Enzyme and Microbial Technology 41 (2007) 876880

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)
Serpil Ozmihci, Fikret Kargi
Department of Environmental Engineering, Dokuz Eylul University, Buca, Izmir, Turkey Received 25 April 2007; received in revised form 11 July 2007; accepted 18 July 2007

Abstract Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l1 while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the efuent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l1 . Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l1 and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v1 ) and productivity (0.54 gE l1 h1 ) were obtained with the feed sugar content of 100 g l1 or 125 g l1 . The ethanol yield coefcient (YP/S ) was also maximum (0.49 gE gS1 ) when the feed sugar was between 100 and 125 g l1 . The growth yield coefcient (YX/S ) decreased steadily from 0.123 to 0.063 gX gS1 when the feed sugar increased from 55 to 200 g l1 due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l1 under the specied experimental conditions. 2007 Elsevier Inc. All rights reserved.
Keywords: Cheese whey powder (CWP); Continuous ethanol fermentation; Feed sugar content; Kluyveromyces marxianus

1. Introduction The world production of cheese whey keeps increasing steadily and is estimated to be over 108 tonnes per year yielding an important source of environmental pollution [1]. Approximately, 10 l cheese whey is produced from 1 kg cheese with high carbohydrate, protein and lipid contents as a liquid waste. Due to high COD content (approximately 80 g l1 ) cheese whey is usually considered as a high strength wastewater from environmental point of view. For this reason biological treatment of cheese whey by conventional activated sludge processes is very expensive (approximately 50 cents/kg COD). Anaerobic treatment of cheese whey is economically more attractive due to production of energy rich methane. Production of valuable chemicals from cheese whey has been considered as an attractive option by many investigators. Because of rich nutrient content, cheese whey has been used for production of different chem-

Corresponding author. Tel.: +90 232 4127109; fax: +90 232 4531143. E-mail address: kret.kargi@deu.edu.tr (F. Kargi).

icals such as organic acids (lactic, acetic), alcohol (ethanol), single cell protein, methane and cheese whey powder [2]. Cheese whey has been used by many investigators as the raw material for ethanol fermentations because of its high carbohydrate content and availability [27]. Typical cheese whey contains 56% lactose (w v1 ), 0.81% protein, and 0.06% fat constituting an inexpensive and nutritionally rich raw material for ethanol fermentations. Direct fermentation of cheese whey to ethanol yields low ethanol concentrations (23%, v v1 ) and, therefore, is not economical because of low lactose content (56%, w v1 ) of raw cheese whey. Cost of raw materials and ethanol separation from dilute fermentation broths are the major expenses in ethanol fermentations. Despite the fact that, cheese whey (CW) is an inexpensive raw material, distillation costs for ethanol separation from dilute fermentation broths (23% EtOH) is still a major cost item in ethanol fermentation of CW [2]. Ultraltration processes used to concentrate lactose in cheese whey before fermentation are expensive (approximately 50 USD/m3 ) which only improve lactose concentration by a factor of 5 [8]. Dry cheese whey powder (CWP) may be an attractive raw material for ethanol production.

0141-0229/$ see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.07.015

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Utilization of CWP instead of cheese whey eliminates costly ultraltration process and provides concentrated source of lactose and other nutrients yielding a more economical process for ethanol production. The cost of cheese whey powder (CWP) production from cheese whey (CW) by spray or drum drying varies between 20 and 40 cents/kg CWP which compensates distillation costs for pure ethanol production from dilute cheese whey. Threoretically, high ethanol concentrations (1012%, v v1 ) can be obtained by fermentation of concentrated CWP solutions (200 g lactose l1 ) to reduce distillation costs. Kluyveromyces species has been the most widely used yeast strains for ethanol fermentation from cheese whey due to galactose fermenting capability of this yeast strain. Ethanol fermentation of cheese whey was realized by different methods including micro-aeration in batch fermentations [9]; continuous fermentations at different hydraulic residence times [10]; use of thermotolerant yeast strains at 45 C [1113]; immobilization of thermotolerant yeasts on delignied cellulosic material [5]; alginate immobilized thermotolerant yeasts [14], and fed-batch fermentation of cheese whey [1517]. The major drawback in almost all cheese whey fermentations was low ethanol yields and, therefore, high ethanol recovery costs. There are a limited number of studies on utilization of cheese whey powder (CWP) solution for ethanol production which were published recently by our group [1821]. CWP is a dried and concentrated form of cheese whey and contains lactose in addition to nitrogen, phosphate and other essential nutrients. The use of CWP instead of cheese whey (CW) for ethanol fermentations has signicant advantages such as elimination of costly ultraltration processes, compact volume, long-term stability and high concentrations of lactose and other nutrients yielding high ethanol concentrations by fermentation. Use of highly concentrated CWP solutions (200300 g l1 CWP) for ethanol fermentations yields high ethanol concentrations and reduces distillation costs for ethanol separation. Continuous ethanol fermentations offer special advantages over batch and fed-batch operations by providing constant efuent quality, high productivity and control over the product concentration by adjusting the feed sugar concentration and the operating HRT. Multistage operation, cell recycle and simultaneous ethanol removal improves the performance of continuous fermentation. Continuous fermentations of ultraltered cheese whey were reported in literature with low ethanol yields [4,6,10]. Effects of hydraulic residence time on continuous ethanol fermentation of CWP were studied by Ozmihci and Kargi [19]. However, there are no literature reports investigating the effects of feed CWP or sugar concentration on continuous ethanol fermentation of CWP to ethanol. Therefore, the major objective of this study is to investigate the effects of feed sugar or CWP concentrations on ethanol fermentation of cheese whey powder (CWP) solution in a continuous fermenter. Sugar concentration in the feed CWP solution was changed between 55 and 200 g l1 while the HRT was kept constant at 54 h. Sugar utilization, ethanol and biomass formation were investigated at different feed sugar concentrations.

2. Materials and methods 2.1. Experimental system

Continuous experiments were performed by using a 5 l fermenter (New Brunswick, Model IIC). The operation was started batch-wise with sterile CWP solution (100 g l1 sugar) inoculated with 1 l pure culture of K. marxianus (DSMZ 7239) which continued until residual sugar was negligible. Continuous operation was started by feeding and removing the CWP solution to the fermenter with a desired ow rate. The volume of the fermentation media in the fermenter was 3 l with a constant HRT of 54 h. Sterilized feed CWP solution was kept in a refrigerator at 4 C to avoid any decomposition and was fed to the reactor under aseptic conditions with a desired ow rate using a peristaltic pump (Watson-Marlow model 323, UK). Samples were withdrawn from the fermenter aseptically every day for pH, ORP, total sugar, biomass (total suspended solids) and ethanol measurements. Na-thioglycolate (200 mg l1 ) was added to the feed CWP solution in order to adjust the ORP to lower than 200 mV. Agitation speed was 100 rpm with N2 gas passage through the fermenter for 15 min every day. pH of feed CWP solution was adjusted to 5 before sterilization. pH of the fermentation media varied between 4.3 and 4.6 during operation while the temperature was controlled at 28 1 C. Every continuous operation lasted until the system reached the steady-state with approximately the same sugar, ethanol and biomass concentrations in the fermenter and the efuent at least for the last 4 days. Every experiment lasted about 810 HRT (430540 h). Control experiments were performed in the absence of yeast cells to determine non-biological sugar utilization under the same experimental conditions as that of the actual experiments.

2.2. Organisms
Kluyveromyces marxianus strain (DSMZ-7239) obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) in lyophilized form was used in all experiments. The yeast strain was cultivated in the growth medium using an incubator shaker under sterile conditions at pH 5, 28 C and 150 rpm. The inoculum culture was prepared by inoculating 180 ml sterile CWP (50 g l1 ) solution with 20 ml of the pure yeast strain (total 200 ml culture volume) in 500 ml Erlenmeyer asks. The culture was grown in an incubator gyratory shaker, at 150 rpm and at 28 C for 5 days. Then, ve Erlenmeyer asks containing Kluyveromyces marxianus cultures with a total volume of 1 l were used for inoculation of the fermenter before the continuous operation.

2.3. Medium composition

The growth medium used for cultivation of inoculum culture consisted of yeast extract (5 g l1 ), peptone (5 g l1 ), NH4 Cl (2 g l1 ), KH2 PO4 (1 g l1 ), MgSO4 7H2 O (0.3 g l1 ), lactose (30 g l1 ) and 200 mg l1 Na-thioglycolate as the reducing agent at pH 5. The initial oxidationreduction potential (ORP) of the media was nearly 250 mV indicating anaerobic conditions. The feed media in continuous experiments had different CWP contents between 82 and 300 g l1 yielding total sugar (TS) concentrations between 55 and 200 g l1 and also 200 mg l1 Na-thioglycolate (ORP = 250 mV) in deionized water at pH 5. Feed CWP solution was heated to 90 C for deproteinization, the solids were removed and the supernatant was autoclaved at 121 C for 20 min for sterilization. Sterilized feed CWP solution was kept in a refrigerator at 4 C to avoid any decomposition. Cheese whey powder (CWP) was obtained from Pinar Dairy Industry in Izmir, Turkey and was dried at 80 C before use. Dry CWP contained approximately 67% total sugar, 12.5% protein, 2.2% fats, 2% total nitrogen and 1.4% total phosphorous on dry weight basis.

2.4. Analytical methods

The samples were taken from the fermenter everyday and centrifuged at 8000 rpm (7000 g) for 30 min to remove solids from the liquid media. Analyses were carried out on the supernatants after centrifugation. Total reducing sugar concentrations were measured by using the phenolacid method [22]. The samples were analyzed in triplicates and results were reproducible within 3%

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deviation. Ethanol concentrations were measured using a Gas Chromatograph (Varian CP-3800) with an FID detector and a WCOT fused silica capillary column (15 m 0.25 mm ID, 0.25 m lm thickness). The column temperature was set for 75 C for 1 min and raised to 130 C with a rate of 20 C/min yielding a total holding time of 4.75 min. Temperatures of injector and detector were 150 C and 200 C, respectively. Nitrogen was used as the carrier gas with a linear velocity of 25 ml min1 . Total suspended solids (TSS) were also determined by drying 10 ml samples from the feed and the reactor at 105 C until constant weight. Difference in total suspended solids content of the fermenter broth and the feed was considered as the biomass yield during fermentation.

2.5. Calculation methods

The total amounts of sugar utilization, ethanol and biomass formation in continuous experiments were calculated using the following equations: S = Se So , P = Pe Po , X = Xe Xo

Fig. 2. Variation of percent ethanol and ethanol productivity with the feed sugar concentration.

where S, P, X are the total amount of sugar (substrate) utilized, ethanol (product) and the biomass (yeasts) produced for every operation (g l1 ); So , Po and Xo are the feed sugar, ethanol and biomass concentrations (g l1 ); Se , Pe and Xe are the efuent or the reactor sugar, ethanol and biomass concentrations at the steady-state for every operation (g l1 ). The yield coefcients, YP/S (gP g1 S) and YX/S (gX g1 S), were calculated by using the following equations at the steady-state for every experiment. YP/S = P , S YX/S = X S

3. Results and discussion Continuous experiments were performed at six different feed sugar concentrations between 55 and 200 g l1 at a constant HRT of 54 h. Fig. 1 depicts variation of the efuent total sugar concentration and percent sugar utilization with the feed sugar concentration. The efuent sugar increased and percent sugar utilization decreased with increasing feed sugar content due to adverse effects of high sugar concentrations on sugar utilization by the yeast cells. The efuent sugar increased from 15.6 g l1 (So = 55 g l1 ) to 146.3 g l1 (So = 200 g l1 ) and percent sugar utilization decreased from 71.6 to 26.6% when the feed sugar content increased from 55 to 200 g l1 . Apparently high sugar concentrations and other dissolved solids increased the osmotic pressure of the fermentation broth which resulted in considerable activity loss in the yeast cells.

Variations of ethanol concentrations (P) and productivity (DP) with the feed sugar concentration are shown in Fig. 2. Both nal ethanol concentration (P) and productivity (DP) increased with the feed sugar content up to 100 g l1 and reached maximum levels of 3.7% (v v1 ) and 0.54 gE l1 h1 , respectively. Further increases in the feed sugar content resulted in decreases in ethanol yield and productivity due to adverse effects of high osmotic pressure at high sugar concentrations. The optimal feed sugar content resulting in the highest ethanol yield and productivity was 100 g l1 although the results obtained at 125 g l1 feed sugar concentration were close to that obtained at 100 g l1 . Ethanol concentration and the productivity decreased to 2% (v v1 ) and 0.29 gE l1 h1 when the feed sugar content was increased to 200 g l1 . Fig. 3 depicts variation of biomass (yeast) concentration (X) and the biomass productivity (DX) with the feed sugar content at an HRT of 54 h. Both biomass concentration and productivity did not change signicantly for the feed sugar concentrations between 55 and 125 g l1 . However, further increases in the feed sugar content above 125 g l1 resulted in considerable decreases in both biomass concentration and the productivity. Biomass concentration and the productivity decreased to 3.34 gX l1 and 0.062 gX l1 h1 when the feed sugar content was increased to 200 g l1 . Variations of the ethanol (YP/S ) and the growth (YX/S ) yield coefcients with the feed sugar content are depicted in

Fig. 1. Variation of percent sugar utilization and efuent sugar content with the feed sugar concentration.

Fig. 3. Variation of biomass concentration and biomass productivity with the feed sugar concentration.

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Fig. 4. Variation of product and biomass yield coefcients with the feed sugar concentration.

Fig. 4. The ethanol yield coefcient increased from 0.465 to 0.493 gE g1 S (theoretical yield is 0.54 gE g1 lactose) when the feed sugar was increased from 55 to 102 g l1 . Further increases in the feed sugar resulted in decreases in the YP/S and yielded a yield coefcient of 0.3 gE g1 S when the feed sugar was 200 g l1 . The optimal feed sugar content maximizing the ethanol yield coefcient was between100 and 125 g l1 . Unlike ethanol yield, the biomass yield coefcient (YX/S ) decreased almost steadily with the increasing feed sugar content. An increase in the feed sugar content from 55 to 200 g l1 resulted in a decrease in the biomass yield coefcient from 0.123 to 0.063 gX g1 S. Fig. 5 depicts variations of volumetric rates of sugar utilization and product (ethanol) formation with the feed sugar concentration where RS and RP were calculated by using the following equations. Q(So S ) = D(So S ), V Q(P Po ) RP = = D(P Po ) V Rs = where So and S are the feed and efuent sugar concentrations at the steady-state (gS l1 ); Po and P are the feed and efuent ethanol concentrations at the steady-state (gE l1 ) and Po

is zero since the feed is ethanol free; Q and V are the feed ow rate (l h1 ) and the volume of fermentation broth (l). Sugar utilization rate (RS ) increased with increasing feed sugar content up to 100 g l1 (Se = 44 g l1 ) and reached a maximum level of 1.09 gS l1 h1 which decreased considerably with further increases in the feed sugar above 125 g l1 (Se = 66 g l1 ). Ethanol formation rate showed a similar trend and increased with increasing feed sugar content up to 100 g l1 and then decreased with further increases in the feed sugar above 125 g l1 . The optimal feed sugar content was between 100 and 125 g l1 maximizing the rates of sugar utilization and ethanol formation. Substrate inhibition at high sugar concentrations in ethanol fermentation has also been observed by other investigators [910,21]. In this study, substrate inhibition was observed for the feed sugar concentrations above 125 g l1 (since the results with So = 100 g l1 and 125 g l1 were not much different) corresponding to the steady-state sugar concentration in the fermenter of 66 g l1 . Presence of solid cheese whey powder (CWP) and other dissolved nutrients along with sugar in the fermenter broth has also contributed to high osmotic pressure development causing inhibition on the metabolism of the yeast cells. Percent sugar utilization and ethanol formation obtained at the high feed sugar concentrations may be improved by operation with cell recycle in continuous culture. 4. Conclusions Continuous fermentation of cheese whey powder (CWP) solution to ethanol was investigated at different feed sugar concentrations (55200 g l1 ) in order to investigate the effects of high sugar concentrations on ethanol formation. A pure culture of Kluyveromyces marxianus (DSMZ 7239) was used in a continuous fermenter under anaerobic conditions. Sugar utilization, ethanol formation and the yeast growth were quantied at different feed sugar concentrations varying between 55 and 200 g l1 . The steady-state efuent sugar concentration increased and percent sugar removal decreased with increasing feed sugar content due to high osmotic pressure caused by high sugar concentrations. Ethanol concentration (P) and productivity (DP) were maximum (3.7% v v1 , and 0.54 gE l1 h1 ) at the feed sugar concentration of 100 g l1 which decreased with further increases in the feed sugar. Steady-state biomass concentration (X) and productivity (DX) also decreased considerably for the feed sugar contents above 100 g l1 indicating adverse effects of high sugar contents on the yeast growth. The ethanol yield coefcient (YP/S ) was also maximum at the feed sugar content of 100 g l1 and decreased with further increases in the sugar content above 125 g l1 . Biomass yield coefcient decreased steadily with the increasing feed sugar concentration where the decrease was more pronounced at sugar concentrations above 100 g l1 . Similar to the other results, the rate of sugar utilization and ethanol formation was also maximum when the feed sugar content was 100 g l1 . The results obtained with 125 g l1 feed sugar content were not much different from those obtained at 100 g l1 and considerable decreases were observed above 125 g l1 feed sugar. Therefore, the optimal feed sugar content was between 100 and 125 g l1 maximiz-

Fig. 5. Variation of volumetric sugar utilization and product formation rates with the feed sugar concentration.

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