BIOTECHNOLOGY AND BIOENGINEERING

VOL. X, PAGES 33-43 (1968)

Cultivation of the Yeast Cundidu ZipoZyticu on

Hydrocarbons. I. Degradation of *Alkanes in
Batch Fermentation of Gas Oil

11. DOSTALEK, V. MUKK, and OLGA VOLFOVA, Czechoslovak

Academy of Sciences, Institute of Microbiology, Department of Tech-
nical Microbiology, and K. PECKA, Department of Synthetic Fuel and
Petroleum, Institute of Chemical Technology, Prague, Czechoslovakia

Summary
The growth of batch-cultivated yeast C a d i & lipolytim on three kinds of gas
oil using mineral medium was studied. A linear dependence was found between
the production of yeast biomass and the consumption of n-alkanes, while the
decrease of freezing point of gas oil during cultivation had a distinct course. This
disproportion was explained by different degradation of individual n-alkanes
contained in gas oil. The rate of degradation of pentadecane, hexadecane, and
heptadecane was the same during the entire cultivation. On the contrary, in the
first phase the utilization of shorter chain n-alkanes, nonane to tetradecane, was
more rapid while that of longer chain homologs, octadecane to pentacosane,
lagged. Rapid utilization of longer chain n-alkanes did not occur before the
concentration of the other n-alkanes decreased. Only then the rapid decrease
of freezing point appeared.

Introduction
Since the time that Champagnat et al.' described multilateral em-
ployment of yeast cultivation on hydrocarbons, considerable atten-
tion has been paid to this problem. One of the outstanding research
fields is the utilization of yeast on gas oil during which the fodder
yeast biomass is produced and the gas oil simultaneously refined by
decreasing its freezing point. This process is based on the selection
of a suitable yeast culture wThich selectively utilizes the n-alkanes
from gas oil for its growth, thus removing components which cause
a high freezing point.
The gas oil consists of a complex composition of hydrocarbons,
about one fifth of which belongs to the paraffin series. The composi-
33
34 DOSTALEK, MUNK, VOLFOVA, PECKA

tion of the paraffin fraction itself is not homogeneous and varies

according to the raw material used in distillation, as well as according
to the medium temperature of the distillation. As a rule, when the
n-alkanes within the range of nonanes to triacontanes are present, the
concentration of individual n-alkanes is different. This means a
considerable complication of the problem of n-alkane degradation by
yeast during the process of deparaffination. Until now only one
paper has been published on this problem (AIiller and Johnson2),
evaluating the process of deparaffination in accordance with the
analyses of five gas oil samples remaining after the fermentation of
the mixed culture of yeast Candida. Moreover, in a number of
papers the respiration of individual n-alkanes, as well as their utiliza-
tion for growth of microorganisms, was followed. The results of
these observations are too ambiguous to explain the degradation of
individual n-alkanes from their mixture. Miller, Lie, and Johnson3
found that the increase of growth rate within the series of n-alkanes
CI2-Cl4was dependent on the increasing molecular weight of hydro-
carbons. Azoulay, Couchod-Beaumont, and Senez4found practically
the identical growth rate with the alkanes C12-C16 while the multi-
plication rate found on decane was higher. On the other hand,
1IiIler and Johnsons found the highest growth rate with the n-alkanes
C2o-Cn.
This paper deals with the study of yeast biomass production at
simultaneous deparaffination dependence of gas oil, pursuing the
relations between the growth of yeast Candida lipolytica during the
degradation of individual n-alkanes and changes in the freezing point
of gas oil.

Materials and Methods

Yeast Candida lipolytica 4-lA, isolated on oil field was used in this
experiment. The medium was composed of: KH2PO4,7.0 g. ;KH4C1,
5.0 g. ; ,IfgS04, 0.2 g. ; KaCI, 0.1 g. ; gas oil, 80-200 ml. ; tap water,
1.Ooo ml.
The inoculum was prepared using a 3-day continuous culture of
Candida lipolytica, working volume 1.300 ml., dilution rate D = 0.12
on the medium containing 10% gas oil. The produced biomass was
centrifuged and used for inoculation of the fermentor amounting to
2 g. of dry weight per 1.000 ml. of working volume.
The fermentation was performed in a fermentor of 100 1. working
BIOTECHNOLOGY A N D BIOENGINEERING, VOL. X, ISSUE 1
 CULTIVATION OF YEAST OK HYDROCARBONS. I 35

volume, stirred by a turbine type stirrer at 950 rpm, aerated at 1.5

l./min./l. The temperature was 30°C. and the pH was controlled
automatically at 4.5 by addition of KOH.
The determination of the yeast biomass increase was made partly
by direct determination of dry matter in sintered glass funnel, partly
by calculation based on determination of the residual nitrogen in the
filtrate of the medium. Freezing point of the gas oil was determined
by means of the method prescribed by the Czechoslovak standard
(CSK 656176).
The total content of n-alkanes both in dry matter and in product
was determined by the method utilizing the ability of n-alkanes to
form urea adductions. For this determination a modification of the
analytical method by Veselov6was employed.
Gas chromatography was used for determination of individual
n-alkanes, and that after decomposition of urea adduction left over
after determination of the total content of n-alkanes. A Carlo Erba
apparatus, type Fractovap D, employing dual system columns 1 m.
long, inner diameter 5 mm., filled with Chromosorb W, grained 60-80
mesh, wetted by 3y0 methylsilicose polymer SE-30 was used. The
analyses ranged from 110 to 270"C., by gradually increasing the
temperature by 8"C./min., using the flame ionization detector.
Three samples of gas oil from a distillation fraction of sulfonic
Romashkin crude oil (U.S.S.R.), medium boiling point 315OC.,
density 0.835, were used for cultivation. The content of n-alkanes
and the initial freezing point of individual samples are as follows:
sample A, 19.9% w/w, -14°C.; sample B, 19.87%, 0°C.; sample C,
17.30~,, - 11°C. The concentration of individual n-alkanes in the
employed gas oil samples is to be seen in Figure 1.
The concentration of gas oil throughout individual experiments
was chosen to achieve during 24 hr. cultivation a decrease of freezing
point to a value lower than -5O"C., when in deparaffined gas oil the
presence of paraffin was not detectable. In experiment A the culti-
vation medium contained 20.0 g. of n-alkanes per liter, in experiment
B, 14.8 g./l., in experiment C, 28.8 g./l.
Results and Discussion
The experiments showed that when multiplicating the yeast
Candida lipolytica in the medium containing gas oil the concentration
of n-alkanes decreased at simultaneous decline of the freezing point
36 DOSTALEK, MUNK, VOLFOVA, PECKA

Fig. 1. Concentration of individual n-alkanes in the medium during batch

cultivation; (2)individual n-alkanes (number of C atoms) ; (y) concentration of
n-alkanes in g./l. of the medium.
Individusl column: Sample A: column (1)initial gas oil; (2)increase of biomass
2.5 g./l.; (3) increase of biomass 6.5 g./l.: (4) increase of biomass 14.0 g./l. Sam-
ple B: column (1)initial gas oil; (%')biomass increase 2.5 g./l.; (3)biomass increase
6.9 g./l.; (4) biomass increase 10.1 g./l. Sample C: column (1) initial gas oil;
(2) biomass increase 9.5 g./l.; (3) biomass increase 19.0 g./l.; (4) biomass increase
19.5 g./l.

(Fig. 2). Evaluating the consumption of n-alkanes in relation to the

increase of yeast biomass, in all cases almost a linear dependence was
found. The causes of the deviation from linearity, resulting in the
decrease of the yield in initial and final cultivation phases, are dis-
cussed later. Quite different dependence was found between the
increase of the yeast biomass and the decrease of the freezing point.
The curve representing this dependence is characterized by a long-
lasting phase of slow decrease of the freezing point. Throughout this
phase, the prevailing amount of the total biomass comes into exist-
ence. This phase is followed by rapid decrease of the freezing point;
the increase of the biomass is lowered substantially. The relation
between these two compared values is striking when employing a high
BIOTECHNOLOGY AND BIOENGINEERING. VOL. X. ISSUE 1
 CULTIVATION OF YEAST ON HYDROCARBONS. I 37

concentration of the gas oil in the medium (Fig. 3). The results of
the experiments show that there is no direct relation between the
concentration of n-alkanes and the freezing point throughout cultiva-
tion. The disproportions in the changes of both quantities become
especially evident under employment of higher concentrations of gas
oil, i.e., at a high concentration of n-alkanes in the medium. Taking
into consideration that the freezing point is given not only by the
total amount of paraffinic hydrocarbons in gas oil, but also by the
representation of individual n-alkanes, we can gather that the yeast
does not degrade all n-alkanes simultaneously.
To verify this hypothesis, the samples of gas oil taken during the

16

~~~ 8 L 8 12

Biomass gll
16 20
I
15

25

35

L5
5

Fig. 2. Consumption of n-alkanes and decrease of freezing point in batch

cultivation on gas oil: (2)increase of biomass in grams of dry matter per liter of
the medium; ( y ~ total
) concentration of n-alkanes in grams per liter of the medium;
(y2) freezing point of gas oil.
38 DOSTALEK, MUNK, VOLFOVA, PECKA

1 2 3
L 8 12 16 20 2L
Biomass gll

Fig. 3. Decrease of the freezing point at different concentrations of gas oil

in medium: (2)increase of biomass in grams of dry matter per liter of the medium
(y) freezing point of gas oil; ( 1 ) medium containing 8% gas oil; (8) medium con-
taining 12’34 gas oil; (3) medium containing 20y0 gas oil.

cultivation were chemically analyzed. The results are given in

Figure 1. The intervals for sampling are indicated by the points in
Figure 2 . From the results of the analyses it is obvious that through-
out the cultivation the degradation of all n-alkanes does not occur at
the same rate.
During the first fermentation phases, the decrease of n-alkanes of
lower molecular weight is obviously more rapid than that those of
higher molecular weight. This causes the shift of the n-alkane
spectrum. In the period preceding the rapid decrease of the freezing
point (Fig. 1, column 4), the highest concentration of heptadecane
and the relative increase of the content of longer chain n-alkanes are
found regardless of whether in the initial crude oil tridecane ( A ) ,
tetradecane (C) or hexadecane ( B ) prevailed.
The comparison of the rate of degradation of individual n-alkanes
is given in Figure 4. The results indicate that the relative rate of
n-alkane degradation decreases in proportion to increasing molecular
weight of n-alkanes. However, during the further fermentation
course in which the gradual decrease of concentration of paraffinic
fraction of gas oil occurs the changes in the rate of degradation of
individual n-alkanes take place (Fig. 5 ) .
There are three characteristic types of the course of degradation
of individual n-alkanes. The consumption of the paraffin of lower
molecular weight is more rapid in the first fermentation phase, later
on the rate of their consumption decreases. On the other hand, the
BIOTECHNOLOGY AND BIOENGINEERING, VOL. X. ISSUE 1
 CULTIVATIOS OF YEAST O X HYDROCARBONS. I 39
--
>
3
- 100
5
g
1
80

60
L

," 40
._
*
0

p 20
c
0,

E
u
2 4 6 8 2 4 6 2 4 6 8 1 0
Time of cultivation (hours)

Fig. 4. Relative rate of n-alkane consumption in the first phase of cultivation:

(2)fermentation time (hr.); (y) concentration of individual n-alkanes in per cent
(initial concentration of each n-alkane = 100%).

-
w

'
3

a
80
C

9
U
60

-
t
0 40
C
._

0
V L
2 4 6 8 1 0
Consumption of N-alkanes gli

Fig. 5. Relative consumption of n-alkanes during cultivation: (2) total con-

sumption of n-alkanes in grams per liter of the medium; (y) concentration of
individual n-alkanes in per cent w/v (initial concentration of each n-alkane
= 100%).

longer chain n-alkanes are degraded slowly at first and only in the
further phases does their consumption increase. Between these two
groups such n-paraffins are to be found, the degradation of which
occurs at constant rate throughout the entire fermentation process.
The n-alkanes were divided into three groups, according to the charac-
teristic course of degradation, and their relation to the course of gas
oil deparaffination was studied. The first group includes the paraf-
fins of lower molecular weight, from decane to tetradecane. Into
the second group pentadecane, hexadecane, and heptadecane are
40 DOSTALEK, MUNK, VOLFOVA, PECKA

included. The third group is for the n-alkanes from oktadecane

upwards. The relative rate of degradation of individual n-alkane
groups in relation to the total consumption of n-alkanes is seen in
Figure 6. With all kinds of gas oil studied, characteristic course of
degradation of individual groups of n-alkanes were found. This
observation suggests that the initial spectrum of the gas oils did not
influence the course of degradation of the individual groups of
n-alkanes. On the contrary, the absolute concentration of hydro-
carbons of the individual groups can influence the relative relation
between the course of the curve characterizing the degradation of
individual groups of n-alkanes. This is why the absolute values of
individual groups of n-alkanes were compared (Fig. 7). I n all cases
the characteristic linear course of the degradation of n-alkanes of the
second group was found. The degradation of n-alkanes of the third
group are degraded after initial lag to the contrary of the shorter
chain homologs. At the same time, the results achieved show that
the course of degradation of n-alkanes belonging to the third group
is influenced by the concentration of n-alkanes of the first group.
The gas oil A (Fig. 7) has a high concentration of n-alkanes of the
first group and the phase of rapid degradation lasts a long time.
Throughout this period the n-alkanes of the third group showed a
long lag phase. A contrast is found with gas oil B (Fig. 7), in which,
with respect to the low concentration of the n-alkanes of the first
group, the phase of their rapid degradation is short and thus the
n-alkanes of the third group are degraded rapidly, after a shorter lag.

g 100
c
0
5
0
I:
-0
60

.G
c
20
c
,t?
4 12 20 28 4 12 20 28 4 12 20 28
"
0 Cmsumption of N-alkanes gll
u

Fig. 6. Relative consumption of different groups of n-alkanes during cultiva-

tion: ( 1 ) decane to tetradecane; (2) pentadecane to heptadecane; ( 3 ) octadecane
to pentacosane; (2) total consumption of n-alkanes in grams per liter of the
medium; (y) concentration of individual n-alkanes in per cent w/v (initial con-
centration of each group of n-alkanes = 1 0 0 ~ o ) .
BIOTECHNOLOGY A N D BIOENGINEERING, VOL. X, ISSUE 1
 CULTIVATION OF YEAST ON HYDROCARBONS. I 41

10 t

L 12 20 28
Consumption ot N-alkanes gll

Fig. 7. Consumption of different groups and decrease of the freezing point in

the course of cultivation: ( 1 ) decane t o tetradecane; (2)pentadecane to hepta-
decane; (3) octadecane to pentacosane; (4) freezing point; (2)total consumption
of n-alkanes in grams per liter of the medium; (yl) concentration of n-alkanes
(groups) in grams per liter of the medium; ( y ~ freezing
) point of gas oil “C.

When comparing the curve of degradation of n-alkanes of the

third group with the curve representing the changes of freezing point,
it can be concluded that the course of degradation of the longer chain
n-alkanes has a decisive influence on the changes in the freezing point.
A complete survey of the gas oil deparaffination and of the freezing
point changes in relation to yeast biomass production is given in
Figure 8.
It is also obvious that a rapid decrease of the freezing point occurs
within-the period of the rapid degradation of the n-alkanes of the
third group, i.e., of the n-alkanes having the highest freezing point.
On the contrary, in the initial fermentation phases shorter chain
hydrocarbons of the first group are degraded preferentially and a t
the highest rate because of their low freezing point. The change of
their concentration does not influence the freezing point of gas oil.
42 DOSTALEK, MUSK, VOLFOVA, PECKA

Biomass gll

Fig. 8. Consumption of n-alkanes and changes of the freezing point in relation

to culture multiplication: ( I ) decane to tetradecane; ( 2 ) pentadecane t o hepta-
decane; (5)octadecane to pentacosane; ( 4 ) freezing point; (2)increase of yeast
biomass in grams of dry matter per liter of medium; (y,) relative concentration
of individual n-alkanes groups per cent w/v (initial concentration of n-alkanes of
each group = 100%);(yz) freezing point of gas oil, "C.

As found by Miller and JohnsonZ15the longer chain n-alkanes (from

nonadecane to tetracosane) are utilized best of all. The authors
employed mostly samples of the gas oil with a high content of longer
chain n-alkanes and concluded mainly the results of the balance
analyses. This conclusion is not valid, for example, for the gas oil,
fraction C, experiment 6, which is almost of the same composition as
that used by us. In the above experiment, the n-alkanes Cl2-CI4
were consumed quantitatively and the final concentration of Cl6-Cl8
homologs is lower than that of Cl9-CZ4n-paraffins. This result is in
conformity with our observations. We did not make any studies of
the biomass yield on individual n-alkanes contained in gas oil. Con-
sidering that in the initial and final fermentation phases lower
biomass yield was found (Fig. 2 ) , it can be concluded that in the
BIOTECHNOLOGY A N D BIOENGINEERING. VOL. X. ISSUE 1
 CULTIVATION OF YEAST OK HYDROCARBONS. I 13

biomass formation both n-alkanes of the first group and n-alkanes of

the third group are utilized to a lesser extent than pentadecane,
hexadecane, and heptadecane. However, this dependence requires
a direct proof as the eventuality that the lower yield may be in
coincidence with the growth phases in question cannot be excluded.
As follows from the results achieved, the course of deparaffination
of gas oil throughout cultivation of Candida lipolytica depends on the
composition and concentration of the paraffinic fraction of gas oil.
The presence of n-alkanes higher than heptadecane is of decisive
influence as their concentration and course of degradation affect the
decrease of the freezing point of gas oil. However, rapid consump
tion of n-alkanes of this group occurs only after the prevailing amount
of n-alkanes, lower than pentadecane, has been consumed and this is
also why the concentration of the hydrocarbons belonging to this
group is of importance for the deparaffination process. At a lower
concentration of the lower chain n-alkanes in the medium, i.e. , when
employing a gas oil with a low concentration of n-alkanes or under
low concentration of gas oil, the phase of degradation of n-alkanes
of the third group takes place shortly after beginning fermentation
and the freezing point of gas oil decreases rapidly (Fig. 3). At very
low concentrations of gas oil in the medium practically all n-alkanes
are degraded at the same time and the course of freezing point de-
crease approaches the total consumption of n-alkanes and is almost
linearly dependent on the biomass production. On the other hand,
when employing high concentrations of gas oil with high concentra-
tion of the lower chain n-paraffins these are preferentially degraded
in the first fermentation phases, while the concentration of longer
chain n-alkanes changes in this phase only slightly. This causes a
disproportion between the slightly changing freezing point of the
gas oil in the first fermentation stage and the increase of biomass and
decrease of the total concentration of n-alkanes.

References
1. A. Champagnat, Ch. Vernet, B. Laine, and J. Filoss, Il'atitre, 197, 13 (1963).
2. T. J . Miller and M. J. Johnson, Biotechnol. Bzoeng., 8, 549 (1966).
3. T. L. Miller, S. Lie, and M. J. Johnson, Biotechnol. Bioeng., 6, 299 (1964).
4. E. Azoulay, P. Couchoud-Beaumont, and J. C. Senez, Ann. Znst. Pastew,
107, 520 (1964).
5. T. J. Miller and hl. J. Johnson, Biotechnol. Bzoeny., 8, 567 (1966).
6. V. V. Veselov, Chim. Technol. Topliv Masel, 5 , 47 (1960).