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Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels
Fuel xxx (2011) xxx–xxx
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Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels Amrita Ranjan a, Vijayanand S. Moholkar a,b,⇑ a b
Centre for Energy, Indian Institute of Technology, Guwahati, Assam 781 039, India Department of Chemical Engineering, Indian Institute of Technology, Guwahati, Assam 781 039, India
a r t i c l e
i n f o
Article history: Received 8 March 2011 Received in revised form 23 March 2011 Accepted 24 March 2011 Available online 6 April 2011 Keywords: Alcoholic biofuels Hydrolyzate ABE solvents Biobutanol ABE fermentation
a b s t r a c t Fast depletion of fossil fuels with high fluctuating market prices has made the hunt for alternate resources for the production of transportation fuels mandatory. Our approach is to utilize rice straw as feedstock for the production of alcoholic biofuels. In this paper, we have compared the effect of various pretreatment processes on rice straw, viz. physical (steam under pressure) and chemical (acid) and enzymatic treatments as a precursor to ABE fermentation for production of biobutanol. Glucose analysis was done by quantification of glucose content spectrophotometrically via Glucose assay kit. The rice straw hydrolyzate produced through pretreatment was allowed to undergo anaerobic fermentation, using C. acetobutylicum MTCC 481. The yield and productivity of ABE solvents (acetone, butanol and ethanol) was calculated using HPLC. The ABE yield produced using hydrolyzate obtained through enzyme assisted acid hydrolysis, viz. acetone: 0.11, butanol: 0.861, ethanol: 0.05, was found to be the best among all experiments. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Research for an alternate and renewable liquid transportation fuel has been a zealous worldwide activity in both industry and academia. One potential technology from the time of World I and II for the conversion of biomass to alcoholic biofuel (basically butanol and ethanol) is via the route of ABE fermentation [1–4]. It is generally achieved by the pretreatment of fibrous biomass via mechanical, physical, thermal, chemical or enzymatic route, which results in conversion of complex cellulose and hemicellulose to soluble glucose and other reducing sugars. Some of the extensively used feedstocks are corn stover, corn steep liquor, wheat bran, wheat straw, apple pomace, Jerusalem artichokes etc. [5–7]. India being a developing country with population of over 1 billion is incapable of utilizing most of these feedstocks because of the food and fuel shortage [8]. Lignocellulosic materials could form alternate economical renewable feedstock for fermentation, existing in large quantities
Abbreviations: RS, rice straw; RSH, rice straw hydrolyzate; ABE, acetone, butanol and ethanol. ⇑ Corresponding author at: Department of Chemical Engineering, Indian Institute of Technology, Guwahati, Assam 781 039, India. Tel.: +91 361 258 2258; fax: +91 361 269 0762. E-mail addresses: [email protected] (A. Ranjan), [email protected] (V.S. Moholkar).
[9]. Rice straw is one of the most copious lignocellulosic residues available in the world [7,10]. Worldwide 731 million tons of rice straw is produce annually (Africa: 20.9 million tons, Asia: 667.6 million tons, Europe: 3.9 million tons, America: 37.2 million tons and Oceania: 1.7 million tons) [11]. Nearly 600 million tons of agricultural residues has been produced by India annually; out of which, approximately 300 million tons of it remain unused, and is destroyed by simple burning [2]. Seven sister states in the northeastern India, viz. Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland and Tripura are predominantly rice producing regions. Reportedly 7.81% of their total area is used for rice production and they share 6.07% of total rice production of India. In these states, rice crop is grown in 89.46% of total area under food grain, and contributes to 92.32% of the total food grain production. Rice straw contains about 40% of the nitrogen (N), 30–35% of the phosphorus (P), 80–85% of the potassium (K), and 40–50% of the sulfur (S), which make it potential nutritional source for microbial utilization [2]. Utilization of surplus rice straw for the production of alcoholic biofuels will be both valuable and beneficial use, which will contribute to economic energy production and self-reliance of the country in meeting demand of transportation fuels [12]. The matter of utilization of agro-residues in terms of feasibility, sustainability and economics for biofuel production (alcoholic fuel and biodiesel) has been addressed by several researchers [13–23]. Rice straw is a domestic feedstock that has potential to produce considerable quantities of alcoholic fuels viz., acetone butanol and
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Please cite this article in press as: Ranjan A, Moholkar VS. Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel (2011), doi:10.1016/j.fuel.2011.03.030
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A. Ranjan, V.S. Moholkar / Fuel xxx (2011) xxx–xxx
ethanol, and other bioenergy based biofuels [24,25]. Majorly rice straw contains cellulose 37%, hemicelluloses 24% and lignin 14%. The purpose of this paper is to comparatively assess hydrolysis of cellulosic and hemicellulosic fraction of rice straw to simple sugars using steam under pressure, acid and enzyme blend [3,26]. This process is a precursor to main ABE fermentation using the rice straw hydrolyzate as major substrate. The yardsticks for the comparative assessment would be yield and productivity of acetone, butanol and ethanol solvents. Such a study could give us an idea of the techno-economic feasibility of ABE fermentation with rice straw as feedstock, and could also form basis for optimization of the process. Previous authors have addressed the subject of use of rice straw and similar agro-feedstock (such as wheat straw, corn straw, bagasse) after hydrolysis for alcoholic fuel production via fermentation. Roslan et al. [27] has reported production of bioethanol from rice straw sacchharified with crude cellulase to release 90% of glucose in solid state fermentation. Rivers and Emert [28] have analyzed the effect of substrate concentration, cellulose crystallinity and particle size on the yields of enzymatic hydrolysis for the agro-residues, viz. bagasse and rice straw. This study revealed that nature of lignocellulose matrix is a major limiting factor in enzymatic hydrolysis. Hsu et al. [29] has studied effect of dilute acid treatment of rice straw prior to enzyme hydrolysis on the yield of hydrolysis. Acid pretreatment was found to increase the pore volume of the solids due to release of acid soluble lignin, which enhanced the yield of enzymatic hydrolysis. Marchal et al. [30] reported that hydrolysates obtained by enzymatic saccharification of wheat straw and corn stover, pretreated by steam explosion (in neutral or acidic conditions) were non fermentable by clostridium. To restore fermentability of these hydrolyzates heat treatment with Ca(OH)2 or MgCO3 at neutral pH was necessary. Qureshi et al. [31] has compared use of acid and enzyme hydrolyzed corn fiber for butanol fermentation. Sulfuric acid treated corn fiber hydrolyzate was found to inhibit cell growth and butanol production, and treatment with XAD-4 resin was necessary to remove some of the inhibitory compounds. On the contrary enzyme treated corn fiber hydrolyzate of wheat straw obtained from acid pretreatment and enzyme hydrolysis has been studied by Qureshi et al. [32]. Supplementation of hydrolyzate with glucose was found to increase productivity of fermentation. Ethanol production from wheat straw using acid pretreatment and enzymatic saccharification using recombinant strain of E. coli FBR5 has been studied by Saha et al. [33]. Detoxification of hydrolyzate obtained from acid pretreated biomass increased yield with simultaneous reduction in fermentation time.
were incubated anaerobically inside an anaerobic culture bag system (Himedia) till active growth was seen (72 h). Actively growing cultures (after lag phase, 18–20 h) of the Clostridia were added subsequently to experimental flasks. 2.2. Rice straw (RS) Rice straw was obtained from local farmer (irrigated location; Guwahati, Assam, India). Collected rice straw is agricultural waste used as packaging material during shifting of some furniture’s and equipments. The crude composition of rice has been reported to be cellulose: 32–47%; Hemicellulose: 19–27%; lignin: 5–24%; ashes: 18.8% and sugar composition as; glucose: 41–43.4%; xylose: 4.8– 20.2%; arabinose: 2.7–4.5%; mannose: 1.8%; galactose: 0.4% [34]. Strips of rice straw were cut into small pieces of length 4–5 cm. These were washed with water and then dried at 80 °C in a hot air oven. Size of dried RS was further reduced by in a mixer grinder. 2.3. Rice straw hydrolyzate (RSH) Hydrolyzate of RS was prepared using three methods as described below: 2.3.1. Steam explosion 5% w/w mixture of RS, i.e. 15 g RS added to 250 mL double distilled water, was taken in a 500 mL screw-capped Erlenmeyer flask. The flask was autoclaved at 121 °C for 30 min at 15 lb pressure, and was then allowed to cool to room temperature. RS suspension was filtered using a sterile cotton cloth. pH of the hydrolyzate thus obtained was 1.0. 2.3.2. Acid pretreatment 5% w/w mixture of RS, i.e. 15 g RS added to 250 mL acidified double distilled water (1% H2SO4), was taken in a 500 mL of screw-capped Erlenmeyer flask. The flask was kept in an orbital shaker incubator for 24 h at 60 °C at 200 rpm. The flask was then autoclaved at 121 °C for 15 min at 15 lb pressure, and was allowed to cool at room temperature. RS suspension was filtered using a sterile cotton cloth. pH of the hydrolyzate thus obtained was 0.8.
2. Material and methods
2.3.3. Enzyme assisted hydrolysis A commercial enzyme blend was generous gift from Varuna Biocell, Varanasi, India. 6 ppm of enzyme was added to 1% acid hydrolyzed rice straw and was incubated at 60 °C for 24 h at 100 rpm. The flask was then autoclaved at 121 °C for 15 min at 15 lb pressure, and was allowed to cool at room temperature. RS suspension was filtered using a sterile cotton cloth. pH of the hydrolyzate thus obtained was 0.8.
2.1. Culture maintenance and growth
2.4. Fermentation
All the chemicals used were of analytical grade procured either from Merck (Germany) or Himedia (India). Lyophilized Clostridium acetobutylicum MTCC 481 has been procured from microbial type culture collection, IMTECH, Chandigarh, India. It was maintained as spore suspension in sterile water. This culture has been rejuvenated in RCA (Reinforced Clostridial Agar) and RCM (Broth) culture media at 37 °C. The inoculums were prepared in RCM containing (g/L): glucose, 5.0; yeast extract, 3.0; starch, 1.0; beef extract, 10.0; peptone, 10.0; sodium chloride, 5.0; sodium acetate, 3.0; Agar,0.5 and cysteine hydrochloride, 0.5; pH 6.5 ± 0.1. 100 mL medium were autoclaved at 121 °C and inoculated in 250 mL screw capped Erlenmeyer flasks, and then incubated for 72 h at 37 ± 0.5 °C at 120 rpm. In addition, Cooked Meat Medium (CMM) is also used for the growth and maintenance of clostridia. These
Batch fermentation experiments were carried out in 250 mL of screw-capped Erlenmeyer flasks under anaerobic conditions. Anaerobic condition in the flask was generated by addition of 0.5% cysteine hydrochloride to the RS hydrolyzate. The fibrous remains collected after sieving the filtrate through cotton cloth was dried at 70 °C in a hot air oven, and then was weighed. The reduced weight of rice straw after pretreatment was noted and was considered for the final yield calculation. An initial sample (0 h) was taken immediately after pretreatment for sugar analysis. Regular samples (10 mL per day) were taken to study Clostridium acetobutylicum’s growth curve, so as to detect the growth stage at which respective products were produced. Experiments were run for nearly 120 h (5 days). At the end of fermentation final sample were taken for determination of ABE production and sugar utilization.
Please cite this article in press as: Ranjan A, Moholkar VS. Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel (2011), doi:10.1016/j.fuel.2011.03.030
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2.5. Analysis The optical density of the cells for growth studies was measured at 600 nm using spectrophotometer (Thermo Fischer) after appropriate dilution in water. Glucose was analyzed using Glucose (GO) assay kit procured from Sigma Aldrich, USA (GAGO20–1KT). All the samples were filtered with 0.2 lm filter and diluted appropriately for the qualitative and quantitative determination of the fermentation products viz., acetone, butanol and ethanol, by using HPLC equipped with a refractive index detector (Series 200, Perkin Elmer). A C18 column (5 lm 250 mm 4.6 mm, Chromatopak) was used with the eluent being acetonitrile (HPLC grade) at a flow rate 0.3 mL/min. 2.6. Estimation of yield and productivity
Glucose Concentration (g L-1)
A. Ranjan, V.S. Moholkar / Fuel xxx (2011) xxx–xxx
Absorbance at 600 nm
2.0
1.6
1.2 Steam Acid Enzyme
0.8
0.4
40
60
Concentration after fermentation
250 200 150 100 50 Untreated
Steam Exploded Acid Treatment
Enzymatic Treatment
80
100
120
Time (h) Fig. 1. Growth cycle of C.acetobutylicum in (a) steam exploded RSH, (b) acid treated RSH, (c) enzyme assisted RSH.
2.5
Concentration (g L-1)
It is reported that 1 kg of rice straw will contain nearly 390 g cellulose [3]. Hydrolysis is directly affected by porosity (available surface area) of lignocellulosic biomass, cellulose fiber crystallinity, and lignin and hemicellulose content. Pretreatment of fibrous feedstock is required in order to remove lignin and hemicelluloses fraction, Dilute-acid hydrolysis gives elevated reaction rates and significantly advanced cellulose hydrolysis [4,34,35]. It is reported that depending on the feedstock and the physical and chemical conditions used, up to 95% of the hemicellulosic sugars can be recovered by dilute-acid hydrolysis from the lignocellulosic feedstock [5]. The results of the experiments are presented in Figs. 1– 3. Fig. 1 depicts the growth cycle of C. acetobutylicum in (a) steam exploded RSH, (b) acid treated RSH, (c) enzyme assisted RSH. Three methods of hydrolyzate preparation used in the present study have resulted in release of varied amount of glucose from the processed rice straw. Fig. 2 gives a comparative illustration of glucose released (g L 1) after pretreatment and fermentation in untreated, steam exploded, acid treated and enzyme pretreated RS. Overall yields and productivities of the three solvents (acetone, butanol and ethanol) obtained in three types of hydrolyzates have been summarized in Table 1. One additional set of untreated blank experimental flask was also set, in which rice straw was immersed in distilled water and kept at 60 °C for 1 day at 120 rpm. This flask
20
300
Concentration after pretreatment
Fig. 2. Comparative analysis of glucose (g L 1) released after pretreatment and fermentation in untreated, steam exploded, acid treated and enzyme pretreated RS.
3. Results and discussion
0
350
0
ABE productivity was calculated as total ABE concentration reported from analysis in g L 1 divided by the fermentation time and is expressed as g L 1 h 1. ABE yield was calculated as per liter of ABE produced divided by amount of substrate added per liter (g/g).
0.0
400
Steam explosion Acid treatment Enzymatic treatment
2.0
1.5
1.0
0.5
0.0 Acetone
Butanol
Ethanol
Fig. 3. Comparative analysis of concentration of ABE production in (g L 1) through anaerobic clostrial fermentation in steam exploded, acid treated and enzyme pretreated RS.
has resulted in 8.9 g L 1 of glucose released from the feedstock. Among the three sets of experiments; steam explosion resulted in 8.91 g L 1 of glucose, 1% of acid treatment resulted in 361 g L 1 of glucose and acid treatment assisted with enzyme action resulted in 380 g L 1 of glucose released from the rice straw. Glucose released from the untreated rice straw and the steam exploded rice straw was observed to be very similar. Glucose utilization during fermentation by clostridial culture of the three hydrolyzates was as follows: steam explosion hydrolyzate = 0.68 g L 1; acid treated hydrolyzate = 282.05 g L 1 and enzyme assisted acid treated hydrolyzate = 313.7 g L 1. This indicates only 7.69% of available glucose utilization by clostridia for the production of ABE solvents using steam exploded rice straw hydrolyzate. On the other hand, acid hydrolyzed rice straw with high amount of sugar has shown 78.14% utilization of available glucose. Finally, acid assisted enzyme treatment has shown maximum amount, i.e. 82.52% of available glucose utilization. However, based on the glucose yields with various hydrolysis methods used in this work, 1% acid treatment assisted enzymatic treatment is the best for pretreatment of rice straw prior to ABE fermentation. Second and the major objective of the work was the production of ABE solvents using rice straw hydrolysates. The hydrolyzates of rice straw obtained with three methods were allowed to undergo anaerobic fermentation for 120 h (refer to Fig. 1 that depicts clostridial growth cycle with different feedstocks), and samples were taken out at regular intervals to study the ABE production at various growth stages of C. acetobutylicum. Fig. 3 presents an analysis of concentration of ABE production in (g L 1) through anaerobic clostrial fermentation in steam exploded, acid treated and enzyme
Please cite this article in press as: Ranjan A, Moholkar VS. Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel (2011), doi:10.1016/j.fuel.2011.03.030
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A. Ranjan, V.S. Moholkar / Fuel xxx (2011) xxx–xxx
Table 1 Comparative analysis of yield (g g assisted acid pretreated RS. Sr. no.
1
Sample
Autoclaved RS Acid treated RS Enzyme assisted acid treated RS
h
1
) in ABE production in through anaerobic clostrial fermentation in steam exploded, acid treated and enzyme
Acetone Yield (g g
1. 2. 3.
1
) and productivity (g L
0.116 0.199 0.341
Butanol 1
)
Productivity (g L
1
h
0.002 0.003 0.006
pretreated RS. For the first 48 h (2 days) no solvent (acetone or butanol or ethanol) production was observed. The lag phase of C. acetobutylicum, which is believed to be the phase at which clostridia adapts its nutritional environment, lasts for nearly 11– 12 h. Lag phase or exponential growth phase, during which bacteria show rapid growth in terms of count and size, continues for 17–18 h. This phase is acidogenic phase, during which bacteria produces acid and other precursors required for the production of solvents [3]. After the end of 30 h of its life cycle, clostridia were observed to enter in a long stationary phase, during which the microorganism starts producing solvents (acetone, butanol and ethanol). This stage is known as solventogenesis [3]. Solventogenesis stage starts with the sole production of acetone. Till 86 h only acetone production has been observed. The samples taken during 5th day of growth cycle reported to contain acetone butanol and ethanol in considerable amount in all the three set of experiments. Fermentation in steam exploded RS hydrolyzate resulted in large amount of acetone production during the initial (4.8– 5.8 g L 1) and mid stationary phase (7.7 g L 1), which was found to be drastically reduced towards the end of stationary phase (0.2 g L 1); while fermentation in acid hydrolyzate RS resulted in nearly 7 g L 1 during initial and 5–7 g L 1 acetone in mid stationary phase. At the end of stationary phase, only 0.4 g L 1 of acetone was noted. On the other hand, fermentation of enzyme assisted acid hydrolyzate RS resulted in nearly 7 g L 1 of acetone during initial and mid stationary phase, which was finally found to be drastically reduced to nearly 0.7 g L 1. Ethanol and butanol were not observed during the initial and mid stationary phase of all three set of experiments. Finally, at the end of stationary phase (120 h) 1.72 g L 1 of butanol and 0.11 g L 1 of ethanol were produced through steam exploded rice straw, 1.6 g L 1 of butanol and 0.12 g L 1 of ethanol were produced through acidified RS hydrolyzate, and 2.1 g L 1 of butanol and 0.2 g L 1 of ethanol were produced through acid-assisted enzyme hydrolyzate of RS. Table 1 explains the yield and productivity of ABE solvents by C. acetobutylicum MTCC 481, after utilizing three sets of RSH. Noticeably, glucose produced from the steam exploded RS was comparative to untreated rice straw, but the yield of ABE produced from it is observed to be pretty high (acetone: 0.11, butanol: 0.861, ethanol: 0.05). It can be explained by the fact that xylose is considered to be the dominant sugar of hemicellulose polymers of rice straw [36–39], which get converted to solvents in absence of glucose. Because of the partial/incomplete hydrolysis of cellulosic fraction of RS, very less amount of glucose gets released in the hydrolysate broth. It has been reported that the clostridial metabolism shifts to Pentose Phosphate pathway (PPP) and starts utilizing pentose sugar preferably xylose, when the hexose sugars were present in limited condition. As C. acetobutylicum can utilize hexoses and xylose sugars (preferably hexose), hydrolyzates prepared by all three methods in this study are suitable substrates for fermentation [11,40,41]. The other possible reason for getting good ABE yield from steam hydrolyzate in spite of very less amount of glucose released, can be due to the cellulolytic activity (i.e. the ability of clostridia to simultaneously break down cellulose into simple sugars along with fermentation under nutrient limitation) of
1
)
Yield (g g
Ethanol 1
)
0.861 0.803 1.042
Productivity (g L 0.014 0.013 0.017
1
h
1
)
Yield (g g 0.055 0.06 0.093
1
)
Productivity (g L
1
h
1
)
0.001 0.001 0.002
C. acetobutylicum, which was reported by Lee et al. [34] under limited nutrient conditions [9]. 4. Conclusion Rice straw forms one of the most abundant agro-residue in India. An estimate given by Karimi et al. [11] and Hameed and El– Khairey [42] puts total global annual rice straw production at 731 million tons, out of which largest contribution is made by Asia (667.6 million tons). This feedstock has thus tremendous potential for alcoholic biofuel production [43–47]. Among the three methods of rice straw pretreatment (hydrolysis) prior to fermentation, enzyme assisted acid treated RSH has liberated highest amount of glucose (nearly 38%). Clostridium acetobutylicum MTCC 481 was capable of utilizing all the three kinds of RS hydrolyzates (steam exploded, acid treated, and enzyme assisted acid treated), and the best ABE productivity and yield was achieved by using enzyme assisted acid pretreated RSH as feedstock. We may also conclude that for 72–86 h of growth cycle acetone production was dominant, while towards the extended growth phase butanol production was found to be dominating. Acknowledgment Mrs. Amrita Ranjan acknowledges Ministry of New and Renewable Energy (MNRE), Government of India for providing financial assistance as Junior Research Fellowship under Renewable Energy Fellowship program. References [1] Jones DT, Woods DR. Acetone–butanol fermentation revisited. Microbiol Rev 1986;50:484–524. [2] Dobermann A, Fairhurst TH. Rice straw management. Better Crops Int 2001;16:7–11 [special supplement]. [3] Chiao JS, Sun ZH. History of acetone–butanol–ethanol fermentation industry in China: development of continuous production technology. J Mol Microbiol Biotechnol 2007;13:12–4. [4] McNeil B, Kristiansen B. The acetone butanol fermentation. Adv Appl Microbiol 1986;31:61–92. [5] Ezeji TC, Qureshi N, Karcher P, Blaschek HP. Production of butanol from corn. Chemical Industries (Boca Raton, FL, USA). Alcoholic Fuels 2006;112:99–122. [6] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 2002;83:1–11. [7] Kim S, Dale BE. Global potential bioethanol production from wasted crops and crop residues. Biomass Bioenergy 2004;26:361–75. [8] Eide A. The right to food and the impact of liquid biofuels (Agrofuels). Food and Agriculture Organization of the United Nations Rome 2008:1–54. [9] Millati R, Niklasson C, Taherzadeh MJ. Effect of pH, time and temperature of overliming on detoxification of dilute-acid hydrolyzates for fermentation by Saccharomyces cerevisiae. Process Biochem 2002;38:515–22. [10] Faveri DD, Torre P, Perego P, Converti A. Statistical investigation on the effects of starting xylose concentration and oxygen mass flow-rate on xylitol production from rice straw hydrolyzate by response surface methodology. J Food Eng 2004;65:383–9. [11] Karimi K, Emtiazi G, Taherzadeh MJ. Ethanol production from dilute–acid pretreated rice straw by simultaneous saccharification and fermentation with Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. Enzyme Microb Technol 2006;40:138–44. [12] Binod P, Sindhu R, Singhania RR, Vikram S, Devi L, Nagalakshmi S, et al. Bioethanol production from rice straw: an overview. Bioresour Technol 2010;101:4767–74.
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Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels Amrita Ranjan a, Vijayanand S. Moholkar a,b,⇑ a b
Centre for Energy, Indian Institute of Technology, Guwahati, Assam 781 039, India Department of Chemical Engineering, Indian Institute of Technology, Guwahati, Assam 781 039, India
a r t i c l e
i n f o
Article history: Received 8 March 2011 Received in revised form 23 March 2011 Accepted 24 March 2011 Available online 6 April 2011 Keywords: Alcoholic biofuels Hydrolyzate ABE solvents Biobutanol ABE fermentation
a b s t r a c t Fast depletion of fossil fuels with high fluctuating market prices has made the hunt for alternate resources for the production of transportation fuels mandatory. Our approach is to utilize rice straw as feedstock for the production of alcoholic biofuels. In this paper, we have compared the effect of various pretreatment processes on rice straw, viz. physical (steam under pressure) and chemical (acid) and enzymatic treatments as a precursor to ABE fermentation for production of biobutanol. Glucose analysis was done by quantification of glucose content spectrophotometrically via Glucose assay kit. The rice straw hydrolyzate produced through pretreatment was allowed to undergo anaerobic fermentation, using C. acetobutylicum MTCC 481. The yield and productivity of ABE solvents (acetone, butanol and ethanol) was calculated using HPLC. The ABE yield produced using hydrolyzate obtained through enzyme assisted acid hydrolysis, viz. acetone: 0.11, butanol: 0.861, ethanol: 0.05, was found to be the best among all experiments. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Research for an alternate and renewable liquid transportation fuel has been a zealous worldwide activity in both industry and academia. One potential technology from the time of World I and II for the conversion of biomass to alcoholic biofuel (basically butanol and ethanol) is via the route of ABE fermentation [1–4]. It is generally achieved by the pretreatment of fibrous biomass via mechanical, physical, thermal, chemical or enzymatic route, which results in conversion of complex cellulose and hemicellulose to soluble glucose and other reducing sugars. Some of the extensively used feedstocks are corn stover, corn steep liquor, wheat bran, wheat straw, apple pomace, Jerusalem artichokes etc. [5–7]. India being a developing country with population of over 1 billion is incapable of utilizing most of these feedstocks because of the food and fuel shortage [8]. Lignocellulosic materials could form alternate economical renewable feedstock for fermentation, existing in large quantities
Abbreviations: RS, rice straw; RSH, rice straw hydrolyzate; ABE, acetone, butanol and ethanol. ⇑ Corresponding author at: Department of Chemical Engineering, Indian Institute of Technology, Guwahati, Assam 781 039, India. Tel.: +91 361 258 2258; fax: +91 361 269 0762. E-mail addresses: [email protected] (A. Ranjan), [email protected] (V.S. Moholkar).
[9]. Rice straw is one of the most copious lignocellulosic residues available in the world [7,10]. Worldwide 731 million tons of rice straw is produce annually (Africa: 20.9 million tons, Asia: 667.6 million tons, Europe: 3.9 million tons, America: 37.2 million tons and Oceania: 1.7 million tons) [11]. Nearly 600 million tons of agricultural residues has been produced by India annually; out of which, approximately 300 million tons of it remain unused, and is destroyed by simple burning [2]. Seven sister states in the northeastern India, viz. Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland and Tripura are predominantly rice producing regions. Reportedly 7.81% of their total area is used for rice production and they share 6.07% of total rice production of India. In these states, rice crop is grown in 89.46% of total area under food grain, and contributes to 92.32% of the total food grain production. Rice straw contains about 40% of the nitrogen (N), 30–35% of the phosphorus (P), 80–85% of the potassium (K), and 40–50% of the sulfur (S), which make it potential nutritional source for microbial utilization [2]. Utilization of surplus rice straw for the production of alcoholic biofuels will be both valuable and beneficial use, which will contribute to economic energy production and self-reliance of the country in meeting demand of transportation fuels [12]. The matter of utilization of agro-residues in terms of feasibility, sustainability and economics for biofuel production (alcoholic fuel and biodiesel) has been addressed by several researchers [13–23]. Rice straw is a domestic feedstock that has potential to produce considerable quantities of alcoholic fuels viz., acetone butanol and
0016-2361/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fuel.2011.03.030
Please cite this article in press as: Ranjan A, Moholkar VS. Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel (2011), doi:10.1016/j.fuel.2011.03.030
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A. Ranjan, V.S. Moholkar / Fuel xxx (2011) xxx–xxx
ethanol, and other bioenergy based biofuels [24,25]. Majorly rice straw contains cellulose 37%, hemicelluloses 24% and lignin 14%. The purpose of this paper is to comparatively assess hydrolysis of cellulosic and hemicellulosic fraction of rice straw to simple sugars using steam under pressure, acid and enzyme blend [3,26]. This process is a precursor to main ABE fermentation using the rice straw hydrolyzate as major substrate. The yardsticks for the comparative assessment would be yield and productivity of acetone, butanol and ethanol solvents. Such a study could give us an idea of the techno-economic feasibility of ABE fermentation with rice straw as feedstock, and could also form basis for optimization of the process. Previous authors have addressed the subject of use of rice straw and similar agro-feedstock (such as wheat straw, corn straw, bagasse) after hydrolysis for alcoholic fuel production via fermentation. Roslan et al. [27] has reported production of bioethanol from rice straw sacchharified with crude cellulase to release 90% of glucose in solid state fermentation. Rivers and Emert [28] have analyzed the effect of substrate concentration, cellulose crystallinity and particle size on the yields of enzymatic hydrolysis for the agro-residues, viz. bagasse and rice straw. This study revealed that nature of lignocellulose matrix is a major limiting factor in enzymatic hydrolysis. Hsu et al. [29] has studied effect of dilute acid treatment of rice straw prior to enzyme hydrolysis on the yield of hydrolysis. Acid pretreatment was found to increase the pore volume of the solids due to release of acid soluble lignin, which enhanced the yield of enzymatic hydrolysis. Marchal et al. [30] reported that hydrolysates obtained by enzymatic saccharification of wheat straw and corn stover, pretreated by steam explosion (in neutral or acidic conditions) were non fermentable by clostridium. To restore fermentability of these hydrolyzates heat treatment with Ca(OH)2 or MgCO3 at neutral pH was necessary. Qureshi et al. [31] has compared use of acid and enzyme hydrolyzed corn fiber for butanol fermentation. Sulfuric acid treated corn fiber hydrolyzate was found to inhibit cell growth and butanol production, and treatment with XAD-4 resin was necessary to remove some of the inhibitory compounds. On the contrary enzyme treated corn fiber hydrolyzate of wheat straw obtained from acid pretreatment and enzyme hydrolysis has been studied by Qureshi et al. [32]. Supplementation of hydrolyzate with glucose was found to increase productivity of fermentation. Ethanol production from wheat straw using acid pretreatment and enzymatic saccharification using recombinant strain of E. coli FBR5 has been studied by Saha et al. [33]. Detoxification of hydrolyzate obtained from acid pretreated biomass increased yield with simultaneous reduction in fermentation time.
were incubated anaerobically inside an anaerobic culture bag system (Himedia) till active growth was seen (72 h). Actively growing cultures (after lag phase, 18–20 h) of the Clostridia were added subsequently to experimental flasks. 2.2. Rice straw (RS) Rice straw was obtained from local farmer (irrigated location; Guwahati, Assam, India). Collected rice straw is agricultural waste used as packaging material during shifting of some furniture’s and equipments. The crude composition of rice has been reported to be cellulose: 32–47%; Hemicellulose: 19–27%; lignin: 5–24%; ashes: 18.8% and sugar composition as; glucose: 41–43.4%; xylose: 4.8– 20.2%; arabinose: 2.7–4.5%; mannose: 1.8%; galactose: 0.4% [34]. Strips of rice straw were cut into small pieces of length 4–5 cm. These were washed with water and then dried at 80 °C in a hot air oven. Size of dried RS was further reduced by in a mixer grinder. 2.3. Rice straw hydrolyzate (RSH) Hydrolyzate of RS was prepared using three methods as described below: 2.3.1. Steam explosion 5% w/w mixture of RS, i.e. 15 g RS added to 250 mL double distilled water, was taken in a 500 mL screw-capped Erlenmeyer flask. The flask was autoclaved at 121 °C for 30 min at 15 lb pressure, and was then allowed to cool to room temperature. RS suspension was filtered using a sterile cotton cloth. pH of the hydrolyzate thus obtained was 1.0. 2.3.2. Acid pretreatment 5% w/w mixture of RS, i.e. 15 g RS added to 250 mL acidified double distilled water (1% H2SO4), was taken in a 500 mL of screw-capped Erlenmeyer flask. The flask was kept in an orbital shaker incubator for 24 h at 60 °C at 200 rpm. The flask was then autoclaved at 121 °C for 15 min at 15 lb pressure, and was allowed to cool at room temperature. RS suspension was filtered using a sterile cotton cloth. pH of the hydrolyzate thus obtained was 0.8.
2. Material and methods
2.3.3. Enzyme assisted hydrolysis A commercial enzyme blend was generous gift from Varuna Biocell, Varanasi, India. 6 ppm of enzyme was added to 1% acid hydrolyzed rice straw and was incubated at 60 °C for 24 h at 100 rpm. The flask was then autoclaved at 121 °C for 15 min at 15 lb pressure, and was allowed to cool at room temperature. RS suspension was filtered using a sterile cotton cloth. pH of the hydrolyzate thus obtained was 0.8.
2.1. Culture maintenance and growth
2.4. Fermentation
All the chemicals used were of analytical grade procured either from Merck (Germany) or Himedia (India). Lyophilized Clostridium acetobutylicum MTCC 481 has been procured from microbial type culture collection, IMTECH, Chandigarh, India. It was maintained as spore suspension in sterile water. This culture has been rejuvenated in RCA (Reinforced Clostridial Agar) and RCM (Broth) culture media at 37 °C. The inoculums were prepared in RCM containing (g/L): glucose, 5.0; yeast extract, 3.0; starch, 1.0; beef extract, 10.0; peptone, 10.0; sodium chloride, 5.0; sodium acetate, 3.0; Agar,0.5 and cysteine hydrochloride, 0.5; pH 6.5 ± 0.1. 100 mL medium were autoclaved at 121 °C and inoculated in 250 mL screw capped Erlenmeyer flasks, and then incubated for 72 h at 37 ± 0.5 °C at 120 rpm. In addition, Cooked Meat Medium (CMM) is also used for the growth and maintenance of clostridia. These
Batch fermentation experiments were carried out in 250 mL of screw-capped Erlenmeyer flasks under anaerobic conditions. Anaerobic condition in the flask was generated by addition of 0.5% cysteine hydrochloride to the RS hydrolyzate. The fibrous remains collected after sieving the filtrate through cotton cloth was dried at 70 °C in a hot air oven, and then was weighed. The reduced weight of rice straw after pretreatment was noted and was considered for the final yield calculation. An initial sample (0 h) was taken immediately after pretreatment for sugar analysis. Regular samples (10 mL per day) were taken to study Clostridium acetobutylicum’s growth curve, so as to detect the growth stage at which respective products were produced. Experiments were run for nearly 120 h (5 days). At the end of fermentation final sample were taken for determination of ABE production and sugar utilization.
Please cite this article in press as: Ranjan A, Moholkar VS. Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel (2011), doi:10.1016/j.fuel.2011.03.030
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2.5. Analysis The optical density of the cells for growth studies was measured at 600 nm using spectrophotometer (Thermo Fischer) after appropriate dilution in water. Glucose was analyzed using Glucose (GO) assay kit procured from Sigma Aldrich, USA (GAGO20–1KT). All the samples were filtered with 0.2 lm filter and diluted appropriately for the qualitative and quantitative determination of the fermentation products viz., acetone, butanol and ethanol, by using HPLC equipped with a refractive index detector (Series 200, Perkin Elmer). A C18 column (5 lm 250 mm 4.6 mm, Chromatopak) was used with the eluent being acetonitrile (HPLC grade) at a flow rate 0.3 mL/min. 2.6. Estimation of yield and productivity
Glucose Concentration (g L-1)
A. Ranjan, V.S. Moholkar / Fuel xxx (2011) xxx–xxx
Absorbance at 600 nm
2.0
1.6
1.2 Steam Acid Enzyme
0.8
0.4
40
60
Concentration after fermentation
250 200 150 100 50 Untreated
Steam Exploded Acid Treatment
Enzymatic Treatment
80
100
120
Time (h) Fig. 1. Growth cycle of C.acetobutylicum in (a) steam exploded RSH, (b) acid treated RSH, (c) enzyme assisted RSH.
2.5
Concentration (g L-1)
It is reported that 1 kg of rice straw will contain nearly 390 g cellulose [3]. Hydrolysis is directly affected by porosity (available surface area) of lignocellulosic biomass, cellulose fiber crystallinity, and lignin and hemicellulose content. Pretreatment of fibrous feedstock is required in order to remove lignin and hemicelluloses fraction, Dilute-acid hydrolysis gives elevated reaction rates and significantly advanced cellulose hydrolysis [4,34,35]. It is reported that depending on the feedstock and the physical and chemical conditions used, up to 95% of the hemicellulosic sugars can be recovered by dilute-acid hydrolysis from the lignocellulosic feedstock [5]. The results of the experiments are presented in Figs. 1– 3. Fig. 1 depicts the growth cycle of C. acetobutylicum in (a) steam exploded RSH, (b) acid treated RSH, (c) enzyme assisted RSH. Three methods of hydrolyzate preparation used in the present study have resulted in release of varied amount of glucose from the processed rice straw. Fig. 2 gives a comparative illustration of glucose released (g L 1) after pretreatment and fermentation in untreated, steam exploded, acid treated and enzyme pretreated RS. Overall yields and productivities of the three solvents (acetone, butanol and ethanol) obtained in three types of hydrolyzates have been summarized in Table 1. One additional set of untreated blank experimental flask was also set, in which rice straw was immersed in distilled water and kept at 60 °C for 1 day at 120 rpm. This flask
20
300
Concentration after pretreatment
Fig. 2. Comparative analysis of glucose (g L 1) released after pretreatment and fermentation in untreated, steam exploded, acid treated and enzyme pretreated RS.
3. Results and discussion
0
350
0
ABE productivity was calculated as total ABE concentration reported from analysis in g L 1 divided by the fermentation time and is expressed as g L 1 h 1. ABE yield was calculated as per liter of ABE produced divided by amount of substrate added per liter (g/g).
0.0
400
Steam explosion Acid treatment Enzymatic treatment
2.0
1.5
1.0
0.5
0.0 Acetone
Butanol
Ethanol
Fig. 3. Comparative analysis of concentration of ABE production in (g L 1) through anaerobic clostrial fermentation in steam exploded, acid treated and enzyme pretreated RS.
has resulted in 8.9 g L 1 of glucose released from the feedstock. Among the three sets of experiments; steam explosion resulted in 8.91 g L 1 of glucose, 1% of acid treatment resulted in 361 g L 1 of glucose and acid treatment assisted with enzyme action resulted in 380 g L 1 of glucose released from the rice straw. Glucose released from the untreated rice straw and the steam exploded rice straw was observed to be very similar. Glucose utilization during fermentation by clostridial culture of the three hydrolyzates was as follows: steam explosion hydrolyzate = 0.68 g L 1; acid treated hydrolyzate = 282.05 g L 1 and enzyme assisted acid treated hydrolyzate = 313.7 g L 1. This indicates only 7.69% of available glucose utilization by clostridia for the production of ABE solvents using steam exploded rice straw hydrolyzate. On the other hand, acid hydrolyzed rice straw with high amount of sugar has shown 78.14% utilization of available glucose. Finally, acid assisted enzyme treatment has shown maximum amount, i.e. 82.52% of available glucose utilization. However, based on the glucose yields with various hydrolysis methods used in this work, 1% acid treatment assisted enzymatic treatment is the best for pretreatment of rice straw prior to ABE fermentation. Second and the major objective of the work was the production of ABE solvents using rice straw hydrolysates. The hydrolyzates of rice straw obtained with three methods were allowed to undergo anaerobic fermentation for 120 h (refer to Fig. 1 that depicts clostridial growth cycle with different feedstocks), and samples were taken out at regular intervals to study the ABE production at various growth stages of C. acetobutylicum. Fig. 3 presents an analysis of concentration of ABE production in (g L 1) through anaerobic clostrial fermentation in steam exploded, acid treated and enzyme
Please cite this article in press as: Ranjan A, Moholkar VS. Comparative study of various pretreatment techniques for rice straw saccharification for the production of alcoholic biofuels. Fuel (2011), doi:10.1016/j.fuel.2011.03.030
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Table 1 Comparative analysis of yield (g g assisted acid pretreated RS. Sr. no.
1
Sample
Autoclaved RS Acid treated RS Enzyme assisted acid treated RS
h
1
) in ABE production in through anaerobic clostrial fermentation in steam exploded, acid treated and enzyme
Acetone Yield (g g
1. 2. 3.
1
) and productivity (g L
0.116 0.199 0.341
Butanol 1
)
Productivity (g L
1
h
0.002 0.003 0.006
pretreated RS. For the first 48 h (2 days) no solvent (acetone or butanol or ethanol) production was observed. The lag phase of C. acetobutylicum, which is believed to be the phase at which clostridia adapts its nutritional environment, lasts for nearly 11– 12 h. Lag phase or exponential growth phase, during which bacteria show rapid growth in terms of count and size, continues for 17–18 h. This phase is acidogenic phase, during which bacteria produces acid and other precursors required for the production of solvents [3]. After the end of 30 h of its life cycle, clostridia were observed to enter in a long stationary phase, during which the microorganism starts producing solvents (acetone, butanol and ethanol). This stage is known as solventogenesis [3]. Solventogenesis stage starts with the sole production of acetone. Till 86 h only acetone production has been observed. The samples taken during 5th day of growth cycle reported to contain acetone butanol and ethanol in considerable amount in all the three set of experiments. Fermentation in steam exploded RS hydrolyzate resulted in large amount of acetone production during the initial (4.8– 5.8 g L 1) and mid stationary phase (7.7 g L 1), which was found to be drastically reduced towards the end of stationary phase (0.2 g L 1); while fermentation in acid hydrolyzate RS resulted in nearly 7 g L 1 during initial and 5–7 g L 1 acetone in mid stationary phase. At the end of stationary phase, only 0.4 g L 1 of acetone was noted. On the other hand, fermentation of enzyme assisted acid hydrolyzate RS resulted in nearly 7 g L 1 of acetone during initial and mid stationary phase, which was finally found to be drastically reduced to nearly 0.7 g L 1. Ethanol and butanol were not observed during the initial and mid stationary phase of all three set of experiments. Finally, at the end of stationary phase (120 h) 1.72 g L 1 of butanol and 0.11 g L 1 of ethanol were produced through steam exploded rice straw, 1.6 g L 1 of butanol and 0.12 g L 1 of ethanol were produced through acidified RS hydrolyzate, and 2.1 g L 1 of butanol and 0.2 g L 1 of ethanol were produced through acid-assisted enzyme hydrolyzate of RS. Table 1 explains the yield and productivity of ABE solvents by C. acetobutylicum MTCC 481, after utilizing three sets of RSH. Noticeably, glucose produced from the steam exploded RS was comparative to untreated rice straw, but the yield of ABE produced from it is observed to be pretty high (acetone: 0.11, butanol: 0.861, ethanol: 0.05). It can be explained by the fact that xylose is considered to be the dominant sugar of hemicellulose polymers of rice straw [36–39], which get converted to solvents in absence of glucose. Because of the partial/incomplete hydrolysis of cellulosic fraction of RS, very less amount of glucose gets released in the hydrolysate broth. It has been reported that the clostridial metabolism shifts to Pentose Phosphate pathway (PPP) and starts utilizing pentose sugar preferably xylose, when the hexose sugars were present in limited condition. As C. acetobutylicum can utilize hexoses and xylose sugars (preferably hexose), hydrolyzates prepared by all three methods in this study are suitable substrates for fermentation [11,40,41]. The other possible reason for getting good ABE yield from steam hydrolyzate in spite of very less amount of glucose released, can be due to the cellulolytic activity (i.e. the ability of clostridia to simultaneously break down cellulose into simple sugars along with fermentation under nutrient limitation) of
1
)
Yield (g g
Ethanol 1
)
0.861 0.803 1.042
Productivity (g L 0.014 0.013 0.017
1
h
1
)
Yield (g g 0.055 0.06 0.093
1
)
Productivity (g L
1
h
1
)
0.001 0.001 0.002
C. acetobutylicum, which was reported by Lee et al. [34] under limited nutrient conditions [9]. 4. Conclusion Rice straw forms one of the most abundant agro-residue in India. An estimate given by Karimi et al. [11] and Hameed and El– Khairey [42] puts total global annual rice straw production at 731 million tons, out of which largest contribution is made by Asia (667.6 million tons). This feedstock has thus tremendous potential for alcoholic biofuel production [43–47]. Among the three methods of rice straw pretreatment (hydrolysis) prior to fermentation, enzyme assisted acid treated RSH has liberated highest amount of glucose (nearly 38%). Clostridium acetobutylicum MTCC 481 was capable of utilizing all the three kinds of RS hydrolyzates (steam exploded, acid treated, and enzyme assisted acid treated), and the best ABE productivity and yield was achieved by using enzyme assisted acid pretreated RSH as feedstock. We may also conclude that for 72–86 h of growth cycle acetone production was dominant, while towards the extended growth phase butanol production was found to be dominating. Acknowledgment Mrs. Amrita Ranjan acknowledges Ministry of New and Renewable Energy (MNRE), Government of India for providing financial assistance as Junior Research Fellowship under Renewable Energy Fellowship program. References [1] Jones DT, Woods DR. Acetone–butanol fermentation revisited. Microbiol Rev 1986;50:484–524. [2] Dobermann A, Fairhurst TH. Rice straw management. Better Crops Int 2001;16:7–11 [special supplement]. [3] Chiao JS, Sun ZH. History of acetone–butanol–ethanol fermentation industry in China: development of continuous production technology. J Mol Microbiol Biotechnol 2007;13:12–4. [4] McNeil B, Kristiansen B. The acetone butanol fermentation. Adv Appl Microbiol 1986;31:61–92. [5] Ezeji TC, Qureshi N, Karcher P, Blaschek HP. Production of butanol from corn. Chemical Industries (Boca Raton, FL, USA). Alcoholic Fuels 2006;112:99–122. [6] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 2002;83:1–11. [7] Kim S, Dale BE. Global potential bioethanol production from wasted crops and crop residues. Biomass Bioenergy 2004;26:361–75. [8] Eide A. The right to food and the impact of liquid biofuels (Agrofuels). Food and Agriculture Organization of the United Nations Rome 2008:1–54. [9] Millati R, Niklasson C, Taherzadeh MJ. Effect of pH, time and temperature of overliming on detoxification of dilute-acid hydrolyzates for fermentation by Saccharomyces cerevisiae. Process Biochem 2002;38:515–22. [10] Faveri DD, Torre P, Perego P, Converti A. Statistical investigation on the effects of starting xylose concentration and oxygen mass flow-rate on xylitol production from rice straw hydrolyzate by response surface methodology. J Food Eng 2004;65:383–9. [11] Karimi K, Emtiazi G, Taherzadeh MJ. Ethanol production from dilute–acid pretreated rice straw by simultaneous saccharification and fermentation with Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. Enzyme Microb Technol 2006;40:138–44. [12] Binod P, Sindhu R, Singhania RR, Vikram S, Devi L, Nagalakshmi S, et al. Bioethanol production from rice straw: an overview. Bioresour Technol 2010;101:4767–74.
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