Original Research

Utilization of glucose and xylose by Pichia stiptis and Saccharomyces cerevisiae for ethanol fermentation

Alok Kumar Dubey1, Parveen Kumar Gupta1, Neelam Garg 2*

1Cellulose and Paper Division, Forest Research Institute, Dehradun (India)
2Department of Microbiology, Kurukshetra University, Kurukshetra, Haryana (India)

J Innov Biol (2014) Volume 2, Issue 1: Pages: 222-225

Abstract: Lignocellulosic biomass is most abundantly available natural raw material consisting of cellulose, hemicellulose and lignin. Glucose and xylose the main sugar constituents of cellulose and hemicellulose polymer respectively are used for bioethanol production. The ability of microorganisms to produce ethanol by fermenting both xylose (pentoses) and glucose (hexoses) present in lignocellulosic biomass is not widespread. In present study two different types of yeast strains Pichia stipitis NCIM 3499 and Saccharomyces cerevisiae NCIM 3570 were evaluated for utilizing xylose and glucose for ethanol production. Pichia stipitis 3499 produced maximum 3.97±0.21 g/l and 7.94±0.19 g/l of ethanol from glucose and xylose after 40 h fermentation period. Pichia stipitis NCIM 3499 was found to have ability to ferment both glucose and xylose whereas Saccharomyces cerevisiae NCIM 3570 fermented only glucose to ethanol.

Received: 09 January 2015
Accepted: 27 February 2015
Published: 14 March 2015


Corresponding Author:
Garg N.,
Department of Microbiology, Kurukshetra University, Kurukshetra, Haryana (India)
email: nlmgarg@yahoo.com

Keywords: Pichia stipitis, Saccharomyces cerevisiae, Fermentation, Ethanol

IntroductionMaterial & MethodsResults & DiscussionFigures & TablesReferences

The depletion of natural resources and hike in petrol cost leads to search for alternate fuel to replace fossil fuels. In this context, ethanol derived from biomass is advocated as a potential alternative to conventional fossil fuels. An alternate ethanol fuel could be used in many unmodified petrol engines with gasoline (Hansen et al. 2005). Ethanol is mainly produced by fermentation process, although it can also be manufactured synthetically from petroleum (hydration of ethylene). In 1995, about 93% of world ethanol was produced by fermentation and 7% by synthetic method. Today fuel ethanol in the United State and Brazil is made from corn starch, sugarcane bagasse and other food based raw materials. In the developing country like India, the possible competition with food is one of the risky factors when using food based feed stocks for ethanol production. In this regard, the lignocellulosic biomass, which is not a part of human food chain, is envisaged to supply a significant portion of the feed stocks for ethanol production on long term basis due to their low cost and abundance.
Lignocellulosic biomass is mainly composed of cellulose, hemicellulose and lignin. Cellulose is a polymer of six-carbon sugar i.e. glucose. Hemicellulose is a branched polymer comprised of xylose and other five carbon sugars. Lignin consists of basic phenyl propane unit. Advanced bioethanol technology allows fuel ethanol production from cellulose and hemicellulose, greatly expanding the renewable and sustainable resource base available for fuel ethanol production. (Jeffries and Kurtsman, 1994; Preez, 1994). Two main processes, involved in the production of bioethanol from lignocellulosic biomass are: the hydrolysis of the carbohydrate fraction of lignocellulosic biomass i.e. cellulosic and hemicellulosic fraction to produce monomeric sugars (mainly glucose and xylose) and the subsequent fermentation of the sugars to ethanol.
Many efforts have been made by different workers to improve the efficiency of ethanol fermentation from lignocellulosic biomass on industrial scale by developing new native bacteria, yeasts, and fungi or genetically engineered microorganisms and improved processes. Sacharomyces cerevisiae is the most exploited microorganism which ferments hexoses (glucose) to ethanol quite easily. This yeast has high ethanol tolerance, yields and rates of fermentation, but its inability to ferment xylose, the second most abundant constituent sugar of lignocelluloses in nature, limits its use in bioethanol production (Kotter and Ciriacy, 1993). The xylose fraction, on the other hand constitutes 10-40 % of the total carbohydrate (Ladisch et al. 1983). Hence, the total ethanol yield could theoretically be increased by 25% through the use of an efficient xylose fermenting yeast that could convert both hexose and pentose sugar to ethanol (Bjorling and Lindman, 1989). Most studied xylose fermenting yeasts are Pachysolen tannophilus and Candida sp. (Jeffries, 1985) however Pichia stipitis has shown promise for industrial application because it ferments xylose rapidly with high ethanol yield (Preez and Prior, 1985).
Various researches have been focused on solving the problem of fermentation of sugars, present in lignocellulosic hydrolyzates. The fermenting organism must be able to ferment all monosaccharides present in the hydrolyzates. The fermentation processes are usually both capital and operative cost-intensive. Hence, the present study was undertaken to determine the potential of Pichia stipitis for ethanol fermentation from glucose and xylose and compare it with that of Saccharomyces cerevisiae.

Procurement of yeast strains and cultivation
Pichia stipitis NCIM 3499 and Saccharomyces cerevisiae NCIM 3570 were procured from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory (NCL), Pune and used in all the experiments. Pichia stiptis NCIM 3499 was cultivated on medium Yeast Extract Peptone Xylose (YEPX) constituting (g/l) xylose 20.0, yeast extract 3.0, malt extract 3.0, peptone 5.0, agar 20.0 and Saccharomyces cerevisiae NCIM 3570 was cultivated on Yeast Extract Peptone Glucose (YEPG) medium containing (g/l) glucose 20.0, yeast extract 3.0, malt extract 3.0, peptone 5.0, agar 20.0 at pH 5.0 and temperature 30ºC for 3 days (Fig. 1). Both the cultures were maintained on agar slants, stored at 4ºC and subcultured regularly.

Fermentation with Pichia stipitis NCIM 3499 and Saccharomyces cerevisiae NCIM 3570
For the determination of fermentability of both sugars such as glucose and xylose sugars were examined individually with Pichia stipitis and Saccharomyces cerevisiae in 5.0 L lab fermentor (Sartorius, Germany) containing 1.0 L of each fermentation medium (pH 5.0) comprising 20.0 g/l of desired test sugars i.e. xylose and glucose and nutritional supplements as described by Amartey and Jeffries, 1996 (g/l) NH4Cl 0.5, KH2PO4 2.0, MgSO4•7H2O 0.5, yeast extract 1.5, CaCl2•2H2O 0.1, FeCl3•2H2O 0.1, ZnSO4•7H2O 0.001. Inocula were prepared in 100 ml of same fermentation medium at 30ºC for 24 h. Both the test sugars medium were inoculated with 5 % (v/v) of respective inoculums and fermentation was carried out at 30ºC and 150 rpm. Samples were withdrawn at regular time intervals of 8 h and centrifuged at 10,000 rpm for 10 minutes at 4ºC. The supernatant was used for the estimation of sugars and ethanol content.

Analytical determinations
The concentration of total reducing sugars (TRS), xylose and ethanol were analyzed by using UV Spectrophotometer. Total reducing sugars, was estimated by 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959) and was calculated by observing absorbance at 550 nm against standard curve of glucose. The xylose concentration was estimated by þ-bromoanaline method (Bala et al. 2004) by using xylose standard curve and absorbance at 520 nm. Ethanol concentration was determined by chromic acid method (Caputi et al. 1968) by using absolute ethanol as standard and absorbance at 585 nm. The concentration of all the standards was expressed in g/l so that the data of experimental results were expressed in g/l.

Pichia stipitis NCIM 3499 and Saccharomyces cerevisiae NCIM 3570 were tested for fermentation of glucose and xylose separately. Pichia stipitis NCIM 3499 metabolized maximum glucose upto 40 h with 7.43 g/l of residual sugars and maximum xylose upto 32 h with 1.59 g/l of residual sugars after that sugar were almost constant. This yeast also produced maximum ethanol content of 3.97±0.21 g/l and 7.94±0.19 g/l after 40 h of fermentation from glucose and xylose as depicted in figure 2 and 3. Saccharomyces cerevisiae produced maximum 9.83±0.20 g/l of ethanol by utilizing 95.2% of glucose within comparatively short period of 24 h with only 0.96 g/l of residual sugars (Fig. 4). However Saccharomyces cerevisiae consumed only 3.61 g/l of xylose with no ethanol production (Table1). It might be due to absence of genes in Saccharomyces cerevisiae which encode for xylose reductase and xylitol dehydogenase, the key enzymes responsible for ethanol production from xylose (Karhumaa et al. 2006; Taherzadeh and Karimi, 2007). Table 1 shows the comparison of fermentation of both the sugars by Pichia stipitis NCIM 3499 and Saccharomyces cerevisiae NCIM 3570. Glucose was fermented with 80.71 % and 49.38% fermentation efficiency by Saccharomyces cerevisiae and Pichia stipitis respectively. However Pichia stipitis could ferment xylose with higher fermentation efficiency (77.16%). These results indicate that Pichia stipitis can ferment both glucose and xylose for ethanol production. Our results are in line with Chung et al. (2013) and Xavier et al. (2010) who have also described the ethanol production both from xylose and glucose sugar by Pichia stipitis. While studying on ethanol fermentation in Pichia stipitis CBS 6054, Biswas et al. (2013) demonstrated that glucose uptake was rapid than xylose. Aloisio et al. (2014) has also indicated that there is a lag of 4-5 h in xylose uptake as compared to glucose uptake in Pichia stipitis. Biswas et al. (2013) further reported that Pichia stipitis CBS 6054 converted 27.2±0.5 g/l of glucose to 10.1±0.1 g/l of ethanol after 36 h whereas 25.6±0.4 g/l of xylose was converted to 8.5±0.2 g/l of ethanol after longer time duration of 82 h. On the contrary, in our study maximum ethanol was produced from glucose (3.97g/l) and xylose (7.94 g/l) in the same time i.e. 40 h utilizing 12.57g/l and 18.41g/l of sugar respectively.

Conclusion
To realize the industrial ethanol production from lignocellulosic biomass, it is essential to obtain microbial strains capable of converting all the major sugars present in lignocellulosic hydrolyzate. The present study concludes that Pichia stiptis NCIM 3499 has the potential to ferment both the glucose and xylose for production of ethanol whereas Saccharomyces cerevisiae NCIM 3570 could ferment only glucose for ethanol production. As hemicellulosic fraction comprising of pentoses such as xylose is also a major part of a lignocellulosic biomass, its conversion to ethanol can improve the efficiency of the process. So the use of Pichia stiptis NCIM 3499 or in addition to Saccharomyces cerevisiae NCIM 3570 may improve the fermentation process by utilizing the two main constituent sugars of lignocellulosic hydrolyzates obtain from cellulosic and hemicellulosic fraction of lignocellulosic biomass.

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