Production of fructose from highly concentrated date extracts using Saccharomyces cerevisiae
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- Putra, M.D., Abasaeed, A.E., Al-Zahrani, S.M. et al. Biotechnol Lett (2014) 36: 531. doi:10.1007/s10529-013-1388-y
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Large amounts of low-quality dates produced worldwide are wasted. Here, highly concentrated fructose syrups were produced via selective fermentation of date extracts with Saccharomyces cerevisiae. Syrups with 95.4–99.9 % (w/w) fructose yields were obtained from date extracts having an initial sugar range of 49–374 g/l without media supplementation; the corresponding ethanol yields were between 69 and 52 % (w/w). At 470 g initial sugars/l, fructose and ethanol yields were 84 and 47 % (w/w), respectively, and the product contained 62 % (w/w) fructose, which is higher than the widely available commercial 42 and 55 % (w/w) high fructose corn syrups. The commercial potential for conversion of waste dates to high-value products is thus demonstrated.
KeywordsDatesEthanolFructoseHigh fructose syrupSaccharomyces cerevisiaeSelective fermentation
Palm dates (Phoenix dactylifera) are grown in many parts of the world and in 2010 the worldwide production was about 7.9 million tons (Jain 2012). Unfortunately, most of the dates produced are wasted (Moshaf et al. 2011). Dates contain mainly equal amounts of fructose and glucose with small amounts of sucrose, protein, minerals and vitamins (Baliga et al. 2011). Fructose is the sweetest natural sugar, 30 % sweeter than sucrose and 80 % sweeter than glucose. Because they are isomers, glucose and fructose are very difficult to separate when present in a mixture. Fructan-rich natural materials, such as Jerusalem artichoke, chicory and dahlia, tubers can be hydrolyzed to produce fructose (Abasaeed and Lee 1995), which is widely used in the food, beverage, confectionery and pharmaceutical industries (Johnson et al. 2009). It is, therefore, critically important to find new ways to use the natural, sustainable and unused resource of dates.
Because of equilibrium limitations, industrial enzymatic isomerization processes produce high fructose syrups (HFS) that contain only 42 % (w/w) fructose (Gaily et al. 2010; Zhang et al. 2004). 90 % (w/w) HFS can be produced via multistage chromatographic processes or membrane technology (Paugam et al. 1996) but both processes suffer from high cost and separation difficulties. A promising process for the production of fructose is selective fermentation of sugars to ethanol (Koren and Duvnjak 1992). Preliminary economic analysis has shown the viability of this process (Carvalho et al. 2008). Using high initial sugar concentration for the production of ethanol minimizes subsequent distillation costs and avoids osmo-sensitive contaminants in the fermentation broth (Jones et al. 1981).
Many microbial strains have been used to ferment sucrose media or glucose/fructose mixtures (Atiyeh and Duvnjak 2001). However, a fermentation process using most of these strains showed significant losses of fructose (up to 50 % w/w in some cases) and formation of undesired by-products, e.g., sorbitol, >4 % (w/w) (Carvalho et al. 2008). Sacharomyces cerevisiae showed high fructose and ethanol yields (Atiyeh and Duvnjak 2001).
Co-production of fructose and ethanol from date extracts at high initial sugar concentrations (>140 g/l) is challenging. Therefore, the objective of the present study was to examine the ability of S. cerevisiae to produce high fructose syrups and ethanol from highly concentrated date extracts without nutrient addition during fermentation.
Materials and methods
Sugars were extracted from dates using deionized water at 40 °C for 2 h. The ratio of pitted dates to water was 2:5 (w/w). The extract was centrifuged at 9500×g for 6 min to remove suspended solids and fibers. The final date syrup, without supplement addition, was then autoclaved at 121 °C for 15 min. The initial sugar concentration (ISC) in the prepared date syrups was between 49 and 470 g/l.
Microorganism and media
The inoculum was prepared by aseptically transferring a loopful of S. cerevisiae ATCC 36858 from an agar slant to a 500 ml Erlenmeyer flask containing 100 ml medium. The Yeast Malt Broth used consisted of 3 g bacto-yeast extract, 3 g bacto-malt extract, 5 g bacto-peptone and 10 g bacto-dextrose and de-ionized water (up to 1 l). The medium was then autoclaved. The transferred yeast was further propagated for 48 h at 30 °C with shaking.
Fermentation experiments were carried out in 500 ml Erlenmeyer flasks (100 ml working volume) placed in a rotary shaker at 30 °C. Liquid samples were withdrawn periodically to measure cell mass, sucrose, fructose, glucose, ethanol, glycerol and sorbitol concentrations. One set of experiments was conducted in a 1 l fermentor with an ISC of 139 g/l for comparison with the results of the 500 ml flasks.
Cell mass concentration (biomass), was determined using the dry weight method. Samples were dried overnight at 105 °C. Cell count and viability were determined using a cell counter system.
Sugars, ethanol, glycerol and sorbitol concentrations were determined using HPLC equipped with an RI detector and Aminex column. The column was at 40 °C and 1 mM H2SO4 was used as the mobile phase at 0.8 ml/min.
Results and discussion
Selective fermentation of date syrups
Effect of initial sugar concentrations in date syrups
Performance of S. cerevisiae in producing fructose, ethanol and biomass from date syrups at various initial sugar concentrations
Initial sugar (g/l)
Initial biomass (g/l)
Biomass yielda (g/g)
Fructose yieldb (%) (w/w)
Ethanol productivitya (g l−1 h−1)
Ethanol yielda (%) (w/w)
Significant drops in ethanol yield and productivity were observed with increasing ISC. At the lowest ISC, the ethanol yield was 52 % (w/w) of its theoretical value because of the relatively high glucose consumption during the short lag phase, as manifested by a much higher biomass yield compared with those obtained at 139 g/l. The biomass yield dropped with increasing ISC due to substrate inhibition (Jones et al. 1981). Ethanol yields obtained in the present study were lower than those obtained by Atiyeh and Duvnjak (2001) but were slightly higher than those obtained by the same authors (Atiyeh and Duvnjak 2002) at high ISC.
For comparative purposes, identical experiments at 139 g initial sugar/l were conducted in a 1 l fermentor. Slightly better results were obtained than for experiments performed in the flasks. This slight improvement was attributed to better mixing and also to the continuous and uninterrupted operation of the system when taking samples (Nuanpeng et al. 2011).
Fructose and by-products
Performance of S. cerevisiae in consumption of sucrose and production of fructose and by-products during the selective fermentation process
Initial sugar (g/l)
Fructose lossa (%) (w/w)
Fructose fraction (%) (w/w)
Sucrose hydrolyzedb (%) (w/w)
The economic viability of any process is affected by formation of by-products, because of downstream separation costs. One of the shortcomings of the production of fructose by selective fermentation is the formation of sorbitol. In processes using Z. mobilis ATCC 39676 (Doelle and Greenfield 1985), ATCC 53431 (Bringer-Meyer et al. 1985) and ATCC 53432 (Suntinanalert et al. 1986), at least 35, 60 and 86 g sorbitol/l was formed, respectively. In the present work, only a small amount (<2 g/l) of sorbitol was formed after complete consumption of glucose.
Glycerol increased with ISC but the amounts of glycerol produced were still lower than those reported: e.g., at an ISC of about 270 g/l, processes using media comprising date syrup (290 g/l in Table 2), sucrose [271 g/l in Atiyeh and Duvnjak (2001)], and sugar beet molasses [242 g/l in Atiyeh and Duvnjak (2002)] resulted in glycerol of 4.8, 7.2 and 16.1 g/l, respectively. More glycerol was found in beet molasses because of its higher mineral content (Maiorella et al. 1984). The presence of glycerol affects ethanol yield (Oura 1977) and, because glycerol increased (Table 2) with ISC, the ethanol yield decreased (Table 1).
In conclusion, unused dates constitute a natural and sustainable raw material for the production of high fructose syrups (>90 % w/w) via selective fermentation of the glucose component in date extracts by S. cerevisiae.
The authors extend their appreciation to the Deanship of Scientific Research and the Research Center at the College of Engineering, King Saud University, for supporting this work.