Comparative study of riboflavin

production from two microorganisms:

Eremothecium ashbyii and Ashbya
gossypii
Tijen O z b a s and Tulin Kutsal

Chemical Engineering Department, Hacettepe University, Ankara, Turkey

(Received 2 December 1985; revised 21 May 1986)

The conversion of the various substrates to riboflavin via fermentation by Eremothecium ashbyii and
Ashbya gossypii was investigated. Optimum operation temperature and initial pH of the medium
were determined as 30°C and 6.5 respectively for both microorganisms. In order to examine the
effects of different substrates and their initial concentrations, glucose, glycerol, sunflower oil, whey
and various combinations of these were utilized in the experiments. Maximum specific growth rates
and riboflavin yields were obtained in the media which contained glucose and sunflower oil together
as the substrate.

Keywords: Riboflavin;Eremothecium ashbyii; Ashbya gossypii; various substrates

Introduction
Riboflavin, also called vitamin Bz, is a water soluble vitamin
which exhibits a yellowish-green fluorescence. It is widely
used in the food enrichment, pharmaceutical and food
supplement industries. ~
Riboflavin can be produced by chemical synthesis or by
biochemical processes. Although crystallized riboflavin for
therapeutic purposes is still made by chemical synthesis,
various commercially competitive fermentation processes
have been recently developed for riboflavin production. In
fact, vitamin B2 concentrates for the enrichment of poultry
and livestock feeds can be prepared more cheaply by fer-
mentation processes, z,2
Riboflavin can be synthesized by many microorganisms.
Bacteria, moulds, algae and protozoa are the microorganisms
which can potentially produce riboflavin. Among these, two
closely related ascomycetes, Eremothecium ashbyii and
Ashbya gossypii are the most important)
In this study, the effects of the initial pH of fermenta-
tion media, of temperature, different substrates and initial
substrate concentrations on growth of microorganisms and
production of riboflavin by E. ashbyii and A. gossypii were
investigated. Whey (disposed of in large quantities in Tur-
key), glucose, glycerol, sunflower oil and different combina-
tions of these were utilized as substrate in the experiments.
Materials a n d m e t h o d s
Organisms
Eremothecium ashbyii NRRL Y-1363 and Ashbya
gossypii NRRL Y-1056 were used for riboflavin production
in this study.
Culture m e d i a
Cultures of b~: ashbyii and A. gossypii were grown on
solid agar media in slant tubes and Petri dishes. The com-
position of the culture medium was as follows (g 1-1):
glucose, 20.0; peptone, 5.0; yeast extract, 5.0; malt extract,
5.0; MgSO4.7H20, 0.2; K2HPO4, 0.2. 4's The pH of the
medium was adjusted to the desired value with 0.5 M
H2SO4. Both liquid and solid media were sterilized at
121°C in an autoclave before inoculation. The initial sub-
strate concentrations were changed by making the amount
of initial carbon in the medium equivalent to 20.0 g 1-1
glucose while the amounts of other constituents were kept
constant.
Growth conditions
The experiments were conducted in shaking flasks.
Erlenmeyer flasks (250 ml with 100 ml working volume)
were shaken at constant temperature and stirring rate in a
water bath shaker. 4,s
M e t h o d s o f assay
In order to determine the amounts of microorganisms
and riboflavin produced, 10 ml samples were drawn at con-
stant time intervals from the flasks and centrifuged. The
supernatants of centrifuged samples were used in the ribo-
flavin assay. The concentration of riboflavin produced was
determined at 445 nm by means of a Bausch and Lomb
spectrophotometer (Spectronic 20). The absorbance of the
supernatant was monitored and the concentration of
riboflavin was determined from a calibration curve. The
cells from the centrifuged samples were resuspended in
distilled water, and the absorbance read at 500 nm. The

0141-0229/86/100593-04 $03.00
© 1986 Butterworth & Co. (Publishers) Ltd Enzyme Microb. Technol., 1986,' vol. 8, October 593
Papers
amount of microorganisms was determined from the
calibration curve.
Results
Experiments were carried out to determine optimum
growth rates and riboflavin production in the various
media.
l;Tfec t of initial pH
Table 1 shows the effects of initial pH on specific
growth and riboflavin production rates with t:'. ashbyii
and A. gossypii. The optimum initial pH value was found
to be 6.5 for both microorganisms.
In riboflavin fermentations with E. ashbyii and A.
gossypii, the pH of the medium decreases with time and
drops to approximately 4.5. At this point the micro-
organisms are growing rapidly but only synthesize insig-
nificant amounts of riboflavin. Then both riboflavin
production and pH increase and riboflavin formation is
terminated at approximately 9.5.
l:'Jfect o f temperature
For 1-. ashbyii and A. gossypii maximum specific growth
rates and riboflavin production rates were obtained at 30°(i
in all medium compositions (Table 2).
Table 1 E f f e c t of initial pH on the specific growth and ribo-
flavin production rates f o r E. ashbyii and A. gossypii (30 ° C,
stirring rate 1 0 0 r e v m i n - I , s G 0 = 2 0 g 1 - 1 )
E. ashbyii A. gossypii
Effect o f initial glucose concentration
For the growth of both microorganisms the main carbon
source is glucose. The initial glucose concentration effects
were investigated from 5 to 30 g 1-1 for E. ashbyff and from
10 to 30 g 1-1 forA. gossypii. For riboflavin production the
optimum initial glucose concentrations were shown to be
20 g 1-1 for both microorganisms (Figures 1 and 2).
Effect o f substrate
After the optimum glucose concentration was determined
for growth and riboflavin production, glycerol, sunflower
oil, whey and various combinations of these substrates were
utilized and their effects investigated. The initial substrate
concentrations were changed by making the amount of
initial carbon of the medium equivalent to 20 g 1-1 glucose
while the amounts of the other media constituents were
unchanged.
The change of specific riboflavin production rates with
initial substrate concentrations for E. ashbyii and A. goso,pii
are shown in Figures 1 and 2 respectively. The effects of
the different substrates and their initial concentrations on
the specific growth rates and riboflavin yields are given in
Table 3.
Sunflower oil was added to the medium containing whey
and its effects were investigated for both microorganisms.
These experiments were carried out similarly to those with
glucose media containing sunflower oil. For constant initial
carbon concentration it was observed that both product
formation and the growth of the microorganism were
decreased using lactose with sunflower oil for l:2 ashbyii.
l:or instance, although the riboflavin yield YP/Sco = 3.8
was obtained in the medium containing 10.0 g 1-1 whey

/aa 103 x v /~a 103 x v SGo( g FI )-,,,-

InitialpH (h- j ) (gg-I h-I) (h-I) (gg-I h-i) 20 15 IO 5 0
I I I I I

6.0 0.1556 1.29 0.0779 0.86

SG~o (g i-~)-,.-

6.5 0.1854 3.57 0.1538 1.25 Sso (g F~) -,.-

0 5 i0 15 20
7.0 0.1797 1.19 0.1050 0.39 I I I I
7.5 0.1099 0.28 0.0747 0.31

aCalculated in the region of exponential growth (Monad kinetic

model)

Table 2 E f f e c t of temperature on the specific growth rate and

riboflavin production rate for E. ashbyii and A. gossypii (stirring
rate 100 rev rain -= , SG0 = 20 g I -z , initial pH = 6.5)

E. ashbyii A. 9ossypii

a a
t # 103 x v # 103 x v
(OC) (h-l) (gg-Z h-Z) (h-l) (gg-, h l )

20 0.0271 0.64 0.1002 0.56 I0

25 0.0440 0.94 0.1180 0.61
28 0.1041 2.50 0.1361 1.00
I
0 5 l0 15 20 25 30
30 0.1854 3.57 0.1538 1.25
SG0 (g I -I) ..~
32 0.1341 1.88 0.1260 0.87 I I l l I
0 5 10 15 20
35 0.0659 0.60 0.0959 0.60
Swo [g r L)
40 0.0584 0.05 0.0793 0.21
Figure 1 Changeof specific riboflavin production rates with initial
acalculated in the region of exponential growth (Monad kinetic substrate concentrations for E. ashbyii, o, SG0; • SWo; ~, SGo +
model) SGIo;B,SG o +Ss 0

594 Enzyme Microb. Technol., 1986, vol. 8, October

 Riboflavin production by E. a s h b y i i and A. gossypii: T. Ozbas and 7-. Kutsal
Table 3 Effects of various substrates on specific growth rates and riboflavin yields for E. ashbyii and A. gossypii (30 ° C, stirring rate 100 rev
rain -I ,initial pH = 6.5)

E. ashbyii A. gossypii
Initial
substrate a a
concentration #
Substrates (g I -I ) (h-= ) YP/SCo (h-I) YP/Sco

Glucose 20.0 0.1854 3.6 0.1538 3.1

Glycerol 20.0 0.1733 3.1 0.1315 1.3
Sunflower oil 20.0 0.1669 3.0 0.2245 2.9
Glucose + glycerol 5.0 + 15.0 0.2044 4.5 0.1979 1.7
Glucose + glycerol 7.5 + 12.5 0.2006 3.7 0.2153 1.9
Glucose + sunflower oil 5.0 + 15.0 0.2438 9.2 0.2323 5.2
Glucose + sunflower oil 10.0 + 10.0 0.2358 6.8 0.2635 8.8
Whey 12.5 0.1379 6.5 - --
Whey 30.0 -- - 0.1529 2.2

acalculated in the region of exponential growth (Monod kinetic model)

$Go (g I - I ) " - Table 4 Effect of adding sunflower oil to medium containing

whey on the riboflavin yield for E.ashbyii and A gossypii (30°C,
20 15 I0 5 0 stirring rate 100 rev rain -1 , initial pH = 6.5)
i 1 I
t I SGIo (g i.i)
Initial substrate
concn (g I -a )
SS0 (g I-I ) whey + Initial carbon E. ashbyii A. gossypii
0 5 I0 15 20 sunflower oil concn (g I -1 ) yp/sc0 yp/sc0
I I 1 I

12.5+0.0 4.0 6.5

10.0+2.0 4.0 3.8
,1 4 0
5.0+5.0 4.0 3.6

25.0+0.0 7.0 2.1

20.0+2.5 7.0 3.7
i 3.0' 30.0+0.0 8.5 2.2
22.0+5.0 8.5 6.8

~ 20

The results showed that for the medium containing whey or

glucose, adding sunflower oil to the medium increased the
0 i.O growth of the microorganism and riboflavin production for
A. gossypii (Table 4).

0 5 I0 15 20 25 30
$60 (g I-I ) - , ,
Conclusions
In the studies on the effects of various substrates on ribo-
I I I I I I
0 I0 20 3O 4O 50 flavin production with E. ashbyii and A. gossypii, the
Swo (g i-~) --.- maximum specific growth and riboflavin production rates
were obtained in media which contained both glucose and
Figure 2 Change of specific riboflavin production rates with initial
sunflower oil as substrate. Although higher riboflavin yields
substrate concentrations for A. gossypii, c, SG0 ; o, SWo; z~, SG ° + were obtained with E. ashbyii in media containing whey
SGI0; m, SG0 + SSo compared to that containing glucose, lower yields were
attained with A. gossypfi under the same experimental
conditions. This may be due to the fact that whey is mostly
lactose and the assimilation rate of this disaccharide by
and 2.0 g 1-t sunflower oil, the yield was YP/Sco = 6.5 in A. gossypii is lower than that of glucose. Since whey is a
the medium containing 12.5 g 1-1 whey. Riboflavin yield, disposal substrate in Turkey, its use in riboflavin biosyn-
YP/sco = 6.8, was obtained in the medium containing thesis by these microorganisms will necessitate enzymatic
22.0 g 1-l whey and 5.0 g 1-1 sunflower oil for A. gossypii. conversion of lactose to monosaccharides by lactase.

Enzyme Microb. Technol., 1986, v o l . 8, O c t o b e r 595

Papers
Nomenclature References

Sc 0 initial carbon c o n c e n t r a t i o n (g 1-l ) 1 Kirk, R. E. and Othmer, D. F. in Encyclopedia o1 Chemical

S% initial glucose concentration (g 1-' ) Technology (Herman, F. M., Mc Kctta, J. J. Jr., Othmcr, D. I'.
and Standcn, A., eds) Wiley Interscience, New York, 1968,
SGlo initial glycerol concentration (g 1-1 )
vol. 17, pp. 445 458
SSo initial sunflower oil concentration (g 1-1 ) 2 Lago, B. D. and Kaplan, L. Adv. Biotechnol. 1981, 3,241 246
Sw0 initial whey concentration (g 1-1 ) 3 Goodwin, T. W. in The Biosynthesis of Vitamins and Related
t temperature (°C) Compounds (Goodwin, T. W., cd.) Academic Press, I.ondon,
riboflavin yield [(g/g initial carbon concentration) 1963, pp. 24 35
YP/Sc o 4 Ozbas, T., MSc Thesis, Itacctlepc University, Ankara, 1985
x 100] 5 Ozbas, T., Kutsal, T. and ~a~lar, A. ir Pre~'. 3rd European
specific growth rate of microorganism (h-1 ), Congress on B~'otechnolog3, (Behrens, D., cd.) Vcrlag Chemic
specific p r o d u c t i o n rate [(g riboflavin (g micro- GmbH, Wcinhcim,1984,vol. l,pp. 389- 394 "
organism) -1 h -l ]

596 Enzyme Microb. Technol., 1986, vol. 8, October