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Influence of alkaline pre-treatment conditions on structural features and methane production from ensiled sorghum forage
Chemical Engineering Journal 211–212 (2012) 488–492
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Influence of alkaline pre-treatment conditions on structural features and methane production from ensiled sorghum forage C. Sambusiti a,b,⇑, E. Ficara a, F. Malpei a, J.P. Steyer b, H. Carrère b a b
Politecnico di Milano, DIIAR, Environmental Section, Piazza L. da Vinci, 32, 20133 Milano, Italy INRA, UR0050, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, 11100 Narbonne, France
h i g h l i g h t s " NaOH pre-treatment reduced the content of lignin and hemicelluloses of sorghum. " NaOH pre-treatment enhanced TOC and proteins solubilisation of sorghum. " The methane yield was not affected by NaOH dosage, temperature and contact time. " Digestion kinetics increased with NaOH dosage, temperature and contact time.
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Article history: Received 12 July 2012 Received in revised form 25 September 2012 Accepted 26 September 2012 Available online 4 October 2012 Keywords: Anaerobic digestion Ensiled sorghum forage Lignocellulosic biomass Sodium hydroxide pre-treatment Structural features
a b s t r a c t Alkaline pre-treatment has been widely applied to lignocellulosic biomass but the tested conditions are quite variable in literature. Results are also quite scattered even when similar substrates are compared. Therefore the aim of this study was to test different alkaline dosages (4% and 10% gNaOH/gTS), temperatures (40 °C and 55 °C), and contact times (12 h and 24 h) in order to investigate the influence of the pretreatment conditions on the structural features and methane production from ensiled sorghum forage. This study confirms the positive effect of NaOH pre-treatment on fibre reduction, total organic carbon and proteins solubilisation, and thereafter the anaerobic degradability of ensiled sorghum forage. An increase in methane yield, with respect to untreated sample (from 8% to 19%), was observed at all pretreatment conditions tested. Nevertheless, no significant differences on methane yield were observed by varying NaOH dosage, temperature, and contact time. The increase of sodium hydroxide dosage led to an increase of the soluble total organic carbon (TOC) (from 12% to 29%) and proteins (from 56% to 72%), at each temperature and contact time tested. By increasing the NaOH dosage, a reduction of hemicelluloses (from 37% to 70%) and lignin contents (from 26% to 70%), and an increase of the anaerobic digestion kinetics (with a maximum increase of 43% for samples treated at 55 °C for 24 h), were also observed. Finally, the anaerobic digestion kinetics were improved with the increase of contact time (up to 13%) and temperature (up to 20%). Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Nowadays some 80% of the world’s overall energy supply is derived from fossil fuels [1]. The Renewable Energy Directive adopted Abbreviations: ADF, acid detergent fibre; ADL, acid detergent lignin; BMP, Biochemical Methane Potential; COD, chemical oxygen demand; NDF, neutral detergent fibre; SMA, specific methane activity; TKN, total kjeldahl nitrogen; TOC, total organic carbon; TS, total solids; VS, volatile solids. ⇑ Corresponding author at: Politecnico di Milano, DIIAR, Environmental Section, Piazza L. da Vinci, 32, 20133 Milano, Italy. Tel.: +39 (0) 223996433; fax: +39 (0) 223996499. E-mail addresses: [email protected] (C. Sambusiti), elena.ficara@ polimi.it (E. Ficara), [email protected] (F. Malpei), [email protected] (J.P. Steyer), [email protected] (H. Carrère). 1385-8947/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cej.2012.09.103
in 2009 focuses on achieving a 20% share of renewable energies in the EU’s energy mix by 2020. Among the renewable energy resources, biomass contributes by some 3–13% to the total world energy supplies of the industrialised countries. In developing countries this proportion is much higher [1]. Biomasses, both residual (such as agro-industrial wastes and crop residues) and specifically grown energy crops offer a huge potential for the production of renewable energy, as heat and electricity. Their use could be beneficial to reduce pollution and greenhouse gas emissions and to reduce the dependence on oil and gas. Anaerobic digestion is considered to be a sustainable way to combine renewable energy generation with sustainable waste
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treatment. The evaluation of biogas and methane production through anaerobic digestion from energy crops or agricultural wastes is not new and is being prompted in recent years, when the number of anaerobic digesters in the EU has increased dramatically. In early 2010, about 5900 biogas plants with an installed electrical capacity of 2300 MWel were operational. Within the next five years, more than 3000 biogas plants with an electrical capacity of more than 1700 MWel will be constructed [2]. Among energy crops, sorghum, with a world cultivated land of 40 million ha in 2009 [3] and with a hectare yield as high as 25 t (dry weight) per year, represents an interesting substrate for methane production. Sorghum is a genus with many species and subspecies, including grain sorghums, grass sorghums, and sweet sorghums. Among them, Sudan grass (Sorghum sudanense) is one of the most commonly used energy crop for biogas production plants [4,5]. The main challenge in using lignocellulosic crops, such as sorghum, for biogas production, is their structure and composition. Crop biomasses mainly consist of cellulose, hemicelluloses and lignin. It is well known that cellulose and hemicelluloses (holocelluloses) are degradable by anaerobic microorganisms; nevertheless, their association with lignin, which acts as a physical barrier, prevents their degradation [6]. Moreover, the crystalline structure of cellulose prevents penetration by micro-organisms or extracellular enzymes [7]. However, physical structure and composition of lignocellulosic materials can be altered through various methods of pre-treatment. An ideal pre-treatment prior to anaerobic digestion would increase surface area, reduce lignin content and the crystallinity of cellulose, making them more accessible to anaerobic micro-organisms and therefore more easily biodegradable [7,8]. Various methods of pre-treatment have been quite intensively investigated for facilitating the enzymatic hydrolysis and consequent ethanol production from lignocellulosic substrates [9], but there is less information available on the effects of pre-treating crop biomass for methane production [10]. Pre-treatments include physical, chemical, thermal, biological processes or combination of them. Physical pre-treatment such as chopping, grinding, and milling, leads to a reduction in the particle size of the biomass, thus reducing the degree of cristallinity of cellulose and the degree of polymerisation of cellulose and hemicelluloses, increasing the surface area of cellulose. Nevertheless, energy requirement remains a limiting factor of this type of treatment, especially when biomass has high moisture content [11]. Biological pre-treatment (fungi, enzymes) is an energy saving and environmental friendly method of pre-treatment but relative low efficiency, potential loss of carbohydrates and long residence time are the three major disadvantages for fungal pre-treatment. Thermal pre-treatments (steam explosion, ammonia fibre explosion (AFEX), wet oxidation, hydrothermal) are also efficient in solubilising crops but they are energy intensive. Finally, among chemical pre-treatments (acid, alkali, organic solvents and oxidant agents), alkaline pre-treatments (NaOH, KOH, lime, ammonia, and urea) are efficient in altering the structure of lignin, solubilising hemicelluloses fraction and increasing efficiently the accessibility of cellulose by a swelling and a partial decrystallization of cellulose [12–14]. Sodium hydroxide pre-treatment has been studied for many years and it has been shown to disrupt the lignin structure of the biomass, thus increasing the enzymatic accessibility to cellulose and hemicelluloses. NaOH pre-treatment was also found efficient in the release of soluble organic carbon and proteins. Xie and co-workers [15] found an increase of soluble COD up to almost 30%, by soaking grass silage in a NaOH solution (7.5% gNaOH/gVS) for 12–24 h at 60 °C. Sun et al. [16] found a protein solubilisation of about 38%, by soaking 2.5 g of wheat straw in 100 mL of NaOH solution (1.5%NaOH) at 20 °C for 6 h.
In general, the applied NaOH pre-treatment conditions are quite variable in literature, however the tested temperature varied between 10 °C and 200 °C, the NaOH dosages varied between 0.1% and 10%, and contact times included between few minutes to 5 days and generally decreased at increasing pre-treatment temperature. Results are also quite scattered even when similar substrates are compared, and this fact suggests that no definite consensus on the effectiveness of alkaline pre-treatments for the improvement of the anaerobic biodegradability of agro-waste and energy crops has yet been attained, as also suggested by Us and Perendeci [16] and Fdez Guelfo et al. [17]. Common substrates used for alkaline tests are straws, grasses, bagasses corn stovers, and sunflower stalks. Sun and co-workers [18] studied the effects of different alkaline pre-treatments on wheat straw. They found that best results for delignification and solubilisation of hemicelluloses were obtained by soaking 2.5 g of straw in 100 mL of NaOH solution (1.5%NaOH) for 144 h at 20 °C, which resulted in 60% release of lignin and 80% release of hemicelluloses. Recently, Zhu and co-workers [19] showed the effectiveness of sodium hydroxide pre-treatment to increase biogas production from corn stover by 37%. Zhao and co-workers [20] showed the effectiveness of sodium hydroxide pre-treatment for hardwoods, wheat straw, switch grass, and softwoods with less than 26% lignin content. Finally, Monlau and co-workers [21] studied the effects of NaOH pretreatments on sunflower stalks at fixed concentration (4 gNaOH/ 100gTS) and time (24 h). The pre-treatment temperature ranged between 30 °C, 55 °C, and 80 °C. The highest methane production (259 ± 6 mLCH4/gVS) was reached at 55 °C. The aim of this study was to test different alkaline dosages (4% and 10% gNaOH/gTS), temperatures (40 °C and 55 °C), and contact times (12 h and 24 h) in order to investigate the influence of the pre-treatment conditions on the structural features and methane potential from ensiled sorghum forage. The pre-treatment temperatures, dosages and contact times were chosen, according to the best pre-treatment results of our previous studies [22], obtained on ensiled sorghum forage and according to some literature suggestions on agricultural substrates [19,21]. 2. Materials and methods 2.1. Sorghum Ensiled sorghum forage ( Sorghum sudanense hybrid) used for animal feed, was collected from a farm near Cremona (Lombardy region, Italy). Sorghum was harvested and then it was left in field for about 4–5 days before ensilage. After collection, it was dried at 60 °C for two days to a moisture content of less than 10%, and ground to 1 mm particles by a cutting mill (Retsch) and finally stored at ambient temperature prior to use. The main characteristics of sorghum, after drying and milling, are given in Table 1.
Table 1 Composition of ensiled sorghum forage after drying and milling. Values correspond to mean ± standard deviation of measurement performed in duplicate. Characteristics
Mean ± SD
TS (% wet weight) VS (%TS) Cellulose (%TS) Hemicelluloses (%TS) Lignin (ADL) (%TS) N-TKN (%TS) Protein (%TS) TOC (%TS) C/N
93.5 ± 0.4 77.6 ± 1.2 47.5 ± 1.0 27.4 ± 1.0 7.0 ± 0.0 1.42 ± 0.01 8.9 ± 0.1 41.6 ± 1.0 29
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2.2. Analytical determinations
2.4. Biochemical Methane Potential (BMP) test
TS and VS were determined according to the APHA standard method [23]. TKN was analysed by the Kjeldhal standard method. Proteins were determined by multiplying TKN6.25. TOC was analysed with a Carbon TOC-V module (Shimadzu). NDF, ADF and ADL were determined according to the Van Soest method [24] with a FIBERBAG system (Gerhardt). It is based on sequential extraction under neutral and acid detergent, followed by strong acid extraction. Different fractions are: (a) soluble in neutral detergent fraction (1-NDF); (b) hemicelluloses (NDF–ADF) which is extracted by acid detergent; (c) cellulose (ADF–ADL) which is extracted by 76% sulphuric acid; (d) lignin (ADL). Biogas composition was determined using a gas chromatograph (PERKIN Clarus 480) equipped with two different capillary columns: (i) RtUBond column (30 m 0.32 mm 10 lm) that enables the separation of CO2 and H2S from the other gases, which were separated with: (ii) RtMolsieve 5A column (30 m 0.32 mm 30 lm) in H2S, O2, N2, CH4. The carrier gas was helium. The temperatures were 65 °C for the oven and 200 °C for the injector and the detector. Detection was done using a thermal conductivity detector. The volume of injected biogas was 200 lL. The calibration was done with a standard gas composed of 25% of CO2, 0.1% of H2S, 0.5% of O2, 10% of N2 and 64.4% of CH4. All analytical determinations were performed in duplicate.
BMP tests were performed under mesophilic conditions (35 ± 0.5 °C). Tests were performed in batch mode, using plasma flasks closed with rubber septa. The total volume of each flask was 500 mL, with a working volume of 400 mL. The inoculum used for BMP tests was a granular sludge from a mesophilic anaerobic digester treating the effluent from a sugar factory. The sludge contained 142 gTS/L and 118 gVS/L, and had a maximum SMA of 47 mLCH4/ gVS/d, as measured by degrading 1 g/L of ethanol as COD. This inoculum was kept under endogenous anaerobic conditions at 35 °C for about 7 days to reduce non-specific biogas generation. Sorghum samples (raw and pre-treated before sieve-separation) were introduced into the flasks with the inoculum, obtaining a substrate to inoculum ratio between 0.9 and 1 gVS/gVS, as suggested [15,25]. Finally, a macro nutrient solution, an oligoelement solution, and a phosphate buffer solution (adapted from [26–28] were added to obtain a 400 mL of working volume. The samples pre-treated with 10 gNaOH/100gTS had a final pH of 12 and they were neutralised with a concentrated HCl solution to pH = 7 prior the BMP test. On the contrary, samples pre-treated with 4 gNaOH/100gTS had a final pH of 7–8, and no further neutralization was necessary. BMP tests were performed in duplicate and the test duration was 35–36 days. The methane yield (mLCH4/gVS) was calculated according to the following equation:
BMP ¼ V CH4;s V CH4;blank =VSs
2.3. Alkali pre-treatment of sorghum forage Pre-treatment tests were carried out in 500 mL digestion flasks, closed with rubber septa. In each flask, sorghum samples were soaked in a NaOH solution at different NaOH dosages (4% and 10% gNaOH/gTS), contact times (12 h and 24 h), and temperatures (40 °C and 55 °C). During the experiments, samples were continuously agitated for complete mixing, by using a heater incubator (INNOVA). As mentioned before, the conditions of pre-treatment were determined according to previous results and to some literature suggestions on agricultural substrates [19,21,22] and are summarised in Table 2. Results of control samples, soaked in tap water without NaOH addition, were included. After pre-treatment, samples were filtered through a sieve of 0.25 mm of pore size. The sieve-separated solid and the liquid fractions were taken for compositional analyses.
2.3.1. Calculations The reduction yield of the fibrous fractions in the solid phase separated after pre-treatment was calculated according to the following equation:
Reduction yield ½% ¼ ðMi Mf Þ=M i 100
ð1Þ
where Mi (g) is the mass of the fibrous component in the untreated samples and Mf (g) is the mass of the fibrous component in the substrate after pre-treatment.
ð2Þ
where (VCH4,s VCH4,blank) (mLCH4) is the net volume (at standard temperature and pressure, STP) of methane measured at the end of the test; VSs (gVS) is the mass of volatile solids from substrate. All gaseous volumes hereafter reported are referred at STP conditions.
2.4.1. Kinetic study The anaerobic degradation process was assumed to follow a first order kinetic as it is the case of slowly degradable lignocellulosic substrates for which the disintegration and hydrolysis are the limiting steps [29]. Gas production in the BMP test started very rapidly, without any apparent lag phase. Therefore, to quantify the kinetic advantage of the pre-treatment on anaerobic digestion, the first order kinetic constants were calculated by using leastsquares fit of methane yield data during time (t), by using a simple model which not includes the lag phase (Eq. (3)):
BMP ¼ BMPt!1 ð1 expðkh tÞÞ
ð3Þ
where BMP (mLCH4/gVS) is the cumulative methane yield, calculated according to Eq. (2); BMPt?1 (mLCH4/gVS) is the ultimate methane yield of the substrate; kh (d1) is the first order kinetic constant and t (d) is the digestion time. This model has been frequently applied to anaerobic digestion systems to correlate the methane yield with the digestion time [29,30]. Least-squares fit of methane production data was performed by using TableCurve Windows v.1.10 (Systat Software Inc.).
Table 2 Alkaline pre-treatment conditions. # Test
Temperature (°C)
Time (h)
Dose of NaOH (gNaOH/100gTS)
1 2 3 4 5 6
55 55 55 55 40 40
12 12 24 24 24 24
10 4 4 10 4 10
3. Results and discussion In this study, the pre-treatment efficiency was evaluated with respect to the reduction of fibrous fractions (cellulose, hemicelluloses and lignin) of the solid fraction, the release of soluble total organic carbon (TOCs) and proteins of the liquid fraction, and thereafter the anaerobic degradability of ensiled sorghum forage.
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3.1. Effect of pre-treatment on the fibrous composition of sorghum forage In lignocellulosic biomass, such as sorghum, lignin is tightly bound to cellulose and hemicelluloses, thus rendering a fraction of these carbohydrate polymers inaccessible to further hydrolysis and fermentation [31]. Sodium hydroxide pre-treatment was reported to cause changes in the chemical composition and structure as well as in the physical characteristics of lignocellulosic biomasses [32]. Fig. 1 shows the reduction yield (Eq. (1)) of cellulose, hemicelluloses and lignin after a sodium hydroxide pre-treatment at 40 °C for 24 h and 55 °C for 12 and 24 h. As shown in Fig. 1, an increase in NaOH dosage (from 4% to 10% NaOH) led to a significant lignin reduction from 26% (±8%) to 67% (±4%), by soaking sample for 24 h at 40 °C; from 20% (±6%) to
70% (±8%), by soaking sample for 24 h at 55 °C; from 29% (±5%) to 63% (±4%), by soaking sample for 12 h at 55 °C. An increase in NaOH dosage (from 4% to 10% NaOH) led to a reduction of hemicelluloses content from 57% (±3%) to 73% (±1%) and from 49% (±6%) to 71% (±1%) by soaking sample for 24 h at 55 °C and at 40 °C, respectively. By soaking sample for 12 h at 55 °C, the reductions of hemicelluloses content were 37% (±2%) and 68% (±2%) at 4% and 10% of NaOH dosage, respectively. On the contrary, no significant effect of the NaOH dosage was observed on the reduction of cellulose content. By soaking samples for 12 and 24 h, at 55 °C, with 10% NaOH dosage, no significant differences in terms of cellulose (24% (±2%) and 19% (±1%)), hemicelluloses (68% (±2%) and 73% (±1%)) and lignin reduction (63 (±4%) and 70% (±8%)), were observed. Nevertheless, a contact time of 24 h with 4% NaOH appeared favourable for the reduction of hemicelluloses (hemicelluloses reduction yields were 37 ± 2% and 57 ± 3% for 12 and 24 h, respectively), but further tests would be necessary to confirm this result. Finally, no significant effect of the pre-treatment temperature on the reduction yield of fibrous fractions was observed since similar reduction yields were found at 40 °C and 55 °C (Fig. 1). 3.2. Effect of pre-treatment on total organic carbon and proteins solubilisation Alkaline pre-treatment resulted in the release of soluble organic carbon and proteins in the liquid phase separated after the pre-treatment and results are shown in Table 3. A limited carbon released was measured under neutral (11% and 12%) and mildly alkaline (15% and 17%) conditions, as observed in previous studies [21]. On the contrary, a significant higher solubilisation (up to 29%) was observed for samples soaked with 10% NaOH dosage. By increasing the contact time and temperature, no significant effect was observed in terms of TOC solubilisation. As for proteins, a significant high solubilisation was observed for all tested conditions, even in those samples soaked in tap water. At the highest dosage, the best solubilisation of proteins (up to 70–72%) was observed. By increasing the pre-treatment temperature, no significant improvement of proteins solubilisation was also observed. 3.3. Biochemical methane potential of raw and pre-treated substrate Methane yields of untreated and pre-treated ensiled sorghum forage, calculated according to Eq. (2), are summarised in Table 4. The methane yield of untreated sorghum was calculated as 266 ± 10 mLCH4/gVS. This value is well in agreement with literature values, ranging from 260 to 390 mLCH4/gVS [33,34]. By both the lowest (4%) and highest (10%) NaOH dosage, an increase in methane yield, compared to untreated sample, was observed: (a) from 8% up to 17% at 40 °C for 24 h; (b) from 10% up to 12% at 55 °C for 24 h; (c) from 10% up to 19% at 55 °C for 12 h.
Table 3 Soluble TOC and soluble proteins (% of the initial total content) after pre-treatment for all conditions tested. Time, temperature, solid to liquid ratio
Fig. 1. Fibre reduction yield after alkaline pre-treatment at (1a) 40 °C for 24 h; (1b) 55 °C for 24 h; (1c) 55 °C for 12 h.
NaOH dosage (g/100gTS) 0
4
10
Soluble TOC 12 h, 55 °C 24 h, 55 °C 24 h, 40 °C
12% 12% 12%
17% 17% 15%
29% 23% 22%
Soluble proteins 12 h, 55 °C 24 h, 55 °C 24 h, 40 °C
n.d 56% 56%
n.d 60% 61%
n.d 70% 72%
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Table 4 BMP (mLCH4/gVS) and kh (d1) values and their relative increase with respect to untreated sorghum. Values correspond to mean ± standard deviation of measurement performed in duplicate.
Untreated sorghum 40 °C, 24 h, 4%NaOH 40 °C, 24 h, 10%NaOH 55 °C, 24 h, 4%NaOH 55 °C, 24 h, 10%NaOH 55 °C, 12 h, 4%NaOH 55 °C, 12 h, 10%NaOH
[5]
BMP (mLCH4/gVS)
kh (d1)
R2
[6]
266 ± 10 287 ± 4 (+8%) 311 ± 8 (+17%) 292 ± 3 (+10%) 298 ± 12 (+12%) 293 ± 10 (+10%) 316 ± 9 (+19%)
0.11 ± 0.00 0.11 ± 0.00 0.15 ± 0.00 0.13 ± 0.00 0.18 ± 0.01 0.13 ± 0.01 0.16 ± 0.00
0.9819 0.9922 0.9952 0.9952 0.9698 0.9863 0.9930
[7]
(+0%) (+35%) (+12%) (+55%) (+15%) (+42%)
[8]
[9] [10]
Nevertheless, no significant differences in methane yields were observed by varying the alkaline dosage, the temperature, and the contact time of pre-treatment. The first order kinetic model was successful in interpreting the experimental production trend, as demonstrated by the high R2 values, suggesting that such a simple methanisation model can be used in practice to describe the complex anaerobic degradation processes for those substrates, such as lignocellulosic ones, for which hydrolysis is the rate-limiting step. The experimental results make it evident the positive effect of the alkaline pre-treatment on the anaerobic digestion kinetics (Table 4). The first order kinetic constant increased (up to 55%), by soaking sample in an alkaline solution. An increase of NaOH dosage and temperature had a positive effect on the kinetic constant: an increase up to 43–20% was observed by increasing the NaOH dosage and the temperature, respectively. The increase in the first order kinetic constant would result in an increase in biogas production in full scale anaerobic digesters, although the actual increase would depend on the reactor fluid-dynamic and on its average retention time.
[11]
4. Conclusion
[21]
This study confirms that NaOH pre-treatment positively affects cellulose, hemicelluloses and lignin reduction, total organic carbon and proteins solubilisation, and thereafter the anaerobic degradability of ensiled sorghum forage. At the conditions tested, the increase of sodium hydroxide dosage improved the TOC and proteins solubilisation and the reduction of hemicelluloses and lignin. On the contrary, no or negative effects of contact time and temperature were observed in terms of TOC and proteins solubilisation and fibre reduction. Moreover, the ultimate methane yield was not significantly improved by increasing the NaOH dosage, the pre-treatment temperature nor by prolonging the pretreatment time. As for the anaerobic digestion kinetic, it coherently increased with the NaOH dosage and was also positively affected by the pre-treatment temperature and by the contact time. Acknowledgements This research work has been conceived and supported in the context of Fabbrica della Bioenergia which is gratefully acknowledged. References [1] R. Braun, P. Weiland, A. Wellinger, Biogas from Energy Crop Digestion, IEA Bionergy Task 37, 2010,. [3] FAO: Food and Agriculture Organization of United States, 2012.
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Karimi, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review, Int. J. Mol. Sci. 9 (2008) 1621–1651. D.P. Chynoweth, C.E. Turick, J.M. Owens, D.E. Jerger, M.W. Peck, Biochemical methane potential of biomass and waste feedstocks, Biomass. Bioenergy. 5 (1993) 95–111. A. Bauer, C. Leonhartsberger, P. Bösch, B. Amon, A. Friedl, T. Amon, Analysis of methane yields from energy crops and agricultural by-products and estimation of energy potential from sustainable crop rotation systems in EU27, Clean. Technol. Environ. 12 (2010) 153–161.
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Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej
Influence of alkaline pre-treatment conditions on structural features and methane production from ensiled sorghum forage C. Sambusiti a,b,⇑, E. Ficara a, F. Malpei a, J.P. Steyer b, H. Carrère b a b
Politecnico di Milano, DIIAR, Environmental Section, Piazza L. da Vinci, 32, 20133 Milano, Italy INRA, UR0050, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, 11100 Narbonne, France
h i g h l i g h t s " NaOH pre-treatment reduced the content of lignin and hemicelluloses of sorghum. " NaOH pre-treatment enhanced TOC and proteins solubilisation of sorghum. " The methane yield was not affected by NaOH dosage, temperature and contact time. " Digestion kinetics increased with NaOH dosage, temperature and contact time.
a r t i c l e
i n f o
Article history: Received 12 July 2012 Received in revised form 25 September 2012 Accepted 26 September 2012 Available online 4 October 2012 Keywords: Anaerobic digestion Ensiled sorghum forage Lignocellulosic biomass Sodium hydroxide pre-treatment Structural features
a b s t r a c t Alkaline pre-treatment has been widely applied to lignocellulosic biomass but the tested conditions are quite variable in literature. Results are also quite scattered even when similar substrates are compared. Therefore the aim of this study was to test different alkaline dosages (4% and 10% gNaOH/gTS), temperatures (40 °C and 55 °C), and contact times (12 h and 24 h) in order to investigate the influence of the pretreatment conditions on the structural features and methane production from ensiled sorghum forage. This study confirms the positive effect of NaOH pre-treatment on fibre reduction, total organic carbon and proteins solubilisation, and thereafter the anaerobic degradability of ensiled sorghum forage. An increase in methane yield, with respect to untreated sample (from 8% to 19%), was observed at all pretreatment conditions tested. Nevertheless, no significant differences on methane yield were observed by varying NaOH dosage, temperature, and contact time. The increase of sodium hydroxide dosage led to an increase of the soluble total organic carbon (TOC) (from 12% to 29%) and proteins (from 56% to 72%), at each temperature and contact time tested. By increasing the NaOH dosage, a reduction of hemicelluloses (from 37% to 70%) and lignin contents (from 26% to 70%), and an increase of the anaerobic digestion kinetics (with a maximum increase of 43% for samples treated at 55 °C for 24 h), were also observed. Finally, the anaerobic digestion kinetics were improved with the increase of contact time (up to 13%) and temperature (up to 20%). Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Nowadays some 80% of the world’s overall energy supply is derived from fossil fuels [1]. The Renewable Energy Directive adopted Abbreviations: ADF, acid detergent fibre; ADL, acid detergent lignin; BMP, Biochemical Methane Potential; COD, chemical oxygen demand; NDF, neutral detergent fibre; SMA, specific methane activity; TKN, total kjeldahl nitrogen; TOC, total organic carbon; TS, total solids; VS, volatile solids. ⇑ Corresponding author at: Politecnico di Milano, DIIAR, Environmental Section, Piazza L. da Vinci, 32, 20133 Milano, Italy. Tel.: +39 (0) 223996433; fax: +39 (0) 223996499. E-mail addresses: [email protected] (C. Sambusiti), elena.ficara@ polimi.it (E. Ficara), [email protected] (F. Malpei), [email protected] (J.P. Steyer), [email protected] (H. Carrère). 1385-8947/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cej.2012.09.103
in 2009 focuses on achieving a 20% share of renewable energies in the EU’s energy mix by 2020. Among the renewable energy resources, biomass contributes by some 3–13% to the total world energy supplies of the industrialised countries. In developing countries this proportion is much higher [1]. Biomasses, both residual (such as agro-industrial wastes and crop residues) and specifically grown energy crops offer a huge potential for the production of renewable energy, as heat and electricity. Their use could be beneficial to reduce pollution and greenhouse gas emissions and to reduce the dependence on oil and gas. Anaerobic digestion is considered to be a sustainable way to combine renewable energy generation with sustainable waste
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treatment. The evaluation of biogas and methane production through anaerobic digestion from energy crops or agricultural wastes is not new and is being prompted in recent years, when the number of anaerobic digesters in the EU has increased dramatically. In early 2010, about 5900 biogas plants with an installed electrical capacity of 2300 MWel were operational. Within the next five years, more than 3000 biogas plants with an electrical capacity of more than 1700 MWel will be constructed [2]. Among energy crops, sorghum, with a world cultivated land of 40 million ha in 2009 [3] and with a hectare yield as high as 25 t (dry weight) per year, represents an interesting substrate for methane production. Sorghum is a genus with many species and subspecies, including grain sorghums, grass sorghums, and sweet sorghums. Among them, Sudan grass (Sorghum sudanense) is one of the most commonly used energy crop for biogas production plants [4,5]. The main challenge in using lignocellulosic crops, such as sorghum, for biogas production, is their structure and composition. Crop biomasses mainly consist of cellulose, hemicelluloses and lignin. It is well known that cellulose and hemicelluloses (holocelluloses) are degradable by anaerobic microorganisms; nevertheless, their association with lignin, which acts as a physical barrier, prevents their degradation [6]. Moreover, the crystalline structure of cellulose prevents penetration by micro-organisms or extracellular enzymes [7]. However, physical structure and composition of lignocellulosic materials can be altered through various methods of pre-treatment. An ideal pre-treatment prior to anaerobic digestion would increase surface area, reduce lignin content and the crystallinity of cellulose, making them more accessible to anaerobic micro-organisms and therefore more easily biodegradable [7,8]. Various methods of pre-treatment have been quite intensively investigated for facilitating the enzymatic hydrolysis and consequent ethanol production from lignocellulosic substrates [9], but there is less information available on the effects of pre-treating crop biomass for methane production [10]. Pre-treatments include physical, chemical, thermal, biological processes or combination of them. Physical pre-treatment such as chopping, grinding, and milling, leads to a reduction in the particle size of the biomass, thus reducing the degree of cristallinity of cellulose and the degree of polymerisation of cellulose and hemicelluloses, increasing the surface area of cellulose. Nevertheless, energy requirement remains a limiting factor of this type of treatment, especially when biomass has high moisture content [11]. Biological pre-treatment (fungi, enzymes) is an energy saving and environmental friendly method of pre-treatment but relative low efficiency, potential loss of carbohydrates and long residence time are the three major disadvantages for fungal pre-treatment. Thermal pre-treatments (steam explosion, ammonia fibre explosion (AFEX), wet oxidation, hydrothermal) are also efficient in solubilising crops but they are energy intensive. Finally, among chemical pre-treatments (acid, alkali, organic solvents and oxidant agents), alkaline pre-treatments (NaOH, KOH, lime, ammonia, and urea) are efficient in altering the structure of lignin, solubilising hemicelluloses fraction and increasing efficiently the accessibility of cellulose by a swelling and a partial decrystallization of cellulose [12–14]. Sodium hydroxide pre-treatment has been studied for many years and it has been shown to disrupt the lignin structure of the biomass, thus increasing the enzymatic accessibility to cellulose and hemicelluloses. NaOH pre-treatment was also found efficient in the release of soluble organic carbon and proteins. Xie and co-workers [15] found an increase of soluble COD up to almost 30%, by soaking grass silage in a NaOH solution (7.5% gNaOH/gVS) for 12–24 h at 60 °C. Sun et al. [16] found a protein solubilisation of about 38%, by soaking 2.5 g of wheat straw in 100 mL of NaOH solution (1.5%NaOH) at 20 °C for 6 h.
In general, the applied NaOH pre-treatment conditions are quite variable in literature, however the tested temperature varied between 10 °C and 200 °C, the NaOH dosages varied between 0.1% and 10%, and contact times included between few minutes to 5 days and generally decreased at increasing pre-treatment temperature. Results are also quite scattered even when similar substrates are compared, and this fact suggests that no definite consensus on the effectiveness of alkaline pre-treatments for the improvement of the anaerobic biodegradability of agro-waste and energy crops has yet been attained, as also suggested by Us and Perendeci [16] and Fdez Guelfo et al. [17]. Common substrates used for alkaline tests are straws, grasses, bagasses corn stovers, and sunflower stalks. Sun and co-workers [18] studied the effects of different alkaline pre-treatments on wheat straw. They found that best results for delignification and solubilisation of hemicelluloses were obtained by soaking 2.5 g of straw in 100 mL of NaOH solution (1.5%NaOH) for 144 h at 20 °C, which resulted in 60% release of lignin and 80% release of hemicelluloses. Recently, Zhu and co-workers [19] showed the effectiveness of sodium hydroxide pre-treatment to increase biogas production from corn stover by 37%. Zhao and co-workers [20] showed the effectiveness of sodium hydroxide pre-treatment for hardwoods, wheat straw, switch grass, and softwoods with less than 26% lignin content. Finally, Monlau and co-workers [21] studied the effects of NaOH pretreatments on sunflower stalks at fixed concentration (4 gNaOH/ 100gTS) and time (24 h). The pre-treatment temperature ranged between 30 °C, 55 °C, and 80 °C. The highest methane production (259 ± 6 mLCH4/gVS) was reached at 55 °C. The aim of this study was to test different alkaline dosages (4% and 10% gNaOH/gTS), temperatures (40 °C and 55 °C), and contact times (12 h and 24 h) in order to investigate the influence of the pre-treatment conditions on the structural features and methane potential from ensiled sorghum forage. The pre-treatment temperatures, dosages and contact times were chosen, according to the best pre-treatment results of our previous studies [22], obtained on ensiled sorghum forage and according to some literature suggestions on agricultural substrates [19,21]. 2. Materials and methods 2.1. Sorghum Ensiled sorghum forage ( Sorghum sudanense hybrid) used for animal feed, was collected from a farm near Cremona (Lombardy region, Italy). Sorghum was harvested and then it was left in field for about 4–5 days before ensilage. After collection, it was dried at 60 °C for two days to a moisture content of less than 10%, and ground to 1 mm particles by a cutting mill (Retsch) and finally stored at ambient temperature prior to use. The main characteristics of sorghum, after drying and milling, are given in Table 1.
Table 1 Composition of ensiled sorghum forage after drying and milling. Values correspond to mean ± standard deviation of measurement performed in duplicate. Characteristics
Mean ± SD
TS (% wet weight) VS (%TS) Cellulose (%TS) Hemicelluloses (%TS) Lignin (ADL) (%TS) N-TKN (%TS) Protein (%TS) TOC (%TS) C/N
93.5 ± 0.4 77.6 ± 1.2 47.5 ± 1.0 27.4 ± 1.0 7.0 ± 0.0 1.42 ± 0.01 8.9 ± 0.1 41.6 ± 1.0 29
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2.2. Analytical determinations
2.4. Biochemical Methane Potential (BMP) test
TS and VS were determined according to the APHA standard method [23]. TKN was analysed by the Kjeldhal standard method. Proteins were determined by multiplying TKN6.25. TOC was analysed with a Carbon TOC-V module (Shimadzu). NDF, ADF and ADL were determined according to the Van Soest method [24] with a FIBERBAG system (Gerhardt). It is based on sequential extraction under neutral and acid detergent, followed by strong acid extraction. Different fractions are: (a) soluble in neutral detergent fraction (1-NDF); (b) hemicelluloses (NDF–ADF) which is extracted by acid detergent; (c) cellulose (ADF–ADL) which is extracted by 76% sulphuric acid; (d) lignin (ADL). Biogas composition was determined using a gas chromatograph (PERKIN Clarus 480) equipped with two different capillary columns: (i) RtUBond column (30 m 0.32 mm 10 lm) that enables the separation of CO2 and H2S from the other gases, which were separated with: (ii) RtMolsieve 5A column (30 m 0.32 mm 30 lm) in H2S, O2, N2, CH4. The carrier gas was helium. The temperatures were 65 °C for the oven and 200 °C for the injector and the detector. Detection was done using a thermal conductivity detector. The volume of injected biogas was 200 lL. The calibration was done with a standard gas composed of 25% of CO2, 0.1% of H2S, 0.5% of O2, 10% of N2 and 64.4% of CH4. All analytical determinations were performed in duplicate.
BMP tests were performed under mesophilic conditions (35 ± 0.5 °C). Tests were performed in batch mode, using plasma flasks closed with rubber septa. The total volume of each flask was 500 mL, with a working volume of 400 mL. The inoculum used for BMP tests was a granular sludge from a mesophilic anaerobic digester treating the effluent from a sugar factory. The sludge contained 142 gTS/L and 118 gVS/L, and had a maximum SMA of 47 mLCH4/ gVS/d, as measured by degrading 1 g/L of ethanol as COD. This inoculum was kept under endogenous anaerobic conditions at 35 °C for about 7 days to reduce non-specific biogas generation. Sorghum samples (raw and pre-treated before sieve-separation) were introduced into the flasks with the inoculum, obtaining a substrate to inoculum ratio between 0.9 and 1 gVS/gVS, as suggested [15,25]. Finally, a macro nutrient solution, an oligoelement solution, and a phosphate buffer solution (adapted from [26–28] were added to obtain a 400 mL of working volume. The samples pre-treated with 10 gNaOH/100gTS had a final pH of 12 and they were neutralised with a concentrated HCl solution to pH = 7 prior the BMP test. On the contrary, samples pre-treated with 4 gNaOH/100gTS had a final pH of 7–8, and no further neutralization was necessary. BMP tests were performed in duplicate and the test duration was 35–36 days. The methane yield (mLCH4/gVS) was calculated according to the following equation:
BMP ¼ V CH4;s V CH4;blank =VSs
2.3. Alkali pre-treatment of sorghum forage Pre-treatment tests were carried out in 500 mL digestion flasks, closed with rubber septa. In each flask, sorghum samples were soaked in a NaOH solution at different NaOH dosages (4% and 10% gNaOH/gTS), contact times (12 h and 24 h), and temperatures (40 °C and 55 °C). During the experiments, samples were continuously agitated for complete mixing, by using a heater incubator (INNOVA). As mentioned before, the conditions of pre-treatment were determined according to previous results and to some literature suggestions on agricultural substrates [19,21,22] and are summarised in Table 2. Results of control samples, soaked in tap water without NaOH addition, were included. After pre-treatment, samples were filtered through a sieve of 0.25 mm of pore size. The sieve-separated solid and the liquid fractions were taken for compositional analyses.
2.3.1. Calculations The reduction yield of the fibrous fractions in the solid phase separated after pre-treatment was calculated according to the following equation:
Reduction yield ½% ¼ ðMi Mf Þ=M i 100
ð1Þ
where Mi (g) is the mass of the fibrous component in the untreated samples and Mf (g) is the mass of the fibrous component in the substrate after pre-treatment.
ð2Þ
where (VCH4,s VCH4,blank) (mLCH4) is the net volume (at standard temperature and pressure, STP) of methane measured at the end of the test; VSs (gVS) is the mass of volatile solids from substrate. All gaseous volumes hereafter reported are referred at STP conditions.
2.4.1. Kinetic study The anaerobic degradation process was assumed to follow a first order kinetic as it is the case of slowly degradable lignocellulosic substrates for which the disintegration and hydrolysis are the limiting steps [29]. Gas production in the BMP test started very rapidly, without any apparent lag phase. Therefore, to quantify the kinetic advantage of the pre-treatment on anaerobic digestion, the first order kinetic constants were calculated by using leastsquares fit of methane yield data during time (t), by using a simple model which not includes the lag phase (Eq. (3)):
BMP ¼ BMPt!1 ð1 expðkh tÞÞ
ð3Þ
where BMP (mLCH4/gVS) is the cumulative methane yield, calculated according to Eq. (2); BMPt?1 (mLCH4/gVS) is the ultimate methane yield of the substrate; kh (d1) is the first order kinetic constant and t (d) is the digestion time. This model has been frequently applied to anaerobic digestion systems to correlate the methane yield with the digestion time [29,30]. Least-squares fit of methane production data was performed by using TableCurve Windows v.1.10 (Systat Software Inc.).
Table 2 Alkaline pre-treatment conditions. # Test
Temperature (°C)
Time (h)
Dose of NaOH (gNaOH/100gTS)
1 2 3 4 5 6
55 55 55 55 40 40
12 12 24 24 24 24
10 4 4 10 4 10
3. Results and discussion In this study, the pre-treatment efficiency was evaluated with respect to the reduction of fibrous fractions (cellulose, hemicelluloses and lignin) of the solid fraction, the release of soluble total organic carbon (TOCs) and proteins of the liquid fraction, and thereafter the anaerobic degradability of ensiled sorghum forage.
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3.1. Effect of pre-treatment on the fibrous composition of sorghum forage In lignocellulosic biomass, such as sorghum, lignin is tightly bound to cellulose and hemicelluloses, thus rendering a fraction of these carbohydrate polymers inaccessible to further hydrolysis and fermentation [31]. Sodium hydroxide pre-treatment was reported to cause changes in the chemical composition and structure as well as in the physical characteristics of lignocellulosic biomasses [32]. Fig. 1 shows the reduction yield (Eq. (1)) of cellulose, hemicelluloses and lignin after a sodium hydroxide pre-treatment at 40 °C for 24 h and 55 °C for 12 and 24 h. As shown in Fig. 1, an increase in NaOH dosage (from 4% to 10% NaOH) led to a significant lignin reduction from 26% (±8%) to 67% (±4%), by soaking sample for 24 h at 40 °C; from 20% (±6%) to
70% (±8%), by soaking sample for 24 h at 55 °C; from 29% (±5%) to 63% (±4%), by soaking sample for 12 h at 55 °C. An increase in NaOH dosage (from 4% to 10% NaOH) led to a reduction of hemicelluloses content from 57% (±3%) to 73% (±1%) and from 49% (±6%) to 71% (±1%) by soaking sample for 24 h at 55 °C and at 40 °C, respectively. By soaking sample for 12 h at 55 °C, the reductions of hemicelluloses content were 37% (±2%) and 68% (±2%) at 4% and 10% of NaOH dosage, respectively. On the contrary, no significant effect of the NaOH dosage was observed on the reduction of cellulose content. By soaking samples for 12 and 24 h, at 55 °C, with 10% NaOH dosage, no significant differences in terms of cellulose (24% (±2%) and 19% (±1%)), hemicelluloses (68% (±2%) and 73% (±1%)) and lignin reduction (63 (±4%) and 70% (±8%)), were observed. Nevertheless, a contact time of 24 h with 4% NaOH appeared favourable for the reduction of hemicelluloses (hemicelluloses reduction yields were 37 ± 2% and 57 ± 3% for 12 and 24 h, respectively), but further tests would be necessary to confirm this result. Finally, no significant effect of the pre-treatment temperature on the reduction yield of fibrous fractions was observed since similar reduction yields were found at 40 °C and 55 °C (Fig. 1). 3.2. Effect of pre-treatment on total organic carbon and proteins solubilisation Alkaline pre-treatment resulted in the release of soluble organic carbon and proteins in the liquid phase separated after the pre-treatment and results are shown in Table 3. A limited carbon released was measured under neutral (11% and 12%) and mildly alkaline (15% and 17%) conditions, as observed in previous studies [21]. On the contrary, a significant higher solubilisation (up to 29%) was observed for samples soaked with 10% NaOH dosage. By increasing the contact time and temperature, no significant effect was observed in terms of TOC solubilisation. As for proteins, a significant high solubilisation was observed for all tested conditions, even in those samples soaked in tap water. At the highest dosage, the best solubilisation of proteins (up to 70–72%) was observed. By increasing the pre-treatment temperature, no significant improvement of proteins solubilisation was also observed. 3.3. Biochemical methane potential of raw and pre-treated substrate Methane yields of untreated and pre-treated ensiled sorghum forage, calculated according to Eq. (2), are summarised in Table 4. The methane yield of untreated sorghum was calculated as 266 ± 10 mLCH4/gVS. This value is well in agreement with literature values, ranging from 260 to 390 mLCH4/gVS [33,34]. By both the lowest (4%) and highest (10%) NaOH dosage, an increase in methane yield, compared to untreated sample, was observed: (a) from 8% up to 17% at 40 °C for 24 h; (b) from 10% up to 12% at 55 °C for 24 h; (c) from 10% up to 19% at 55 °C for 12 h.
Table 3 Soluble TOC and soluble proteins (% of the initial total content) after pre-treatment for all conditions tested. Time, temperature, solid to liquid ratio
Fig. 1. Fibre reduction yield after alkaline pre-treatment at (1a) 40 °C for 24 h; (1b) 55 °C for 24 h; (1c) 55 °C for 12 h.
NaOH dosage (g/100gTS) 0
4
10
Soluble TOC 12 h, 55 °C 24 h, 55 °C 24 h, 40 °C
12% 12% 12%
17% 17% 15%
29% 23% 22%
Soluble proteins 12 h, 55 °C 24 h, 55 °C 24 h, 40 °C
n.d 56% 56%
n.d 60% 61%
n.d 70% 72%
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Table 4 BMP (mLCH4/gVS) and kh (d1) values and their relative increase with respect to untreated sorghum. Values correspond to mean ± standard deviation of measurement performed in duplicate.
Untreated sorghum 40 °C, 24 h, 4%NaOH 40 °C, 24 h, 10%NaOH 55 °C, 24 h, 4%NaOH 55 °C, 24 h, 10%NaOH 55 °C, 12 h, 4%NaOH 55 °C, 12 h, 10%NaOH
[5]
BMP (mLCH4/gVS)
kh (d1)
R2
[6]
266 ± 10 287 ± 4 (+8%) 311 ± 8 (+17%) 292 ± 3 (+10%) 298 ± 12 (+12%) 293 ± 10 (+10%) 316 ± 9 (+19%)
0.11 ± 0.00 0.11 ± 0.00 0.15 ± 0.00 0.13 ± 0.00 0.18 ± 0.01 0.13 ± 0.01 0.16 ± 0.00
0.9819 0.9922 0.9952 0.9952 0.9698 0.9863 0.9930
[7]
(+0%) (+35%) (+12%) (+55%) (+15%) (+42%)
[8]
[9] [10]
Nevertheless, no significant differences in methane yields were observed by varying the alkaline dosage, the temperature, and the contact time of pre-treatment. The first order kinetic model was successful in interpreting the experimental production trend, as demonstrated by the high R2 values, suggesting that such a simple methanisation model can be used in practice to describe the complex anaerobic degradation processes for those substrates, such as lignocellulosic ones, for which hydrolysis is the rate-limiting step. The experimental results make it evident the positive effect of the alkaline pre-treatment on the anaerobic digestion kinetics (Table 4). The first order kinetic constant increased (up to 55%), by soaking sample in an alkaline solution. An increase of NaOH dosage and temperature had a positive effect on the kinetic constant: an increase up to 43–20% was observed by increasing the NaOH dosage and the temperature, respectively. The increase in the first order kinetic constant would result in an increase in biogas production in full scale anaerobic digesters, although the actual increase would depend on the reactor fluid-dynamic and on its average retention time.
[11]
4. Conclusion
[21]
This study confirms that NaOH pre-treatment positively affects cellulose, hemicelluloses and lignin reduction, total organic carbon and proteins solubilisation, and thereafter the anaerobic degradability of ensiled sorghum forage. At the conditions tested, the increase of sodium hydroxide dosage improved the TOC and proteins solubilisation and the reduction of hemicelluloses and lignin. On the contrary, no or negative effects of contact time and temperature were observed in terms of TOC and proteins solubilisation and fibre reduction. Moreover, the ultimate methane yield was not significantly improved by increasing the NaOH dosage, the pre-treatment temperature nor by prolonging the pretreatment time. As for the anaerobic digestion kinetic, it coherently increased with the NaOH dosage and was also positively affected by the pre-treatment temperature and by the contact time. Acknowledgements This research work has been conceived and supported in the context of Fabbrica della Bioenergia which is gratefully acknowledged. References [1] R. Braun, P. Weiland, A. Wellinger, Biogas from Energy Crop Digestion, IEA Bionergy Task 37, 2010,
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