. Daily gas production and methane concentrations in the one-stage leach bed process (run 1) and in the leach bed–UASB processes, without (runs 2and 4) and with (run 3) pH adjustment. Dashed lines mark the time when the leach bed reactors were disconnected from the UASB. In run 4, values for gasproduction in UASB on day 3 are out of scale (3466 ml d�1 CH4 and 2683 ml d�1 CO2, respectively). D CH4 production; · CO2 production; h CH4

concentration.

Table 4Substrate methane potential, specific methane yields, VS removals and digestate methane potentials in the one-stage leach bed process (run 1) and in theleach bed–UASB processes, without (runs 2 and 4) and with (run 3) pH adjustment (average values of replicates ± standard deviations, where applicable)

Run 1 2 3 4

Substrate methane potential m3 CH4 kg�1 VSadded 0.300 ± 0.003 0.300 ± 0.003 0.300 ± 0.003 0.300 ± 0.003m3 CH4 t�1 ww 72 ± 1 72 ± 1 72 ± 1 72 ± 1

Specific methane yield m3 CH4 kg�1 VSadded 0.060 0.197 0.103 0.107m3 CH4 t�1 ww 15 47 25 26% of substrate methane potential 20 66 34 36

VS removal % 34 55 39 42Digestate methane potential m3 CH4 kg�1 VSadded 0.204 ± 0.013 0.141 ± 0.025 0.160 ± 0.012 n.d.

m3 CH4 t�1 ww 21 ± 1 22 ± 4 19 ± 1 n.d.m3 CH4 kg�1 VSoriginal

a 0.152 ± 0.010 0.091 ± 0.016 0.115 ± 0.008 n.d.Reactor + digestate methane yield of substrate methane potential, % 71 96 73 n.d.

n.d. = not determined.a Calculated per VS of substrate originally added to the reactor, taking into account the VS removal during reactor operation.


3.5 g l�1 on day 23, thereafter decreasing to 1.8 g l�1 at theend of the run while in the LB3 SCOD remained between1.5 and 1.8 g l�1 (Fig. 2). The VFAs were mostly higher inLB2 effluent than in LB3 (Fig. 2). The proportion of TVFA

of SCOD was highest, 75%, on day 13 in LB2 effluent,whereas in LB3, the corresponding figure was 42% on day 7.

In both LB2 and LB3 the methane content in the gasand methane production remained low until days 6–7

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uctio

n (%

)Fig. 4. Reduction in VS and heat content, and amounts of analysedfractions in untreated grass silage (day 0) and in digestion residues afterdifferent periods of digestion in the leach bed–UASB process (run 4).


and then started to increase, methane content reaching 47%in LB2 on day 34% and 43% in LB3 on day 31 (Fig. 3).

Both UASB reactors removed COD initially by morethan 90%, but the removal decreased to 45–55% whenthe influent COD dropped to ca. 1 g l�1 (days 9–10). InUASB3 the COD reduction fluctuated more than inUASB2. VFAs were not present in the UASB2 effluents,while acetate and propionate were detected in the UASB3effluents in concentrations up to 0.3 g l�1 (data not shown).Methane concentration in the gas was higher in the UASB2(60–72%) than in UASB3 (46–60%). Also methane produc-tion was mostly higher in UASB2 than in UASB3, andcontinued in both UASBs even after they were discon-nected from the LBs (Fig. 3).

VS removal amounted to 55% in LB2 and 39% in LB3during the 55 and 31 days runs, respectively (Table 4).The total specific methane yields were 0.197 and0.103 m3 CH4 kg�1 VSadded in runs 2 and 3, respectively.Of these methane yields, 80% and 76% in runs 2 and 3,respectively, originated from the UASB. The methanepotentials of the LB2 and LB3 digestates were 0.141 and0.160 m3 CH4 kg�1 digestate VSadded, respectively, andthe sum of methane yields in LBs and digestates amountedto 96% and 73% of the methane potential in grass in runs 2and 3, respectively (Table 4).

3.2.2. Characterisation of the residues (run 4)

Six LBs installed in parallel and connected to a commonUASB were operated in run 4. The LBs were first operatedwith internal recirculation for 24 h, after which the SCODin the LB effluent had reached a level of 37 g l�1, and circu-lation to the UASB was initiated. Circulation to the UASBwas continued until day 17, when the SCOD in the effluentfrom leach bed reactors had dropped to below 2 g l�1 andthe pH of the LB effluent was 7.5 (Fig. 2). After the UASBwas disconnected, the SCOD in the effluent from the leachbed reactors slightly increased, peaking at 3.3 g l�1 on day20, thereafter varying between 1.5 and 2.6 g l�1 until theend of the run. VFA concentrations followed a pattern verysimilar to that of COD, acetate and propionate peaking at1.8 and 0.5 g l�1, respectively, on day 3, TVFA correspond-ing to 2.8 g COD l�1 and decreasing steadily from then onto <1 g COD l�1 by day 14. After the UASB was discon-nected, the pH in the leach bed effluents varied between7.1 and 7.7 for the rest of the run (Fig. 2).

The COD reduction in the UASB was 96% to 99%until day 10 and then decreased to 47% to 49% as theSCOD in the leachate declined. Methane concentrationsin the gas produced in UASB varied between 49% and70%, while the methane concentration in the gas fromthe leach bed reactors remained below 1% until day 10,then started to increase slowly, reaching 14% on day 41(Fig. 3). The total specific methane yield in run 4 was0.107 m3 CH4 kg�1 VSadded and 26 m3 CH4 t�1 ww after49 days of operation, corresponding to 36% of the meth-ane potential in grass silage (Table 4). Of this methane

yield, 98% orginated from the UASB, and 2% from leachbed reactors.

The extent of VS removal was determined each time areactor was terminated. After 1 day of leachate recircula-tion, VS removal had reached 16% (Fig. 4). After day 1,the reduction in VS slowed down, reaching 30% by the timemethanogenesis had begun in the leach bed reactors (day17). Total VS removal in run 4 amounted to 42%. Thereduction in heat content correlated well with the VS rem-ovals, amounting to 45% at the end of the run (Fig. 4).

The composition of grass was analysed on day 0 andafter 1, 10 and 49 days of digestion. Seventeen percent ofgrass TS initially consisted of lignin (Klason lignin and acidsoluble lignin), 45% of carbohydrates, 8% of extractivesand 10% of proteins (Table 3, Fig. 4). After 1 day of diges-tion, 11% of Klason lignin and 24% of acid soluble ligninhad been removed from the solid residue, whereas proteins,extractives and carbohydrates had degraded by 34%, 12%and 10% (Fig. 4). After 10 days of digestion extractiveswere the most rapidly removed component, their removalreaching 59%. At the end of digestion (after 49 days),74%, 51% and 39% of extractives, proteins and carbohy-drates, respectively, had been removed from the solid resi-due, whereas the removal of Klason lignin and acid solublelignin from the solid residue amounted to 17% and 58%,respectively (Fig. 4). The residue after completion of diges-tion consisted of 23% (from TS) of lignin, 50% of carbohy-drates, 4% of extractives and 9% of proteins.

4. Discussion

Anaerobic digestion of grass silage in leach bed reactors,with and without a second stage UASB reactor, was eval-


uated, and the highest methane yields were obtained in thetwo-stage process without pH adjustment. With this pro-cess 66% of the total methane potential in grass silagewas obtained within the 55 days solids retention time,whereas in the one-stage leach bed process only 20% ofthe methane potential in grass silage was extracted duringthe corresponding period. In the two-stage process, 76–98% of the total methane yield originated from the UASB,which clearly shows the advantage of applying a secondstage methanogenic reactor in combination with a leachbed process.

The methane yields and VS removals obtained in thepresent study in the two-stage anaerobic digestion processemploying batch leach bed reactors in the first stage were ofthe same order of magnitude as those reported by Yu et al.(2002), who obtained a 0.165 m3 CH4 kg�1 VSadded meth-ane yield and 67% VS removal, and Cirne et al. (in press),who reported a 0.27 m3 CH4 kg�1 VSadded methane yieldand 60% VS removal, in laboratory batch leach bed pro-cesses connected to anaerobic filters digesting grass waste(Yu et al., 2002) and grass silage (Cirne et al., in press,Table 2). Lehtomaki and Bjornsson (2006) obtained 59%VS removal and a methane yield of 0.39 m3 CH4 kg�1

VSadded after 50 days of digestion of grass silage in pilotbatch leach bed processes connected to anaerobic filters(Table 2). The higher methane yields reported in the latterstudy compared with those obtained in the present studywere most likely due to differences in the composition ofthe grass mixtures used as substrates, since the grass usedin the present study had lower biodegradability, as indi-cated by its lower methane potential and higher lignin con-centration (Lehtomaki and Bjornsson, 2006). Lignin isknown to be poorly degraded in anaerobic conditions,and the intense cross-linking of lignin with cellulose andhemicellulose also limits the degradation of these fibre frac-tions (Fan et al., 1981). Nutrient restriction on microbialdegradation due to the lower nitrogen content of the grassmay also have been a cause for the lower methane yields inthe present study.

The volumetric methane yields in one- and two-stageleach bed processes were low compared with previouslyreported yields in either batch or continuously fed wet pro-cesses. We have previously operated wet processes (contin-uously stirred tank reactors, CSTRs) co-digesting grasssilage, similar to the one used in the present study, withcow manure with up to 40% of grass in the feed VS, andobtained up to 53% VS removal and a methane yield of0.268 m3 CH4 kg�1 VSadded, corresponding to 105% of thetotal methane potential in the substrates (Lehtomakiet al., in press), whereas in the present study, up to 66%of the total methane potential in grass was obtained inthe leach bed–UASB process. The higher methane yieldsobtained with co-digestion can be partly explained by syn-ergy effects due to a more balanced nutrient composition inthe feed, but also by microbial adaptation, which is likelyto be enforced by the semi-continuous feeding in CSTRs(Lehtomaki et al., in press) as opposed to the batch pro-

cesses applied in the present study. Furthermore, in thetwo-stage process described in this report, no inoculumaddition was done in the first stage. Inoculating the batchreactors with digestate from previous runs would enablecontinuous adaptation of microbes to the degradation ofthe substrate and would be likely to enhance the extentof degradation and methane production also in batchprocesses.

In the one-stage leach bed process, 83% of the extractedCOD was converted to methane, whereas the correspond-ing figure for the two-stage operation was 92–95%. Thelow COD extraction rate in the one-stage operation wasapparently due to the high SCOD and VFA concentrationsin the recirculated leachate (SCOD and TVFA up to 15and 7 g l�1, respectively), which can cause inhibition ofhydrolysis and acidogenesis (Vavilin et al., 2003), whereasin the two-stage operation, the UASB efficiently removedSCOD and VFA from the leachate (up to 99% SCODreduction), as a result of which the UASB effluent returnedto the batch leach bed reactor was low in SCOD and VFA(mostly <1 g l�1), resulting in turn in more efficient extrac-tion of grass SCOD. VFA accumulation was apparentlythe cause of the lower methane yield and lower VS removalalso in run 4 with six parallel leach bed reactors, where thelower L/S ratio (8) applied resulted in higher SCOD andVFA concentrations in the leachate as opposed to the cor-responding run with a L/S ratio twice as high (17 in run 2).

Grasses are primarily composed of cellulose, hemicellu-loses and lignin, the polysaccharides and lignin accountingtogether for 62% of the grass TS, as analysed in the presentstudy. The carbohydrate and lignin content of grass in thepresent study was close to that previously reported for bor-eal timothy-based grasses (carbohydrates 37–43% TS, lig-nin 16–19% TS, Viinikainen et al., submitted forpublication). In total, 39% of the carbohydrates wereremoved in the leach bed–UASB process within the 49 daysof operation. Proteins were the most rapidly hydrolysablecomponent in grass, as they were degraded to the highestextent after 1 day of liquid recirculation, whereas extrac-tives were the most solubilised component after 10 and49 days of operation. The apparent loss of lignin in leachbed digesters fed with grass silage was most probably dueto solubilisation rather than degradation, as also suggestedby Kivaisi et al. (1990), as lignin is known to be refractoryand poorly degraded in anaerobic conditions (Fan et al.,1981). However, in the present study it was shown thatmore than half of the acid soluble lignin was solubilisedafter 49 days of digestion in a leach bed digester fed withgrass silage, whereas Klason lignin was the most recalci-trant component of those determined in the present study.

In the two-stage operation, adjustment of the pH ofinfluent to the leach bed reactor to 6 with HCl led to inhibi-tion in both the leach bed reactor and the UASB. Inhibitionof hydrolysis and acidogenesis in the leach bed process wereindicated by the low SCOD values and the low share ofTVFA of SCOD in the leachate, whereas inhibition of met-hanogesis in the UASB was indicated by the presence of


VFAs in the UASB effluent and by the lower and fluctuat-ing methane concentration in the gas from the UASB (vary-ing between 46% and 60%) compared with that in thecorresponding run without pH adjustment, despite the sim-ilar UASB loading rates in the two experiments. The low VSremoval and the high post-methanation potential of dige-state from the run with pH adjustment (run 3) indicated amuch lower extent of degradation than in the correspondingexperiment without pH adjustment (run 2), with the resultthat the total specific methane yield from the run with pHadjustment (run 3) remained much lower than in the corre-sponding run without pH adjustment (run 2) despite thesimilar UASB loading rates. Due to the problems in theUASB, the run with pH adjustment (run 3) was terminatedafter only 31 days of operation. pH values of around 6 havebeen reported optimal for the functioning of the extracellu-lar cellulase enzymes produced by hydrolytic bacteria (Sleatand Mah, 1987), and therefore it was assumed that pHadjustment to 6 could be advantageous in a leach bed pro-cess. However, lowering the pH below neutral did notclearly enhance the rate of hydrolysis in this and some pre-vious experiments (Veeken et al., 2000; Dinamarca et al.,2003; Babel et al., 2004). Moreover, chloride ion has beenreported to give rise to toxic effects in anaerobic wastewatertreatment (Mendez et al., 1992; Vijayaraghavan andRamanujam, 1999), and thus it is possible that the lowmethane yields and VS removal in run 3 were caused byinhibitory effects due to the application of hydrochloric acidin pH adjustment. However, Wujcik and Jewell (1980)found no inhibitory effect due to increased chloride concen-trations (added as NaCl) in high solid digesters digestingnewsprint paper and dairy manure, and Zhang et al.(2005) did not report any inhibitory effects in hydrolysisand acidogenesis of kitchen waste when hydrochloric acidwas used in pH adjustment.

The inoculation ratio applied in the one-stage leach bedprocesses digesting grass silage (6% of inoculum of totalVS) was apparently too low for an efficient extraction ofthe methane potential in the substrate, as indicated bythe low specific methane yield and VS removal during reac-tor operation, as well as by the high post-methanationpotential in the digestate. Torres-Castillo et al. (1995, Table1) studied digestion of barley straw in batch leach bed reac-tors with varying inoculum concentrations (2–12% of VS),and the highest gas production was obtained in the reactorwhere the share of inoculum was highest (12% of VS:0.226 m3 CH4 kg�1 VSadded). However, the difference ingas production between the reactors inoculated with 12%and 6% of inoculum (0.211 m3 CH4 kg�1 VSadded) was onlyminor and overshadowed by the lower volumetric gas pro-duction at the higher inoculum application ratios. There-fore, the authors recommended the use of 6% ofinoculum of total VS (Torres-Castillo et al., 1995). In diges-tion of wheat straw in batch leach bed reactors with vary-ing inoculum concentrations (5–20% of inoculum of totalVS), the difference in reactor performance using a largeor small addition of inoculum was insignificant after a

few days of hydrolysis, and an inoculum concentration ofup to 5% was suggested sufficient for a proper start-up(Llabres-Luengo and Mata-Alvarez, 1988). However, grasssilage is a more biodegradable substrate than straw, as indi-cated by the higher methane potential and the higheramounts of readily available soluble compounds in grasscompared with straw (Lehtomaki et al., in press). There-fore, the inoculation ratio previously recommended forthe digestion of straw was too low for that of grass silage,pointing to the need to optimise the substrate/inoculumratios for batch processes digesting energy crops.

The present results showed that the digestates still con-tained degradable material with significant methane poten-tial, which, if completely recovered, would correspond toup to 0.204 m3 CH4 kg�1 VSadded of digestate and 32–72%of the total methane production (sum of methane produc-tion in reactors and in post-methanation), the proportionbeing highest after digestion of grass in the one-stage leachbed process. If not recovered, part of this post-methanationpotential can be lost as atmospheric methane emissionsdue to spontaneous degradation, the extent of which wouldbe strongly dependant on the ambient temperatures(Kaparaju and Rintala, 2003). Digestates from CSTRsco-digesting energy crops and crop residues with cowmanure had post-methanation potentials of 0.133–0.197 m3 CH4 kg�1 VSadded and 3–8 m3 CH4 t�1 ww ofdigestate after 100 days post-methanation at 35 �C (Lehto-maki et al., in press). Thus, the post-methanation poten-tials of digestates from one- and two-stage leach bedprocesses were of the same order of magnitude as thosefrom CSTRs when calculated per VS of digestate. How-ever, owing to the high TS concentrations (12–17% in thepresent study) of digestates from leach bed processes com-pared with those from CSTRs (3–5% according to Lehto-maki et al. (in press)), the values for post-methanationpotential in the present study were of an order of magni-tude higher than those obtained in post-methanation ofdigestates from CSTRs when calculated per wet weight.Applying post-methanation enabled long total retentiontimes (131–155 days) in the present experiment, yieldingin total 71–96% of the grass methane potential as measuredin the batch methane potential assays with 94 days reten-tion time.

5. Conclusions

Anaerobic digestion of grass silage in leach bed reactors,with and without a second stage UASB reactor, was evalu-ated, and the highest methane yields were obtained in thetwo-stage process without pH adjustment. With this process66% of the total methane potential in grass silage wasobtained within the 55 days solids retention time, whereasin the one-stage leach bed process only 20% of the methanepotential in grass silage was extracted during the corre-sponding period. In the two-stage process, up to 98% ofthe total methane yield originated from the UASB, demon-strating the advantage of applying a second stage methano-


genic reactor in combination with a leach bed process. Inthe two-stage operation, adjustment of the pH of influentto the leach bed reactor to 6 with HCl inhibited both hydro-lysis/acidification and methanogenesis. The leach bed–UASB process removed 39% of the carbohydrates, whilemore than half of the acid soluble lignin was solubilised,whereas Klason lignin was the most recalcitrant componentof those determined in the present study. The digestatesstill contained degradable material with significantmethane potential, which, if completely recovered, wouldcorrespond to 0.141–0.204 m3 CH4 kg�1 VSadded and 19 to22 m3 CH4 t�1 ww of digestate.

Acknowledgements

The authors wish to thank EU Sixth Framework Pro-gramme (Project SES6-CT-2004-502824) and the FinnishGraduate School for Energy Technology for providingfunding for this work, and farmer E. Kalmari for kindlyproviding the substrates and Lannen Tehtaat plc for pro-viding the granular sludge. Furthermore, Ms. L. Malkkiand Ms. S. Rissanen are acknowledged for their help inmaintaining the reactors and conducting the laboratoryanalyses.

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