Production of Fermentable Sugars from Recycled Paper Sludge for Alcohol Production Nantanat Kulsuwan Division of Biochemical Technology, School of Bioresources and Technology, King Mongkut's University of Technology, Thailand Jirasak Kongkiattikajorn Division of Biochemical Technology, School of Bioresources and Technology, King Mongkut's University of Technology, Thailand E-mail: jirasak.kon@kmutt.ac.th Abstract Recycled paper sludge (RPS) is basically made up of secondary poor- quality nonrecyclable paper fibres. The effects of operating variables including the concentration of diluted acid and the enzymatic digestibility were optimized for fermentable sugars production. RPS was dried and milled into small pieces by blender, ultra-centrifugal mill and disc mill. RPS was pretreated using aqueousdiluted acid solution at high temperatures to enable production of the maximum amount of fermentable sugars for enzymatic hydrolysis. The results showed that 2.0% (w/v) RPS was pretreated by diluted sulfuric acid at 135ºC under pressure 15 lb/inch2 for 30 min producing highest yield of reducing sugars while 0.1 M hydrochloric acid and 0.25 M acetic acid pretreatment produced lower yields of reducing sugars under the same conditions. The optimal reaction conditions, which resulted in an pretreated disc milled- RPS with 0.1 M sulfuric acid and enzymatic digestibility of 66.67 %, were found to be at ph 5.0, 55 ºC, 24 h. The conditions for simultaneous saccharification and fermentation (SSF) were studied at 30 C, 37 C and 42 C for 48 hr adding with Accellulase (Genecor), yeast inoculum 10 g/l and substrate concentration 2.0% (w/v). Under the optimal conditions, the total ethanol concentration in SSF of disc milled RPS pretreated with aqueous-diluted acid solution at high temperatures reached 8.10 g/l and an overall yield of 99.5% was obtained, respectively. Keywords: Paper sludge, reducing sugars, ethanol I. INTRODUCTION Solid wastes generated from industrial sources are heterogeneous in composition, ranging from inert inorganic (such as produced in mining and collieries) to organic (in industries producing basic consumer products) and may include even hazardous constituents (as in pesticide industry). RPS is the largest by-product of the pulp and paper industry and disposal of RPS is a major solid waste problem for the industry [1, 2]. It was predicted that a global shift in paper and paperboard production would result in the Asia-Pacific region emerging as a major producer of RPS. Global production of RPS was predicted to rise over the next 50 years by between 48 and 86% over current levels [3]. The nature of RPS generated from paper industries is mainly depends on the raw materials used in different unit processes. RPS generated from the industrial sources contains a large number of ingredients, some of which are toxic. Solid waste is generated from the both large and small categories of paper mills. Producing a value added product, from the cellulose present in the RPS, could International Journal of the Computer, the Internet and Management Vol.20 No.3 (September-December, 2012) pp 57-62 57
Nantanat Kulsuwan, and Jirasak Kongkiattikajorn provide an economically more attractive option. Due to the high and rather accessible cellulose content (50 60%) of RPS, it could be a potential feedstock for a value added product [4]. One way to perform enzymatic hydrolysis and fermentation is simultaneous saccharification and fermentation (SSF). This was chosen in the present study to minimize the end-product inhibition of the. The enzymes used to hydrolyse the cellulose and the remaining hemicelluloses after the pretreatment. SSF has been shown to be superior to separate hydrolysis and fermentation at elevated dry-matter contents when the ethanol yield is the main priority. Combining two process steps also results in a lower capital cost, and the fact that the ethanol concentration is high during SSF reduces the risk of contamination [5]. The aim of the study was to obtain soluble reducing sugars by diluted acid and enzymatic hydrolysis of RPS and to determine the optimal conditions of each treatment and approach SSF by Kluyveromyces marxinus 5049 for RPS conversion to ethanol was implemented and compared for process efficiency at different cultivated temperature in terms of product yield and production rate. II. MATERIALS AND METHODS Substrate No more than 3RPS from the factory of paper production was washed by tap water and dried at 90 C in hot-air oven for 3 days. The dry RPS was milled into small pieces by blender, ultra-centrifugal mill and disc mill. Dilute acid pretreatment of RPS The following condition were used in the pretreatment of RPS by dilute acid. The concentration of RPS was 2.0% (w/v) in 0.01-0.25 M of sulfuric acid, hydrochloric acid and acetic acid. The temperature was varied from 135 C and reaction time was 30 min. Fermentation The pretreated RPS suspension were neutralized to ph 5.5 for fermentation process. The enzyme solution (filtersterilized Accellulase enzyme solubilized in citrate-phosphate buffer ph 5.0) was used for hydrolysis of pretreated RPS. The pretreated sample was supplemented with additional nutrients to give a base medium composition of 1 g/l MgSO4 7H2 O, 2 g/l (NH4 )2 SO4, 0.5 g/l KH2PO4, 1 g/l yeast extract. The enzymatic hydrolysis was run at the same time as fermentations that were carried out in 125 ml Erlenmeyer flasks containing 50 ml of this medium. The culture was incubated in incubator shaker with 50 rpm for 48 hr at 30 C, 37 C and 42 C. Analysis The amount of released glucose was measured using glucose oxidase/peroxidase assay. The amount of reducing sugar produced was determined using the dinitrosalicylic acid (DNS) method. Ethanol concentration was measured by gaschromatography (GC-17A, Shimadzu) using a chromosorb 101 column and a flame ionization detector. III. RESULTS AND DISCUSSION 2.0% RPS in diluted acids were pretreated under varying conditions and then determined %reducing sugars yield was determined. Figure 1 shows that optimal % reducing sugar yields could be obtained at 32.6, 39.5 and 7.2% from pretreatment at 135 C for 30 min with 0.1 M sulfuric acid, 0.05 M hydrochloric acid and 0.25 M acetic acid%, respectively. Thus, dilute sulfuric acid produced highest yield of reducing sugars. On the other hand, acetic acid could produce as much reducing sugars as did dilute sulfuric acid and hydrochloric acid but was needed at the concentration that was much higher than the other two acids. From the results, the diluted sulfuric acid seemed to be suitable for RPS pretreatment. The production of reducing sugars and glucose from un-milled RPS pretreated either with distilled water ph 5.5 or 0.1 M sulfuric acid at 135 C under pressure of 15 lb/inch2 for 30 min as shown in Table 1, resulting the yield of reducing sugars and glucose from 58
Production of Fermentable Sugars from Recycled Paper Sludge for Alcohol Production The pretreated RPS after enzymatic hydrolysis for 48 h at 50 C. The highest yield of reducing sugars was 5.76 g/l obtained from diluted acid pretreatment after enzymatic hydrolysis. So, the dilute-acid pretreatment is more efficient in the improving enzymatic hydrolysis than just steam. The main objective of the biomass pretreatment is to make the cellulose more accessible to enzyme. Fig. 1 Percentage of reducing sugars yield from 2.0 % disc mill RPS pretreated by various concentration of diluted acid at 135 C for 30 min.,, stand for sulfuric acid, hydrochloric acid and acetic acid, respectively. TABLE 1 YIELD OF SUGAR FROM ENZYMATIC HYDROLYSIS OF BLENDER RPS. Pretreatment distilled water Reducing sugars (g/l) 0.77 0.1 M sulfuric acid a 1.83 0.1 M sulfuric acid 5.76 a without enzymatic hydrolysis The dried RPS of this substrate composed of 60.09 % cellulose, 11.18 % hemicelluloses, 9.74 % lignin. Generally, biomass cannot be saccharified by enzymes without a pretreatment because the lignin in plant cell walls forms a barrier against enzyme attack [6]. An ideal pretreatment would reduce the lignin content and the crystallization of cellulose while increasing the surface area for enzymatic hydrolysis. The milling pretreatment either with blender rotating at 1,000 rpm or ultra-centrifugal mill rotating at 6,000 rpm or Disc mill rotating at 12,000 rpm was investigated for enable production of the fermentable sugars from enzymatic hydrolysis. From the results, the highest amounts of reducing sugars was obtained from disc mill PRS in the concentration of 10.08 g/l (66.67% reducing sugars yield). So, this pretreatment method suggested the effectiveness on hemicelluloses and lignin components and resulted in efficient hydrolysis. TABLE 2 YIELD OF SUGAR FROM ENZYMATIC HYDROLYSIS OF PRETREATED RPS. Pretreatment Reducing sugars (g/l) Blender 5.76 Ultra-centrifugal mill 6.04 Disc mill 10.08 The ethanol yields, reducing sugars and glucose conversions obtained after 48 h of fermentation for the pretreated RPS with disc mill by K. marxinus 5049 at 30 C, 37 C and 42 C, ph 5.0 in fermentation process by SSF. Ethanol production at 30 C, 37 C and 42 C are summarized in Fig. 2 Fig. 4 and Table 3. Ethanol production from RPS drydefibrated with disc mill was produced by K. marxinus 5049 at 30 C as shown in Fig. 2, SSF of the pretreated disc mill dry-defibrated RPS with diluted acid pretreatment was use as substrate for ethanol production by SSF of K. marxinus 5049. Glucose in hydrolysate was utilized completely in 20 hr, with 6.42 g/l maximum ethanol production. Ethanol yield were 0.32 g/g dry RPS (Table 3). The strain exhibited high flocculation ability, good osmo and thermo tolerance and ferment higher concentrations of sugars yield appreciable amount ofethanol. These attributes are requisite criteria for selecting yeast as candidates of industrial production of ethanol [7]. Fig. 3-4 shows the yield of reducing sugars, glucose ethanol and dry mass by K. marxinus 5049 at 37 C and 42 C. Glucose in hydrolysate was utilized completely in 24 hr, with maximum ethanol production of 8.02 g/l and 8.10 g/l, respectively. Ethanol was 0.40 and 0.41 g/g dry RPS, respectively, (Table 3). International Journal of the Computer, the Internet and Management Vol.20 No.3 (September-December, 2012) pp 57-62 59
Nantanat Kulsuwan, and Jirasak Kongkiattikajorn These results suggest that this thermotolerant yeast K. marxinus 5049 could perform the SSF at high temperature. In the performance of ethanol production from RPS using SSF process were carried out by K. marxinus 5049. SSF reduces equipment cost by performing the hydrolysis and fermentation in a single reactor and eliminates the need for expensive capable of with standing strong acid or other chemicals. SSF technique provides the possibility to overcome the main disadvantage of enzymatic hydrolysis i.e. decreasing the enzyme loading and therefore the production cost. In spite of the economical advantage of SSF over separate hydrolysis and fermentation (SHF), the critical problem with SSF is the difference in temperature optimum of cellulases and the fermenting microorganism. Fig. 2 Simultaneous saccharification and fermentation of RPS for ethanol production with K. marxinus 5049 at 30 C.,,, stand for reducing sugars, glucose, ethanol, and cell g/l, respectively. Fig. 3 Simultaneous saccharification and fermentation of RPS for ethanol production with K. marxinus 5049 at 37 C.,,, stand for reducing sugars, glucose, ethanol, and cell g/l, respectively Fig. 4 Simultaneous saccharification and fermentation of RPS for ethanol production with K. marxinus 5049 at 42 C.,,, stand for reducing sugars, glucose, ethanol, and cell g/l, respectively. Table 3 reports the ethanol yield (obtained from a balance of the glucose converted to ethanol, based on total sugar available in the raw material) and ethanol productivity for SSF. These results were obtained after 48 h of SSF at 30 C, 37 C and 42 C with 0.06 g of cellulase composite/g of biomass and 10 g/l of K. marxinus 5049. The effectiveness of the combined pretreatment (disc mill and diluted acid pretreatment at high temperature under vapor pressure of 15 lb/inch2) becomes clear when the data are presented in terms of ethanol productivity (Fig. 2 - Fig. 4): the maximum ethanol productivity is attained in a shorter time in the presence of 10 g/l yeast. TABLE 3 COMPARISON OF ETHANOL PRODUCTION AT 30 C, 37 C AND 42 C BY K. MARXINUS 5049 Temperature C E P max Q E Y p/s 30 C 0.32 6..42 0.41 0.41 37 C 0.40 8.02 0.51 0.51 42 C 0.41 8.10 0.50 0.51 CE Ethanol yield per unit biomass (g(g-biomass)-1) P max Maximum ethanol production (g/l) QE Ethanol production rate (g/l/hour) Yp/s Product (ethanol) yield coefficient (g(gtotal sugar)-1) The present results clarify that the complex pretreatment is sufficient for promoting the fermentation of RPS. A substantial increase in the bioconversion of cellulose to ethanol can be obtained by disc mill pretreatment prior to enzymatic hydrolysis and enabled satisfactory ethanol yields and productivities 60
Production of Fermentable Sugars from Recycled Paper Sludge for Alcohol Production to be achieved. The most efficient method of pretreatment was found to be disc mill combined with using aqueous-diluted acid solution at high temperatures of RPS is confirmed to be an effective method for improving the enzymatic saccharification of the cellulose content of biomass. The use SSF is advantageous in the fermentation step as overall enzymatic activity can be balanced. When the recycled paper is used for the process of production of paper, the residue is RPS. Hydrolysis of the cellulosic fraction results in hydrolysate rich in hexoses and pentoses [8]. From this study, the best enzymatic reaction was found to be Accellulase (cellulolytic enzyme from Genecor company), producing the high production of reducing sugars. This might be due to large amount of cellulose in the RPS. Cellulose is known to be highly resistant to enzymatic hydrolysis but these difficulties can be overcome by employing a suitable chemical or mechanic al pretreatment prior to hydrolysis [9]. However, one of the drawbacks of this process is that the enzymatic hydrolysis come from high cost of enzyme employed. From the experiment, diluted acid pretreatment also increase the sugar yield. However, in this experiment, the RPS pretreated by milling to flour was taken part in hydrolysis by these hydrolytic enzymes to produce reducing sugars. IV. CONCLUSIONS The results showed that three diluted acids; sulfuric acid, hydrochloric acid and acetic acid; were evaluated for the acid hydrolysis for the production of reducing sugars from RPS. 0.1 M sulfuric acid was found to yield significantly more reducing sugars than hydrochloric acid and acetic acid. Under optimal conditions, Accellulase (Genecor) was capable of producing the amount of reducing sugars RPS. It was found that RPS pretreated by disc mill combined with using aqueous-diluted acid solution at high temperatures produced highest reducing sugars yield after enzymatic hydrolysis. The disc mill -pretreatment combined with aqueous-diluted acid solution at high temperatures of RPS is an effective method for improving the enzymatic saccharification of the cellulose content of biomass. The use SSF is advantageous in the fermentation step as overall enzymatic activity can be balanced. ACKNOWLEDGMENT The authors gratefully acknowledge the financial support given by the National Research University, Thailand. REFERENCES [1] A. Battaglia, N. Calace, E. Nardi., B.M. Petronio, and M. Pietroletti, Paper mill sludge-soil mixture: Kinetic and thermodynamic tests of cadmium and lead sorption capability, Microchem. J, vol. 75, pp. 97 102. 2003, [2] X. Geng, J. Deng, and S.Y. Zhang, Effects of hot-pressing parameters and wax content on the properties of fiberboard made from paper mill sludge, Wood Fiber Sci. vol. 38, no. 4, pp. 736 741. 2006, [3] W. Mabee, and D.N. Roy, Modeling the role of papermill sludge in the organic carbon cycle of paper products, Environ. Rev, vol. 11, no.1, pp. 1 16. 2003. [4] L.R. Lynd, K. Lyford, C.R. South, and K. Levenson, Evaluation of paper sludge for amenability to enzymatic hydrolysis and conversion to ethanol, TAPPI J, vol. 84, pp. 50-69. 2001 [5] C.E. Wyman, D.D. Spindler, and K. Grohmann, Simultaneous saccharification and fermentation of several lignocellulosic feedstocks to fuel ethanol, Biomass Bioenergy, vol. 3, no. 5, pp. 301 307, 1992. [6] V.J.H. Sewalt, W. Ni, J.W. Blount, H. G. Jung, S. A. Masoud, P.A. Howles, C. Lamb, and R.A. Dixon, Reduced lignin content and altered lignin composition in transgenic tobacco down-regulated in expression of L- phenylalanine ammonia-lyase or International Journal of the Computer, the Internet and Management Vol.20 No.3 (September-December, 2012) pp 57-62 61
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