Fibers and Polymers 2019, Vol.20, No.

1, 80-85 ISSN 1229-9197 (print version)

DOI 10.1007/s12221-019-8742-1 ISSN 1875-0052 (electronic version)

Using Saccharomyces cerevisiae Strains as Biocatalyst for Indigo Reduction

Younsook Shin1*, Kyunghee Son1, and Dong Il Yoo2*
1
Department of Clothing and Textiles/Human Ecology Research Institute, Chonnam National University,
Gwangju 61186, Korea
2
School of Polymer Science and Engineering, Chonnam National University, Gwangju 61186, Korea
(Received August 12, 2018; Revised October 12, 2018; Accepted October 18, 2018)

Abstract: The aim of this study is to investigate the efficacy of S. (Saccharomyces) cerevisiae strains to reduce natural indigo
and to develop an eco-friendly reduction process of indigo as an alternative of chemical reductant. S. cerevisiae strains from
baker’s yeast powder and Korean rice wine respectively were cultured, and used for carrying out the reduction of indigo. The
reducing-activity toward natural indigo was evaluated quantitatively by dyeing test to measure color strength (K/S value)
onto ramie fabric. The changes in K/S value and pH were monitored on the time-based mesurements. The time required to
start reduction and maximum reduction, and duration were also evaluated. The time to reach the highest reduction level, i.e.,
the highest K/S value, of strain I (2-3 days) was shorter than that of strain II (3-4 days). The strain I from baker’s yeast
showed higher reducing-activity, resulting stronger color yield on the fabric, and longer duration period than the strain II from
Korean rice wine. Initial pH decreased drastically from 11.2 to 7-9 for one week with the progress of reduction reaction. K/S
value increased to maximum (12-13) at first 2-4 days and decreased rapidly to 6-8, and then maintained for more than one
week. Among reaction variables, controlling pH was the most critical to get maximum color strength when we used S.
cerevisiae as biocatalyst.
Keywords: Indigo, Reduction, Enzyme, Saccharomyces cerevisiae, Biocatalyst

Introduction have used rice wine (makgoli) for activating fermentation of

People have been applied indigo material in various
industries including textile dyeing, cosmetics, printing and
medicinal products. It is insoluble in water and thus, the
reduction of indigo into leuco-indigo is an important process.
Chemical reducing agents such as sodium dithionite, sodium
sulfide etc., are preferred to use in industry due to their
strong reduction power. For long period of time, many
attempts have been made to replace chemical reduction
method with more environmentally friendly alternatives,
such as the biotechnological reduction [1-5], the electrochemical
reduction [6-8], and the reduction using organic reducing
agents [9,10] and extracts from food wastes or by-products
[11,12].
The replacement of the conventional chemical reduction
process with microbial (biotechnological) process including
bacteria and enzymes could be a very attractive alternative
from ecological point of view. Enzymes as highly specific
biocatalysts instead of whole cells like bacteria would be
considerably easier to handle in this application [13]. The
main advantages of the enzymatic technology are no cost of
treating the effluent, no toxicity, mild temperatures of
processing as claimed to be eco-friendly. The disadvantage
of the enzymatic process is in the dependence of enzyme
activity on the temperature and pH [7,14]. In this context, we
reviewed the traditional indigo dyeing process in Korea,
which basically relies on the bacterial reduction through a
complex fermentative process [7,15]. Indigo dyers in Korea
*Corresponding author: yshin@jnu.ac.kr
*Corresponding author: diyoo@jnu.ac.kr
indigo. It is thought that yeast in rice wine may help indigo
reduction.
Yeast, as same as S. (Saccharomyces) cerevisiae, is the
most widely exploited and commercially significant micro-
organism. Researchers regard yeast as useful biocatalyst,
since it possesses relatively rigid cell walls that enable the
structures retained in the presence of various organic
compounds and solvents [16]. People have been applied
yeast for many centuries in brewing, baking, distilling and
wine making [17] and for the production of ethanol from
biomass as a renewable alternative energy resourse [18]. S.
cerevisiae strains are also known to produce aldo-keto
reductases, which catalyze the reduction of carbonyl groups
[19-21]. They are exploited in a variety of reactions, such as
bio-dehydrogenation, hydrolysis, oxidation and reduction
[16]. However, published studies regarding the use of yeast
are hardly available as reductive biocatalyst for indigo
bioconversion.
The reaction variables used in the microbial reduction are
such as pH, temperature, elapsed time, aerobic/anaerobic
environment and nutrients. The mechanism of bacterial
indigo reduction still remains unknown, but the indigo
reducing species like C. Isatidis indicate possible mechanism
for biotechnological process [7]. Many enzymes are known
that could be used for the reduction of vat dyes. To enhance
catalytic activities, some enzymes require a coenzyme such
as nicotinamide adenine dinucleotide phosphate (NAD(P)H)
from which a hydride is transferred to the substrate with
carbonyl group (coupled-enzyme system). The simple process
makes use of a single enzyme which simultaneously
transforms the substrate with carbonyl groups (coupled-

80
Biocatalyst for Indigo Reduction
substrate system) [7]. Supercritical fluids are used to discover
a range of novel chemical processes. The use of alcohol
dehydrogeneses (cells of fungus, Geotrichum candidum) in
supercritical carbon dioxide is also intensive investigation
[22].
The enzymatic process has a lot of advantages, such as the
mild process resulting in less damage onto the fibers, lower
energy consumption and shorter time of treatment needed
[7]. Several enzymes of catalyzing the oxidation-reduction
reactions have been evaluated in the environmental and
biological field to remove pollutants and to catalyze a great
variety of redox processes with no hazardous side effects
[23-25]. Emzymatic reduction could be a useful substitute
for existing environmentally harmful chemicals based
reduction technologies and a faster alternative to time-
consuming bacterial reduction. However, though the enzymatic
methods give some benefits to indigo reduction, there are
still lot of obstacles to be satisfactory alternative to chemical
reduction process [7]. Listed below are some important
findings in view of microbial (bacterial and enzymatic)
reduction: C. isatidis was isolated from woad dye vat,
generated redox potentials from -474 mV to -602 mV (about
100 mV more negative than those of other bacteria examined),
within 1 day at 40 oC and pH 9 [26]. A. psychrotolerans sp.
Nov. (psychrotolerant, obligatory alkalibacterium) was
isloated from a fermented polygonum indigo using a
Japanese method [27]. Alkalibacterium sp. and Pseudomonas
sp. (facultative alkaliphilic bacteria, reduce indigo at pH 10
under anoxic condition) were also isolated from Korean
fermentation liquor [28]. Quite recently, we isolated Dietzia
sp. KDB1 (KC433534), Nestrenkonia sp. KDB2 (KC433535),
Nestrenkonia sp. KDB3 (KCKC433536), Nestrenkonia sp.
KDB4 (KC433537) from Korean fermentation liquor [4,5].
Mediated NADH-dependent reductases (enzymes) isolated
from B. subtilis systematically substituted azo and indigoid
compounds with the reduction potential (-580 mV) to reduce
indigo completely in a temperature range from 55-60 oC at
pH 7 and pH 11 [21,24,25,29-31]. Formate dehydrogenase
(FDH) from C. boidinii involves the use of formate as an
innocuous substrate and the production of carbon dioxide
[21]. As the enzyme, S. Cervisiae has been used to mediate
enantioselective reduction [32-36], to apply in asymmetric
organic synthesis with high optical yield [37], to carry out
respiratory-fermentative catabolism [38,39], to reduce
chemicals in biosynthesis [40-43], and to produce pro-chiral
ketone [44].
S. cervisiae strains as biocatalyst give us several distinct
characteristics, such as process robustness, able to grow
anaerobically, high tolerance to low pH, high sugar and
ethanol concentrations that lower the risk of contamination
in industrial fermentation [45]. This study aims to investigate
the efficacy of developing an eco-friendly indigo reduction
using S. cerevisiae as a biocatalyst. We cultured S. cerevisiae
strains I and II, separated from baker’s yeast and rice wine
Fibers and Polymers 2019, Vol.20, No.1 81
respectively, in YPD agar media for preparing indigo reduction
bath. The indigo reducing-activities of S. cerevisiae strains I
and II were studied quantitatively by evaluating color
strength (K/S value) onto ramie fabric through dyeing test.
The changes in K/S value and pH were monitored on the
time-based mesurements. Reduction duration including
maximum reduction was also evaluated.
Experimental
Materials
Baker’s yeast and Korean rice wine (makgoli) were
purchaed commercially from a local market. 100 % ramie
fabric were purchased commercially. Natural indigo(indigo
content; 10.66 %) was made from Polygonum tinctorium.
All the chemicals and reagents were of analytical grade.
Cultivation of Yeast Strains
S. cerevisiae strains in Korean rice wine and baker’s yeast
were cultured in YPD agar media at 30 oC for 16 h before
inoculation. YPD agar media consisted of (g/l): yeast extract
10, peptone 10, dextrose 20. Baker’s yeast solution was
made with sterilized distilled water. Second cell cultivation
was carried out in YPD liquid medium on a rotary shaker at
200 rpm and 30 oC for 16 h. The optical density (O.D.) of
liquid culture was adjusted to 2.5-2.8 at λ=600 nm. The
cultured cells were centrifuged at 4 oC for 20 min and used
for indigo reduction. For convience, S. cerevisiae strains
from baker’s yeast and rice wine were called by S. cerevisiae
I and S. cerevisiae II, respectively.
Preparation of Indigo Reduction Bath
To investigate the indigo reducing-activity of S. cerevisiae
strains, indigo reduction bath was made of natural indigo
2.5 g and cultured cell 0.17-2.0 (wet weight) in 35 ml of
0.2 % Na2CO3 (pH 11.02). The indigo bath was maintained
at 32 oC.
Evaluation of Reducing-activity by Dyeing Test
For investigating reduction level of indigo, dyeing was
carried out in reduction media. Ramie fabric samples were
impregnated in the supernatants of indigo reduction bath for
20 min, removed from the bath and oxidized in open air for
15 min to get blue color of indigo, rinsed, neutralized in
0.1 % acetic acid solution, washed and dried. The reducing-
activity was evaluated in terms of color strength (K/S value)
of fabrics dyed in reduction media. Color strength and pH of
reduction bath were monotored by daily measurement over a
period up to 26 days.
The color strength (K/S value) was calculated from the
reflectance of the samples using Kubelka-Munk equation.
K/S = (1 − R)2 / 2R
where R is the reflection of the dyed sample.
82 Fibers and Polymers 2019, Vol.20, No.1 Younsook Shin et al.

Reflectance of the dyed sample was measured at the
maximum adsorption wavelength (λmax; 640 nm) using a
Macbeth Coloreye 3100 spectrophotometer.
Results and Discussion
Indigo Reduction with Saccharomyces cerevisiae I
The indigo reducing-activity of S. cerevisiae I, isolated
from baker’s yeast solution, was evaluated with varied
amount of strains, 0.5-2.0 g. The results are presented in
Figure 1(a). It took 1-2 days to exhibit obvious reducing-
activity. And it was taken approximately 3-4 days to reach
the highest reduction level, as shown highest K/S value.
Since K/S value is used as an estimation of the color strength
onto fabric after dyeing, K/S value indicates the amount of
leuco-indigo absorbed on fabrics as a result of reduction of
indigo. With more amount of strains, time required to reach
maximum reduction got shorter. Also, higher K/S value and
longer duration of reduction were obtained with more
amount of the strains added. Maximum K/S values were
10.21, 12.64, and 12.79 for 0.5 g, 1.0 g, and 2.0 g of the
strains added, respectively. Reduction duration was maintained
for 6, 16, and 24 days depending on the amount of strains.
The more strains used, the longer reduction duration
maintained, as expected.
The changes in pH of reduction bath were monitored and
the results are presented in Figure 1(b). The pH of reduction
medium was decreased continously as reduction proceeded
by the strains. The decrease in pH is associated with the
formation of protons accompanied by the growth of strains
[21]. The pH values of bath at maximum activity were 9.14,
8.71, and 8.25 at different amount of strains, respectively. S.
cerevisiae I was active up to approximately pH 8. A larger
decrease in pH could be resulted by more metabolites
produced with more strains.
The results of dyeing test with S. cerevisiae I are
summarized in Table 1. It indicates that with more amount of
the strains, reduction tend to start in a shorter time and
reaches to maximum level in a shorter time, and continues
for longer time.
Indigo Reduction with Saccharomyces cerevisiae II
S. cerevisiae II was isolated from Korean rice wine,
containing brewer’s yeast. Its indigo reducing-activity was
evaluated with varied amount of strain, 0.5-2.0 g. The results
are presented in Figure 2(a). With natural indigo, it took for
1-2 days from the time of vat setting to exhibit obvious
reducing-activity depending on the amount of strains. And
the time to reach the highest reduction level, i.e., the highest
K/S value, was taken approximately for 2-3 days depending
on the amount of strain. Times required to show clear
activity and to reach maximum K/S value got shorter as the
amount of the strains increased. Also, with more amount of
the strains, higher K/S value was obtained and reduction was

Figure 1. Changes in indigo-reducing activity (K/S) (a) and pH (b) of reduction bath with S. cerevisiae I.

Table 1. Indigo reduction with Saccharomyces cerevisiae I from baker’s yeast

0.5 g 1.0 g 2.0 g
Reduction Time Time Time
pH K/S Dyed sample pH K/S Dyed sample pH K/S Dyed sample
(day) (day) (day)
Start 2 9.58 4.16 1 9.75 0.95 1 9.19 1.00

Peak 4 9.14 10.21 4 8.71 12.64 3 8.25 12.79

End 6 8.81 1.57 16 7.80 1.35 24 7.68 1.23

Biocatalyst for Indigo Reduction Fibers and Polymers 2019, Vol.20, No.1 83

Figure 2. Changes in color yield (K/S) (a) and pH (b) of reduction bath with S. cerevisiae II.

Table 2. Indigo reduction with Saccharomyces cerevisiae II from Korean rice wine
0.5 g 1.0 g 2.0 g
Reduction Time Time Time
pH K/S Dyed sample pH K/S Dyed sample pH K/S Dyed sample
(day) (day) (day)
Start 2 9.31 4.64 1 9.61 0.73 1 9.03 0.56

Peak 2 9.31 4.64 2 8.98 7.49 3 8.37 12.07

End 4 9.17 1.63 10 8.03 1.22 26 7.92 0.67

continued for longer time. Maximum K/S values obtained

were 4.64, 7.49, and 12.07 with 0.5 g, 1.0 g, and 2.0 g of
strains added, respectively. Reduction duration was maintained
for 4, 10, and 26 days depending on the amount of the strains
added. We got the longer reduction duration with the more
strains used, as same as S. cerevisiae I.
Changes in the pH of reduction bath are presented in
Figure 2(b). The pH of bath with natural indigo was changed
continously as time passes. The pH values of reduction bath
showing maximum activity were 9.31, 8.98, and 8.37 at the
amount of strain II, 0.5 g, 1.0 g, and 2.0 g, respectively. The
results of dyeing test with S. cerevisiae II are summarized in
Table 2. With more amount of the strain, higher color
strength was obtained and reduction maintained for longer
time. With 0.5 g of the strains, no clear reduction was
observed after day one and then maximum activity was
Scheme 1. Reduction and oxidation mechanism of indigo.
shown in the next day, rendering the highest color strength
(K/S value) on the fabric sample. The reduction started
between the days 1-2 and the end of reduction was observed value increased to maximum (approximately 12-13) until the
after day four. days 2-4 and decreased rapidly to the K/S of 6-8, and then
maintained for more than one week. We reported previously
Controlling pH in Microbial Reduction similar results when some bacterial strains separated from
As shown in Figure 1 and Figure 2, initial pH of 11.2 fermented indigo vat were applied [5].
shifted to pH 7-9 and the decrease of pH value for one week As far as we know from cumulated researches, trans-
was drastic with the progress of reduction reaction. K/S formation of indigo in chemical reduction process seems to
84 Fibers and Polymers 2019, Vol.20, No.1
occur as in Scheme 1. In the first stage of indigo reduction,
indigo is reduced to acid leuco compound at pH < 5.5. When
acid leuco compound is reacted with alkali, it changes to
mono-substituted and di-substituted sodium salts of leuco
compound at pH 5.5-11 and pH > 11, respectively [46,47].
In this study, indigo dyeing was proceeded when indigo
molecule is reduced to mono-substituted sodium salt in the
pH range of 7-9. Cumulated results confirm that controlling
pH is critical to get maximum color strength when micro-
organsim is employed as biocatalyst [5]. However it is not
well understood at the moment why drastic pH decrease is
observed at the initial stage of microbial reduction.
Conclusion
We applied S. cerevisiae strains to reduce indigo for the
development of an eco-friendly bioprocess. S. cerevisiae
strains I and II separated from baker’s yeast powder and
Korean rice wine, respectively, cultured, and used for
carrying out the reduction of indigo. Their indigo reducing-
activities were evaluated by color strength (K/S value) onto
fabric through dyeing with indigo reduction medium. And
the changes in K/S value and pH of reduction bath were
monitored.
With more amount of strains, the time required to start and
to reach maximum level of reduction appeared in a shorter
time, and the reaction continued for longer time. Also, we
obtained higher K/S value and longer duration of reduction
with more amount of the strains. The time to reach the
highest reduction level, i.e., the highest K/S value, of S.
cerevisiae strains I (2-3 days) was shorter than that of S.
cerevisiae strains II (3-4 days). The S. cerevisiae strain I
from baker’s yeast showed higher reducing-activity,
resulting higher color strength on the fabric, and longer
duration period than the S. cerevisiae strain II from Korean
rice wine. Initial pH of 11.2 shifted to pH 7-9 and the
decrease of pH value for one week was drastic with the
progress of reduction reaction. K/S value increased to
maximum until 2-4 days and decreased rapidly to the K/S of
6-8, and then maintained for more than one week. Controlling
pH of reduction medium is critical to get maximum color
strength when we employ microorgansim as biocatalyst.
Acknowledgements
We are grateful for the financial support of the Korea
Research Foundation (KRF) grant funded by the Korea
government (MSIP) (No. 2017R1A2B4009555).
References
1. A. N. Padden, P. John, M. D. Collins, R. Hutson, and A. R.
Hall, J. Archaeol. Sci., 27, 953 (2000).
2. M. Bozic, M. Díaz-González, T. Tzanov, G. M. Guebitz,
Younsook Shin et al.
and V. Kokol, Enzyme Microb. Technol., 45, 317 (2009).
3. Y. Shin, K. Son, and D. I. Yoo, Color. Tech., 26, 237
(2014).
4. E. S. Choi, E. B. Lee, H. A. Choi, K. Son, G. J. Kim, and Y.
Shin, Kor. Soc. Biotech. Bioeng. J., 28, 295 (2013).
5. Y. Shin, K. Son, and D. I. Yoo, Fiber. Polym., 17, 1000
(2016).
6. T. Bechtold and A. Turcanu, J. Clean. Prod., 17, 1669
(2009).
7. M. Bozic and V. Kokol, Dyes and Pigm., 76, 299 (2008).
8. A. Roessler and X. Jin, Dyes and Pigm. 59, 223 (2003).
9. R. S. Blackburn and A. Harvey, Environ. Sci. Technol., 38,
4034 (2004).
10. N. Meksi, M. B. Ticha, M. Kechida, and M. F. Mhenni, J.
Cleaner Prod., 24, 149 (2012).
11. Y. Shin, M. Choi, and D. I. Yoo, Fiber. Polym., 14, 2027
(2013).
12. Y. Shin, M. Choi, and D. I. Yoo, Fashion and Textiles, 1, 6
(2014).
13. R. G. Compton, S. J. Perkin, D. P. Gamblin, J. Davis, F.
Marken, A. N. Padden, and P. John, New J. Chem., 24, 179
(2000).
14. A. K. Patra, A. Madhu, and N. Bala, Fashion and Textiles,
5, 1 (2018).
15. A. Osimani, L. Aquilanti, G. Baldini, G. Silvestri, A. Butta,
and F. Clementi, J. Ind. Microbiol. Biotechnol., 39, 1309
(2012).
16. G. K. Khor and M. H. Uzi, Yeast, 28, 93 (2011).
17. C. R. Landry, J. P. Townsend, D. L. Hartl, and D. Cavalier,
Mol. Ecol., 15, 575 (2006).
18. M. D. Putra, A. K. Sulieman, A. E. Abasaeed, M. H. Gaily,
S. M. Al-Zahrani, M. A. Zeinelabdeen, and H. K. Atiyeh,
J. Clean. Prod., 162, 420 (2017).
19. G. Ford and E. M. Ellis, Chemico-Biol. Inter., 130-132, 685
(2001).
20. Q. Chang, T. A. Griest, T. M. Harter, and J. M. Petrash,
Biochim. et Biophys. Acta, 1773, 321 (2007).
21. W. Kroutil, H. Mang, K. Edegger, and K. Faber, Curr.
Opin. Chem. Biol., 8, 120 (2004).
22. T. Masuda, K. Watanabe, T. Harada, and K. Nakamura,
Catal. Today, 96, 103 (2004).
23. M. D. Leonida, Curr. Med. Chem., 8, 345 (2001).
24. S. Pricelius, C. Held, S. Sollner, S. Deller, M. Murkovic,
R. Ulrich, M. Horfrichter, A. Cavaco-Paulo, P. Macheroux,
and G. M. Guebitz, Enzyme Microb. Technol., 40, 1732
(2007).
25. E. Siu, K. Won, and C. B. Park, Biotechnol. Prog., 23, 293
(2007).
26. A. N. Padden, V. M. Dillon, J. Edmonds, M. D. Collins, N.
Alvarez, and P. John, Int. J. System. Bacteriol., 49, 1025
(1999).
27. I. Yumoto, K. Hiroda, Y. Nodasaka, Y. Yokata, T. Hoshino,
and K. Nakajima, Int. J. Sys. Evol. Microbiol., 54, 2379
(2004).
Biocatalyst for Indigo Reduction
28. S. Park, J. Y. Ryu, J. Seo, and H. G. Hur, J. Korean Soc.
Appl. Biol. Chem., 55, 83 (2012).
29. M. Bozic, V. Kokol, and G. M. Guebitz, Text. Res. J., 79,
895 (2009).
30. M. Bozic, S. Pricelius, G. M. Guebitz, and V. Kokol, Biol.
Prod. Proc. Eng., 85, 563 (2010).
31. S. Pricelius, C. Held, S. Sollner, M. Murkovic, M. Bozic,
V. Kokol, A. Cavaco-Paulo, and G. M. Guebitz, Appl.
Microbiol. Biotechnol., 77, 321 (2007).
32. S. Rodriguez, M. M. Kayser, and J. D. Stewart, J. Am.
Chem. Soc., 123, 1547 (2001).
33. R. Csuk and B. I. Glanzer, Chem. Rev., 91, 49 (1991).
34. E. Santaniello, P. Ferrabochi, P. Grisenti, and A. Manzocchi,
Chem. Rev., 92, 1071 (1992).
35. P. D’Arrigo, G. Pedrocchi-Fantoni, and S. Servi, Adv. Appl.
Microbiol., 44, 81 (1997).
36. S. M. Roberts, J. Chem. Soc., Perkin Trans., 1, 611 (2000).
37. K. Nakamura and T. Matsuda, Curr. Org. Chem., 10, 1217
(2006).
Fibers and Polymers 2019, Vol.20, No.1 85
38. A. Frechter, G. F. Fuhrmann, and O. Kappelli, Adv. Micro.
Physiol., 22, 123 (1981).
39. B. M. Bakker, K. M. Overkamp, A. J. A. van Maris, P.
Kotter, A. H. Luttik, J. P. van Dijken, and J. T. Pronk,
FEMS Microbiol. Rev., 25, 15 (20010).
40. T. Kometani, E. Kitasuji, and R. Matsuno, Chem. Lett., 8,
1465 (1989).
41. T. Kometani, H. Yoshi, Y. Takeuchi, and R. Matsuno, J.
Ferment. Bioeng., 76, 414 (1993).
42. T. Kometani, Y. Sakai, U. Hisae, H. Yoshi, and R. Matsuno,
Biosci. Biotechnol. Biochem., 61, 1370 (1997).
43. C. H. Wong and G. M. Whitesides, “Enzymes in Synthetic
Organic Chemistry”, Pergamon Press, Oxford, 1994.
44. C. E. Perles, P. J. S. Moran, and P. L. O. Volpe, J. Mol. Cat.
B: Enzym., 52-53, 82 (2008).
45. E. Nevoigt, Microbiol. Mol. Biol. Rev., 72, 379 (2008).
46. G. A. Baig, Color Technol., 128, 114 (2012).
47. G. A. Baig, AUTEX Res. J., 10, 21 (2010).