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Microbial production of citric acid was performed by Yarrowia lipolytica NBRC 1658 and a domestic strain
in the media containing glucose or fructose as substrates. The study was carried out in a batch system. The non-competitive
substrate inhibition model was proposed for growth of the yeasts in both substrate media. Higher dry mass and lower Ks
values were obtained in glucose media when compared to fructose media. The highest citric acid concentration (65.1 g/L)
was obtained with the domestic strain in the medium containing 200 g/L fructose. In the media containing fructose,
maximum citric acid concentration and productivity values determined with the domestic strain were aproximately two-fold
greater than those obtained with NBRC 1658 strain. The required initial concentration of glucose or fructose at which best
citric acid production properties were observed changed between 100-200 g/L for both of the strains. The ratio of citric acid
to isocitric acid was found to change between 11.70-16.62.
Keywords: Batch fermentation, citric acid, growth kinetics, production kinetics, Yarrowia lipolytica, yeast
such as, molasses, n-paraffins, hexadecanes, edible oils, was obtained by the domestic Y. lipolytica 57 which
starch hydrolysates and pure or raw glycerol, have been was chosen for further studies. At the beginning of the
used to produce citric acid from Y. lipolytica7,10,13. The present study, identification of Y. lipolytica 57 and
optimization of fermentation conditions are of primary Y. lipolytica NBRC 1658 was confirmed with the use
importance in the development of any fermentation of rapid API ID 32C (bioMérieux, France) test system
process owing to their impact on the economy and and also molecular methods (data not shown). The
practability of the process2. Work to improve yeast strains were kept as stock cultures at 4°C on
fermentative production of citric acid is continually in yeast extract malt extract (YM) agar consisting of
progress. Different techniques of production, using a (in g/L): yeast extract 3, malt extract 3, peptone 5,
variety of substrates, strain selection and development glucose 10, and agar 15. Cultures stored in YM agar
are continuously studied. were activated in the same medium by maintaining
The aim of the present study was to investigate consecutive transfers.
growth and citric acid production kinetics of two
Growth and Fermentation Media
different Y. lipolytica strains, at different initial
The inocula used in the experiments were prepared
concentrations of glucose and fructose as substrates, in
a batch system. This study was also planned to by incubation of the cultures at 28°C for 24 h in a
demonstrate the potential of citric acid production of a modified growth medium containing (in g/L): glucose,
novel strain by comparing its production characteristics 30; yeast extract, 2; NH4Cl, 2; KH2PO4, 0.5; and
with Y. lipolytica NBRC 1658, a citric acid producer. MgSO4.7H2O, 116. The experiments were carried out in
a fermentation medium containing (in g/L): glucose,
Materials and Methods 0-242 or fructose, 0-200; NH4Cl, 2; KH2PO4, 1;
Yeast Strains MgSO4.7H2O, 1; yeast extract, 1; CaCO3, 4015. CaCO3
Y. lipolytica NBRC 1658 was obtained from was used in the fermentation medium as a buffering
National Institute of Technology and Evaluation agent. The media were sterilized in an autoclave at
(NITE), Biological Resource Center, Japan. The strain 121°C for 15 min. Initial pH of the fermentation
NBRC 1658 was defined as a “citric acid producer” medium was adjusted to 5.2 by using 1 N HCl.
by the culture collection. Y. lipolytica IFO 1195 was
Equipment and Fermentation Conditions
obtained from the same culture collection in Japan.
Fermentations were carried out in water bath
The domestic strain Y. lipolytica 57 was obtained
shakers using 300 mL cotton-plugged flasks
from the culture collection of Food Engineering
containing 100 mL of fermentation medium. The
Department, Faculty of Engineering, Ankara
yeasts were inoculated separately to fermentation
University, Turkey. The selection of this strain was
media at an inoculum volume percentage of 5%.
performed with a preliminary study in which citric
Experiments were carried out at constant temperature
acid production abilities of 30 domestic strains
of 30°C, with a shaking rate of 100 strokes/min. All
belonging to different species, such as, Candida
of the runs were performed in duplicate. Mean values
guillermondii (7), C. pelliculosa (4), C. intermedia
of the results were plotted on the graphs.
(2), C. parapsilosi (4), Rhodotorula glutinis (8),
Saccharomyces cerevisiae (3), Pichia anomala (1) Biomass Determination
and Y. lipolytica (1), as well as the strain Y. lipolytica Dry mass (x) of the yeast was determined
IFO 1195 were investigated. The domestic yeast spectrophotometrically by using wet mass-absorbance
strains belonging to the culture collection of Food and wet mass-dry mass calibration curves which had
Engineering Department, Hacettepe University, been prepared before. During the experiments,
Turkey, had been isolated from some high-sugar samples were taken out from the fermentation media
foods (honey, fruit yoghurt, dried apricot, dried fig, at a time interval of 24-48 h. 6 N HCl was added into
date, glucose syrup, etc.) in a previous study the samples in order to dissolve CaCO315. Samples
performed by Senses-Ergul and Ozbas14. Screening of were centrifuged at 5000 rpm for 25 min. Precipitate
citric acid production capabilities of the strains were was used for determination of dry mass
performed in a fermentation medium containing spectrophotometrically at 660 nm (UV 210PC
(in g/L): glucose, 100; NH4Cl, 2; KH2PO4, 1; Shimadzu and Bausch & Lomb Spectronic 20).
MgSO4.7H2O, 1; yeast extract, 1; and CaCO3, 4015. Of Supernatant was used for citric acid, isocitric acid and
the tested strains, the highest citric acid production substrate analysis.
410 INDIAN J BIOTECHNOL, OCTOBER 2009
1 dP
qp = ... (1)
x dt
Cc m
YP/So = 100 ... (2)
So - S
Statistical Analysis
Calculations of kinetic constants and rates were
performed by using Origin 6.0 program package
(Microcal Software, Inc., Northampton, MA01060,
USA). Equations expressing the changes of specific
growth and citric acid production rates with initial
glucose or fructose concentration were derived from
non-linear regression analysis by using the same
package program.
0.120 SGo
µ= ... (6)
S 2
57.55 + SGo + Go
71.40
R2 = 0.799
Maximum value of υm was obtained at 100 g/L
initial glucose concentration for the novel endogenic
strain. Eq. 7 shows the variation of specific citric acid
production rate with initial glucose concentration for
this strain.
Glucose concentrations determined at specific time shown on Table 1. For the strain NBRC 1658,
intervals are shown on Fig. 3. Calculated values of maximum values of citric acid concentration and
glucose consumption rates and conversion ratios (xG) productivity were obtained at 150 g/L initial glucose
are also represented on the figure. It was found that in concentration, while maximum citric acid yield was
the medium with 20 g/L initial glucose concentration, obtained in the medium with 100 g/L glucose. Similar
all of the glucose was consumed in 94 h for the strain results were obtained for the domestic strain that the
NBRC 1658. When initial concentration of glucose highest citric acid concentration was determined at
was 50 g/L, no glucose was determined in the 150 g/L initial glucose concentration. Maximum values
fermentation medium after 112 h. In the media for product yield and productivity were obtained in the
containing 100 and 150 g/L glucose, no residual medium containing 100 g/L glucose. NBRC 1658 and
glucose was determined at the end of the fermentation, the domestic strain reached maximum productivity in
so that obtained glucose conversion ratios were 1 in 160 and 280 h, respectively. Cell yield of NBRC 1658
those media. Unconsumed glucose concentrations were strain was calculated as 0.162 g dm/g glucose, while it
found as 83.3 g/L and 128.8 g/L in the media was 0.134 g dm/g glucose for the domestic strain.
containing 200 and 242 g/L glucose, respectively. In The effects of initial fructose concentration on cell
the experiments performed with the novel domestic growth and citric acid production is represented in
strain, whole glucose was consumed in the media with Fig. 4. High values for maximum dry mass were
20, 50, 100 and 150 g/L initial glucose concentrations. obtained at initial concentrations of 150 and 200 g/L for
When initial glucose concentration was 200 and both of the strains. Maximum citric acid concentration
235 g/L, residual glucose concentration was was obtained as 33.70 g/L at 150 g/L initial fructose
determined as 92.4 and 131.2 g/L, respectively. concentration for the strain NBRC 1658. Citric acid
The effects of initial glucose concentration on production was very low at low initial fructose
maximum citric acid concentration, maximum dry concentrations. For the domestic strain, maximum citric
mass, product yield and maximum productivity are acid concentration was obtained as 65.10 g/L at 200 g/L
initial fructose concentration. Between 50-200 g/L initial
fructose concentration, citric acid production of this
strain was found higher than that of NBRC 1658 strain.
The same comment can be made for productivity and
Table 1—Effects of initial glucose concentration (SGo) on
maximum citric acid concentration (Cc m), maximum dry mass
(xm), product yield (YP/So), and maximum productivity (rc m) for
Y. lipolytica NBRC 1658 and Y. lipolytica 57
SGo Cc m xm YP/So rc m
(g/L) (g/L) (g/L) (%) [g citric acid/(L.h)]
citric acid production with this alternative substrate is novel “citric acid producer”. Demonstrating citric acid
limited. Hamissa and Abou-Zeid15 reported that 23.75 production amount at different media with this novel
g/L citric acid was obtained with a Y. lipolytica strain strain, the results are also informative in determining
in a medium containing 100 g/L fructose. In the culture conditions for scale-up in a commercial
present study, determined citric acid concentrations, production process.
especially, in fructose media are relatively high when
compared to those reported in the literature. Since Acknowledgement
glucose is a cheaper substrate, its usage may be The authors wish to thank the Scientific and
advantageous in a citric acid production process in the Technological Research Council of Turkey for
mean of scale up. This can be valid for the strain providing financial support (Project no. TOGTAG-
NBRC 1658, since similar results were obtained in 3337).
glucose and fructose media. But the domestic strain
surprisingly produced very high amounts of citric acid References
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