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International Journal of Agricultural Science and Research (IJASR) ISSN 2250-0057 Vol. 3, Issue 4, Oct 2013, 93-102 © TJPRC Pvt. Ltd.

ETHANOL PRODUCTION FROM DEGRAINED SUNFLOWER HEAD WASTE BY ZYMOMONAS MOBILIS AND SACCHAROMYCES CEREVISIAE GEETHA S1, KUMAR A2 & DEIVEEKASUNDARAM M3 1

Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India

2

Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Nova Scotia, Canada

3

Department of Microbiology, Faculty of Agriculture, Annamalai University, Chidambaram, Tamil Nadu, India

ABSTRACT This work was conducted to study the potential of degrained sunflower head waste as substrate for ethanol production and to optimize the bioprocess for higher yields of ethanol. The various pretreatment methods viz., the aqueous hydrolysis, heat treatment, acid hydrolysis, alkali hydrolysis, steaming and fungal enzyme hydrolysis resulted in the significant release of fermentable sugars from the degrained sunflower head waste. The combination of acid @ 0.2N + autoclaving at 121°C and 15lb pressure yielded the highest quantity of reducing sugars. The increase in reducing sugars was significant upto 10hr of incubation period. Hence, acid + steam pretreatment was followed for further studies on ethanol production. The Zymomonas mobilis 2427 and Saccharomyces cerevisiae AU-05 were tested to study their efficiency to ferment hydrolysed sunflower head waste for alcohol production.The fermentation period of 5 days, pH of 6.5, temperature of 30°C and 2.0 % inoculum level were found optimum for production of ethanol. Zymomonas mobilis 2427 was found to be better than Saccharomyces cerevisiae AU-05 to ferment the sugars released from sunflower head waste. The studies on the supplementation of nitrogen sources showed that urea at 500 mgL -1 favoured alcohol production (24.40 gL-1). Thus, sunflower head waste an agricultural residue on hydrolysis can serve as one of the potential substrate for alcohol fermentation employing the bacterium Zymomonas mobilis 2427.

KEYWORDS: Sunflower Head Waste, Pretreatment, Ethanol, Zymomonas mobilis, Saccharomyces cerevisiae INTRODUCTION At present, the transportation sector is almost entirely dependent on petroleum-based fuel, it being responsible for around 60% of the world oil consumption. Biofuels represent an alternative to petroleum-based fuel; in particular, bioethanol is the most widely used biofuel for transportation (Balat, 2010). Nowadays, industrial bioethanol production is mainly focused on corn, wheat and sugarcane, as well as on highly abundant agricultural wastes. The use of residual biomass for bioethanol productions has the added advantage of transforming a waste material into a valorized product. The increase in the prices of fuel and possibility of shortfalls has led to an extensive evaluation of alternative sources of energy to meet the global energy demand. Microbial processes have proved useful for the production of alternate energy products from renewable resources. Alcoholic fermentation is one of the most important examples. As a consequence, ethanol is the most promising liquid fuel since it can be readily produced from various agriculture-based renewable materials, like sugarcane juice, molasses, potatoes, corn and barley etc (Esser and Karsch, 1997). Currently, yeast Saccharomyces cerevisiae is used as the major ethanol producing microorganism worldwide. Despite its extensive use, it has a number of disadvantages, such as high aeration cost, high biomass production and low temperature and ethanol tolerances (Saigal, 1993). Therefore, efforts have been made to improve the existing technologies through the raw materials and alternate strains for ethanol production. Zymomonas mobilis has emerged as a potential bacterium for ethanol production. Production


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of value added products from agro-industrial and food processing wastes is now a focusing area, as it reduces pollution in the environment in addition to energy generation. The annual availability of these wastes amounts to 1.05 billion tons (Anonymous, 2004). The yeast Saccharomyces cerevisiae and facultative bacterium Zymomonas mobilis are better candidates for industrial alcohol production. Z. mobilis possesses advantages over S. cerevisiae with respect to ethanol productivity and tolerance. Zymomonas mobilis, a gram-negative ethanol producing bacterium, has been of considerable interest in recent years for ethanol production because it gives a near-theoretical yield of ethanol from glucose and fructose. It is an osmoand ethanol-tolerant bacterium that has shown higher specific rates of glucose uptake and ethanol production via the Entner-Doudoroff pathway under anaerobic conditions (Roger et al., 1982, 1997; Yamshita et al., 2008﴿. Fermentation technologies utilizing strains of Z. mobilis in place of the traditional yeast, have been proposed by a number of authors, as they have been shown to ferment under fully anaerobic conditions with faster specific rates of glucose uptake and ethanol production as well as ethanol yields close to theoretical (Rogers et al., 1979; Doelle et al., 1993; Davis et al., 2006). Currently, use of low cost substrates as potential sources for ethanol production by Z. mobilis has been considered in industry. Some investigators have attempted to study the consequence of using some low, cost materials such as thippi, starch cassava, tapioca, starch from feedstock, sago starch, waste starch stream paper sludge and wheat stillage on ethanol production by Z. mobilis (Davis et al., 2006; Yamshita et al., 2008; Patle and Lal, 2008﴿. The sunflower (Helianthus annus) is an annual plant in the family Asteraceae, with a large flower head (inflorescence). Sunflower is the fourth source of oil-seeds world-wide, representing around 26,049,793 ha of cultivated land (FAOSTAT, 2011). Stalks, heads and leaves, left in the fields after harvesting, have no suitable use and are burnt causing environmental pollution. The huge volume annually generated, 3-7 tonnes of dry matter/ha (Marechal and Rigal, 1999), makes these lignocelluloses residues a major low-cost source of sugars that can be converted into valuable products, such as ethanol, by means of hydrolysis (either acid or enzymatic) and fermentation of the sugars released. Looking to the potentiality of the substrate that would be made available, the present investigation was taken up to exploit so suitable bacterial and yeast strains and to optimize the parameters for maximum ethanol production.

MATERIALS AND METHODS Strains and Medium Zymomonas mobilis 2427 obtained from IMTECH, Chandigarh and Saccharomyces cerevisiae from Annamalai University were used for ethanol production throughout the experiments. The Zymomonas mobilis was maintained on RM medium (glucose-20gL-1, yeast extract-10gL-1, potassium dihydrogen phosphate-20gL-1). The yeast culture was maintained on MYPG medium (yeast extract-3gL-1, malt extract-3gL-1, peptone-5gL-1, glucose-10gL-1). The overnight grown bacterial and yeast cultures in broth were used as an inoculum. Substrate for Fermentation The degrained sunflower heads were cut into small bits. These bits were then sun dried for 3-4 days. The dried sunflower head bits were finely ground in an electric grinder and sieved through a mesh sieve of 56 m and the powder was collected and they have analysed for different components by standard methods. Pretreatment of the Substrate Twenty five grams of sunflower head waste powder was slurried with 100 ml water in 250 ml Erlenmeyer flasks. The suspension was autoclaved at 121°C for 30, 60, 90, 120, 150 min, the contents were cooled and incubated at room temperature. And also the suspension was treated with sulphuric acid alone (at a concentration of 0.05, 0.1 and 0.2 N


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acidity) or along with autoclaving at 121 ºC for 30 min and sodium hydroxide (at an alkali concentration of 0.5, 1.0 and 1.5% alkalinity). The treated powder mixture was squeezed through muslin cloth and centrifuged at 3000xg for 15 min. The clear supernatent of the hydrolysate was taken for the estimation of reducing sugars. The fungi cultures Viz., Trichoderma reesei, Pencillium restrictum and Aspergillus niger were prepared and added at room temperature (28  2°C) to the suspension.

OPTIMIZATION OF MEDIUM COMPONENTS AND CULTURE CONDITIONS To find a suitable carbon source for ethanol production by Zymomonas mobilis and Saccharomyces cerevisiae, different sugar solutions of glucose, sucrose and maltose (10, 15, 20 and 25% (w/v) concentration) and different nitrogen sources of urea and ammonium sulphate (at concentrations of 500, 1000, 1500 and 2000 mg/1000 ml) were optimized. It was allowed to ferment for 48 hr. Samples were withdrawn at different intervals (0, 8, 20, 28, 36 and 48 hr) and the cell density was measured by optical density at 625nm using spectrophotometer. To examine the effects of fermentation time, temperature and pH on ethanol production, The acid hydrolysate was inoculated @ 5% bacterial and yeast inoculums with different fermentation time (4,5,7 and 9 days), pH (3.5, 4.5, 5.5 and 6.5). After determination of optimum pH which was 4.5, hydrolysate was incubated at different temperatures viz., 25, 30 and 37ºC for 7 days. After complete fermentation, the samples were analysed for ethanol and residual sugar content. The head extract was further studied to know the effect of various levels of inoculum on ethanol production. The head extract (100ml of pH 5.0) was inoculated with 1.0, 1.5 and 2.0% of inoculum levels and kept for fermentation at 30°C for 7 days and samples were analysed for residual sugar and ethanol yield. Alcohol Fermentation The sunflower head hydrolysate obtained after the saccharification with acid/alkali/enzyme was filtered and centrifuged to remove unhydrolysed materials where as in case of acid hydrolysate, the pH of the supernatent was adjusted to 5.0 with 10% ammonium hydroxide solution before inoculation. The alkali hydrolysate, the pH of the supernatant was adjusted to 5.0 with 10% sulphuric acid solution before inoculation. The clear supernatant was then enriched with 0.2% urea as nitrogen source and the fermentation was carried out using Zymomonas mobilis, Saccharomyces cerevisiae and their combination (5% w/v) after multiplication. The fermentation was allowed to continue for 3-4 days at ambient temperature ( 28-30°C). Recovery of Alcohol The fermented sunflower head waste powder hydrolysate was distilled to recover the total ethanol yield by using laboratory model distillation unit after filtering the fermented hydrolysate through muslin cloth.

ANALYTICAL METHODS The reducing sugars were estimated by Dinitro salicylic acid (DNSA) method as described by Miller (1959).The ethanol was estimated by colorimetric method as described by Caputi et al. (1968). The results obtained were analysed statistically using Randomised Block design as described by Panse and Sukhatme (1985).

RESULTS AND DISCUSSIONS The pulp portion of the sunflower head waste contains cellulose (35%), Hemicellulose (38%), Lignin (24%), Ash (5%), Protein (5%), Pectin (23%), Moisture (12.8%), Total solids (87.2%) and Potassium (23.7%).


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EFFECT OF PRETREATMENT ON THE RELEASE OF FERMENTABLE SUGARS The highest reducing sugar content recorded in sunflower head waste was 35.42 gL -1 at 121ºC for 150 min, followed by 120 min which showed 33.87 gL-1. Reducing sugar content increased significantly with increase in acid concentration upto 0.2N (36.11 gL-1), but it did not differ significantly with 0.1N acid concentration (35.98 gL -1). The highest reducing sugar content was recorded in both 0.1 and 0.2N acid concentration at 9 th and 10th hour of incubation period. At 10th hr of incubation, the reducing sugar of 35.98 gL-1 and 36.11 gL-1 were recorded at 0.1, 0.2 N acid respectively (Figure 1). The optimum condition for maximum extraction of sugar was 0.2N acid from 8-10 hr of incubation. Dilute acid pretreatment, ammonia fibre explosion and lime pretreatment have emerged as particularly effective chemical methods (Himmel et al., 1997; Dale et al., 1996; Kaar and Holtzapple, 2000). Singh et al. (2002) studied the acid hydrolysis of begasse and rice husk for the production of reducing sugars with sulphuric acid. The highest reducing sugar content was recorded at 1.0 and 1.5 percent alkali concentrations, which accounts to 30.12 gL-1 and 30.62 gL-1 respectively. The highest reducing sugar content was recorded at 1.0 and 1.5 percent alkali concentrations at 8, 9 and 10 hours of incubation which was 29.32, 29.77 and 30.12, and 29.92, 30.01 and 30.62 gL -1 respectively (Figure 2). Among fungal cultures, substrate inoculated with Trichoderma reesei recorded highest reducing sugar (29.11 gL-1) followed by Aspergillus niger and Penicillium restrictum which recorded 26.47 gL-1 and 25.89 gL-1 respectively (Figure 3). The data indicated that out of different concentrations of acids 0.2N sulphuric acid with steaming at 121°C for 150 min gave the highest yield of reducing sugars (41.63 gL-1). The yield of reducing sugars progressively increased as the incubation period increased from 30 to 150 min and hydrolysis beyond a period of 150 min didn’t improve the yield of reducing sugars (Figure 4). The impregnation of cellulosic waste with sulphuric acid before steam explosion has been reported by several groups (Grohmann et al., 1985; Schell et al., 1991), and is known to improve sugar recovery. Schwald et al. (1989) reported that sulphuric acid catalysed and steam treated cellulosics indicated the high recovery of hemicellulose derived sugar predominantly the source of monosaccharides.

EFFECT OF SUGARS AND NITROGEN ON CULTURES GROWTH Among the different sugar substrates viz., Glucose, maltose and sucrose used, glucose supported maximum growth of Zymomonas mobilis. The results indicated that glucose was readily fermented in all four concentrations and among the different concentration of sugars, 15% glucose showed maximum growth in Zymomonas mobilis and 10% in Saccharomyces cerevisiae as evidenced from the OD values. There was an increase in cell mass production with increase in concentration from 10-20% in Saccharomyces cerevisiae and 10-15% in Zymomonas mobilis. When the glucose concentration was increased above 15%, a decreasing trend was observed in Zymomonas mobilis cell mass production after 48 hr of incubation (Figure 5). Ratnakumari and Ramakrishna (1988) reported that glucose was found to be the best substrate with regards to maximum ethanol production, substrate utilization and biomass production. Higher growth rate of Zymomonas mobilis in glucose containing medium has been attributed to its higher rate of glucose uptake (Rogers et al., 1982). Doelle and Greenfield (1985) found that highly adapted and efficient strains of Zymomonas mobilis produce 95.5 gL-1 ethanol from 200 gL-1 of sucrose. The results showed that increasing the concentration of nitrogen from 500 to 1000mg the cell density proportionately increased and there after no significant increase in fact reduced the OD value of 1.45 was observed at 48hr of incubation when 500 mg and 2.24 OD with 1000mg ammonium sulphate added. In the case of urea, maximum cell mass density of 1.34 and 2.24 OD was recorded in Saccharomyces cerevisiae and Zymomonas mobilis respectively with 500mg/1000ml used. The growth rate was decreased as the concentration of urea in the medium was increased.


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OPTIMIZATION OF PARAMETERS ON ETHANOL YIELD FROM ACID+STEAM HYDROLYSED SUNFLOWER HEAD WASTE The ethanol yield increased with the supplementation of ammonium sulphate and the highest yield of 23.44 gL-1 was obtained at the concentration of 500 mg. Among the cultures, Zymomonas mobilis gave more ethanol yield(23.44 gL-1) as compared to Saccharomyces cerevisiae (23.22 gL-1) with supplementation of nitrogen source as ammonium sulphate. The ethanol content increased with the supplementation urea also and the yield obtained was 24.99 gL-1 at 500 mg concentration (Figure 6). The ethanol content decreased significantly from 1000mg to 2000mg concentration and least was 18.32 gL-1. The addition of nutrients or supplements could be an alternate source for getting maximum alcohol production. Ammonium sulphate and urea were used to supplement nitrogen source and they were used at various concentrations. Slaughter (1988) reported that ammonium sulphate can support growth itself or with the addition of trace amounts of vitamins. When nitrogen was supplemented as ammonium sulphate using molasses medium at concentrations of 0.1 percent using Zymomonas mobilis strain B-61147, ethanol concentrations at hourly intervals was 65-70 gL-1, 54 gL-1 and 75.1 gL-1, 68.8 gL-1 (Iida et al., 1993). Similarly Amutha and Gunasekaran (2000) obtained higher ethanol yield of 44.22 and 54.9 gL-1 by supplementation of liquefied cassava starch with ammonium sulphate and yeast extract at concentrations of 1 and 10 gL-1 respectively. The ethanol yield was maximum in the treatment with acid hydrolysis followed steaming. Zymomonas mobilis and Saccharomyces cerevisiae inoculation recorded 21.89, 20.52 gL-1 of alcohol respectively. However, the two strains were on par with each other on the alcohol yield. The ethanol yield increased significantly with increase in fermentation period from 4 to 7 days. Further increase in fermentation period did not increase significantly between 4th and 9th days (20.81, 21.20 gL-1 respectively). The ethanol yield recorded on 7th and 9th day was significantly superior as compared to 4th and 5th day of fermentation (Figure 7a). The ethanol yield increased significantly from pH of 4.5 to 5.5 (19.46 and 21.32 gL -1) respectively. Beyond this level the ethanol yield although slightly increased with Zymomonas mobilis (21.43 gL-1) however it was not significant with pH 6.5 and with inoculation of Saccharomyces cerevisiae the ethanol yield was 18.34, 20.21 gL-1 respectively. Beyond this level the ethanol yield decreased slightly (20.00 gL-1) at pH 6.5. At pH 6.5, there was an inhibitory effect on ethanol yield with Saccharomyces cerevisiae inoculated medium (Figure 7b). The ethanol yield increased due to the increase in temperature from 25 to 37 °C (20.36 to 22.34 gL-1) with the inoculation of Zymomonas mobilis. The ethanol yield were 19.31, 21.03 gL-1 respectively at 25, 30°C with Saccharomyces cerevisiae (Figure 7c). The room temperature gave higher ethanol yield (21.68gL-1). The Zymomonas mobilis culture gave better ethanol yield (22.34 gL-1) than the Saccharomyces cerevisiae (21.03 gL-1). The higher ethanol yield was observed at 2.0 percent inoculum size. Among the cultures Zymomonas mobilis recorded significantly higher ethanol yield (26.32 gL-1) and was superior to Saccharomyces cerevisiae, which accounts to 25.91 gL-1. Inoculum size significantly influenced the ethanol yield. The ethanol yield increased with the inoculum size upto 1.5 percent. Further increase decreases the ethanol yield however the decrease was significant. But, the highest ethanol yield of 24.79 gL-1 and 26.11 gL-1 was observed with 1.5 and 2.0 percent inoculum size. Hence the optimum was 1.5 percent of inoculum size and incubation of 5 days (Figure 7d). Mathur et al. (1986) obtained alcohol concentration of 2.18 percent (w/v) from whey inoculated with 1.00 percent of Saccharomyces cerevisiae after 18 hours of fermentation period at pH and temperature of 4.0 and 22ºC respectively. On the contrary, Ramanathan (2000) could achieve maximum alcohol recovery of 5.85 percent at 5 percent yeast concentration during fermentation of yam to ethanol. The increase in


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ethanol yield at 1.5 or 2.00 percent inoculum size can be attributed to increase the cell density responsible for oxidative metabolism thereby maximum conversion of sugars is possible. Due to the limitation in oxygen, fermentative metabolism would then set earlier as compare to that of lower inoculum size. Among the different pretreatments combination of acid + steam pretreatment was found to give more ethanol yield than the other pretreatments. The milling operations overcome the lignin barrier in cellulose matrix (Wilke and Mitra, 1975). The increased yield in this combined pretreatment is due to removal of lignin and increase of pore space by steam pretreatment which aid complete action of cellulase enzyme on substrate and acid solubilised polymers to monomer (Grohmann et al., 1985; Schwald et al., 1989; Schell et al., 1991; Torget et al., 1996).

CONCLUSIONS Alcohol is a viable transportation fuel and can be used to replace gasoline and diesel fuel in many applications, extend energy supplies and provide energy security. Advances in technology have resulted in higher conversion rates and a wider raw material base for ethanol production. The utilization of bio-ethanol as an energy source has stimulated studies on the cost and efficiency of industrial processes for ethanol production. It has been carried out for obtaining efficient fermentative organisms, low cost-fermentation substrates and optimum environmental conditions for fermentation to occur. At present ethanol is produced from molasses which is a byproduct of sugar Industries. Most of the distilleries operate at high cost, as the cost of molasses is very high. Hence it is absolutely necessary to search for alternate sources for ethanol production. So for, common crops recognized as substrate for ethanol are sugarcane, sweet sorghum, sugar beet and potato. The sunflower head waste is a potential unutilized agriculture residue contain considerable amount of nutrients which can form an ideal medium for ethanol production. Therefore, the substrate was evaluated for ethanol production using various means for releasing fermentable sugars and optimizing various parameters including selection of suitable yeast and bacterial strains, pH, temperature, fermentation time, inoculum size and nutrient supplementation etc.

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Amutha, R. & P. Gunasekaran. (2000). Improved ethanol production by a mixed culture of Saccharomyces diastaticus and Zymomonas mobilis from liquefied cassava starch. Indian Journal of. Microbiology, 40, 103-107.

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Caputi, A., Ueda, M., & Brown, T. (1968). Spectrophotometric determination of ethanol in wine. American Journal of Enology and Viticulture, 19(3), 160-165.

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Dale, B. E., Leong, C. K., Pham, T. K., Esquivel, V. M., Rios, I., & Latimer, V. M. (1996). Hydrolysis of lignocellulosics at low enzyme levels: application of the AFEX process. Bioresource Technology, 56(1), 111-116.

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Davis, L., Rogers, P., Pearce, J., & Peiris, P. (2006). Evaluation of Zymomonas -based ethanol production from a hydrolysed waste starch stream. Biomass and Bioenergy, 30(8), 809-814.

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Doelle, H. W., Kirk, L., Crittenden, R., Toh, H., & Doelle, M. B. (1993). Zymomonas mobilis-science and industrial application. Critical reviews in biotechnology, 13(1), 57-98.


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24. Schell, D. J., Torget, R., Power, A., Walter, P. J., Grohmann, K., & Hinman, N. D. (1991). A technical and economic analysis of acid-catalyzed steam explosion and dilute sulfuric acid pretreatments using wheat straw or aspen wood chips. Applied biochemistry and biotechnology, 28(1), 87-97. 25. Schwald, W., Breuil, C., Brownell, H. H., Chan, M., & Saddler, J. M. (1989). Assessment of pretreatment conditions to obtain fast complete hydrolysis on high substrate concentrations. Applied Biochemistry and biotechnology, 20(1), 29-44. 26. Saigal, D. (1993). Yeast strain development for ethanol production, Indian Journal of Microbiology, 33, 159-168 27. Singh, S.K., V. Kumar, R. Gera, S.S. Dhamija and D. Singh. 2002. Grain alcohol production using thermotolerant yeasts. In: Proceedings of the 43rd annual conference of Association of Microbiologists of India, held at Haryana Agricultural University, Hisar, India, December 11-13, p.130. 28. Slaughter, J.C. 1988. Nitrogen metabolism. In: physiology of Industrial Fungi (D.R. Berry. (Eds) Black well sceintific publications, Oxford, London, p.61. 29. Torget, R., Hatzis, C., Hayward, T. K., Hsu, T. A., & Philippidis, G. (1996, January). Optimization of reverseflow, two-temperature, dilute-acid pretreatment to enhance biomass conversion to ethanol. In Seventeenth Symposium on Biotechnology for Fuels and Chemicals (pp. 85-101). Humana Press. 30. Wilke, CR and Mitra, G, (1975). Process development studies on the enzymatic hydrolysis of cellulose. In: CR Wilke (Editor), Cellulose as a Chemical and Energy Re- source. Biotechnology and bioengineering symposium, 5, pp. 253—274 31. Yamashita, Y., Kurosumi, A., Sasaki, C., & Nakamura, Y. (2008). Ethanol production from paper sludge by immobilized Zymomonas mobilis. Biochemical Engineering Journal, 42(3), 314-319.

APPENDICES

Figure 1: Effect of Acid Treatment on Releasing Reducing Sugar

Figure 2: Effect of Alkali Treatment on Releasing Reducing Sugar


Ethanol Production from Degrained Sunflower Head Waste by Zymomonas mobilis and Saccharomyces cerevisiae

Figure 3: Effect of Fungal Treatment on Releasing Reducing Sugar

Figure 4: Effect of Acid + Steaming Treatment on Releasing Reducing Sugar

Figure 5: Effect of Sugars on Zymomonas mobilis Culture Growth

Figure 6: Effect of Nutrients on Ethanol Production from Sunflower Head Waste

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Figure 7a

Figure 7b

Figure 7c

Figure 7d Figure 7: Effect of Fermentation Periods, pH, Temperature, Inoculum Size on Ethanol Production


12 ethanol production full