International Journal of Environment and Bioenergy, 2012, 3(2): International Journal of Environment and Bioenergy

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1 International Journal of Environment and Bioenergy, 2012, 3(2): International Journal of Environment and Bioenergy Journal homepage: ISSN: Florida, USA Article Acid Hydrolysis Technique and Yeast Adaptation to Increase Red Macroalgae Bioethanol Production Dwi Setyaningsih 1, *, Sri Windarwati 1, Indah Khayati 1, Neli Muna 1, Pandit Hernowo 2 1 Surfactant and Bioenergy Research Center, Bogor Agricultural University, 16144, Indonesia 2 Department of Agroindustrial Technology, Bogor Agricultural University, 16002, Indonesia * Author to whom correspondence should be addressed; dwisetya.sbrc@gmail.com; Tel.: ; Fax: Article history: Received 12 June 2012, Received in revised form 7 August 2012, Accepted 12 August 2012, Published 17 August Abstract: Macroalgae or seaweed is considered being promising feedstock for bioethanol production because of high polysaccharides content. Those can be converted into fermentable sugar through acid and enzymatic hydrolysis. The research objective is to develop bioethanol production from red macroalgae through acid hydrolysis and yeast adaptation. Polysaccharides of red algae Euchema cottonii consist of 26.9% carbohydrates, 2.93% hemicelluloses and 10.3% cellulose. Acid hydrolysis at temperature of 121 o C, 1.5 bars, at 15.0% dry seaweed produced hydrolysates with reducing sugar content in the range of % (w/v). Continued enzymatic hydrolysis increased reducing sugar up to 5.02% (w/v). Native yeast growth on acid hydrolysates which had 3.15% reducing sugar showed low metabolismc activity. Adaptation of S. cerevisiae yeast can improve the ability of yeasts to produce ethanol from acid seaweed hydrolysate. Adaptation of S. cerevisiae IPBCC resulted in the highest cell density from adaptation I to IV, followed by bulk yeast and commercial yeast. With the long process of adaptation, the cells density tends to decrease and the residual reducing sugar tends to increase. The highest ethanol production of adapted IPBCC yeast was 2.20% (v/v, ethanol in fermentation broth), with 80.3% substrate efficiency and 48.9% fermentation efficiency. While the highest ethanol production of adapted bulk yeast is 1.96% (v/v, ethanol in fermentation broth), which has 85.05% substrate efficiency and 42.71% fermentation efficiency. Keywords: acid hydrolysis; red seaweed; bioethanol; yeast adaptation.

2 99 1. Introduction The worldwide annual marine algae production is approximately 14 million tons, and expected to increase more than 22 million tons in 2020 (Kim et al., 2008). Macroalgae, such as Ulva lactuca, Gelidium amansii, Laminaria japonica, Sargassum fulvellum, and E. cottonii are considered being the promising feedstock for bioethanol in terms of sustainability and environmental conservation (Goh and Lee, 2010; John et al., 2011, Kim et al., 2011). This is because the cultivation is not limited by agricultural expansion over terrestrial plants. The growth rate of algae is tremendously high (Borines et al., 2011), but the biomass accumulation in the ocean is low as it has very short live and will be degraded quickly. If the algae is not harvested, then it will be dead and recycled. The utilization of algae biomass to fulfill demand of bioenergy feedstock is very strategic, since it only use simple inputs: sea water, sunlight and carbon dioxide. Furthermore, macroalgae also assist in reducing carbon dioxide in the atmosphere and supplying oxygen to the sea. Polysaccharide fibers from macroalgae have different characteristic from terrestrial plant, since there were almost no lignin (Yanagisawa et al., 2011) and built from specific monosaccharides such as galactose, glucoronic acid, alginic acid, fucose, xylose and mannitol. There were also highly sulphated sugar. Therefore, special techniques need to be developed to hydrolyze and convert polysaccharide from macroalgae into bioethanol. Dried macroalgae should be desalinated to reduce ash content because salinity would cause problem during fermentation. Cutting and chopping in some different particle sizes should be done before autoclaved with different acid concentration. The dilute sulfuric acid was added to loosen the fiber of polysaccharides and facilitate enzyme penetration. Neutralization and detoxification of the acid hydrolysates was the subsequence step to remove toxic compounds formed during heating. Fermentation process of hydrolysates is the critical step, since the monosaccharides consist of galactose, anhydro galactose, fucose, glucuronic acid, and other simple sugar besides glucose. This substrate is not a normal substrate for Saccharomyces cerevisiae, an ethanol producing yeast. The strategy to overcome this problem is to adapt the S. cerevisiae against hydrolysates, so that some important enzymes for galactose metabolism will be induced. The objective of this study was to develop bioethanol production technique from macroalgae through acid hydrolysis, neutralization and yeast adaptation 2. Materials and Methods 2.1. Raw Materials Red algae E. cottonii was used as feedstocks. Dry seaweed was desalinated, chopped, dried and milled to obtain the samples in the form of dry powder. Characters of moisture, ash, carbohydrate,

3 100 protein, hemicelluloses, cellulose, lignin, silica, and fiber content were determined by standard method for food analysis. E. cottoni comprised of 26.87% carbohydrate, 2.93% hemicelluloses and 10.31% cellulose. S. cerevisiae yeast was obtained from IPB Culture Collection (IPBCC ) which was maintained on PDA medium at 4 o C prior to fermentation in acid hydrolysates Acid Hydrolysis Acid hydrolysis was done by dilute sulfuric acid concentration ranged from 1 to 3%, for min at temperature of 121 o C, 1.5 bar. Analyses of acid hydrolysates were done after neutralization or detoxification step to measure reducing sugar (DNS method), total sugar and solid residue (gravimetry). The best treatment was chosen statistically using Complete Randomized Design based on those parameters Enzymatic Hydrolysis Commercial cellulase from Novozymes (Denmark), Sinobios (China) and local cellulose from paper industry were used for treatment after acid hydrolysis. Cellulase complex from Novozymes with activity 1000 BHU(2)/g and enzyme complex (100 FBG/g~ PGU/g) were dosed at 5% (ml/g biomass) and 0.4% (ml/g biomass), respectively. Cellulase from Sinobios, ETHOL GE and CEL 150 were dosed at 0.007g/g biomass (1 ml, 25g/250 ml) and g/g biomass (0.75 ml, 0.25g/250 ml). Meanwhile local cellulase from paper industry (36.8IU/mL) was dosed at 6-24 IU/g dry biomass. The enzymatic hydrolysis was performed at waterbath shaker at o C for h Hydrolysates Neutralization After acid and enzymatic hydrolysis of E. cottonii (2% H 2 SO 4 for 30 min), the liquid was then separated from its solid by vacuum filtration. Hydrolysates was neutralized with bases. The bases treatment was done by saturated calcium oxide solution (1:3 in water), 20% sodium hydroxide solution and ammonium hydroxide (12%), then stirred continuosly for 30 min and allowed to settle. Calcium and other precipitates were separated by vacuum filtration. The ph of filtrate was set at To adsorb chemical inhibitors like acids, furfural and hydroxymethyl furfural (HMF), some activated charcoal was added at concentration of 10% (w/v), stirred for about 15 min and filtrated. This resulted hydrolysates then used in yeast adaptation and ethanol fermentation Yeast Adaptation S. cerevisiae yeast was chosen to be adapted against neutralized E. cottonii acid hydrolysates, because it s ethanol production capacity is well recognized. Adaptation procedure was held by batch

4 101 culture. Two types of adaptation were done called quick and slow adaptation. For quick adaptation, 10% (v/v) of S. cerevisiae culture added to fermentation media which contains macroalgae hydrolysates. Incubation lasted for 48 h with agitation at rpm in the first 24 h. The cell growth was counted by hemocytometer and PDA plating. A 10% of the broth was then subculturing into the fresh media and incubated for the second adaptation cycle. This cycle repeated until 8 times. For slow adaptation, the broth was plating after completed one incubation period and the best colony was selected from PDA plate after 48 h incubation at 30 o C. The selected colony was inoculated at fresh YMGP media and used as starter for the next cycle. This cycle repeated until 9 times Fermentation Stock cultures of S. cerevisiae (native and adapted cultures) were maintained in YMGP media, incubated at room temperature for h as a starter. Starter then cultivated in media containing E. cottonii hydrolysate or glucose for h at room temperature (30 o C). The ethanol concentration was analyzed at the end of fermentation. Broth was distilled to obtain ±10% distillate volume and ethanol concentration was determined by densitometer (Anton Paar). Fermentation and substrate efficiencies were calculated according to formulation below: Substrate efficiency (%) = 100% Fermentation efficiency (%) = 100% 3. Results and Discussion 3.1. Acid Hydrolysis of E. cottonii Acid hydrolysis is the use of acid to break down the long chain polysaccharides into short chains or simple carbohydrates. Polysaccharides breakdown with acid solution will occur randomly and most of the sugars produced are reducing sugars, in contrast to the more specific enzyme breakdown that will produce a uniform sugar. Acquisition of sugar in the acid hydrolysis was influenced by the type of material, acid and solid concentration. The polysaccharide of red algae E. cottonii mostly consist of carrageenans which are water soluble linear polysaccharides composed of alternating α(1-3) and β(1-4) linked D-galactose residues. Three primary forms (κ-λ-і) of carrageenan are identified based on the modification of the disaccharide repeating unit resulting from the occurrence of ester sulphate, or anhydride formation in the 4-linked residue (Karlsson and Singh, 1999).

5 102 Acid hydrolysis of E. cottonii (Fig. 1-2) showed the highest total dissolved solids (TDS) in the range of o Brix from 10-15% (w/v) solid concentration of dry seaweed powder, or 50-60% (w/v) solid concentration of fresh seaweed. Reducing sugar of E. cottonii hydrolysates increased correspondingly with the process time at concentration of 0 and 1%, but it will decrease after 60 min at concentration of 2 and 3% (Fig. 1). Longer hydrolysis time might cause monosaccharides decomposition at high acid concentration. The highest reducing sugar obtained from hydrolysis with 2% sulfuric acid for 45 min, resulted in 32.8% (w/w) reducing sugars, and 19.21% (w/w) total solids residue. The comparable result was also found by Khambaty et al (2011) from hydrolysis of 250 g red algae Kappaphycus alvarezii (100 o C, 0.9 N H 2 SO 4 ) which yielded 26.2% (w/w) sugar residue. Figure 1. Reducing sugar of E. cottonii acid hydrolysates. Figure 2. Total solid residue of E. cottonii acid hydrolysates Enzymatic Hydrolysis of E. cottonii The use of commercial cellulase enzymes can increase reducing sugar content of acid hydrolysates ranged from 5 to 64% from the initial reducing sugar (5.02 %, w/v) (Figs. 3-5). The highest one was obtained from Cellulase Complex.

6 103 Figure 3. Enzymatic hydrolysis using enzyme complex and cellulose complex from Novozymes. Fig. 3 showed that the highest reducing sugar concentration was obtained from cellulase complex hydrolysis for 48 h (5.02%, w/v; control ph 6 was 3.06%, w/v), meanwhile the highest total sugar content was obtained from cellulase complex hydrolysis with addition of β-glucosidase after 48 h hydrolysis (5.05%, w/v; control ph 6 was 3.62%, w/v). Enzymatic hydrolysis with Enzyme complex produced reducing and total sugar in lower concentration, due to its enzyme composition which more suitable for lignocelluloses hydrolysis with high content of pectin such as arabinase, β-glucanase, cellulase, hemicellulase, pectinase and xylanase (Novozymes product catalog). Enzymatic hydrolysis using cellulase enzyme from paper industry (Fig. 4) with dose of 7.5 ml for 24 h, and dose of 10 ml for 24 h produced the highest reducing sugar (4.36%, w/v) and total sugar (5.03%, w/v), respectively. The longer of hydrolysis time will decrease reducing and total sugar concentration might be due to sugar decomposition. Fig. 5 showed reducing and total sugar concentration after enzymatic hydrolysis with cellulases from Sinobios. Hydrolysis using ETHOL-GE (dose of 0.75g) for 48 h produced the highest concentration of reducing sugar and total sugar, resulted in 3.49% and 4.35% (w/v), respectively. Figure 4. Enzymatic hydrolysis using cellulase enzyme from paper industry.

7 104 Figure 5. Enzymatic hydrolysis using cellulases from Sinobios S. cerevisiae Growth on Acid Hydrolysates of E. cottonii One of the factors affected microbial growth was nutrition. The compatibility of nutrition provided in the media with the microbial specific need for its growth is very important. Acid hydrolysates of E. cottonii is a new media for S. cerevisiae, so that they need an adaptation process to survive and able to use sugar derived carrageenans, such as anhydrogalactose, fucose, galactose-4- sulphate (Knutsen and Grasdalen, 1992), and oligosaccharides with 4-O-sulfo-D-galactopyranose residue (Yu et al, 2002). Neutralized acid hydrolysates of E. cottonii which were use for yeast adaptation had total soluble solid of 12 o Brix. We evaluated two kind of hydrolysates, one was pure hydrolysates and second was mix of hydrolysates and YMGP media at ratio of 1:1. Fermentation was held at temperature of 32 o C for 3 days (72 h) with shaking at rpm in the first 24 h. During fermentation, formation of CO 2 and reducing sugar consumption were measured. Fig. 6 showed that at pure hydrolysates, formation rate of CO 2 was slower and reducing sugar at the end of fermentation was relatively high (2.85%), compare with mixed 50% hydrolysates in YMGP. In this mixed media, residual sugar was only 0.75% (b/v). Figure 6. Concentration of reducing sugar at seaweed hydrolysates fermentation

8 105 Glucose concentration in YMGP media is 4%, it acts as primary substrate before yeast can ferment sugar produced from acid hydrolysis. Glucose provides nutrition for growth and adaptation to produce induced enzymes. In S. cerevisiae, galactose is metabolised to glucose 6-phosphate by five enzymes of Leloir pathway. This pathway is necessary as the initial enzyme at glycolysis unable to recognise galactose (Timson, 2007). S. cerevisiae for ethanol fermentation came from different sources, which are IPB culture collection (IPBCC), commercial baker s yeast, bulk yeast, and traditional tapai (fermented cassava or rice) yeast. The yeast was grown in the mixed media at o C with shaking at the first 24 h and incubation continued till 100 h. Growth and sugar residue curves were obtained from sampling every 6 and 12 h (Fig. 7). All the yeast showed almost the same growth curve; line below is yeast which grown at only YMGP media as control. They reached the stationary phase after h incubation. Although traditional tape yeast had very low cell growth, this still was used for adaptation because we expect a new potential wild type yeast. Figure 7. Courses of cell number and reducing sugar during S. cerevisiae fermentation from different sources. The growth of S. cerevisiae from commercial and bulk yeast showed the same pattern of reducing sugar consumption. Yeast from IPBCC grew slower, this matched with its reducing sugar curve. Control media (YMGP only) showed rapid decline of reducing sugar because of no growth inhibitor from acid hydrolysis Adaptation of S. cerevisiae on Acid Hydrolysates of E. cottonii Neutralized E. cottonii acid hydrolysate without addition of standard media (YMGP) can support yeast growth as well, and the condition was influenced by the sugar concentration. Therefore,

9 106 to increase sugar consumption against seaweed hydrolysates, the yeast adaptation was conducted using diluted acid hydrolysate in water to achieve various soluble solid concentrations (5; 7.5; 10 and 12 o Brix). High soluble solid content (12 o Brix) showed inhibition of yeast growth as seen from the slow formation of CO 2 and reducing sugar levels were still high at the end of fermentation. Concentration of 7.5 o Brix indicated the best formation of CO 2. This concentration was chosen for further adaptation process. Result of the quick and slow adaptation can be seen at Table 1. Quick adaptation of S. cerevisiae IPBCC in diluted acid hydrolysates (7.5 o Brix) resulted in the highest cell density from adaptation I to IV, followed by bulk yeast and commercial baker s yeast. With the long process of adaptation, the cells density tends to decrease. Different from quick adaptation, slow adaptation showed homogenous cell density of S. cerevisiae from all sources and cycles of adaptation, approximately Base on the growth, it can be concluded that IPBCC strain had the highest cell density, followed by bulk and commercial baker s yeast. Table 1. Observations of cell number for quick and slow adaptation Adaptation Number Cell concentration (cell/ml) Commercial Bulk IPBCC Traditional tape Quick adaptation I II III IV V VI VII VIII Slow Adaptation I II III IV V VI VII VIII IX Fig. 8 showed the residual reducing sugar after 72 h fermentation of quick adaptation cycles. Sugar residue increased until cycle number VI for commercial yeast and bulk yeast, while for IPBCC at cycle number VIII and then decreased. There was a high consumption of reducing sugar at the early

10 107 fermentation cycles. It could be related to the energy need for detoxifying inhibitors and producing galactose metabolizing enzymes. In the next cycles, there was a decrease on sugar consumption, which indicated that some adaptation process had been done. At the end of the cycles, there was an increase of sugar consumption, but the cell density decreased. Probably only adapted cells will survive and grow well to produce ethanol. Figure 8. Residual reducing sugar of S. cerevisiae at quick adaptation cycles Residual reducing sugar at slow adaptation (Fig. 9) showed that sugar consumptions were relatively stable at every cycle compared with quick adaptation. The lowest residual sugar came from cycle VI (0.589 %, b/v), and the highest was IX (1.42 %, b/v). At slow adaptation, only good colonies at PDA plating were elected to the next cycles. Table 2 showed that the adaptation process can improve the ability of yeast to produce ethanol. In IPBCC culture, adaptation can increase substrate efficiency (59.05%) by 9 and 4%, to 64.17% and 61.22% for quick and slow adaptation, respectively. Table 2. Fermentation parameters of ethanol production from native and adapted culture Treatment IPBCC Bulk Yeast Native Quick adaptation VIII Slow Adaptation IX Native Quick adaptation VIII Slow Adaptation IX Substrate eff. (%) ± ± ± ± ± ± 0.41 Fermentation eff. (%) ± ± ± ± ± ± 4.83

11 108 Figure 9. Residual reducing sugar of S. cerevisiae at slow adaptation cycles Fermentation of Adapted S. cerevisiae for Bioethanol Production In order to achieve higher ethanol yield, the effect of several fermentation conditions were studied. Table 3 showed the fermentation parameters of ethanol production of adapted IPBCC and bulk yeast with some conditions, such as shaking, flask covering, dosing of starter and fermentation time. Table 3. Fermentation parameters of ethanol production Treatment IPBCC Bulk Yeast Substrate Fermentation Ethanol yield Substrate Fermentation Etanol yield efficiency (%) efficiency (%) (% v/v) efficiency (%) efficiency (%) (% v/v) a. Shaker Shaker 70 rpm ± ± ± ± ± ± 0.02 No shaker ± ± ± ± ± ± 0.11 b. Flask Cover Cover ± ± ± ± ± ± 0.21 No cover ± ± ± ± ± ± 0.08 c. Starter volume 2,5% ± ± ± ± ± ± ,0% ± ± ± ± ± ± ,5% ± ± ± ± ± ± ,0% ± ± ± ± ± ± 0.04 d. Time of fermentation 3 days ± ± ± ± ± ± days ± ± ± ± ± ± days ± ± ± ± ± ± days ± ± ± ± ± ± 0.03

12 109 Fermentation parameter of IPBCC yeast was significantly different from bulk yeast. The use of shaking and flask covering on adapted IPBCC yeast showed no significant different in fermentation efficiency. An amount of 10% (v/v) starter volume resulted in the highest ethanol production, but not significantly different with addition of 7.5% starter volume. Four days were the best incubation time for IPBCC yeast culture. Adapted bulk yeast showed different results. Shaking at 70 rpm for the first 24 h resulted in better fermentation efficiency although the value was not significantly different with no shaking, whereas covering flask also did not give positive effect. The highest efficiency of fermentation was obtained with the addition of 7.5% (v/v) starter volume, significantly different with the addition of 2.5% and 5%. On bulk yeast, fermentation time give no significant different, although five days fermentation was considered to be better. 4. Conclusions E. cottonii dried powder at 15% solid concentration can be hydrolysed by 2% sulfuric acid autoclaved for 45 min. Average reducing sugar was 4.53% (w/v) after neutralized by saturated calcium oxide. The quick and slow adaptation on acid hydrolysates can improve IPBCC and bulk yeast performance to produce ethanol from E. cottonii hydrolysates. In IPBCC culture, quick adaptation can increase substrate efficiency about 9%, while slow adaptation can increase up to 4%, from 59.05% to 64.17% and to 61.22% for quick and slow adaptation, respectively. The highest ethanol production of adapted yeast IPBCC cycle IX was 2.20% (v/v) ethanol in fermentation broth, with the calculated substrate efficiency was 80.28% and 48.86% fermentation efficiency. While the highest ethanol production of adapted bulk yeast cycle IX was 1.963% (v/v) ethanol in fermentation broth, which had 85.05% substrate efficiency and 42.71% fermentation efficiency. Acknowledgment This work was supported by Directorate of Research and Community Service, Directorate General of Higher Education, Ministry of National Education through Unggulan Strategis Nasional Grant References Borines, M. G., de Leon, R. L., and McHenry, M. P. (2011). Bioethanol production from farming nonfood macroalgae in Pacific island nations: Chemical constituents, bioethanol yields, and prospective species in the Philippines. Renew. Sustain. Energy Rev., 15:

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