ap-3-12.dvi Acta Polytechnica Vol. 52 No. 3/2012 Production of Biocellulosic Ethanol from Wheat Straw Ismail, W. Ali1, Braim, R. Rasul1, Ketuly, K. Aziz2, Awang Bujag, D. Siti Shamsiah2, Arifin, Zainudin2 1 Salahaddin University, Science Education College, Chemistry Department, Erbil, Kurdistan Region, Iraq 2 Malaya University, Science Faculty, Chemistry Department, Malaysia Correspondence to: wshyarali@yahoo.com, wshyarali@esc-ush.com (Ismail, W. Ali) Abstract Wheat straw is an abundant lignocellulosic feedstock in many parts of the world, and has been selected for producing ethanol in an economically feasible manner. It contains a mixture of sugars (hexoses and pentoses). Two-stage acid hydrolysis was carried out with concentrates of perchloric acid, using wheat straw. The hydrolysate was concentrated by vacuum evaporation to increase the concentration of fermentable sugars, and was detoxified by over-liming to decrease the concentration of fermentation inhibitors. After two-stage acid hydrolysis, the sugars and the inhibitors were measured. The ethanol yields obtained from by converting hexoses and pentoses in the hydrolysate with the co-culture of Saccharomyces cerevisiae and Pichia stipites were higher than the ethanol yields produced with a monoculture of S. cerevisiae. Various conditions for hysdrolysis and fermentation were investigated. The ethanol concentration was 11.42 g/l in 42 h of incubation, with a yield of 0.475 g/g, productivity of 0.272 g/l·h, and fermentation efficiency of 92.955 %, using a co-culture of Saccharomyces cerevisiae and Pichia stipites. Keywords: wheat straw, two-stage acid hydrolysis, bioethanol, co-culture, fermentation. 1 Introduction Lignocellulosic materials are renewable, largely un- used, and abundantly available sources of raw mate- rials for the production of ethanol fuel. A potential source for future low-cost ethanol production is to utilize lignocellulosic materials such as crop residues, grasses, sawdust, wood chips, oil palm empty fruit bunches, trunks, and fronds [1]. These materials con- tain sugars polymerized in the form of cellulose and hemicellulose, which can be liberated by hydrolysis and subsequently fermented to ethanol by microor- ganisms [2]. Hydrolysis can be performed with the use of enzymes or chemicals to further decompose the starch or cellulose to simple sugars [3]. The cel- lulose hydrolysis step is a major element in the total production cost of ethanol from lignocellulosic mate- rial [4]. Three groups of enzymes, i.e. endoglucanase, exo-glucanase and β-glucosidase, are involved in the cellulose-to-glucose process, with synergetic interac- tions [5]. Enzymatic hydrolysis or saccharification is mainly limited by a number of factors, includ- ing the crystallinity of the cellulose, the degree of polymerization, the moisture content, the available surface area, etc. [6]. Acid hydrolysis is becoming more popular, due to its lower cost and greater ef- fectiveness than enzymatic hydrolysis [7]. The ligno- cellulosic material is subjected to strong concentra- tions of hydrochloric or sulfuric acid in order to begin the breakdown and separation of the materials [4]. Acid hydrolysis can be divided into two groups: (a) concentrated-acid hydrolysis and (b) dilute-acid hydrolysis. Concentrated-acid processes are gener- ally reported to give a higher sugar yield (e.g. 90 % of the theoretical glucose yield), and consequently a higher ethanol yield, than dilute-acid processes [3]. In addition, concentrated-acid processes can oper- ate at low temperature, which is a clear advantage over dilute-acid processes [8]. There are other stud- ies that apply hydrothermal processes [9], steam ex- plosion [10], wet oxidation [11], alkaline peroxide [12] and ammonia fiber explosion [13] for biomass pre- treatment in the ethanol process. The sugars formed in the hydrolysate are fer- mented into ethanol. The most common microor- ganisms for this purpose are Saccharomyces cere- visiae and Zymomonas mobilis, which are not me- tabolized pentoses. However, almost one-third of the reducing sugars obtained from hydrolyzed lignocel- lulosic materials are pentoses, composed primarily of D-xylose [14]. Yeasts that have the ability to convert xylose to ethanol have been reported, and one of the earliest identified with this unique capa- bility was Pichia stiptis [15]. For efficient conver- sion of all sugars to ethanol, a co-culture of OVB 11 (Sacchromyces cerevisiae) and Pichia stipitis NCIM 348 is used to co-ferment hexoses and pentoses to ethanol [14]. The average yield of wheat straw is 589.670 to 635.029 g/g of wheat grain [16]. Wheat straw con- 28 Acta Polytechnica Vol. 52 No. 3/2012 tains 35–45 % cellulose, 20–30 % hemicellulose, and 8–15 % lignin, and can also serve as a low-cost attrac- tive feedstock for fuel alcohol production [17]. Sev- eral studies are available on the production of ethanol from wheat straw hydrolysates [18–22]. In this work, wheat straw was treated with different concentra- tions of perchloric acid in two-stage hydrolysis to fer- mentable sugars. In addition, vacuum evaporation and overliming of the hydrolysate were optimized. The fermentability of the hydrolysate was evaluated using a monoculture of baker’s yeast and a co-culture of baker’s yeast and P. stipitis. The optimum yields were obtained from variations of acid concentration, temperature and time. The ethanol that was pro- duced was 11.42 g/l in 42 h of incubation, with a yield of 0.475 g/g, productivity of 0.272 g/l · h, and fermentation efficiency of 92.955 %. Perchloric acid was used because of its double function as an oxi- dising agent and a hydrolysing agent, and it can be recyclyed from its KClO4 salt. 2 Materials and methods 2.1 Wheat straw and reagents Sun-dried wheat straw, cultivated in June 2011, was obtained from the local harvest in Erbil, Kurdistan- Iraq. It was further air-dried in an oven at 70 ◦C for 12 h before it was milled in a hammer mill, and particles smaller than 0.12 mm were collected for fur- ther use in the experiments. The following reagents were obtained from Sigma-Aldrich: Perchloric acid (70 % w/w), potassium hydroxide pellets, glucose, arabinose, xylose, ethanol, lime and charcoal. 2.2 Microorganisms and Culture Media Hexose yeast; Dry baker’s yeast (Saccharomyces cere- visiae), widely used in the bakery and brewery indus- tries (Mauri-Pan, Instant yeast, AB Mauri Malaysia Sdn. Bhd.), was used for ethanol fermentation of glu- cose. The yeast (10 g/l) was inoculated in YM broth (pH 6.0), which consisted of glucose (10 g/l), peptone (5 g/l), yeast extract (3 g/l), malt extract (3 g/l) and distilled water (up to 1l). This culture was incubated at 30 C for 48 h. Pentose yeast; Pichia stipitis (CBS 5773), was purchased from the CBS culture collection center, Netherlands, and was used for ethanol fermentation of xylose. It was maintained on a GPYA (ATCC 144) agar plate (glucose (40 g/l), yeast extract (5 g/l), peptone (5 g/l), agar (15 g/l)) medium. It was grown in GPYA medium at 30 C, 100 rpm for 48 h, but this growing process was done after xylose (40 g/l) got subtituted for glucose in the medium. The pre- culture yeast cells were collected by centrifugation at 6 000 g for 10 min. The cells were washed twice with distilled water prior to inoculation. 2.3 Pretreatment 2.3.1 Acid hydrolysis The powdered wheat straw (10 g) was treated with perchloric acid (17.5 %) with a solid-to-liquid ratio of 1 : 4 at 100 C for 20 min. The mixture was cooled in an ice bath and filtered. The residual wheat straw from the filtration was kept for the second stage of hydrolysis. The filtrate was neutralised with KOH (10M), KClO4 was precipitated, and the solution was filtered. The residual wheat straw was hydrolysed with HClO4 (35 %) at 100 C for 40 min. The fil- trate from this second stage was treated as above. The sugars and the by-products were measured by HPLC. 2.3.2 Concentrating the sugars The combined filtrates from above (550 ml) were con- centrated by vacuum distillation (Buchi Rotavap R- 215) at 80 ◦C. Sugars, phenolics and furans were mea- sured by HPLC. 2.3.3 Detoxification The concentrated filtrate was treated with calcium oxide with stirring, until pH 10. This mixture was incubated for half an hour, followed by centrifuga- tion (3 000 g, 20 min) and filtration. Later, the pH of the hydrolysate was brought down to pH 6 by HClO4 (10M) [23]. Activated charcoal (3.5 g) was added to the hydrolysates and stirred for 1 h. The mixture was centrifuged and filtered. The sugars and the by- products were measured by HPLC. 2.4 Fermentation Monoculture (S. cerevisiae); The detoxified hy- drolysates (100 ml) were taken along with supple- mentation of 0.1 % (w/v) yeast extract, peptone, NH4Cl, KH2PO4 and 0.05 % of MgSO4 · 7H2O, MnSO4, CaCl2·2H2O, FeCl3·2H2O and ZnSO4·7H2O in a 250 ml conical flask, adjusting the pH to 5.5, and autoclaved at 115 ◦C for 15 min [23]. After the medium had been cooled to room temperature it was transferred to a 500 ml jar (Laboratory Fermentor, B. E. MARUBISHI (THAILAND CO., LTD) under sterile conditions. Then the baker’s yeast starter cul- ture (10 ml) was inoculated, and incubated anaero- bically at 30 ◦C, 200 rpm for 72 h. Samples from the medium were withdrawn periodically at various intervals from the replicated fermenter jars and cen- trifuged at 600 g for 10 min at 10 ◦C and analyzed for ethanol. 29 Acta Polytechnica Vol. 52 No. 3/2012 The fermentation efficiency was calculated using the following formula [23]. FE% = Practical yield Theoretical yield × 100, where the practical yield is the ethanol that is pro- duced, and the theoretical yield is 0.511 per gram of sugar consumed. Co-culture (S. cerevisiae and P. stipitis); The detoxified hydrolysate (100 ml) was taken along with supplementation of 0.1 % (w/v) yeast extract, pep- tone, NH4Cl, KH2PO4 and 0.05 % of MgSO4 ·7H2O, MnSO4, CaCl2·2H2O, FeCl3·2H2O and ZnSO4·7H2O in a 250 ml conical flask, adjusting pH to 5.5, and autoclaved at 115 ◦C for 15 min [21]. After the medium had been cooled to room temperature it was transferred to a 500 ml jar (Laboratory Fermentor, B. E. MARUBISHI (THAILAND CO., LTD) under sterile conditions. After it had been transferred, the baker’s yeast starter culture (10 ml) was inoculated and incubated anaerobically at 30 ◦C, 200 rpm for 24 h. Then, P. stipitis inoculum was added at a rate of (10 g/l) and fermentation was allowed to con- tinue at 30 ◦C, 300 rpm for 72 h at an aeration rate of 5 ml/min. Samples from the medium were with- drawn periodically at various intervals from the repli- cated fermenter jars, centrifuged at 600 g for 10 min at 10 ◦C and analyzed for ethanol by HPLC. 2.5 Analytical methods Sugars, furfural, HMF, ethyl vanillin, syringalde- hyde, acetic acid and ethanol were analysed using the Waters HPLC system with a refractive index as a detector (Waters 600E Multisolvent Delivery System, Waters 717plus Autosampler, Waters TCM column heater- item 2989 HPLC,Waters 2414 Refractive In- dex Detector) equipped with a Rezex column (ROR- Organic Acid 00F-0138-K0, 8 % H, 150×7.8 mm) and a Rezex micro-guard cartridige column (03B-0138- K0, 50 × 7.8 mm). The eluent was H2SO4 (5 mM) at a flow rate of 0.6 ml/min, and the injection volume was 10 μl. 3 Results and discusion 3.1 Acid hydrolysis Perchloric acid was used because of its double func- tion as an oxidising agent and as a hydrolysing agent. In addition to the acid hydrolyzing effect of HClO4, its oxidizing function helps in delignification and re- quires less time and energy than other acids pretreat- ments that are used [24]. In addition, neutralisation of the access HClO4 with KOH leads to precepitation of the insoluble KClO4, and this can be recycled to HClO4. First stage of acid hydrolysis; The effects of tem- perature and time on the acid hydrolysis of wheat straw were studied. In this study, the ratio of pow- dered wheat straw to the volume of perchloric acid was 1 : 4, and the concentration of perchloric acid was 17.5 %. The effects of four different temper- atures (50, 70, 90, 100 ◦C) on the hydrolysis of wheat straw were investigated. The effects of different heat- ing times from 10 min to 60 min were also investi- gated at each of the above temperatures. Reducing sugars from the hydrolysis increased as the tempera- ture and the heating time increased, as shown in Fig- ures 1 and 2. However, at 100 ◦C, the concentration of arabinose decreased slightly after 20 min of hy- drolysis, and the xylose concentration approximately stabilized. This indicated that the hemicellulose hy- drolysis of wheat straw was almost complete when it was hydrolysed at 100 ◦C for 20 min. The concen- trations of by-products such as furfural, HMF, ethyl vanillin, syringaldehyde and acetic acid increased as the temperature and time increased, as shown in Fig- ures 2 and 3. This indicates why no degradation products were observed at 50 ◦C. Based on the re- sults, the optimum conditions for the first stage were when hydrolysis was conducted at 100 ◦C for 20 min. Table 1: Effects of HClO4 concentrations on hydrolysis at 100 ◦C and for 60 min HClO4 Glucose Xylose Arabinose acetic acid HMF Furfural Ethyl vanillin Syringaldehyde % g/l g/l g/l g/l g/l g/l g/l g/l 17.5 4.226 4.926 0.958 0.749 0.345 0.195 0.26 0.21 20 5.097 4.906 0.946 1.099 0.364 0.308 0.29 0.24 25 6.123 4.876 0.923 1.783 0.407 0.396 0.29 0.28 30 6.502 4.812 0.902 2.117 0.446 0.442 0.28 0.30 35 6.765 4.656 0.889 2.378 0.479 0.422 0.27 0.29 40 6.796 4.520 0.867 2.524 0.490 0.382 0.25 0.27 30 Acta Polytechnica Vol. 52 No. 3/2012 Figure 1: Effects of the tempera- tures and the heating times on hy- drolysis (the first stage), where X is xylose and G is glucose Figure 2: Effects of the tempera- tures and the heating times on hy- drolysis (the first stage), where AR is arabinose and AA is acetic Figure 3: Effects of the tempera- tures and the heating times on hy- drolysis (the first stage), where H is HMF, S is syringaldehyde, E is ethyl vanilin and F is furfural Table 2: Effects of heating time on the hydrolysis of wheat straw residual using 35 % HClO4 at 100 ◦C Time Glucose Xylose Arabinose Acetic acid Ethyl vanillin Syringaldehyde HMF Furfural (min) g/l g/l g/l g/l g/l g/l g/l g/l 10 1.586 0.012 0 0.030 0.10 0.00 0 0 20 2.631 0.087 0.000 0.057 0.27 0.02 0 0 30 3.108 0.188 0.002 0.084 0.63 0.05 0 0 40 3.325 0.238 0.003 0.092 1 0.84 0.08 0 0 50 3.419 0.270 0.003 0.134 1.02 0.12 0 0 60 3.414 0.266 0.004 0.168 1.10 0.16 0 0 The effects of perchloric acid concentration on the hydrolysis were investigated. The perchloric acid concentration ranged from 17.5 % to 40 % (w/w). The glucose concentration from hydrolysis increased as the perchloric concentration increased, until a level of 35 % was reached, after which it stabilized. In- versely, however, the xylose and arabinose concen- tration decreased, as shown in Table 1. These re- sults show that hemicellulose hydrolysis of wheat straw was approximately complete when it was hy- drolyzed by 17.5 % perchloric acid at 100 ◦C for 60 min, but the cellulose of the wheat straw still remained and continued to hydrolysis. However, the concentration of furfural, ethyl vanillin and sy- ringaldehyde increased when the concentration of perchloric acid began to increase, but later it de- creased. This is because furfural was oxidized to formic acid, ethyl vanillin was oxidized to isovanil- lic acid and syringaldehyde was oxidized to syringic acid by HClO4 [7]. The concentration of HMF con- tinuously increased as the concentration of the acid increased, perhaps because the conversion rate of fur- fural is about four times faster than that of HMF [25]. Based on the results, the optimum concentration of perchloric acid for hydrolysis of cellulose was when 35 % HClO4 was used. This is guaranteed to pro- duce a high yield of glucose. In order to prevent degradation of xylose and arabinose and to minimize the amount of by-products, it was decided to sepa- rate the hydrolysis of hemicellulose and cellulose into two separate stages. Second stage of acid hydrolysis; The effects of var- ious heating times from 10 min to 60 min on the hydrolysis of the wheat straw residual from the first stage filtration were investigated. In this study, 40 ml of 35 % HClO4 and the wheat straw residue were added to the flask of the hydrolysis set, and hydroly- sis was performed at 100 ◦C. The glucose concentra- tion from the hydrolysis increased significantly as the time of heating increased, until 50 min, after which it declined slightly, as shown in Table 2. By con- trast, only very small amounts of xylose, arabinose 31 Acta Polytechnica Vol. 52 No. 3/2012 Figure 4: Effects of the hydro- lysate concentrating and detoxifi- cation on the amount of sugars, where B.C. if before concentrating A.C. is after concentrating, B.D. is before detoxification and A.D. is after detoxification Figure 5: Effects of the hydroly- sate concentrating and detoxifica- tion on the amount of inhibitors, where B.C. is before concentrating, A.C. is after concentrating, B.D. is before detoxification, A.D. is after detoxification, AA is acetic acid, H is HMF, F is furfural, E is ethyl vanilin and S is Syringaldehyde Figure 6: Effect of incubation ti- me on the ethanol production us- ing mono-culture (Baker’s yeast) an co-culture Baker’s yeast and P. stipites and acetic acid were produced, and when the heating time increased their concentration slightly increased. This is further evidence that the hemicellulose hy- drolysis of wheat straw was approximately complete in the first stage of hydrolysis, but the cellulose of the wheat straw still remained and continued to hy- drolyse. Therefore, as shown in Table 2, furfural and HMF were not detected. The concentration of ethyl vanillin and syringaldehyde increased with increased heating time, which may be due to degradation of lignin from the residual, as shown in Table 2. Based on the results, 50 min was selected as the optimum heating time for the second stage of hydrolysis. 3.2 Concentrating the sugars The sugars were concentrated by about twofold from their initial concentrations, as shown in Figure 4. The furfural, HMF, ethyl vanillin, syringaldehyde and acetic acid contents were 0.141, 0.354 1.68, 0.31, 1.27 g/l, respectively, from initial concentrations of 0.126, 0.236, 0.94, 0.25, 0.88 g/l, respectively, as shown in Figure 5. The by-products were concen- trated at different rates. This may be due to degrada- tion, e.g. furfural to acetic acid and formic acid [26]. 3.3 Detoxification Partial neutralization, over-liming and activated charcoal treaments were used to minimize the ef- fect of the microbial inhibitors (by-products) caused by acid hydrolysis, and to improve the formation of ethanol during the fermentation process. This pro- cess led to a reduction by 87.2 %, 86.2 %, 75 %, 84.9 % and 84.4 % in furfural, HMF, acetic acid, ethyl vanillin and syringaldehyde, respectively, as shown in Figure 5. Lower amounts of glucose (3.03 %), xylose (2.95 %) and arabinose (2.79 %) were absorbed, as shown in Figure 4. 3.4 Fermentataion Monoculture ethanol fermentation using baker’s yeast (Saccharomyces cere visiae): The fermentabil- ity of the concentrated and detoxified hydrolysate was evaluated using the baker’s yeast starter culture. The highest concentration of ethanol was 6.516 g/l after 36 h of incubation, as shown in Figure 6. The resulting yield of ethanol was eqiuvalent to 0.271 g/g with volumetric productivity of 0.181 g/l · h and fer- mentation efficiency of 53 %, based on the total fer- mentable sugars 24.044 g/l of the hydrolysate. The ethanol efficiency declined after 36 h of incubation. This may be because most of the xylose remained unfermented by the baker’s yeast, as the hydrolysate contain pentoses and hexoses. Co-culture ethanol fermentation using baker’s yeast and P. stipitis; The fermentability of the con- centrated and detoxifided hydrolysate of wheat straw was evaluated using the co-culture of baker’s yeast and P. stipitis. The highest concentration of ethanol was 11.421 g/l after 42 h as the optimum incuba- tion period for maximum fermentation efficiency of 32 Acta Polytechnica Vol. 52 No. 3/2012 92.955 %, as shown in Figure 6. The resulting yield of ethanol was eqiuvalent to 0.475 g/g, with volumet- ric productivity of 0.272 g/l · h, based on the total fermentable sugars (24.044 g/l) of the hydrolysate. However, the ethanol productivity declined after 42 h of incubation. The higher ethanol yield is attributed to the fermentation of both hexoses and pentoses in the hydrolysate. 4 Conclusion Bioethanol was produced from wheat straw using two different concentrations of perchloric acid hydrolysis in two separate stages. Perchloric acid was used be- cause of its double function as an oxidizing agent and as a hydrolyzing agent, and it can also be recycled from its KClO4 salt. Two-stage hydrolosis was pre- ferred to one-stage hydrolysis because there is less sugar degradation from the hydrolyzed materials in the first stage. Fewer fermentation inhibitors formed, and less heating time was required during two-stage hydrolysis. 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