Bioresource Technology 101 (2010) 95279535

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Theoretical and observed biogas production from plant biomass of different bre contents
Ewa Klimiuk a, Tomasz Pokj a,*, Wojciech Budzynski b, Bogdan Dubis b
a b

Department of Environmental Biotechnology, University of Warmia and Mazury in Olsztyn, Soneczna Str. 45G, 10-709 Olsztyn, Poland Department of Agrotechnology and Crop Management, University of Warmia and Mazury in Olsztyn, M. Oczapowskiego Str. 8, 10-719 Olsztyn, Poland

a r t i c l e

i n f o

a b s t r a c t
The methane productivity of silage of four crop species Zea mays L., Sorghum saccharatum, Miscanthus giganteus and Miscanthus sacchariorus was investigated. The experiments revealed that at a hydraulic retention time of 60 days the volumetric methane yields from the Z. mays L. or S. saccharatum silages were higher than those from the Miscanthus giganteus or M. sacchariorus silages because of the higher crude bre content in Miscanthus spp. However, at comparable lignin concentrations in the feedstock, methane productivity for M. sacchariorus (0.19 0.08 L/g volatile solids) was twice that of Miscanthus giganteus (0.10 0.03 L/g volatile solids). The efciency of cellulose conversion varied from 83.6% (S. saccharatum) to 52.1% (Miscanthus giganteus), and hemicellulose from 88.9% (Z. mays L.) to 59.7% (Miscanthus giganteus). Conversion of cellulose and hemicellulose depended on the ratio of these polysaccharides to the lignin concentration of the feedstock. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 28 February 2010 Received in revised form 14 June 2010 Accepted 25 June 2010 Available online 8 August 2010 Keywords: Anaerobic digestion Energy crops Agricultural biogas plants Theoretical biogas potential

1. Introduction Directive 2009/28/EC of the European Parliament and the Council of 23 April 2009 endorsed a mandatory target of a 20% share of energy from renewable sources in overall community energy consumption by 2020. It also mandated a 10% minimum target to be achieved by all Member States for the share of biofuels in transport petrol and diesel consumption by 2020. Biogas production is a key technology for the sustainable use of agricultural biomass as a renewable energy source. Biogas can be produced from a wide range of crops, animal manures and organic wastes, and thus it offers high exibility and can be adapted to the specic needs of different locations and farm management. After anaerobic digestion, the digestate is a valuable fertiliser for agricultural crops. Germany is a European leader in using biogas technologies, installing more than 3500 biogas plants with an overall electrical capacity of more than 1000 MW during 20022007 (Demirel and Scherer, 2009). According to Parawira et al. (2008), in Sweden during 2004 the biogas-derived energy was 1.4 TWh, with further increases to 25 TWh per year. It is expected that more than half of this amount (14 TWh) will be from the agricultural sector. In Poland, according to the Institute of Renewable Energy data, 156 biogas plants operated in 2006, and only one used agricultural products (Granoszewski and Grabias, 2009). By 2020, however, Polish government projects plan to have installed about 2000

* Corresponding author. Tel.: +48 89 5234161; fax: +48 89 5234131. E-mail address: tomasz.pokoj@uwm.edu.pl (T. Pokj). 0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.130

new biogas plants with an overall electrical capacity of 2000 3000 MW. In an agricultural biogas plant, each group of substrates has a specic potential for biogas production. The best properties are raw-harvested plant material with low lignin content (Zubr and Wise, 1989). The literature indicates that maize (Zea mays L.), grasses (Poaceae), clovers (Trifolium), Sudan grass (Sorghum sudanense) and fodder beet (Beta vulgaris L.) may be suitable for biogas production (Gunaseelan, 1997; Tong et al., 1990; Weiland, 2006). The evaluation of modern biogas plants in Germany, monitored during 20022004, has shown that maize is the most commonly used co-substrate in 80% of all agricultural biogas plants operated with the fermentation of manure (Weiland, 2006). Key parameters affecting biogas yield investigated so far, are the maize variety, time of harvesting, mode of conservation and pre-treatment of the biomass prior to the digestion process (Amon et al., 2007a,b). Although biogas production from maize is the most efcient and technically advanced option, it could result in severe competition between energy and food supplies, which is probably not favourable in the long term. For that reason, a great deal of interest in energy crops has been aroused in recent years. This interest has focused on the use of agricultural wasteland and perennial crops. On large farms, an important criterion in crop selection for biogas production is the specic agriculture land available for production. The variety of agricultural land and the need for a continuous biomass supply for the biogas plants requires diversication in the substrates supplied. The variety of plant biomass used not only improves the operational management of biogas plants, but also favours agroecosystem biodiversity.

9528

E. Klimiuk et al. / Bioresource Technology 101 (2010) 95279535

The complex structure of lignocellulosic materials is known to be a crucial obstacle for transition from traditional cereal crops to lignocellulosic feedstocks. However, in recent years there has been a dynamic development of different methods of structural digestion and recovery of lignin-encrusted sugars (Sun et al., 1995; Zheng et al., 2009). This should increase the range of crop types used for conversion into biogas in the future. This present study investigated biogas productivity from plant biomass of annual crops, i.e. maize (Z. mays L.), sugar sorghum (Sorghum saccharatum) and perennial crops (Miscanthus giganteus and Miscanthus sacchariorus), without using manure or any other co-substrate. The research sought to (i) determine whether the hemicellulose and cellulose conversion into biogas, at determined hydraulic retention times (HRT), depends on the ratio of their concentration to the lignin concentration in the feedstock; (ii) estimate the methane digestion operational parameters, and the specic biogas production rate and biogas yield; and (iii) compare the estimated biogas production using elementary composition feedstock with the experimental values. 2. Methods 2.1. Field and laboratory investigations Maize (Z. mays L.), sugar sorghum (S. saccharatum) and Miscanthus spp. (Miscanthus giganteus and M. sacchariorus) silages were used for biogas production. The plant biomass was obtained from eld experiments performed in 2008 (for Miscanthus spp. this was the second year of use and the third year of vegetation) in the Production and Experimental Station at Bacyny (53350 4900 N, 19510 20.300 E), University of Warmia and Mazury in Olsztyn. The crops were cultivated in typical lessive soil, of medium silt content, composed of medium loam, having the IIIa bonitation class, and agricultural valuation complex 2. The area of each single plot was 100 m2. Harvesting of the maize (mid-early LG 3232) was carried out at the BBCH 89 stage, the sugar sorgum (Sucrosorgo 506) at the milkwaxen stage, the Miscanthus giganteus (clone) when the rst lower leaves were beginning to dry, and the M. sacchariorus at the owering phase. The crops were harvested in the rst decade of October by self-propelled harvesters equipped with cutting drums that chopped the crops into pieces 23 cm in length. Next, the raw-harvested crops were ensiled. Samples of the plant materials were concentrated in 200 L silos lined with foil for 90 d. Formic acid (85%) was added at a ratio of 5 g acid to 1 kg biomass. 2.2. Feedstock preparation For standardisation of feedstock in the digestion process, the silage was chopped in a cutting mill (Retsch SM100, Germany) and passed through 1-mesh screen. Then the silage was stored in plastic bags at 4 C. After chopping, the concentrations of total solids (TS) and volatile solids (VS) in the silage were 0.4248 0.028 g/g and 0.4016 0.022 g/g (Z. mays L.); 0.1412 0.053 g/g and 0.1373 0.047 g/g (S. saccharatum); 0.3093 0.060 g/g and 0.2878 0.055 g/g (Miscanthus giganteus); and 0.7007 0.048 g/ g and 0.6755 0.045 g/g (M. sacchariorus), respectively. 2.3. Experimental set-up and experimental assumptions The experiments were conducted in four parallel anaerobic, continuously stirred tank-reactors (CSTRs) with a working volume of 6 L. The stainless-steel reactors were equipped with a stirrer with adjustable speed of rotation and a water jacket. Properly mounted valves enabled reactor feeding, biogas and digestate collection. The following silages were used as feedstock: Z. mays L. (series 1), S. saccharatum (series 2), Miscanthus giganteus (series

3) and M. sacchariorus (series 4). The experiments were performed in duplicate. Reactors were inoculated with anaerobic sludge from the sludge digestion chambers of a municipal wastewater treatment plant in Olsztyn (North Poland). Before feeding, the feedstock was mixed with tap water to obtain a concentration of total solids of 8%. The silages needed to be mixed with water in different ratios (w/w): 1:5.31 (Z. mays L.), 1:1.77 (S. saccharatum), 1:3.86 (Miscanthus giganteus) and 1:8.76 (M. sacchariorus). The reactors were operated at 39 C. Once a day each reactor was supplied with 100 mL of the feedstock after 100 mL of mixed liquid had been withdrawn. The biogas was collected in Tedlar sample bags. In all the series, a constant HRT of 60 days was assumed. Relatively long HRT resulted from the high bre content of the Miscanthus spp. (Miscanthus giganteus: neutral detergent bre (NDF) = 75.34 5.73% TS, acidic detergent bre (ADF) = 47.48 4.02% TS; M. sacchariorus: NDF = 84.77% 4.25% TS, ADF = 54.25 3.18% TS). 2.4. Analytical methods Analytical process control involved analysis of the silage, feedstock, digestate, the liquid phase of the digestate and biogas production and composition. In the silage and digestate the following parameters were determined: TS, VS, water soluble carbohydrate (by the anthrone method; Daniels et al., 1994), NDF, ADF, lignin (acid detergent lignin (ADL)) (by the Van Soest method; PN-EN ISO 13906:2009) cellulose, hemicellulose, and elementary composition with reference to carbon (C), nitrogen (N) and hydrogen (H). Hemicellulose content was calculated as the difference between NDF and ADF, while cellulose was the difference between ADF and ADL. The C, N and H content of the biomass was measured at the Institute of Organic Chemistry, Polish Academy of Sciences in Warsaw (Poland) using the vario EL III Element Analyzer (Elementar Analysensysteme GmbH, Germany). The TS and VS were determined for the feedstock. In the liquid phase of the feedstock and digestate the following parameters were determined: pH, chemical oxygen demand (COD) by the dichromate method, ammonium nitrogen (N-NH4) and volatile fatty acids (VFAs) by the distillation method, and alkalinity by the titration method. The measurements were performed for ltered supernatant samples, previously centrifuged (8693g for 10 min). The parameters, including pH, alkalinity, TS, VS, COD, N-NH4 and VFAs, were determined according to the standards methods for the examination of water and wastewater (APHA, 1992). 2.5. Biogas characteristics Biogas production and composition were measured daily in averaged samples, collected in Tedlar sample bags. The biogas volume was measured following standard methods (APHA, 1992) using apparatus consisting of a cylinder lled with a saturated solution of sodium chloride combined with an equalising tank equipped with a side tube. The composition of the biogas with reference to maximum methane (CH4) and carbon dioxide (CO2) contents and to minimum oxygen concentration was measured using a GA 2000+ automatic analyser (Geotechnic Instruments, UK). 3. Results and discussion 3.1. Characteristic of the feedstock and degree of organic digestion Results of the chemical analysis of the silages (after dilution with tap water) are given in Table 1.

E. Klimiuk et al. / Bioresource Technology 101 (2010) 95279535 Table 1 Characteristics of the feedstocks fed to reactors in series 14 (standard deviation from the mean value is given in parentheses). Parameter pH Volatile fatty acids Ammonium nitrogen Total solids Volatile solids Unit mg/L mg N-NH4/L % % Series 1 Z. mays L. 4.3 (0.16) 1457 (150.2) 110.6 (10.9) 8.4 (0.79) 7.8 (0.74) Series 2 S. saccharatum 3.75 (0.04) 2648 (133.3) 112 (0.177) 8.5 (0.38) 7.8 (0.61) Series 3 Miscanthus giganteus 7.46 (0.042) 600 (12.7) 66.5 (0.071) 8.8 (1.41) 7.9 (1.32)

9529

Series 4 M. sacchariorus 7.37 (0.113) 274 (2.1) 22.4 (0.092) 8.8 (0.68) 8.5 (0.64)

From the data presented, it can be seen that total solids (TS) in the feedstock did not exceed 8.8%. Silages of Z. mays L. and S. saccharatum were characterised by acidic pH, whereas both Miscanthus species exhibited a slightly alkaline pH, indicating little ensilage. The VFAs and N-NH4 concentrations in the ltered supernatant depended on the silage type. The highest VFAs concentration was for S. saccharatum, and the lowest for M. sacchariorus. Review of literature revealed that miscanthus can be preserved and stored by ensiling (Lewandowski et al., 2000). In present study low degree of ensiling of Miscanthus spp. seemed to result from low concentration of water soluble carbohydrate (WSC) and for M. sacchariorus also low moisture content in fresh plant biomass. The ensiling process is successful at minimum content of WSC equal 50 g/kg (McDonald et al., 1991). Similarly, when dry matter of crop exceeds 500 g/kg, the process of ensiling is also restricted, because of impaired enzyme activity at low moisture content (Adogla-Bessa and Owen, 1995). The WSC and dry matter (TS) content of Miscanthus giganteus and M. sacchariorus used in our research were 7.9 2.27 g/kg, 35 2.83 g/kg and 45.1 1.44 g/kg, 65 4.24 g/kg, respectively, and were kept behind the values needed for adequate ensiling. After digestion, the highest degree of organic removal (expressed as volatile solids concentration) was 77.5 2.6% for S. saccharatum silage (Fig. 1b). S. saccharatum contained a signicant concentration of water soluble carbohydrate (14.9 g/L), considered to be easily available compounds for microorganisms. The sum of the hemicellulose, cellulose and lignin concentrations was 43.0 g/ L, and the conversion degree was 80.2%. Silage of S. saccharatum contained more cellulose than that of Z. mays L., with a lignocellulose concentration of 35.1 g/L, and degree of conversion 74.3%. The degree of conversion of hemicellulose was higher than that for cellulose for all the silages tested (Fig. 1). The lowest efciency of hemicellulose and cellulose removal was for Miscanthus giganteus. Lignin was not degradable during digestion (Fig. 1) and its concentration in the digestate was comparable with its concentration in the feedstock for Z. mays L. and S. saccharatum silages. The average lignin concentration for Miscanthus spp. was higher in the digestate than in the feedstock. These differences were not statistically signicant (t-test for dependent samples: Miscanthus giganteus, P = 0.1233; and M. sacchariorus, P = 0.7811). There is a common view in scientic literature that lignin is not biodegradable under anaerobic conditions. This is because the extracellular enzymes required for depolymerisation of lignin need molecular oxygen, and their oxidative reactions would not be anticipated under anaerobic conditions (Hatakka, 1994; Jeffries, 1994). However, some authors have shown that lignin can be degraded under anoxic conditions, i.e. sulphate-reducing conditions (Pareek et al., 2001). Earlier, Benner et al. (1984) demonstrated that [14C]-lignocellulose preparations and synthetic [14C]-lignin incubated with anoxic sediments were slowly degraded anaerobically to 14CO2 and 14CH4. A review of the literature shows that both cellulose and hemicellulose can be anaerobically converted to CH4 and CO2. However, the rate of degradation, especially of cellulose, depends strongly on its state in the feedstock; if it is lignin-incrusted, then lignin prevents the access of cellulases to the cellulose

bres. If the cellulose is mainly in a crystalline form, then cellulases can attach to it, and hydrolysis can be relatively fast, leading to propionate and butyrate formation (Jrdening and Winter, 2005). The chemistry of grass lignocellulose varies considerably from that of wood (Akin et al., 1995), and explains why it is currently assumed that lignin concentration does not always reect the degree to which lignin inhibits cellulose bioavailability. The lignin in grass is not as restrictive to microorganisms as the lignin in components such as branches (Barlaz, 2006). The present study showed that under dened operational conditions (i.e. constant HRT), there was a relationship between the efciency of cellulose/hemicellulose removal (E) and the chemical composition of the bre in the feedstock, expressed as the ratio of cellulose/hemicellulose to lignin concentration. For crops tested in this study, the relationship is described using the empirical equations:

E f ln C Cel =C ADL or E f ln C Hem =C ADL :

where CCel is the concentration of cellulose; CADL is the concentration of acid detergent lignin; and CHem is the concentration of hemicellulose. Experimental results were tted to regression lines showing the rectilinear regression coefcients for both polysaccharides (Fig. 2). The efciency of hemicellulose removal was higher, as indicated by coefcient b. Moreover, the experiments demonstrated that the effectiveness of cellulose/hemicellulose removal increased together with an increase in the CCel/CADL and CHem/CADL ratios. Low CCel/CADL and CHem/CADL ratios were typical for silages of Miscanthus spp., related to their high lignin contents. For the Z. mays L. and S. saccharatum silages the ratios were high because of signicantly lower lignin concentrations. The CHem/CADL ratio of Miscanthus spp. was approximately 1.31.7 times lower that of Z. mays L. and S. saccharatum and the CCel/CADL ratio of Miscanthus spp. was 1.92.4 times lower that of Z. mays L. and S. saccharatum. However, further investigations are necessary to point out whether empirically determined equations can by applied for other crop species and operational conditions. Lignin concentration was not the only factor affecting the conversion of the organic matter in the tested plants. The theoretical biogas yield (biodegradable fraction, B) resulting from the biodegradation of the silages tested was compared with the empirical results. Chandler et al. (1980) correlated the biodegradability of various agricultural residues, determined by long-term digestion studies, with the lignin content of the substrate, as determined by sequential bre analysis. They developed the following empirical relationship to estimate the B value of an organic substrate from a lignin test:

B 0:83 0:028 ADL

where ADL is expressed as % VS. For Z. mays L. and S. saccharatum silages, the B values calculated from Eq. (1) (74.6% and 73.9% VS, respectively) were comparable with the values obtained experimentally, 75.5% and 77.5% VS, respectively. For Miscanthus giganteus and M. sacchariorus the experimental values were 29.4% and 36.3% VS about half of the

9530

E. Klimiuk et al. / Bioresource Technology 101 (2010) 95279535

a 100
80

100

b 100
80 60

100 80 60 40 20 0
Volatile solids Water soluble Hemicellulose carbohydrates Cellulose Lignin

80

60

60

E (%)

40

40

40 20 0

20

20

0
Volatile solids Water soluble Hemicellulose carbohydrates Cellulose Lignin

c 100
80

100

d 100
80

100

80

80

E (%)

60 g/L

60

60 g/L

60

40

40

40

40

20

20

20

20

0
Volatile solids Water soluble Hemicellulose carbohydrates Cellulose Lignin

0
Volatile solids Water soluble Hemicellulose carbohydrates Cellulose Lignin

silage digestate
Fig. 1. Concentration of volatile solids, water soluble carbohydrate and bre in tested silages and digestate (g/L) and the efciency of their removal (E): (a) series 1 (Z. mays L.); (b) series 2 (Sorghum saccharatum); (c) series 3 (Miscanthus giganteus); and (d) series 4 (M. sacchariorus). Error bars represent standard deviation.

100 80

100 80
y = 39,531x + 14,133 R 2 = 0,992

E Hem (%)

60 40 20 0 1 1.25

60

E Cel

y = 39,86x - 8,0881 R 2 = 0,9941

40 20 0 1.5

1.5 lnC Hem /C ADL

1.75

1.75

2 lnCCel /CADL

2.25

2.5

Fig. 2. The relationships between the efciency of hemicellulose (EHem) and cellulose (ECel) removal and the ratio of hemicellulose and cellulose concentration to lignin concentration (CHem/Ci and CCel/CADL, respectively). Error bars represent standard deviation.

calculated B values of 59.0% and 61.0% VS, respectively. These differences could be the result of an insufciently long HRT for the Miscanthus spp. silage in the CSTR reactor, which limited the degree of conversion. Benner et al. (1984) tested specically radiolabelled [14C-lignin]-lignocellulose and [14C-polysaccharide]-lignocellulose prepared from a variety of marine and freshwater wetland plants including a grass, a sedge, a rush and a hardwood. They demonstrated that lignocellulose derived from herbaceous plants was

degraded more rapidly than lignocellulose from hardwood. After 246 days, 16.9% of the lignin and 30.0% of the polysaccharide components of the lignocellulose derived from the grass were degraded. However, only 1.55% of the lignin and 4.15% of the polysaccharide components of the lignocelullose derived from hardwood were degraded to gaseous end products. Cellulose degradability is not always directly correlated with lignin concentration. Tong et al. (1990) reported the methanogenic conversion of white r and wood grass to be 9% and

E (%)

E (%)

g/L

g/L

E. Klimiuk et al. / Bioresource Technology 101 (2010) 95279535

9531

66%, respectively, despite similar lignin concentration in both substrates. Based on the decomposition of cellulose in grass and paper, Eleazer et al. (1997) showed that lignin concentration alone was not a good predictor of cellulose bioavailability. 3.2. Characteristic of the digestate liquid phase Maintenance of suitable environmental conditions is extremely important for the performance of CH4 digestion. Some of the environmental factors that affect methanogenesis require stringent monitoring and control to avoid process failure. Among them, pH, alkalinity and VFAs concentration have been investigated. These factors play leading roles in building up the selective pressure, which elicits and stabilises a biological community, and they are important mainly because of the critical nature of the syntrophic relationship between the microorganisms in the digester. Anaerobic bacteria, especially methanogens, exhibit a characteristic sensitivity to extremes of pH. In an effective working digester, pH drop can be countered by natural processes, such as alkalinity and the consumption of VFAs by methanogens. Alkalinity is the equilibrium between CO2 and bicarbonate ions that provides buffer capacity in anaerobic digestion, preventing signicant and rapid changes in pH. Therefore, buffering capacity is proportional to bicarbonate concentration. It should be noted that to maintain an adequate buffering capacity, and to keep the pH at a safe and stable level (Fig. 3a), the digesters fed with Z. mays L. and S. saccharatum silages required the addition of 5 M NaOH solution throughout the operation. This explains the higher alkalinity (Fig. 3b), despite the high concentration of VFAs in the digestate liquid phase, of these feedstocks (Fig. 3d). During digestion of Z. mays L. and S. saccharatum silage, VFAs concentration in the digestate liquid phases were 2420.8 612.1 and 1555.7 377.9 mg C2H4O2/L, respectively; higher than for Miscanthus spp. silage. Similarly, the concentration of soluble organic compounds (measured as COD) was highest in the digestate liquid phase from Z. mays L. silage (Fig. 3c). For the other silages, it varied between 1761 285 (Miscanthus giganteus) and 1290 119 mg COD/L (M. sacchariorus). During fermentation, high molecular weight compounds (i.e. polysaccharides) are hydrolysed to monomers, and then digested to organic acids. Acetic acid, H2 and CO2 produced at the acetogenesis phase are utilised by methanogens as substrates and converted into CH4. Consumption of volatile acids is dependent on the equilibrium between acidogens and acetogens and this can be easily upset by changes in the operational and environmental conditions. When the activity of hydrolytic bacteria converting cellulose and hemicellulose to soluble intermediates, and the acetogenic bacteria and methanogens remain at equilibrium, then short-chain acids are converted to CH4 and CO2. Under unsustainable conditions, short-chain carboxylic acids accumulate in the liquid phase of the digestate. If the acids are present at high concentrations they alter the pH and cause microbial stress and methanogenesis inhibition. Siegert and Banks (2005) reported that glucose fermentation was inhibited at total VFAs concentration greater than 4 g/L. To overcome accumulation of VFAs and in order to transform all soluble organic compounds into biogas it seems to employ twostage digestion. Efcacy of such process conguration is conrmed in practice (Weiland, 2006). Acetic acid is usually present at higher concentrations than other fatty acids during anaerobic digestion (Wang et al., 1999). Essentially, the methanogens cannot metabolise the acetate produced by acetogenic organisms until the number of methanogenic organisms has increased sufciently. This is especially true of feedstocks that are rapidly hydrolysed. With poorly-degradable feedstocks, the hydrolysis stage is more likely to be the limiting step, as conrmed in the present study. For the Miscanthus spp. silage, the hydrolysis of polysaccharides proceeded slowly because of lig-

nin incrustation, limiting the rate of further digestion phases. The concentration of organic acids in the digestate liquid phase was low, whereas the pH remained near neutral, without the need to add further chemicals (Fig. 3a). Nitrogen plays an important role in supplying the basic cellular building blocks for cell growth and the synthesis of enzymes and cofactors. Additionally, fermenting nitrogenous compounds contribute to the stable neutral pH of the CH4-digesting liquor by the release of NH ions. The investigations demonstrated that indi4 vidual silages were characterised by different C/N ratios (Table 3). The highest C/N ratio was for the M. sacchariorus silage (C/ N = 103.3), and the lowest (C/N = 19.1) was for S. saccharatum, which had the highest N-NH4 concentration (447.8 51.6 mg/L) in the digestate liquid phase. During anaerobic digestion of organic matter, the rate of C consumption was 2530-times higher than that of N. For that reason, to maintain optimal growth conditions, the C/N ratio should be between 20:1 and 30:1 (Malik et al., 1987). If the organics in the silage were completely converted into biogas, then the N concentration of the Miscanthus spp. would be insufcient for proper microorganism growth. The research showed, however, that the degree of organic conversion into biogas was 29.3% for Miscanthus giganteus and 36.3% for M. sacchariorus, resulting in only partial use of the N, which was reected in the N-NH4 concentration. The N-NH4 concentration in the digestate liquor was high at 297 and 220 mg N-NH4/L for Miscanthus giganteus and M. sacchariorus, respectively (Fig. 3e). 3.3. Biogas production rate and biogas yield At a constant HRT of 60 days, the organic loading rate (OLR) in the following series varied from approximately 1.30 g VS/L d (Z. mays L., S. saccharatum and Miscanthus giganteus) to 1.42 g VS/ L d (M. sacchariorus) (Table 2). The biogas production rate (rb) and methane production rate (rm) for Z. mays L. silage were comparable with the rates for S. saccharatum silage, and were correlated with low concentrations of brous materials, specically lignin. Specic biogas production from silage of M. sacchariorus was higher than for Miscanthus giganteus. The Miscanthus spp. had a similar lignin content; however, the cellulose concentration in M. sacchariorus silage (41.9% TS) was higher than in Miscanthus giganteus (31.9% TS). There was a similar trend for the biogas yield coefcient, Yb, and the methane yield coefcient, Ym. The coefcient values for the Z. mays L. and S. saccharatum silages were nearly twice those for the M. sacchariorus silage. Likewise, for the specic biogas production rate, Miscanthus giganteus was characterised by a lower Yb value than M. sacchariorus. The literature suggests that for Z. mays L. silage the Ym value is 0.280.42 L/g VS, for winter wheat straw 0.28 L/g VS (HRT 82 days), for rice straw 0.23 L/g VS (HRT 73 days) and for rape straw 0.19 L/g VS (HRT 42 days) (Zubr and Wise, 1989). In the present study, biogas production rate and methane yield coefcient of M. sacchariorus were almost twice higher than those of Miscanthus giganteus, while the Ym value (0.19 L/g VS) was the same as for rape straw (Zubr and Wise, 1989). Uellendahl et al. (2008) reported twice higher methane yield for Miscanthus giganteus amounted to 0.2 L/g VS. The difference seemed to result from distinct assumptions of investigation employed by the authors. They provided batch experiments compared with continuously operated reactors in present study. 3.4. Comparing calculated biogas production and experimentally obtained values The amount of biogas produced is closely related to the elementary composition of the biomass. Some models have been

9532

E. Klimiuk et al. / Bioresource Technology 101 (2010) 95279535

8 7.6 7.2

b
Alkalinity (mg CaCO3/L)
Z. mays L. S. saccharatum M. gigantheus M. sacchariflorus

7000 6000 5000 4000 3000 2000 1000 0

Z. mays L. S. saccharatum M. gigantheus M. sacchariflorus

pH
6.8 6.4 6

c
COD (mg O2/L)

8000

d
VFAs (mg C2H4O2/L)
Z. mays L. S. saccharatum M. gigantheus M. sacchariflorus

8000

6000

6000

4000

4000

2000

2000

0
Z. mays L. S. saccharatum M. gigantheus M. sacchariflorus

Ammonium nitrogen (mg N-NH4/L)

600 500 400 300 200 100 0

Z. mays L. S. saccharatum M. gigantheus M. sacchariflorus

Fig. 3. Chemical parameters of digestate liquid phase after digestion of silage of Z. mays L., Sorghum saccharatum, Miscanthus giganteus and M. sacchariorus: (a) pH; (b) alkalinity; (c) chemical oxygen demand (COD); (d) volatile fatty acids (VFAs); and (e) ammonium nitrogen. Error bars represent standard deviation.

Table 2 Parameters characterizing biogas productivity in series 14 (standard deviation from the mean value is given in parentheses). Parameter Feedstock loading (FX) Organic loading rate (OLR) Methane content in biogasa Biogas production rate (rb)a Methane production rate (rm)a Biogas yield coefcient (Yb)a Methane yield coefcient (Ym)a
a

Unit g VS/d g VS/L d % L/L d L/L d L/g VS L/g VS

Series 1 Z. mays L. 7.84 1.31 58.1 0.82 0.47 0.57 0.33 (0.12) (5.7) (0.26) (0.19) (0.21) (0.13)

Series 2 Sorghum saccharatum 7.85 1.31 57.1 0.79 0.44 0.59 0.33 (0.10) (4.8) (0.18) (0.13) (0.14) (0.10)

Series 3 Miscanthus giganteus 7.93 1.32 48.2 0.31 0.13 0.23 0.10 (0.22) (6.3) (0.07) (0.03) (0.06) (0.03)

Series 4 M. Sacchariorus 8.52 1.42 55.3 0.45 0.27 0.32 0.19 (0.12) (5.5) (0.12) (0.10) (0.08) (0.07)

Volume of biogas and methane were expressed at normal temperature (C) and pressure (101 kPa).

developed to estimate biogas composition from the chemical composition of organic substrates in a feedstock, including C, H, N and sulphur, and there are suitable equations available (Nyns, 1986). However, these models do not estimate the CH4 yield when the degree of conversion of organic substrates is not complete, which is common in practice. In the present study, biogas production and composition was estimated as the difference between the theoretical value calculated on the basis of elementary silage composition

(Qtheor), the potential amounts of biogas in the digestate (Qwaste) and in the digestion liquor (Qalkal), according to the following equation:

Q estimated Q theor Q waste Q alkal

where Qestimated is the theoretical biogas production obtained under established experiment conditions, Qtheor is the theoretical biogas production calculated on the basis of feedstock composition, Qwaste

E. Klimiuk et al. / Bioresource Technology 101 (2010) 95279535

9533

Table 3 Elementary composition of silages used in experiments as substrates and digestate (standard deviation from the mean value is given in parentheses).

Sorghum saccharatum

Z: mays L: :

C56:51 H112 O48:07 N 5:3H2 O ! 29:9CH4 26:4CO2 NH3 4 5 C22:33 H40:18 O19:82 N 3:1H2 O ! 10:9CH4 11:5CO2 NH3 C36:53 H51:92 O29:25 N 9:7H2 O ! 17:1CH4

Silage Digestate Silage

The molar composition of the biogas obtained from one mole of silage tested, is expressed by the following equations:

S: saccharatum :

Miscanthus giganteus :

19:5CO2 NH3 6 M: sacchariflorus : C121:87 H192:78 O96:82 N 26H2 O ! 60:5CH4 61:4CO2 NH3 7
The mass of CH4 (F CH4 ;theor ) and CO2 (F CO2 ;theor ) theoretically obtained during silage digestion, assuming complete conversion of organics into biogas, was calculated using the formulas below:

F CH4 ;theor

Unit

MCH4 FX Mfeedstock M CO2 FX Mfeedstock

g CH4 =d

where FX is the organics load fed to the reactor, M CH4 is the molar mass of CH4, MCO2 is the molar mass of CO2, and Mfeedstock is the molar mass of the individual silages. Assuming the density of CH4 as 0.717 g/L and that of CO2 as 1.978 g/L (at 0 C, 101 kPa), the total volume was calculated as

Carbon Hydrogen Oxygen Nitrogen Chemical formula

F CO2 ;theor

g CO2 =d

Element

% % % %

43.13 (0.191) 7.12 (0.021) 48.88 (0.318) 0.89 (0.106) C56.51H112O48.07N

Z. mays L.

40.88 (0.117) 6.28 (0.309) 49.10 (0.244) 3.73 (0.086) C12.78H23.56O11.51N

41.91 (0.042) 6.29 (0.106) 49.62 (0.177) 2.19 (0.113) C22.33H40.18O19.82N

    4a b 2c 3d 4a b 2c 3d H2 O ! CH4 Ca H b O c N d 4 8   4a b 2c 3d 3 CO2 dNH3 8

Digestate

32.70 (0.191) 5.54 (0.028) 58.61 (0.163) 3.16 (0.057) C12.07H24.54O16.23N

3.4.1. Theoretical biogas production (Qtheor) The chemical composition (as % TS) of the silages investigated, determined on the basis of elementary composition, are given in Table 3. DJesus et al. (2006) reported for Z. mays L. silage the following elementary composition: 43.40% C, 6.17% H, 46.70% O and 1.02%, N, and were comparable with the values reported in the present study. The C concentration in S. saccharatum silage was similar to a mean value of 41.9% given by Monti et al. (2008). Sweet sorghum was cultivated in the experimental farm of Bologna University (44330 N, 33m a.s.l) in soil containing: 1.8% organic matter, 27.0% sand, 39.0% silt and 34.0% clay. The crop was sown in late April and harvested in September. A higher C concentration (45.08%), but lower H (5.76%) and N (0.85%) concentrations in S. saccharatum biomass grown in Italy (Umbria region) and also harvested in the autumn but without ensilage was found by Piskorz et al. (1998). The elementary composition of Miscanthus biomass corresponded to values reported by Lewandowski and Kicherer, 1997. According to the authors carbon and hydrogen content (% dry weigh) of Miscanthus giganteus ranged 47.849.7 and 5.645.92, respectively. Cited values referred to plant cultivated in haplic luvisol soil, composed of loamy sand located in Durmersheim (Upper Rhine Valley, Germany), and harvested in February when the plantation was in the third ratoon. To calculate the amounts of CH4 and CO2 that it is possible to obtain from one mole of biomass with a known elementary composition, ORourkes equation was applied (Nyns, 1986):

M. sacchariorus

Miscanthus giganteus

45.09 (0.042) 5.34 (0.127) 48.13 (0.071) 1.44 (0.014) C36.53H51.92O29.25N

Silage

38.95 (0.141) 6.02 (0.014) 52.58 (0.318) 2.46 (0.191) C18.51H34.33O18.74N

Digestate

45.44 (0.113) 5.99 (0.014) 48.14 (0.092) 0.44 (0.035) C121.87H192.78O96.82N

Silage

33.79 (0.134) 5.53 (0.106) 58.19 (0.057) 2.50 (0.028) C15.77H30.94O20.37N

Digestate

is the potential biogas production from the digestate, and Qalkal is the potential amount of CO2 bound in the form of bicarbonates in the digestate liquid phase. Calculated values of Qestimated were then compared with the values obtained experimentally.

9534

E. Klimiuk et al. / Bioresource Technology 101 (2010) 95279535

the sum of the volumes of CH4 and CO2 (Qtheor). The results obtained are presented in Table 4. 3.5. Potential biogas production from digestate (Qwaste) During digestion, part of the organic compounds in the feedstock was not converted into biogas (Fig. 1) and remained in the digestate together with the biomass. The chemical composition of the digestate is given in Table 3. To calculate the potential production of biogas from the digestate after silage digestion, chemical equations were formulated, in the same way as described for the theoretical biogas production:

Z:mays L: :

Q theor B 6:27 L=d 0:746 4:67 L=d;

14

whereas Qexp = 4.61 L/d

S: saccharatum :

Q theor B 6:11 L=d 0:739 4:51 L=d; 15

Z: mays L: :

C12:78 H23:56 O11:51 N 1:9H2 O ! 6:1CH4 6:7CO2 NH3 10 11 12 C12:07 H24:54 O16:23 N 1:4H2 O ! 4:7CH4 7:4CO2 NH3 C18:51 H34:33 O18:74 N 1:3H2 O ! 8:5CH4

S: saccharatum :

Miscanthus giganteus : M: sacchariflorus :

10:0CO2 NH3

C15:77 H30:94 O20:37 N 1:4H2 O ! 6:3CH4 9:5CO2 NH3 13

The data shows that the digestates were characterised by higher potentials of CO2 than CH4 production in comparison with the feedstock (Table 4). 3.6. Potential volume of carbon dioxide in the form of bicarbonates (Qalkal) During digestion, a chemical balance is established between gaseous CO2 escaping from the digester and the soluble carbonates and bicarbonates produced, the concentrations of which correspond to the total alkalinity. To estimate the potential volume of CO2 in the form of bicarbonates (Qalkal), it was assumed that 1 mg CaCO3 of total alkalinity was equivalent to 0.88 mg CO2. The results obtained are shown in Table 4. Calculated biogas production, on the basis of the elementary composition of the feedstock and digestate, together with the results obtained experimentally are shown in Table 4. The value of e (%) indicates the ratio of the observed biogas production determined experimentally (Qexp) to the theoretical biogas production (Qestimated) obtained under experimental conditions. The e values uctuated over a wide range; the lowest (0.65%) was for Z. mays L. and the highest (12.2%) for M. sacchariorus. For the Z. mays L., S. saccharatum and Miscanthus giganteus silages the potential for biogas production (CH4 and CO2) were comparable under the experimental conditions. The experiments revealed that for Z. mays L. and S. saccharatum silages, theoretical biogas production multiplied by the biodegradable fraction content calculated from Eq. (1) (on the basis of the lignin concentration in silages), corresponded to the values obtained experimentally:

whereas Qexp = 4.74 L/d. However, such relationships were not obtained for the Miscanthus spp. Although the degrees of conversion of hemicellulose and cellulose (as weighted means) were comparable with (e.g. Z. mays L.) or higher than (e.g. S. saccharatum) the biofraction content (converted into CH4) (B) calculated from Eq. (1), the average degrees of conversion of cellulose and hemicellulose for Miscanthus silages were lower, with 55.2% for Miscanthus giganteus and 66.0% for M. sacchariorus. As mentioned above, the HRT of the feedstock at 60 days for both Miscanthus spp. was too short and may explain why theoretical biogas production, calculated on the basis of B, was higher than Qexp. According to Weiland (2006), the typical retention times for biogas plants which treat agricultural biomass and other substrates containing brous material are from 60 to 90 days. Thus, the HRT of 60 days of the present study was minimal for the digestion of such substrate types. In the case of Miscanthus spp. silages, cellulose and hemicellulose were the main source of organic compounds, which at longer HRT could be converted into CH4, if a correction coefcient, g, is assumed. The value of g corresponds to the ratio of the organic biodegradable fraction estimated experimentally to B calculated from Eq. (1). After recalculation, the following values were obtained:

Miscanthus giganteus : 0:498 1:95 L=d

whereas Qexp = 1.75 L/d

Q theor B g 6:63 L=d 0:59 16

M: sacchariflorus : 2:61 L=d;

Q theor B g 7:18 L=d 0:61 0:595 17

whereas Qexp = 2.8 L/d. The results indicate that on the basis of the elementary composition of the feedstock and the lignin concentration, it is possible to determine biogas production with high probability, as conrmed for Z. mays L. and S. saccharatum silages. However, this relationship can only be achieved when operational conditions (i.e. HRT) enable a high degree of conversion for the cellulose and hemicellulose. When the degree of conversion is low, the above relationship is not fullled, and the use of a correction coefcient, estimated experimentally, is required. 4. Conclusions The efciency of the cellulose and hemicellulose conversion, depended on the ratio of polysaccharide to lignin concentration in the plant biomass. The empirical equations describing these relationships were determined. The volumetric methane yields from the Z. mays L. or S. saccharatum silages were higher than those from

Table 4 Material balance and the difference between theoretical and experimental biogas (CH4 and CO2) production. Series (silage type) Qtheor (L/d) CH4 Series Series Series Series
a b

Qwaste (L/d) Sum 6.27 6.11 6.63 7.18 CH4 0.70 0.43 2.16 1.59 CO2 0.76 0.66 2.54 2.39 Sum 1.46 1.07 4.70 3.98

Qalkal (L/d) CO2 0.23 0.15 0.12 0.10

Qestimatea (L/d) CH4 2.62 2.54 0.94 1.98 CO2 1.96 2.33 0.87 1.12 Sum 4.58 4.87 1.81 3.10

Qexpb (L/d) CH4 2.64 2.69 1.02 1.52 CO2 1.97 2.05 0.81 1.20 Sum 4.61 4.74 1.83 2.72

e (%)
0.65 3.07 1.09 12.2

CO2 2.95 3.14 3.53 3.61

1 2 3 4

(Z. mays L.) (Sorghum saccharatum) (Miscanthus giganteus) (M. sacchariorus)

3.32 2.97 3.10 3.57

(Qtheor Qwaste Qalkal). Volume of biogas and methane were expressed at normal temperature (0 C) and pressure (101 kPa).

E. Klimiuk et al. / Bioresource Technology 101 (2010) 95279535

9535

Miscanthus giganteus or M. sacchariorus silages because of higher lignin content in Miscanthus spp. The results indicate that anaerobic digestion of silage of Miscanthus spp. under mesophilic conditions needs longer HRT than 60 d or applying methods of lignin-encrusted sugars recovery (e.g. alkaline pre-treatment).

References
Adogla-Bessa, T., Owen, E., 1995. Ensiling of whole-crop wheat with cellulasehemicellulase based enzymes. 1. Effect of crop growth stage and enzyme on silage composition and stability. Anim. Feed Sci. Technol. 55, 335347. Akin, D.E., Rigsby, L.L., Sethuraman, A., Morrison III, W.H., Gamble, G.R., Eriksson, K.E.L., 1995. Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus. Appl. Environ. Microbiol. 61, 15911598. American Public Health Association (APHA), 1992. Standard Methods for the Examination of Water and Wastewater, 18th ed. American Public Health Association, Washington, DC. Amon, T., Amon, B., Kryvoruchko, V., Machmller, A., Hopfner-Sixt, K., Bodiroza, V., Hrbek, R., Friedel, J., Ptsch, E., Wagentristl, H., Schreiner, M., Zollitsch, W., 2007a. Methane production through anaerobic digestion of various energy crops grown in sustainable crop rotations. Bioresource Technol. 98, 32043212. Amon, T., Amon, B., Kryvoruchko, V., Zollitsch, W., Mayer, K., Gruber, L., 2007b. Biogas production from maize and dairy cattle manure inuence of biomass composition on the methane yield. Agr. Ecosyst. Environ. 118, 173182. Barlaz, M.A., 2006. Forest products decomposition in municipal solid waste landlls. Waste Manage. 26, 321333. Benner, R., Maccubbin, A.E., Hodson, R.E., 1984. Anaerobic biodegradation of the lignin and polysaccharide components of lignocellulose and synthetic lignin by sediment microora. Appl. Environ. Microbiol. 47, 9981004. Chandler, J.A., Jewell, W.J., Gossett, J.M., Vansoet, P.J., Robertson, B.J., 1980. Predicting methane formation biodegradability. Biotechnol. Bioeng. Symp. 10, 93107. DJesus, P., Boukis, N., Kraushaar-Czarnetzki, B., Dinjus, E., 2006. Gasication of corn and clover grass in supercritical water. Fuel 85, 10321038. Daniels, L., Hanson, R.S., Phillips, J.A., 1994. Metabolism. Chemical analysis. In: Gerhardt, P., Wood, W.A. (Eds.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, pp. 512554. Demirel, B., Scherer, P., 2009. Bio-methanization of energy crops through monodigestion for continuous production of renewable biogas. Renew. Energ. 34, 29402945. Directive 2009/28/EC of the European Parliament and of the Council of 23 April 2009 on the promotion of the use of energy from renewable sources and amending and subsequently repealing Directives 2001/77/EC and 2003/30/EC. Eleazer, W.E., Odle, III, W.S., Wang, Y.-S., Barlaz, M.A., 1997. Biodegradability of municipal solid waste components in laboratory-scale landlls. Environ. Sci. Technol. 31, 911917. Granoszewski, K., Grabias, M., 2009. Potencja rozwoju instalacji biogazowych w Polsce. Available from: <http://www.ieo.pl>.

Gunaseelan, V.N., 1997. Anaerobic digestion of biomass for methane production: a review. Biomass Bioenerg. 13, 83114. Hatakka, A., 1994. Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiol. Rev. 13, 125135. Jeffries, T.W., 1994. Biodegradation of lignin and hemicelluloses. In: Ratledge, C. (Ed.), Biochemistry of Microbial Degradation. Kluwer Academic Publishers, Netherlands, pp. 233277. Jrdening, H.-J., Winter, J., 2005. Environmental Biotechnology Concepts and Applications. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. Lewandowski, I., Clifton-Brown, J.C., Scurlock, J.M.O., Huisman, W., 2000. Miscanthus: European experience with a novel energy crop. Biomass Bioenerg. 19, 209227. Lewandowski, I., Kicherer, A., 1997. Combustion quality of biomass: practical relevance and experiments to modify the biomass quality of Miscanthus giganteus. Eur. J. Agron. 6, 163177. Malik, R.K., Singh, R., Tauro, P., 1987. Effect of inorganic nitrogen supplementation on biogas production. Biol. Waste. 21 (2), 139142. McDonald, P., Henderson, A.R., Heron, S.J.E., 1991. The Biochemistry of Silage, second ed. Chalcombe Publications, Aberystwyth, UK. Monti, A., Di Virgilio, N., Venturi, G., 2008. Mineral composition and ash content of six major energy crops. Biomass Bioenerg. 32, 216223. Nyns, E.J., 1986. Biomethanation processes. Microb. Degrad. 8, 207267. Parawira, W., Read, J.S., Mattiasson, B., Bjrnsson, L., 2008. Energy production from agricultural residues: high methane yields in pilot-scale two-stage anaerobic digestion. Biomass Bioenerg. 32, 4450. Pareek, S., Azuma, J.-I., Matsui, S., Shimizu, Y., 2001. Degradation of lignin and lignin model compound under sulfate reducing condition. Water Sci. Technol. 44 (2 3), 351358. Piskorz, J., Majerski, P., Radlein, D., Scott, D.S., Bridgwater, A.V., 1998. Fast pyrolysis of sweet sorghum and sweet sorghum bagasse. J. Anal. Appl. Pyrol. 46, 1529. PN-EN ISO 13906:2009. Animal feeding stuffs. Determination of acid detergent bre (ADF) and acid detergent lignin (ADL) contents. Siegert, I., Banks, C., 2005. The effect of volatile fatty acid additions on the anaerobic digestion of cellulose and glucose in batch reactors. Process Biochem. 40, 3412 3418. Sun, R., Lawther, J.M., Banks, W.B., 1995. Inuence of alkaline pre-treatments on the cell wall components of wheat straw. Ind. Crop. Prod. 4, 127145. Tong, X., Smith, L.H., McCarty, P.L., 1990. Methane fermentation of selected lignocellulosic materials. Biomass 21, 239255. Uellendahl, H., Wang, G., Mller, H.B., Jrgensen, U., Skiadas, I.V., Gavala, H.N., Ahring, B.K., 2008. Energy balance and costbenet analysis of biogas production from perennial energy crops pretreated by wet oxidation. Water Sci. Technol. 58.9, 18411847. Wang, Q., Kuninobu, M., Ogawa, H.I., Kato, Y., 1999. Degradation of volatile fatty acids in highly efcient anaerobic digestion. Biomass Bioenerg. 16, 407416. Weiland, P., 2006. Biomass digestion in agriculture: a successful pathway for the energy production and waste treatment in Germany. Eng. Life Sci. 6, 302309. Zheng, M., Li, X., Li, L., Yang, X., He, Y., 2009. Enhancing anaerobic biogasication of corn stover through wet state NaOH pretreatment. Bioresource Technol. 100, 51405145. Zubr, J., Wise, D., 1989. Field crops as renewable sources of convertible energy for methanogenic fermentation. Int. Biosyst. 3, 165171.