BIOETHANOL PRODUCTION FROM BIOMASS BY USING THERMOPHILES

Abstract

In the present study, a thermotolerant microorganism, which utilizes a variety ofsugars, was isolated from soil sample, and characterized and used for further fermentation studies. The strain was identified as Kluyveromyces sp. IIPE453 (KS), a yeast strain, and was found to grow on both glucose and xylose as the substrate. The strain was also found to be tolerant to high concentration of glucose and ethanol. However, the cell mass yield was higher for xylose than that for glucose as substrate. The optimum temperature and pH for growth and fermentations were found to be 50 °C and 5.0, respectively. In a mixture of glucose and xylose, the strain produced both ethanol and xylitol simultaneously. At an initial glucose concentration of 300 g 1* , a maximum ethanol concentration 86.8 g 1"' was obtained. The sugarcane bagasse (SCB) was selected as a lignocellulosic biomass for the production of ethanol. Sulfuric acid (H2S04) was used as a catalyst for the hydrolysis of SCB at a temperature of 121 °C and a solid to liquid ratio of 1:10. The optimum acid concentration was found to be 5.4%(w/w). High temperature was found to promote the formation of furfural and, therefore, further hydrolysis experiments were conducted at a lower temperature of 100 °C. Hydrolysis experiments were also conducted in two stages at a lower temperature. The optimum hydrolysis conditions during first stage of hydrolysis with dilute acid were found to be 8%H2SO4 with a solid to liquid ratio of 1:4for a period of 75 min. The optimum conditions for the second stage of hydrolysis were found to be 40 % H2S04 with a solid to liquid ratio of 1:4 at 80 °C in 60 min. The total recovery of sugars in the first and second stages together was estimated to be ~92 % of the total recoverable sugar. The sugars from the hydrolysate were recovered by using a mixture of amberlite IRA- 904 and alumina in the ratio of 5:2 as anion exchange resin. About 95 % acid free sugars were recovered and about 95 % acid was recovered from the regeneration of the column. The acid free sugars present in the hydrolysate were used for the growth of the strain as well as fermentation to ethanol. The recovered acid was recycled back to the digester for hydrolysis of fresh bagasse. The yeast KS was grown on xylose rich bagasse hydrolysate obtained from the first stage of acid hydrolysis at 100 °C. The maximum cell mass concentration of 5.35 g 1 was obtained with a cell mass yield of 58 g g"1 and the specific growth rate of 0.13 h"1. The batch fermentation of bagasse hydrolysate (obtained from dilute acid hydrolysis at 121 °C), gave a maximum 5.4 g l"1 of ethanol and 11.7 g 1" of xylitol at 50 °C. Batch fermentation of xylose rich bagasse hydrolysate, obtained from the first stage of acid hydrolysis at 100 °C, by KS yielded amaximum ethanol concentration of 3gl"1 and axylitol concentration of 14.8 g1" in the broth. Batch fermentation of glucose rich bagasse hydrolysate, obtained from the second stage ofacid hydrolysis at 80 °C, by KS resulted in amaximum ethanol concentration of14.8 g l"1 in the broth. The continuous fermentation of hydrolysate with cell recycle was also carried out, and the effect of dilution rate, cell mass concentration in the broth, and nitrogen purging rate used for in-situ ethanol recovery was studied. Almost 90 % of ethanol was recovered during fermentation by stripping by air/N2 which resulted in five times higher concentration of ethanol than that obtained in the fermentation broth. The fermentation was carried out for about 20 days with no reduction in the production of ethanol. This showed the stability of the yeast KS for fermentation at 50 °C for bagasse hydrolysate. Various substrates like sugarcane juice, molasses and mahua flower extract were also used for fermentation using the strain KS in a batch fermentation. Ethanol productivities of 2 g l"1 h"\ 2.6 g l"1 h"1 and 3.4 g l"1 h"1, respectively were obtained with juice, molasses and mahua flower extract. In order to see the effect of mixed substrate with enhanced sugar concentration, batch fermentation was carried out using a mixture of bagasse hydrolysate with sugarcane juice, molasses and mahua flower extract. The ethanol productivity was found to have increased with the enhancement in sugar concentration in the mixed substrate. The alcohol dehydrogenase (ADH) was extracted from the KS strain. The effect of ethanol concentration, temperature, pH and thermal stability was studied. The Michaelis- Menten constant (Km) and maximum specific activity were found to be 0.0139 M and 139 U nig"1, respectively. The highest enzyme activity was observed at a pH of 8.8 and at 45 °C temperature. The ADH activity decreased steeply after 50 °C with the total activity loss being observed at a temperature, T > 70 °C. It was observed that the Fe ions have positive impact and promote the enzyme activity upto 25 mg 1" concentration; whereas zinc ions have a negative effect and they retard the activity of the ADH. About 4.2 fold increase in activity was observed in the precipitate when ammonium sulphate was used in the precipitation. The specific activity of the purified ADH was 584.6 Umg"1 and the turnover number of purified ADH was 70x104 min"1. The molecular mass of the ADH was observed to be 45 kDa. It is concluded that the new thermotolerant strain KS shows good activity for growth and fermentation at 50 °C. Bagasse, an abundantly available waste produced from the sugar industry, can be used as asource of various kinds of sugar for fermentation, through two-stage acid hydrolysis reaction