Journal of Scientific

GUPTE&&Industrial Research
NAIR : β-GALACTOSIDASE PRODUCTION AND ETHANOL FERMENTATION FROM WHEY 855
Vol. 69, November 2010, pp. 855-859

β-Galactosidase production and ethanol fermentation from whey using

Kluyveromyces marxianus NCIM 3551
A M Gupte* and J S Nair
Department of Biotechnology and Bioinformatics, Padmashree Dr D Y Patil University, CBD Belapur, Navi Mumbai 400 614,
India

Received 31 May 2010; revised 30 August 2010; accepted 01 September 2010

β-Galactosidase production and ethanol fermentation from whey were studied using Kluyveromyces marxianus NCIM 3551
at laboratory scale. Optimum β-galactosidase production and ethanol fermentation was obtained with 16 h old culture at an
inoculum size of 10% over an incubation period of 20 h at pH 5.0 and at 25°C. Nitrogen supplementation was found to have not
much effect on β-galactosidase production and ethanol fermentation.

Keywords: Alcohol fermentation, β-Galactosidase, Kluyveromyces marxianus NCIM 3551, Whey, Yeast

Introduction Experimental Section

India, leading producer of milk in the world,
produces channa (2 million tonnes) and paneer
(1.5 million tonnes), besides whey1 (75 - 85 %), which
contains 50% of total solids of milk2. Whey (BOD 30,000
- 50,000 ppm) disposal is a serious problem for dairy
industry3. In order to reduce pollution load, whey has
been treated chemically and microbiologically to obtain
commercial products4. Options available for treatment
of whey include aerobic cultivation of microorganisms5,
use of whey as feedstock for ethanol production6 and
production of β-galactosidase7. β-Galactosidase is used
in hydrolysis of lactose into glucose and galactose
during production of low lactose milk for lactose
intolerant population and in prevention of lactose
crystallization during manufacture of concentrated milk
and ice cream8,9. Industrial production of β-galactosidase
is limited due to low productivity and high cost10. To
improve productivity, different microorganisms
(Streptococcus thermophilus, Escherichia coli, Candida
pseudotropicalis and Klyuveromyces sp.) have been
studied for β-galactosidase production11.
This study presents parameter optimization for
β-galactosidase production and ethanol fermentation
using whey as a medium by K. marxianus NCIM 3551
in shake flask cultures.
*Author for correspondence
Tel: +91-22-27563600; Fax: +91-22-39286176
E-mail: garpita@gmail.com
Materials
Whey was prepared from cow’s whole milk
purchased from local market. Milk (200 ml) pH (3.5)
was adjusted with 1 N HCl. It was then boiled for 20
min and cooled. Clear filtrate was obtained by filtering
it through muslin cloth followed by Whatman filter
paper No. 1. Whey (150 ml) thus obtained was sterilized
by autoclaving after adjusting to desired pH using 0.1N
NaOH or 0.1 N HCl. K. marxianus NCIM 3551 was
procured from National Collection of Industrial
Microorganisms, National Chemical Laboratory, Pune
(India).
Maintenance and Preparation of Culture
Yeast culture was revived by incubating at 30°C for
48 h on maintenance medium (MM) containing: glucose,
1; peptone, 0.5; malt extract, 0.3; and yeast extract, 0.3%
w/v. Culture was maintained and preserved at 4°C by
serial subculture at 2 weeks intervals on MM agar slants.
MM (50 ml) was inoculated with a loopful of culture
from agar slants and incubated at 30°C for 18 h on a
rotary shaker at 100 rpm.
Production Medium
Clarified whey was used as production medium for
β-galactosidase and ethanol. Whey medium (50 ml) was
inoculated with 10% (v/v) of starter culture and
incubated on a rotary shaker at 100 rpm. At various time
856 J SCI IND RES VOL 69 NOVEMBER 2010
intervals, samples were drawn from flask and assayed
for β-galactosidase activity and ethanol concentration.
Extraction Methods
SDS-Chloroform Method
Cell suspension (0.1 ml) was mixed with 0.9 ml Z
buffer (0.06 M Na2HPO4, 0.04 M NaH2PO4, 0.01 M KCl
and 0.001 M MgSO4). To this, chloroform (100 ìl) and
0.1% SDS solution (50 µl) was added. Suspension was
centrifuged at 1398 x g to obtain cell free extract12.
Isoamyl Alcohol Method
A volume of re-suspended cells in 0.2 M phosphate
buffer (pH 6.5) containing dry cell mass (10 - 20 mg)
was mixed with isoamyl alcohol (5 ml) and diluted up
to 25 ml with 0.2 M phosphate buffer (pH 6.5). Mixture
was shaken for 15 min at room temperature (RT) to make
cell envelope permeable and used for enzyme assay13.
Liquid Nitrogen Method
Biomass was ground with liquid nitrogen using
mortar-pestle and later centrifuged at 4000 x g for 20
min. Supernatant was used for enzyme assay14.
Toluene Method
Cell suspension (1 ml) was mixed with equal
quantity of cold toluene for 15 min with intermittent
stirring. Mixture was vortexed for 5 min and then
centrifuged at 4000 x g at 5°C for 20 min. Supernatant
obtained was used for enzyme assay15.
Toluene-Acetone Method
Production culture (10 ml) was centrifuged at 4000
x g at 5°C for 20 min. Pellet obtained was dissolved in
2 ml of 0.2 M phosphate buffer (pH 7.0), ground with
alumina(2 g) and toluene: acetone (9:1) solution
(0.1 ml). Suspension was redissolved in buffer (8 ml)
and centrifuged at 4000 x g at 5°C for 10 min.
Supernatant obtained was used for enzyme assay11.
β-Galactosidase Enzyme Assay
Biomass was washed twice with 0.1 M phosphate
buffer (pH 7.0) and re-suspended in same buffer.
Appropriately diluted cell suspension (0.1 ml) was
spectrophotometrically assayed for enzyme activity
using O-nitrophenol-β-D-galactopyranoside (ONPG) as
a substrate7. One unit of enzyme activity is the enzyme
quantity that liberated 1 ìmole of O-nitrophenol per min
under assay conditions. Values expressed are mean ±
standard error of three independent experiments.
7
6
â-galactosidase activity,
µmoles/min/g cells
5
4
3
2
1
0
a b c d e
Fig. 1—Effect of extraction methods for β-D-galactosidase
extraction: a) Chloroform-SDS; b) Isoamyl alcohol; c) Liquid
nitrogen; d) Toluene; and e) Toluene-acetone
Ethanol Estimation
Ethanol was measured using potassium dichromate
(K2Cr2O7) method. Potassium dichromate reagent (10 ml)
was added to 1 ml of whey (1:10 diluted) and was
incubated for 30 min at RT. After incubation, distilled
water (100 ml) and 4 ml of potassium iodide (25%) was
added in each flask. Solution was titrated against 0.1 N
sodium thiosulfate with starch (1%) as an indicator.
Values expressed are mean ± standard error of three
independent experiments.
Results and Discussion
Effect of Extraction Methods on β-Galactosidase Activity
Chloroform-SDS method was found best among
methods tried for enzyme extraction (Fig. 1). Cell
disintegration, applied in production of intracellular
β-galactosidase from Kluyveromyces sp., can be achieved
by application of shear forces (mechanical methods) and
digestion or permeabilization of cell envelopes
(chemical methods)17 . For enzyme extraction using
permeabilization of cells by organic solvents, performance
of organic solvents is dependent on incubation time,
incubation temperature and concentration of both cells
and solvents18. Chloroform-SDS method is reported9 ideal
for extraction of β-galactosidase from yeast cells.
Effect on β-Galactosidase Activity and Ethanol Production of
Following Parameters
Incubation Period
Maximum β-galactosidase activity was observed
after 20 h of incubation (Fig. 2a) and also was found to
be optimal for ethanol production (Fig. 2b). A decrease
in enzyme activity was observed with further increase in
incubation time (24 - 32 h), may be attributed to culture
reaching stationary phase. Earlier studies19-21 have also
reported optimal incubation period (18 - 24 h).
 GUPTE & NAIR : β-GALACTOSIDASE PRODUCTION AND ETHANOL FERMENTATION FROM WHEY 857

7 a) 7 a)
â-galactosidase activity, 6 6

â-galactosidase activity,
µmoles/min/g cells 5
5

µmoles/min/gcells
4 4

3
3

2 2
1
1
0
0
12 16 18 20 24 28 32
4% 6% 8% 10% 12% 16%
Incu b ation pe riod , h
Ino culu m s ize, %

20 b) 20 b)
18
18
16
16
Ethanol produced, g/L

Ethanolproduced, g/l
14
14
12
12
10
10
8
8
6
6
4 4
2
2
0 0
12 16 18 20 24 28 32 4% 6% 8% 10% 12% 16%
Inc u ba tio n pe rio d , h In o c u lu m s ize , %

Fig. 2—Effect of incubation period on: a) β-D-Galactosidase Fig. 3—Effect of inoculum size on: a) β-D-Galactosidase activity;
activity; b) Ethanol production b) Ethanol production
However, incubation period extending up to 30 h is also
reported11.
7 a)
Inoculum Size and Age 6
â-galactosidase activity,
µmoles/min/gcells

Different inoculum levels (4 - 16%) were used as 5

starter culture for β-galactosidase activity and ethanol 4
production. Enzyme activity increased with inoculum 3
size; maximum activity was observed at 16% (Fig. 3a).
2
However, in case of alcohol production, concentration
1
of alcohol produced gradually increased with inoculum
0
size upto 10% and thereafter was found to decrease 12 16 20 24 28
(Fig. 3b). Inoculum age was also found to have an effect
Inoculum age, h
on β-galactosidase production (Fig. 4a). Activity was
maximum with a 16 h culture; increasing from 12 - 20%
and thereafter decreasing. Alcohol produced remained 20 b)
18
almost constant with inoculum age of up to 20 h and 16
Ethanol produced, g/L

decreased thereafter (Fig. 4b). Therefore, a 16 h old 14

12
culture and a 10% inoculum size was used in further 10

experiments to maintain optimal production of 8

6
β-galactosidase and alcohol. Earlier studies used cultures 4

varying in inoculum age from 20 h7,22 to 24 h21. 2

0
12 16 20 24 28

pH In o c u l u m a g e , h

Enzyme activity was constant till pH 4.5, peaked at Fig. 4—Effect of inoculum age on: a) β-D-Galactosidase activity;
pH 5.0, and decreased between pH 5.5 and 6.0 (Fig. 5a). b) Ethanol production
858 J SCI IND RES VOL 69 NOVEMBER 2010

a) 7
7 a)
6 6
â-galactosidase activity,

â-galactosidase activity,
µmoles/min/g cells 5 5

µmoles/min/g cells
4 4

3 3

2 2

1
1
0
0
3.5 4 4.5 5 5.5 6
R.T R.T (static) 37 37 ( static)
pH (shaker) (shaker)
Temperature, 0C
25 b)
20 b)
20
18
Ethanol produced, g/l

16
15

Ethanol produced, g/l

14
12
10
10
8
5
6
4
0
3.5 4 4.5 5 5.5 6 2

pH 0
R.T R.T (static) 37 37 ( static)
Fig. 5—Effect of pH on: a) β-D-Galactosidase activity; b) Ethanol (shaker) (shaker)
0
production Tem perature, C

A) Fig. 6—Effect of temperature on: a) β-D-Galactosidase activity; b)

7 Ethanol production
6
â-galactosidase activity,
µmoles/min/g cells

5
Alcohol production increased with increasing pH up to
4
4.5 and was constant till pH 5.5 and dropped at pH 6.0
3
(Fig. 5b). Earlier studies for optimal β-galactosidase
2 production used pH 4.68 with K. lactis20, pH 5.0 with K.
1 marxianus23, and pH 5.5 with K. marxianus24.
0
a b c d e Temperature
20 B) β-Galactosidase production was better under shake
18 flask conditions (SFC) at RT of 25°C (Fig. 6a).
16 Similarly, alcohol production was also better at RT
Ethanol produced, g/l

14
(25°C) under SFC (Fig. 6b). Temperatures (28 - 31°C)
12
have been used in earlier studies7,20,21,24 for optimal
10

8
β-galactosidase production. Earlier studies7,22,25,26 have
6 also supported SFC for β-galactosidase production.
4

2 Nitrogen Source
0 Effect of supplementing whey with different
a b c d e
nitrogen sources on β-galactosidase (Fig. 7a) and
Fig. 7—Effect of nitrogen supplementation on: A) β-D-Galactosi-
dase activity; B) Ethanol production (a, whey; b, whey + ammo-
ethanol production (Fig. 7b) was studied. Nitrogen
nium sulphate; c, whey + ammonium nitrate; d, whey + urea; and supplementation increases production of β-galactosidase
e, whey + yeast extract) and ethanol by reducing lag period and increasing
 GUPTE & NAIR : β-GALACTOSIDASE PRODUCTION AND ETHANOL FERMENTATION FROM WHEY
bioconversion efficiency of ethanol production27,28.
Ethanol production studied earlier using
Kluyveromyces29,30 (conc. 13 - 22 g/l) is in agreement with
present study.
Conclusions
Optimal production of β-galactosidase and ethanol
fermentation from whey using K. marxianus NCIM
3551was found at following process conditions: pH, 5.0;
temp., 25°C (RT); incubation period, 20 h; and inoculums
size, 10%.
References
1 Aneja R P, Mathur B N, Chandan R C & Banerjee A K,
Technology of Indian milk products, in Handbook on Processed
Technology, Modernization for Professional Enterprises and
Scientists, edited by P R Gupta (Dairy India Publication, New
Delhi) 2002, 133-157.
2 Lund B, Norddahl B & Ahring B, Production of lactic acid from
whey using hydrolysed whey protein as nitrogen source,
Biotechnol Lett, 14 (1992) 851-856.
3 Domingues L, Dantas M M, Lima N & Teixeira J A,
Continuous ethanol fermentation from lactose by a
recombinant flocculating Saccharomyces cerevisiae strain,
Biotech Bioengg, 64 (1999) 692-697.
4 Paul D, Mukhopadhyay R, Chatterjee B P & Guha A K,
Nutritional profile of food yeast Kluyveromyces fragilis biom-
ass grown on whey, Appl Biochem Biotech, 97 (2002) 209-218.
5 Ben Hassan R M & Ghaly A E, Continuous propagation of
Kluyveromyces fragilis in cheese whey for pollution potential
reduction, Appl Biochem Biotech, 47 (1994) 89-105.
6 Christensen A D, Ka´da´r Z F, Popiel P O & Thomsen M H,
Production of bioethanol from organic whey using
Kluyveromyces marxianus, J Ind Microb Biotechnol, (2010)
(in press).
7 Panesar P S, Production of β-D-galactosidase from whey using
Kluyveromyces marxianus, Res J Microbiol, 3 (2008) 24-29.
8 Bonekamp F J & Oosteron J, On the safety of Kluyveromyces
lactis: A review, Appl Microbiol Biotechnol, 41 (1994) 1-3.
9 Panesar P S, Panesar R, Singh R S, Kennedy J F & Kumar H,
Microbial production, immobilization and applications of β-D-
galactosidase, J Chem Technol Biotechnol, 81 (2006) 530-543.
10 Becerra M & Sizo M I G, Yeast β-galactosidase in solid-state
fermentations, Enz Microb Technol, 19 (1996) 39-44.
11 Bansal S, Oberoi H S, Dhillon G S & Patil R T, Production of
β-galactosidase by Kluyveromyces marxianus MTCC 1388 us-
ing whey and effect of four different methods of enzyme
extraction on β-galactosidase activity, Ind J Microbiol, 48 (2008)
337-341.
12 Oberoi H S, Bansal S & Dhillon G S, Enhanced β-galactosidase
production by supplementing whey with cauliflower waste, Int
J Food Sci Technol, 43 (2008) 1499-1504.
13 Barberis S & Gentina J C, Effect of dissolved oxygen level of
lactose production by Kluyveromyces fragilis, J Chem Technol
Biotechnol, 73 (1998) 71-73.
859
14 Liu D L, Yao D S, Liang Y Q, Zhou T H, Song Y P, Zhao L &
Ma L, Production, purification and characterization of an
intracellular aflatoxin-detoxifizyme from Armillariella tabescens
(E-20), Food Chem Toxicol, 39 (2001) 461-466.
15 Gupte A M, Enzyme and cell technology for bioremediation,
Ph D thesis, Bhabha Atomic Research Centre, Mumbai,
India, 2001.
16 Miller G L, Use of DNS acid reagent for the determination of
reducing sugars, Anal Chem, 31 (1959) 426-428.
17 Numanoglu Y & Sungur S, β-galactosidase from Kluyveromyces
lactis cell disruption and enzyme immobilization using
cellulose-gelatin carrier system, Process Biochem, 39 (2004)
705-711.
18 Flores M V, Voget C E & Ertola R J J, Permeabilization of yeast
cells (Kluyveromyces lactis) with organic solvents, Enz Micob
Technol, 16 (1994) 340-346.
19 Pinheiro R, Belo J & Mota M, Growth and β-galactosidase
activity in cultures of Kluyveromyces marxianus under increased
air pressure, Lett Appl Microb, 37 (2003) 438-442.
20 Mathews A O R & Rivas N, Production and partial character-
ization of β-galactosidase from Kluyveromyces lactis grown in
deproteinized whey, Arch Lat Amer Nutr, 53 (2003) 194-201.
21 Ku M A & Hang Y D, Production of yeast lactase from sauerkraut
brine, Biotechnol Lett, 14 (1992) 925-928.
22 Chen K C, Lee T C & Houng J Y, Search method for optimal
medium for the production of lactose by Kluyveromyces fragilis,
Enz Microb Technol, 14 (1992) 659-664.
23 Inchaurrondo V A, Yantorno O M & Voget C E, Yeast growth
and β-galactosidase production during aerobic cultures in
lactose limited synthetic medium, Process Biochem, 29 (1994)
47-54.
24 Artolozaga M J, Jones R, Scheider A L, Furlan S A & Carvallo-
Jones M F, One step partial purification of β-galactosidase from
Kluyveromyces marxianus CDB002 using streamline-DEAE,
Bioseparation, 7 (1998) 137-143.
25 Pedrigue M & Castillo F J, Regulation of β-galactosidase
synthesis in Candida pseudotropicalis, Appl Environ Microbiol,
43 (1982) 303-310.
26 Champluvier B, Kamp B & Rouxhet P G, Preparation and
properties of β-galactosidase confined in cells of Kluyveromyces
sp., Enz Microb Technol, 10 (1988) 611-617.
27 Ghaly A E & Tweel E L, Effect of nutrient supplements
addition on ethanol production from cheese whey using
Candida pseudotropicalica, Appl Biochem Biotechnol, 53 (1995)
107-131.
28 Panesar R, Panesar P S, Singh R S, Kennedy J F & Bera M B,
Process optimization of β-D-galactosidase production using
yeast culture, J Biol Sci, 6 (2006) 193-197.
29 Siso M I G, Ramil E, Cerdan M E & Freire-Picos M A,
Respirofermentative metabolism in Kluyveromyces lactis:
Ethanol production and the Crabtree Effect, Enz Microb Technol,
18 (1996) 585-591.
30 Serpil O & Fikret K, Fermentation of cheese whey powder so-
lution to ethanol in a packed-column bioreactor: Effects of feed
sugar concentration, J Chem Technol Biotechnol, 84 (2009)
106-111.