enzyme and microbial technology, volume 41, issues 6–7, 1 november 2007, pages 876-880.pdf

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Enzyme and Microbial Technology 41 (2007) 876880

Effects of feed sugar concentration on continuous ethanolfermentation 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

whilethe 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 effluent 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 125gl1. The ethanol

yield coefficient (YP/S) was also maximum (0.49 gE gS1) when the feed sugar was between 100 and 125 g l1. The growth yield coefficient (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 specified 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]. Approxi-

mately, 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 envi-

ronmental 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

chemicalsfrom cheesewhey hasbeen 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: [email protected] (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 car-

bohydrate 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 mate-

rial 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 lac-

tose content (56%, w v1) of raw cheese whey. Cost of raw

materials and ethanol separation from dilute fermentationbroths

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 fermenta-

tion broths (23% EtOH) is still a major cost item in ethanol

fermentation of CW [2]. Ultrafiltration processesused to concen-

trate lactose in cheese whey before fermentation are expensive

(approximately 50 USD/m3) which only improve lactose con-

centration 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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S. Ozmihci, F. Kargi / Enzyme and Microbial Technology 41 (2007) 876880 877

Utilization of CWP instead of cheese whey eliminates costly

ultrafiltration process and provides concentrated source of

lactose and other nutrients yielding a more economical pro-

cess 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 com-

pensates distillation costs for pure ethanol production from

dilute cheese whey. Threoretically, high ethanol concentrations

(1012%, v v1) can be obtained by fermentation of concen-

trated 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 fer-

mentation 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 delignified cellulosic material [5];

alginate immobilized thermotolerant yeasts [14], and fed-batchfermentation 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 productionwhich were

published recently by our group [1821]. CWP is a dried and

concentrated form of cheese whey and contains lactose in addi-

tion to nitrogen, phosphate and other essential nutrients. The

use of CWP instead of cheese whey (CW) for ethanol fermenta-

tions has significant advantages such as elimination of costly

ultrafiltration processes, compact volume, long-term stability

and high concentrations of lactose and other nutrients yield-ing 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

effluent quality, high productivity and control over the prod-

uct 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 ultrafil-

tered cheese whey were reported in literature with low ethanol

yields [4,6,10]. Effects of hydraulic residence time on contin-uous ethanol fermentation of CWP were studied by Ozmihci

and Kargi [19]. However, there are no literature reports inves-

tigating 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 fer-

menter. 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 for-

mation were investigated at different feed sugar concentra-

tions.

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. Continu-

ous operation was started by feeding and removing the CWP solution to the

fermenter with a desired flow 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 flow rate using a peristaltic

pump (Watson-Marlow model 323, UK). Samples were withdrawn from the fer-

menter aseptically every day for pH, ORP, total sugar, biomass (total suspended

solids) and ethanol measurements. Na-thioglycolate (200mg l1) was added to

the feed CWP solution in order to adjust the ORP to lower than 200 mV. Agi-

tation speed was 100rpm with N2 gas passage through the fermenter for 15 min

everyday. pH of feed CWP solution was adjusted to 5 beforesterilization. pH of

the fermentation media varied between 4.3 and 4.6 during operation while the

temperature wascontrolled at 28 1 C. Every continuous operationlasted until

the system reached the steady-state with approximately the same sugar, ethanol

and biomass concentrations in the fermenter and the effluent at least for the last

4 days. Every experiment lasted about 810 HRT (430540 h). Control experi-

ments 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 150rpm.

The inoculum culture was prepared by inoculating 180 ml sterile CWP

(50gl1) solution with 20 ml of the pure yeast strain (total 200 ml culture

volume) in 500ml Erlenmeyer flasks. The culture was grown in an incuba-tor gyratory shaker, at 150 rpm and at 28 C for 5 days. Then, five Erlenmeyer

flasks 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 (5g l1), peptone (5 g l1), NH4Cl (2gl1), KH2 PO4 (1 g l

1),

MgSO47H2O (0.3gl1), lactose (30gl1) and 200 mgl1 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

300gl1 yielding total sugar (TS) concentrations between 55 and 200g l1

and also 200mg l1 Na-thioglycolate (ORP =250mV) 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 anydecomposition. Cheese wheypowder(CWP) was obtainedfrom 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. Anal-

yses 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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878 S. Ozmihci, F. Kargi / Enzyme and Microbial Technology 41 (2007) 876880

deviation. Ethanol concentrations were measured using a Gas Chromatograph

(Varian CP-3800) with an FID detector and a WCOT fused silica capillary col-

umn (15 m0.25mm ID, 0.25m film thickness). The column temperature

was set for 75 C for 1 min and raised to 130C with a rate of 20 C/min yield-

ing 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 suspendedsolids (TSS) were also determined by drying10 ml samples

from the feed and the reactor at 105 C until constant weight. Difference in totalsuspended 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

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, Poand Xo are the feed sugar, ethanol and biomass concentrations (g l

1); Se, Peand Xe are the effluent or the reactor sugar, ethanol and biomass concentrations

at the steady-state for every operation (g l1

).The yield coefficients, YP/S (gPg

1S) and YX/S (gXg1S), 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 effluent total sugar

concentration and percent sugar utilization with the feed sugar

concentration. The effluent sugar increased and percent sugar

utilization decreased with increasing feed sugar content due

to adverse effects of high sugar concentrations on sugar uti-

lization by the yeast cells. The effluent sugar increased from

15.6gl1(So = 5 5 g l1) to 146.3gl1 (So =200gl

1) and per-

cent 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.

Fig. 1. Variation of percent sugar utilization and effluent sugar content with the

feed sugar concentration.

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

concentration.

Variations of ethanol concentrations (P) and productivity

(DP) with the feed sugar concentration are shown in Fig. 2. Both

final ethanol concentration (P) and productivity (DP) increased

with the feed sugar content up to 100 g l

1

and reached maxi-mum 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 produc-

tivity was 100g 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 0.29gEl1 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 productivitydid not change significantly 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

200gl1.

Variations of the ethanol (YP/S) and the growth (YX/S)

yield coefficients with the feed sugar content are depicted in

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

feed sugar concentration.


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S. Ozmihci, F. Kargi / Enzyme and Microbial Technology 41 (2007) 876880 879

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

concentration.

Fig. 4. The ethanol yield coefficient increased from 0.465 to

0.493 gE g1S (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 andyielded a yield coefficient of 0.3 gE g1S when the feed sugar

was 200g l1. The optimal feed sugar content maximizing the

ethanol yield coefficient was between100 and 125 g l1. Unlike

ethanol yield, the biomass yield coefficient (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 coefficient from 0.123 to

0.063gXg1S.

Fig. 5 depicts variations of volumetric rates of sugar uti-

lization and product (ethanol) formation with the feed sugar

concentration where RS and RP were calculated by using the

following equations.

Rs =Q(So S)

V= D(So S),

RP =Q(P Po)

V= D(P Po)

where So and S are the feed and effluent sugar concentrations

at the steady-state (gS l1); Po and P are the feed and efflu-

ent ethanol concentrations at the steady-state (gE l1) and Po

Fig. 5. Variation of volumetric sugar utilization and product formation rates

with the feed sugar concentration.

is zero since the feed is ethanol free; Q and V are the feed

flow rate (l h1) and the volume of fermentation broth (l). Sugar

utilization rate (RS) increased with increasing feed sugar con-

tent up to 100gl1 (Se = 4 4 g l1) and reached a maximum

level of 1.09 gS l1 h1 which decreased considerably with fur-

ther increases in the feed sugar above 125 g l1(Se = 6 6 g l1).

Ethanol formationrate showeda similar trend andincreased 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 max-

imizing 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 =100gl1 and125gl1 were not much different) corre-

sponding 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 caus-ing 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 con-

centrations (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 usedin a continuous fermenter under anaerobic conditions. Sugar

utilization, ethanol formation and the yeast growth were quan-

tified at different feed sugar concentrations varying between

55 and 200 g l1. The steady-state effluent 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 produc-

tivity (DP) were maximum (3.7% v v1, and 0.54gEl1 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 consid-

erably for the feed sugar contents above 100 g l1 indicating

adverse effects of high sugar contents on the yeast growth. Theethanol yield coefficient (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 coefficient

decreased steadily with the increasing feed sugar concentration

where the decrease was more pronounced at sugar concen-

trations 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 100g 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-


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880 S. Ozmihci, F. Kargi / Enzyme and Microbial Technology 41 (2007) 876880

ing the rate and extent of ethanol formation from the CWP

solution.

Acknowledgement

This study was supported by the funds from the State Plan-

ning Organization, Ankara and also Dokuz Eylul University,

Izmir, Turkey.

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