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

Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 3267–3278

Anaerobic digestion of grass silage in batch leach bed processes for methane production A. Lehtoma¨ki

*,1,

S. Huttunen, T.M. Lehtinen, J.A. Rintala

University of Jyva¨skyla¨, Department of Biological and Environmental Science, P.O. Box 35, FI-40014 Jyva¨skyla¨, Finland Received 6 April 2007; received in revised form 6 April 2007; accepted 6 April 2007 Available 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 time in the leach bed–UASB process without pH adjustment, whereas in the one-stage leach bed process 20% of the methane potential in grass was extracted. In two-stage operation, adjustment of the pH of influent to the leach bed reactor to 6 with HCl led to inhibition of both hydrolysis/acidogenesis and methanogenesis. In the leach bed–UASB process 39% of the carbohydrates and 58% of the acid soluble lignin were solubilised within the 49 days of operation, whereas Klason lignin was most recalcitrant. The methane potential of the digestates varied 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 digestion of organic materials provides a clean and versatile carrier of renewable energy, as methane can be used in replacement for fossil fuels in both heat and power generation and as a vehicle fuel. Methane production through anaerobic digestion has been evaluated as one of the most energy-efficient and environmentally benign ways of producing vehicle biofuel (LBS, 2002). The European Union (EU) has set a target of increasing the share of biofuels in vehicles to 5.75% by year 2010 in each member state (European Parliament, 2003). The utilisation of energy crops and crop residues for methane production is an interesting option for increasing domestic biofuel production, as it has been estimated that within the agricultural sector in *

Corresponding author. Tel.: +358 14 4451 160; fax: +358 14 4451 199. E-mail address: [email protected].fi (A. Lehtoma¨ki). 1 Presently at Jyva¨skyla¨ Innovation Ltd., P.O. Box 27, FI-40101 Jyva¨skyla¨, Finland. 0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.04.072

the EU, 1500 million tons of biomass could be anaerobically digested each year, half of this potential accounted for by energy crops (Amon et al., 2001). In Finland, for example, the Ministry of Agriculture has estimated that by 2012 up to 500 000 hectares (ha), an area corresponding to about one fourth of all arable land in Finland, could be dedicated to energy crop production (Vainio-Mattila et al., 2005). Energy crops and crop residues can be digested either alone or in co-digestion with other materials, employing either wet or dry processes. Energy crops typically have a high total solids (TS) content of 10–50%, and in order to treat this kind of material in wet processes, the solids must usually be homogenised and diluted with other materials high in water content. In the agricultural sector, the most widely applied solution is to co-digest crop biomass with animal manures in wet processes (Lehtoma¨ki et al., in press). Dilution increases the volume to be treated and thus the energy required for heating and pumping (Ghosh et al., 2000). Furthermore, floating of the crop materials along with crust or scum formation has been reported in

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digestion of crop materials in wet processes (Nordberg and Edstro¨m, 1997). Moreover, suitable materials for co-digestion may not always be available, and water would then have to be used for dilution. The gas production per digester volume (volumetric gas production) can potentially be increased by operating the digesters at a higher solids concentration. Batch high solids reactors, characterised by lower investment costs than those of continuously fed processes, but with comparable operational costs, are currently applied in the agricultural sector to a limited extent (Ko¨ttner, 2002; Weiland, 2003). In these systems, digesters are filled with fresh substrate, with or without addition of inoculum, and allowed to go through all the degradation steps sequentially. Batch reactors are often leach bed processes where solids are hydrolysed by circulating leachate over a bed of organic matter. Recirculation of leachate stimulates the overall degradation owing to more efficient dispersion of inoculum, nutrients and degradation products (Chanakya et al., 1993; Lissens et al., 2001). Digestion of plant biomass in one-stage leach bed processes has been seldom reported in the literature (Table 1), but in batch leach bed processes digesting barley straw, reductions in volatile solids (VS) of 45–60% and methane yields of 0.159–0.226 m3 CH4 kg 1 VSadded were obtained (Torres-Castillo et al., 1995), and in one-stage leach bed processes fed on a weekly basis with various lignocellulosic substrates (such as water hyacinth, straw, bagasse, cane trash etc.) and vegetable wastes, VS removals and biogas yields ranging from 37% to 78% and from 0.26 to 0.95 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 conjunction with a second stage methanogenic reactor, with the leachate generated in the first stage pumped to the methanogenic reactor for further degradation (Ghosh, 1984). Since the leachate has a low solids content, highrate reactors such as upflow anaerobic sludge blanket

reactors (UASBs) or anaerobic filters can be used in the second stage, and a high solid retention time is achieved in these reactors through the formation of granules or attachment of biomass to carriers (Henze and Harremoes, 1983; Lettinga, 1995). Digestion of plant material in processes of this kind has been reported (Table 2), but experiments on digestion of energy crops in these processes are few. Methane yields and VS removals of 0.27 to 0.39 m3 CH4 kg 1 VSadded and 59–60%, respectively, were obtained in two-stage anaerobic digestion of grass silage in batch leach bed processes connected to anaerobic filters, in both laboratory (Cirne et al., in press) and pilot trials (Lehtoma¨ki and Bjo¨rnsson, 2006) (Table 2). In practice, not all of the methane potential in substrates can be extracted in anaerobic digestion within the reactor residence time, and if the digestates are stored in uncovered storage tanks without gas collection, part of this methane can be lost to the atmosphere through spontaneous degradation, contributing to climate change. Post-methanation of digestates in covered storage tanks offers the possibility of both minimizing the potential methane emissions, as well as contributing to an increase in the methane yields (Kaparaju and Rintala, 2003), as up to 15% more biogas has been be obtained in post-methanation of digestates from liquid phase low solids digesters (Weiland, 2003). However, to our knowledge the methane potential of digestates from leach bed processes has not been determined previously. The aim of this study was to evaluate the suitability of leach bed reactors for methane production from grass silage. Operation of a one-stage process consisting of a batch leach bed reactor and a two-stage process with leach bed reactor in connection with an UASB were compared. Furthermore, the effect of adjusting the pH of influent in the first stage of the two-stage process was evaluated, and the extent of degradation of different fractions of VS in various stages of digestion was evaluated both by chemical characterisation and by determining the methane potential of the digestates.

Table 1 Examples of anaerobic digestion of plant material in one-stage leach bed processes, as reported in the literature Feedstock

Mode of feeding

Reactor volume (l)

T (C)

Feed TS (% ww)

VS removal (%)

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

Barley straw Water hyacinth Paddy straw Bagasse Cane trash Synedrella Parthenium Vegetable waste

Batch Weekly

220 2

35 21–27

35–36a 9.4

45–60 n.r.

0.159–.226b 0.348c

1 2

Weekly Weekly Weekly Weekly Weekly Weekly

6 6 6 6 6 11

26 26 26 26 26 35

n.r. n.r. n.r. n.r. n.r. n.r.

56.5 37.1 49.8 68.1 78.1 n.r.

0.48a,c 0.83a,c 0.26a,c 0.95a,c 0.71a,c 0.513–0.869b

3 3 3 3 3 4

n.r. = not reported. 1: Torres-Castillo et al. (1995), 2: Chanakya et al. (1993), 3: Chanakya et al. (1997), 4: Ramasamy and Abbasi (2000). a Values calculated from the data reported. b m3 CH4 kg 1 VSadded. c m3 biogas kg 1 VSadded.

1

VSadded or

References

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

Mode of feeding in first stage

Type of reactor as second stage

Reactor volume first stage/second stage (l)

T (C)

Fruit and vegetable waste Fruit and vegetable waste Fruit and vegetable waste Fruit and vegetable waste Fruit and vegetable waste Fruit and vegetable waste Fruit and vegetable waste Fruit and vegetable waste Potato waste Potato waste Sugar beet leaves Unpeeled potatoes Peeled potatoes Sugar beet leaves, potatoes 1:2 Sugar beet leaves, potatoes 1:3 Grass waste Grass silage Sugar beet Willow Grass silage Sugar beet Rice straw Rice straw Rice straw Water hyacinth

Batch

UASB-AF

1.3/0.5

35

Batch

UASB-AF

1.3/0.5

Batch

UASB-AF

Batch

Feed TS (% ww)

VS removal (%)

Spec. CH4 yield (m3 CH4 kg 1 VSadded)

Refs.

5

83

0.345

1

35

5

82

0.355

1

1.3/0.5

35

5

87

0.368

1

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

Batch Batch Batch Batch

UASB AF AF AF

2.0/0.84 2.0/1.0 7.6/2.6 7.6/2.6

37 37 35–37 35–37

19 19 n.r. n.r.

n.r. n.r. n.r. n.r.

0.39 0.39 0.216a 0.258a

3 3 4 4

Batch Batch

AF AF

7.6/2.6 7.6/2.6

35–37 35–37

n.r. n.r.

n.r. n.r.

0.351a 0.402a

4 4

Batch

AF

7.6/2.6

35–37

n.r.

n.r.

0.402a

4

Batch Batch Batch Batch Batch Batch Batch Batch Batch Weekly

AF AF AF AF AF AF ASBR ASBR ASBR AF

8000/190 7.6/2.6 7.6/2.6 7.6/2.6 0.75/0.9 0.75/0.9 4.0/4.0 4.0/4.0 4.0/4.0 2.0/0.5

Ambient 37 37 37 37 37 35 35 35 n.r.

92 31.8 20.2 49.5 27 24 92 92 92 9.6

67 59 96 46 60 89 44 45 48 n.r.

0.165a 0.39 0.38 0.16 0.27 0.44 0.19a 0.19a 0.21a 0.181b

5 6 6 6 7 7 8 8 8 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: Parawira et al. (submitted for publication), 5: Yu et al. (2002), 6: Lehtoma¨ki and Bjo¨rnsson (2006), 7: Cirne et al. (in press), 8: Zhang and Zhang (1999), 9: Chanakya et al. (1992). a Values calculated from the data reported. b m3 biogas kg 1 TSadded.

2. Methods 2.1. Origin of materials Grass silage was obtained from a farm in central Finland (Kalmari farm, Laukaa) (Table 3). It was prepared at the farm from grass (75% timothy Phleum pratense, 25% meadow fescue Festuca pratensis) harvested at early

flowering stage, chopped with an agricultural precision chopper after 24 h of pre-wilting and ensiled in a bunker silo with the addition of a commercial silage additive (lactic acid bacteria inoculant AIV Bioprofit, containing 60% Lactobacillus rhamsonus and 40% Propionibacterium freudenreichii spp. shermanii (Kemira Growhow Ltd.) with a total 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

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(Table 3). The UASB was inoculated with granular sludge obtained from an internal circulation (IC) reactor treating wastewater from sugar beet and vegetable processing (Sa¨kyla¨, Finland).

Table 3 Characteristics of grass silage and inoculum Parameter

Unit

Grass silage

Inoculum

pH TS VS SCOD Ntot NH4-N Klason lignin LigninAS Carbohydrates Extractives Proteins Higher heat content

% ww % ww mg g 1 TS mg g 1 TS mg g 1 TS % TS % TS % TS % TS % TS MJ kg 1 TS

4.1 25.9 24.0 228 16.9 1.4 13.0 4.1 45.0 8.4 10.4 19.8

7.7 6.6 5.0 189 48.9 17.2 n.d. n.d. n.d. n.d. n.d. n.d.

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) were used. Furthermore, in order to characterise the changes in LB material, six LBs in conjunction with a common UASB (UASB4) were operated (run 4) (Fig. 1). All the LBs (plastic columns) and UASBs (glass columns) had a liquid volume of 1000 ml and they were operated at 35 (±1) C. Leachate from LBs was collected at the bottom of the reactors in a liquid reservoir (R1) and either circulated to an UASB or recycled back to the top of the reactor when internal recirculation was applied. The UASB effluent was collected in another reservoir (R2), from which it was recirculated to LB (Fig. 1). The biogas produced was collected from the top of the reactors and the liquid reservoirs into aluminium gas bags. Before starting the present experiments the UASBs had been inoculated with granular sludge (see Section 2.1) and operated for two months with

n.d. = not determined.

material in a ratio of 0.5% volume/weight, v/w). In the laboratory, the material was further chopped with a garden chopper (Wolf Garten SD 180E) to a particle size of approximately 3 cm and then immediately frozen and stored at 20 C. Before analysis and feeding to the reactors, the samples were allowed to thaw overnight at 4 C. The inoculum used to inoculate the one-stage leach bed reactors and methane potential assays was from a mesophilic farm digester (Laukaa, Finland) treating cow manure and industrial confectionary by-products as substrate

R2 pH adj.

R2

LB1

LB2

R1 pH adj.

R1

U

LB3

U

R1

Run 1

Run 3

Run 2 R2

LB

LB

LB

LB

LB

LB

U

R1

Run 4 Fig. 1. Reactor set-ups in runs 1–4 (U = UASB, pH adj. = pH adjustment). Dashed lines represent the flow of process liquid during internal recirculation.

A. Lehtoma¨ki et al. / Bioresource Technology 99 (2008) 3267–3278

artificial wastewater at an organic loading rate (OLR) of 5 kg chemical oxygen demand (COD) m 3 d 1. The LBs (LB1–LB3) were filled with 50 g VS (208 g wet weight, ww) of grass silage mixed with 3.2 g VS (64 g ww) of inoculum (LB1) or without inoculum (LB2, LB3), after which 750 ml of tap water was added to fill the reactor (an initial liquid/solid (L/S) ratio of 17). After each sampling of leachate an equivalent amount of tap water was added to the liquid reservoir. When internal recirculation was applied, the recirculation rate of the leachate was 750 ml d 1. In two-stage operation the OLR to the UASB was maintained at 5 kg COD m 3 d 1, which determined the flow rate to both UASB and LBs. The pH adjustments of the influents to LBs in runs 1 (pH 7) and 3 (pH 6) were performed automatically with 1 M NaOH and 1 M HCl. In run 4 six parallel LBs were filled with 50 g VS (208 g wet weight) of grass silage without inoculum, after which 250 ml of tap water was added per reactor (1500 ml in total) (initial L/S ratio 8). The leachate from all six LBs was collected in a common reservoir (R1) and circulated from 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 each LB received the same liquid at the same flow rate. The LBs were terminated sequentially during the run to characterise the residual materials. The methane potentials of grass silage and digestates were determined in triplicate batch experiments in 2 l glass bottles (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 of 1 were added into the bottles, distilled water was added to achieve a liquid volume of 1.5 l, and NaHCO3 (3 g l 1) was added as buffer. In assays with digestates, the digestates (1 g VS) and inoculum corresponding to 1 g VS were added into the bottles. The contents of the bottles were flushed with N2/CO2-gas (70%:30%, Aga Ltd.) for 5 min and 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 the methane yield of the inoculum from those of substrates. The assays were terminated when CH4 production became negligible after 94–100 days. The methane potentials of digestates were expressed as m3 CH4 kg 1 VSadded and m3 CH4 kg 1 VSoriginal. The latter was calculated per VS of substrate originally added to the reactor, taking into account the VS removal during reactor operation. 2.3. Analyses and calculations Methane and volatile fatty acids (VFAs) were measured by gas chromatograph (GC) (methane: Perkin Elmer Clarus 500 GC with thermal conductivity detector and Supelco Carboxen 1010 PLOT fused silica capillary column, 30 m · 0.53 mm, and VFA: PE Autosystem XL GC with flame-ionisation detector and PE FFAP column, TM

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30 m · 0.32 mm). Operating conditions were for methane: oven 200 C, injection port 225 C, detector 230 C, and for 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 measurements. TS and VS were determined according to the Standard Methods (APHA, 1998) and COD according to the SFS 5504 (Finnish Standards Association, 1988). Total (Ntot), ammonium nitrogen (NH4-N) and proteins were determined according to the Tecator application note (Perstorp Analytical/Tecator AB, 1995) with a Kjeltec system 1002 distilling unit (Tecator AB), protein content calculated as 6.25 · Ntot. NH4-N and soluble COD (SCOD) from crop samples were analysed after extraction according to SFS-EN 12457-4 (Finnish Standards Association, 2002) and the samples for NH4-N and SCOD determination were filtered with GF50 glass fibre filter papers (Schleicher and Schuell). Extractives were determined by acetone extraction according to the TAPPI Test Method T 280 pm99 (TAPPI, 2000). For lignin and carbohydrate analyses, the acetone-extracted samples were hydrolysed according to the TAPPI Test Method T 249 cm-00 (TAPPI, 2000). Klason lignin content was measured according to the TAPPI Test Method T 222 om-98 (TAPPI, 2000). Acid soluble lignin (ligninAS) in hydrolysis filtrate was quantified spectroscopically (Beckman DU640 Spectrofotometer) on the basis of ultra-violet absorption at 205 nm using an absorptivity value of 110 l g 1 cm 1, and total lignin (lignintot) content was calculated as the sum of Klason lignin and ligninAS. The monosaccharides obtained (arabinose, galactose, mannose and xylose from the hemicellulose components and glucose from cellulose) were per(trimethylsilyl)ated and analysed with GC (HP 5890 Series II GC with 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 detector 300 C. Oven temperature was programmed to begin at 100 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 content was analysed as higher heat content with a bomb calorimeter (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 internal recirculation and pH adjustment of the influent (pH 7), the pH of the LB effluent decreased to 4.8 on day 1, but increased to 6.3 by day 3 and from day 9 onwards varied between 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, after which it started to decrease, falling below 2 g l 1 by day 55 (Fig. 2). VFA concentrations peaked at 5.2 g l 1 (total VFA, TVFA) and accounted to 52% of the SCOD on

A. Lehtoma¨ki et al. / Bioresource Technology 99 (2008) 3267–3278

Run 1 40

8

30

8

30

7

20

6 5

10

3

0

20

40

60

Run 3 9 8 7

40 30 20

4

6 5 4 3

10 0

3

pH

5

4

0

SCOD

6

10

pH SCOD (gl-1)

SCOD (gl-1)

9

7

20

VFA (gl-1)

Run 2

40

9

0

3

0

20

60

Run 4 40

9 8

30

7

20

6 5

10

2

2

1

1

SCOD pH

4 3

0

3

Run 1

40

pH

3272

Run 2 Acetate Propionate Butyrate

0

0 0

20

40

Run 3

3

0

60

20

40

60

Run 4

3

Acetate 2

2

1

1

0 0

20

40

60

Time (d)

Propionate Butyrate

0 0

20

40

60

Time (d)

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 from the 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, then started to increase. Methane concentration varied between 34% and 53% from day 16 onwards, while methane production ranged mainly from ca. 30 to 90 ml d 1 (peaking at 120 ml d 1) and was ca. 30 ml d 1 when the run was terminated (Fig. 3). During the 55 days run, VS removal in the LB1 amounted to 34% and the specific methane yield was 0.060 m3 CH4 kg 1 VSadded and 15 m3 CH4 t 1 ww (tons of wet weight), corresponding to 20% of the methane potential in grass silage (Table 4). The methane potential of the LB1 digestate was 0.204 m3 CH4 kg 1 digestate VSadded, and the sum of methane yields from LB1 and from digestate amounted to 71% of the methane potential 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 then in conjunction with UASB reactors. In run 3, the pH of the influent to the leach bed reactor (LB3) was adjusted to 6. After 1 day of leachate recirculation, SCOD in both LB effluents had increased to 11–12 g l 1, while pH 4.0–4.2, and circulation to the UASB was initiated. Circulation was continued until days 9–10, when the SCOD in both LB effluents had dropped to below 1 g l 1. After the UASB was disconnected, the pH in the LB2 effluent initially decreased from 7.3 (day 9) to 6.1, and remained at 6.6 to 6.8 for the rest of the run while in LB3 effluent pH varied between 5.5 and 5.8. Correspondingly, after UASB disconnection SCOD increased in the LB2 effluent, peaking at

A. Lehtoma¨ki et al. / Bioresource Technology 99 (2008) 3267–3278 Leach bed reactor Run 1

400

3273

UASB 60

300

40

200 20

0 0

20

40 Run 2

400

0 60 60

300 40

0 0

20

40 Run 3

400

0 60

60

300

40

200 20

100 0 0

20

40 Run 4

400

0 60

1200

60

800

40

400

20

0

5

10

15

Run 3

0 20

1600

80

1200

60

800

40

400

20

0 0

5

1600

60

300

80

0

Gas production (ml d—1)

100

CH4 concentration (%)

Gas production (ml d—1)

200 20

Run 2

1600

10 Run 4

15

0 20

CH4 concentration (%)

100

80

1200

60

800

40

400

20

40

200 20

100 0 0

20

40

0 60

0 0

5

Time (d)

10

15

0 20

Time (d)

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 2 and 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 gas production 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 4 Substrate methane potential, specific methane yields, VS removals and digestate methane potentials 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 (average values of replicates ± standard deviations, where applicable) Run Substrate methane potential

3

1

m CH4 kg VSadded m3 CH4 t 1 ww Specific methane yield m3 CH4 kg 1 VSadded m3 CH4 t 1 ww % of substrate methane potential VS removal % Digestate methane potential m3 CH4 kg 1 VSadded m3 CH4 t 1 ww m3 CH4 kg 1 VSoriginala Reactor + digestate methane yield of substrate methane potential, %

1

2

3

4

0.300 ± 0.003 72 ± 1 0.060 15 20 34 0.204 ± 0.013 21 ± 1 0.152 ± 0.010 71

0.300 ± 0.003 72 ± 1 0.197 47 66 55 0.141 ± 0.025 22 ± 4 0.091 ± 0.016 96

0.300 ± 0.003 72 ± 1 0.103 25 34 39 0.160 ± 0.012 19 ± 1 0.115 ± 0.008 73

0.300 ± 0.003 72 ± 1 0.107 26 36 42 n.d. n.d. n.d. 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 the end of the run while in the LB3 SCOD remained between 1.5 and 1.8 g l 1 (Fig. 2). The VFAs were mostly higher in LB2 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 gas and methane production remained low until days 6–7

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 more than 90%, but the removal decreased to 45–55% when the influent COD dropped to ca. 1 g l 1 (days 9–10). In UASB3 the COD reduction fluctuated more than in UASB2. VFAs were not present in the UASB2 effluents, while acetate and propionate were detected in the UASB3 effluents 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 production was mostly higher in UASB2 than in UASB3, and continued in both UASBs even after they were disconnected from the LBs (Fig. 3). VS removal amounted to 55% in LB2 and 39% in LB3 during the 55 and 31 days runs, respectively (Table 4). The total specific methane yields were 0.197 and 0.103 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 methane potentials of the LB2 and LB3 digestates were 0.141 and 0.160 m3 CH4 kg 1 digestate VSadded, respectively, and the sum of methane yields in LBs and digestates amounted to 96% and 73% of the methane potential in grass in runs 2 and 3, respectively (Table 4).

3.2.2. Characterisation of the residues (run 4) Six LBs installed in parallel and connected to a common UASB were operated in run 4. The LBs were first operated with internal recirculation for 24 h, after which the SCOD in the LB effluent had reached a level of 37 g l 1, and circulation to the UASB was initiated. Circulation to the UASB was continued until day 17, when the SCOD in the effluent from leach bed reactors had dropped to below 2 g l 1 and the pH of the LB effluent was 7.5 (Fig. 2). After the UASB was disconnected, the SCOD in the effluent from the leach bed reactors slightly increased, peaking at 3.3 g l 1 on day 20, thereafter varying between 1.5 and 2.6 g l 1 until the end of the run. VFA concentrations followed a pattern very similar to that of COD, acetate and propionate peaking at 1.8 and 0.5 g l 1, respectively, on day 3, TVFA corresponding to 2.8 g COD l 1 and decreasing steadily from then on to <1 g COD l 1 by day 14. After the UASB was disconnected, the pH in the leach bed effluents varied between 7.1 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 the SCOD in the leachate declined. Methane concentrations in the gas produced in UASB varied between 49% and 70%, while the methane concentration in the gas from the 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 was 0.107 m3 CH4 kg 1 VSadded and 26 m3 CH4 t 1 ww after 49 days of operation, corresponding to 36% of the methane potential in grass silage (Table 4). Of this methane

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yield, 98% orginated from the UASB, and 2% from leach bed reactors. The extent of VS removal was determined each time a reactor was terminated. After 1 day of leachate recirculation, VS removal had reached 16% (Fig. 4). After day 1, the reduction in VS slowed down, reaching 30% by the time methanogenesis had begun in the leach bed reactors (day 17). Total VS removal in run 4 amounted to 42%. The reduction in heat content correlated well with the VS removals, amounting to 45% at the end of the run (Fig. 4). The composition of grass was analysed on day 0 and after 1, 10 and 49 days of digestion. Seventeen percent of grass TS initially consisted of lignin (Klason lignin and acid soluble lignin), 45% of carbohydrates, 8% of extractives and 10% of proteins (Table 3, Fig. 4). After 1 day of digestion, 11% of Klason lignin and 24% of acid soluble lignin had 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 extractives were the most rapidly removed component, their removal reaching 59%. At the end of digestion (after 49 days), 74%, 51% and 39% of extractives, proteins and carbohydrates, respectively, had been removed from the solid residue, whereas the removal of Klason lignin and acid soluble lignin from the solid residue amounted to 17% and 58%, respectively (Fig. 4). The residue after completion of digestion consisted of 23% (from TS) of lignin, 50% of carbohydrates, 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-

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uated, and the highest methane yields were obtained in the two-stage process without pH adjustment. With this process 66% of the total methane potential in grass silage was obtained within the 55 days solids retention time, whereas in the one-stage leach bed process only 20% of the methane potential in grass silage was extracted during the 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 second stage methanogenic reactor in combination with a leach bed process. The methane yields and VS removals obtained in the present study in the two-stage anaerobic digestion process employing batch leach bed reactors in the first stage were of the same order of magnitude as those reported by Yu et al. (2002), who obtained a 0.165 m3 CH4 kg 1 VSadded methane yield and 67% VS removal, and Cirne et al. (in press), who reported a 0.27 m3 CH4 kg 1 VSadded methane yield and 60% VS removal, in laboratory batch leach bed processes connected to anaerobic filters digesting grass waste (Yu et al., 2002) and grass silage (Cirne et al., in press, Table 2). Lehtoma¨ki and Bjo¨rnsson (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 pilot batch leach bed processes connected to anaerobic filters (Table 2). The higher methane yields reported in the latter study compared with those obtained in the present study were most likely due to differences in the composition of the grass mixtures used as substrates, since the grass used in the present study had lower biodegradability, as indicated by its lower methane potential and higher lignin concentration (Lehtoma¨ki and Bjo¨rnsson, 2006). Lignin is known to be poorly degraded in anaerobic conditions, and the intense cross-linking of lignin with cellulose and hemicellulose also limits the degradation of these fibre fractions (Fan et al., 1981). Nutrient restriction on microbial degradation due to the lower nitrogen content of the grass may also have been a cause for the lower methane yields in the present study. The volumetric methane yields in one- and two-stage leach bed processes were low compared with previously reported yields in either batch or continuously fed wet processes. We have previously operated wet processes (continuously stirred tank reactors, CSTRs) co-digesting grass silage, similar to the one used in the present study, with cow manure with up to 40% of grass in the feed VS, and obtained up to 53% VS removal and a methane yield of 0.268 m3 CH4 kg 1 VSadded, corresponding to 105% of the total methane potential in the substrates (Lehtoma¨ki et al., in press), whereas in the present study, up to 66% of the total methane potential in grass was obtained in the leach bed–UASB process. The higher methane yields obtained with co-digestion can be partly explained by synergy effects due to a more balanced nutrient composition in the feed, but also by microbial adaptation, which is likely to be enforced by the semi-continuous feeding in CSTRs (Lehtoma¨ki et al., in press) as opposed to the batch pro-

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cesses applied in the present study. Furthermore, in the two-stage process described in this report, no inoculum addition was done in the first stage. Inoculating the batch reactors with digestate from previous runs would enable continuous adaptation of microbes to the degradation of the substrate and would be likely to enhance the extent of degradation and methane production also in batch processes. In the one-stage leach bed process, 83% of the extracted COD was converted to methane, whereas the corresponding figure for the two-stage operation was 92–95%. The low COD extraction rate in the one-stage operation was apparently due to the high SCOD and VFA concentrations in the recirculated leachate (SCOD and TVFA up to 15 and 7 g l 1, respectively), which can cause inhibition of hydrolysis and acidogenesis (Vavilin et al., 2003), whereas in the two-stage operation, the UASB efficiently removed SCOD and VFA from the leachate (up to 99% SCOD reduction), as a result of which the UASB effluent returned to the batch leach bed reactor was low in SCOD and VFA (mostly <1 g l 1), resulting in turn in more efficient extraction of grass SCOD. VFA accumulation was apparently the cause of the lower methane yield and lower VS removal also in run 4 with six parallel leach bed reactors, where the lower L/S ratio (8) applied resulted in higher SCOD and VFA concentrations in the leachate as opposed to the corresponding run with a L/S ratio twice as high (17 in run 2). Grasses are primarily composed of cellulose, hemicelluloses and lignin, the polysaccharides and lignin accounting together for 62% of the grass TS, as analysed in the present study. The carbohydrate and lignin content of grass in the present study was close to that previously reported for boreal timothy-based grasses (carbohydrates 37–43% TS, lignin 16–19% TS, Viinikainen et al., submitted for publication). In total, 39% of the carbohydrates were removed in the leach bed–UASB process within the 49 days of operation. Proteins were the most rapidly hydrolysable component in grass, as they were degraded to the highest extent after 1 day of liquid recirculation, whereas extractives were the most solubilised component after 10 and 49 days of operation. The apparent loss of lignin in leach bed digesters fed with grass silage was most probably due to solubilisation rather than degradation, as also suggested by Kivaisi et al. (1990), as lignin is known to be refractory and poorly degraded in anaerobic conditions (Fan et al., 1981). However, in the present study it was shown that more than half of the acid soluble lignin was solubilised after 49 days of digestion in a leach bed digester fed with grass silage, whereas Klason lignin was the most recalcitrant component of those determined in the present study. In the two-stage operation, adjustment of the pH of influent to the leach bed reactor to 6 with HCl led to inhibition in both the leach bed reactor and the UASB. Inhibition of hydrolysis and acidogenesis in the leach bed process were indicated by the low SCOD values and the low share of TVFA of SCOD in the leachate, whereas inhibition of methanogesis in the UASB was indicated by the presence of

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VFAs in the UASB effluent and by the lower and fluctuating methane concentration in the gas from the UASB (varying between 46% and 60%) compared with that in the corresponding run without pH adjustment, despite the similar UASB loading rates in the two experiments. The low VS removal and the high post-methanation potential of digestate from the run with pH adjustment (run 3) indicated a much lower extent of degradation than in the corresponding experiment without pH adjustment (run 2), with the result that the total specific methane yield from the run with pH adjustment (run 3) remained much lower than in the corresponding run without pH adjustment (run 2) despite the similar UASB loading rates. Due to the problems in the UASB, the run with pH adjustment (run 3) was terminated after only 31 days of operation. pH values of around 6 have been reported optimal for the functioning of the extracellular cellulase enzymes produced by hydrolytic bacteria (Sleat and Mah, 1987), and therefore it was assumed that pH adjustment to 6 could be advantageous in a leach bed process. However, lowering the pH below neutral did not clearly enhance the rate of hydrolysis in this and some previous experiments (Veeken et al., 2000; Dinamarca et al., 2003; Babel et al., 2004). Moreover, chloride ion has been reported to give rise to toxic effects in anaerobic wastewater treatment (Mendez et al., 1992; Vijayaraghavan and Ramanujam, 1999), and thus it is possible that the low methane yields and VS removal in run 3 were caused by inhibitory effects due to the application of hydrochloric acid in pH adjustment. However, Wujcik and Jewell (1980) found no inhibitory effect due to increased chloride concentrations (added as NaCl) in high solid digesters digesting newsprint paper and dairy manure, and Zhang et al. (2005) did not report any inhibitory effects in hydrolysis and acidogenesis of kitchen waste when hydrochloric acid was used in pH adjustment. The inoculation ratio applied in the one-stage leach bed processes digesting grass silage (6% of inoculum of total VS) was apparently too low for an efficient extraction of the methane potential in the substrate, as indicated by the low specific methane yield and VS removal during reactor operation, as well as by the high post-methanation potential in the digestate. Torres-Castillo et al. (1995, Table 1) studied digestion of barley straw in batch leach bed reactors with varying inoculum concentrations (2–12% of VS), and the highest gas production was obtained in the reactor where the share of inoculum was highest (12% of VS: 0.226 m3 CH4 kg 1 VSadded). However, the difference in gas production between the reactors inoculated with 12% and 6% of inoculum (0.211 m3 CH4 kg 1 VSadded) was only minor and overshadowed by the lower volumetric gas production at the higher inoculum application ratios. Therefore, the authors recommended the use of 6% of inoculum of total VS (Torres-Castillo et al., 1995). In digestion of wheat straw in batch leach bed reactors with varying inoculum concentrations (5–20% of inoculum of total VS), the difference in reactor performance using a large or small addition of inoculum was insignificant after a

few days of hydrolysis, and an inoculum concentration of up to 5% was suggested sufficient for a proper start-up (Llabre´s-Luengo and Mata-Alvarez, 1988). However, grass silage is a more biodegradable substrate than straw, as indicated by the higher methane potential and the higher amounts of readily available soluble compounds in grass compared with straw (Lehtoma¨ki et al., in press). Therefore, the inoculation ratio previously recommended for the digestion of straw was too low for that of grass silage, pointing to the need to optimise the substrate/inoculum ratios for batch processes digesting energy crops. The present results showed that the digestates still contained degradable material with significant methane potential, which, if completely recovered, would correspond to up to 0.204 m3 CH4 kg 1 VSadded of digestate and 32–72% of the total methane production (sum of methane production in reactors and in post-methanation), the proportion being highest after digestion of grass in the one-stage leach bed process. If not recovered, part of this post-methanation potential can be lost as atmospheric methane emissions due to spontaneous degradation, the extent of which would be strongly dependant on the ambient temperatures (Kaparaju and Rintala, 2003). Digestates from CSTRs co-digesting energy crops and crop residues with cow manure had post-methanation potentials of 0.133– 0.197 m3 CH4 kg 1 VSadded and 3–8 m3 CH4 t 1 ww of digestate after 100 days post-methanation at 35 C (Lehtoma¨ki et al., in press). Thus, the post-methanation potentials of digestates from one- and two-stage leach bed processes were of the same order of magnitude as those from CSTRs when calculated per VS of digestate. However, owing to the high TS concentrations (12–17% in the present study) of digestates from leach bed processes compared with those from CSTRs (3–5% according to Lehtoma¨ki et al. (in press)), the values for post-methanation potential in the present study were of an order of magnitude higher than those obtained in post-methanation of digestates from CSTRs when calculated per wet weight. Applying post-methanation enabled long total retention times (131–155 days) in the present experiment, yielding in total 71–96% of the grass methane potential as measured in the batch methane potential assays with 94 days retention time. 5. Conclusions Anaerobic digestion of grass silage in leach bed reactors, with and without a second stage UASB reactor, was evaluated, and the highest methane yields were obtained in the two-stage process without pH adjustment. With this process 66% of the total methane potential in grass silage was obtained within the 55 days solids retention time, whereas in the one-stage leach bed process only 20% of the methane potential in grass silage was extracted during the corresponding period. In the two-stage process, up to 98% of the total methane yield originated from the UASB, demonstrating the advantage of applying a second stage methano-

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genic reactor in combination with a leach bed process. In the two-stage operation, adjustment of the pH of influent to the leach bed reactor to 6 with HCl inhibited both hydrolysis/acidification and methanogenesis. The leach bed– UASB process removed 39% of the carbohydrates, while more than half of the acid soluble lignin was solubilised, whereas Klason lignin was the most recalcitrant component of those determined in the present study. The digestates still contained degradable material with significant methane potential, which, if completely recovered, would correspond to 0.141–0.204 m3 CH4 kg 1 VSadded and 19 to 22 m3 CH4 t 1 ww of digestate. Acknowledgements The authors wish to thank EU Sixth Framework Programme (Project SES6-CT-2004-502824) and the Finnish Graduate School for Energy Technology for providing funding for this work, and farmer E. Kalmari for kindly providing the substrates and La¨nnen Tehtaat plc for providing the granular sludge. Furthermore, Ms. L. Malkki and Ms. S. Rissanen are acknowledged for their help in maintaining the reactors and conducting the laboratory analyses. References Amon, T., Hackl, E., Jeremic, D., Amon, B., Boxberger, J., 2001. Biogas production from animal wastes, energy plants and organic wastes. In: van Velsen, A.F.M., Verstraete, W.H. (Eds.), In: Proceedings of the Ninth World Congress Anaerobic Digestion. Technologisch Instituut zw, Antwerp, Belgium, pp. 381–386. APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington DC. Babel, S., Fukushi, K., Sitanrassamee, B., 2004. Effect of acid speciation on solid waste liquefaction in an anaerobic digester. Water Res. 38, 2417–2423. Chanakya, H.N., Borgaonkar, S., Rajan, M.G.C., Wahi, M., 1992. Twophase anaerobic digestion of water hyacinth or urban garbage. Biores. Technol. 42, 123–131. Chanakya, H.N., Borgaonkar, S., Meena, G., Jagadish, K.S., 1993. Solidphase biogas production with garbage or water hyacinth. Biores. Technol. 46, 227–231. Chanakya, H.N., Venkatsubramaniyam, R., Modak, J., 1997. Fermentation and methanogenic characteristics of leafy biomass feedstocks in a solid phase biogas fermentor. Biores. Technol. 62, 71–78. Cirne, D.G., Lehtoma¨ki, A., Bjo¨rnsson, L., Blackall, L.L., in press. Hydrolysis and microbial community analyses in two-stage anaerobic digestion of energy crops. J. Appl. Microbiol. Dinamarca, S., Aroca, G., Chamy, R., Guerrero, L., 2003. The influence of pH in the hydrolytic stage of anaerobic digestion of the organic fraction of urban solid waste. Water Sci. Technol. 48, 249–254. European Parliament, 2003. Directive 2003/30/EC of the European Parliament and of the Council of 8 May 2003 on the promotion of the use of biofuels or other renewable fuels for transport. European Parliament, Brussels, Belgium. Fan, L.T., Gharpuray, M.M., Lee, Y.-H., 1981. Evaluation of pretreatments for enzymatic conversion of agricultural residues. Biotechnol. Bioeng. Symp. 11, 29–45. Finnish Standards Association. 1988. SFS 5504 Determination of chemical oxygen demand (CODCr) in water with closed tube method,

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Viinikainen, T., Lehtoma¨ki, A., Ale´n, R., Rintala, J., 2006. Effect of harvest time on the chemical composition of energy crops and crop residues intended for methane production by anaerobic digestion. submitted for publication. Vijayaraghavan, K., Ramanujam, T.K., 1999. Effect of chloride and condensable tannin in anaerobic degradation of tannery wastewaters. Bioproc. Biosyst. Eng. 20, 499–503. Weiland, P., 2003. Production and energetic use of biogas from energy crops and wastes in Germany. Appl. Biochem. Biotechnol. 109, 263– 274. Wujcik, W.J., Jewell, W.J., 1980. Dry anaerobic fermentation. Biotechnol. Bioeng. Symp. 10, 43–65. Yu, H.W., Samani, Z., Hanson, A., Smith, G., 2002. Energy recovery from grass using two-phase anaerobic digestion. Waste Manage. 22, 1– 5. Zhang, R., Zhang, Z., 1999. Biogasification of rice straw with an anaerobic-phased solids digester system. Biores. Technol. 68, 235– 245. Zhang, B., Zhang, L.-L., Zhang, S.-C., Shi, H.-Z., Cai, W.-M., 2005. The influence of pH on hydrolysis and acidogenesis of kitchen wastes in two-phase anaerobic digestion. Environ. Technol. 26, 329–339.