PRETREATMENT PROCESSES OF MOLASSES FOR THE UTILIZATION IN

FERMENTATION PROCESSES
GZIDE ALIK, MELIHA BERK, FATMA GL BOYACI, PINAR
ALIK, SERPIL TAKA, TUNER H. ZDAMAR
Ankara University Biotechnology Research Center, Industrial
Biotechnology Department,
06100, Ankara, TURKEY

Abstract

The composition of beet molasses was modified by 14 pretreatment processes which

were the combination of physical, physicochemical, and chemical processes, i.e. acid
hydrolysis of sucrose, precipitation of metal ions, removal of organic acids by ion
exchange resins, and entrapment of metal ions in solution. Acid hydrolysis was
favourable for glutamic acid fermentation, while the use of diluted and centrifuged
molasses was advantageous for alkaline protease fermentation.
1. Introduction

Molasses, which are the by-products of the sugar-beet or sugarcane extraction processes
are among the most important raw materials of the fermentation industry; especially for
the production of baker's yeast, citric acid, feed yeasts, acetone/butanol, organic acids,
amino acids, antibiotics, and enzymes. The suitability of molasses for industrial
fermentations cannot be evaluated according to their origin and chemical composition
as different criteria determine the productivity and quality for their use in different
processes. In processes in which they are the sole carbon source, the molasses should
be pretreated and the inhibitors should be removed. For yeast and methanol production
molasses are often simply neutralised with calcium carbonate. For many other processes
they are only boiled in an acidic or alkaline medium and after setting out separated from
the precipitate. For citric acid production the molasses are boiled with potassium
ferrocyanide and generally fermented together with the precipitate (Palacios, 1966,
Kundu et al, 1984, Cejka, 1985, Sharma et al., 1991).
Although beet or cane molasses has been used in the fermentation media for the
production of glutamic acid with Brevibacterium or Corynebacterium strains in
literature the aim were generally to investigate the effect of other operational parameters
21
M. Hofman and P. Thonart (eds.), Engineering and Manufacturing for Biotechnology, 2128.
2001 Kluwer Academic Publishers. Printed in the Netherlands.

Gzide alik, et al

such as the use of surface active agents to increase the membrane permeability in fedbatch operation (Yu-cheng, 1973), shift in operation temperature with temperature
sensitive mutants (Momose and Takagi, 1978), estimation of cell growth with on-line
measurement (Park et al., 1983a,b, 1984, Wu et al., 1989), continuous production
with immobilised biocatalyst (Kim and Ryu 1982). The pretreatment processes, if
applied any, has not been described in the above-mentioned literature.

The effects of various defined and semi-defined media involving simple carbon sources
such as glucose and/or fructose, organic acids, and amino acids, or complex carbon

sources such as casein, corn steep liquor, and starch (alik et al., 1998, alik et al.,
2000), in the production of serine alkaline proteases that are the most important group
of industrial enzymes using Bacillus strains have been investigated, however molasses
has not been used for the protease production processes in the literature.
This work investigates the effects of several pretreatment processes (PP), which
were the combination of physical, physicochemical, and chemical processes,
systematically on the composition of molasses and finally on the efficiency of their use

on two bioprocesses, namely, glutamic acid and serine alkaline protease production.
2. Materials and methods
2.1. PRETREATMENT PROCESSES (PP)
Beet molasses, the composition and properties of which was given in Table 1 was
modified with 14 pretreatment processes that have been designed as the combination of

the following unit processes and abbreviated as PP1 - PP14 as given in Figure 1 (Berk,
1995). Not pretreated, that is, only diluted and centrifuged molasses was abbreviated as

PP0. The details of the physical, physicochemical, and chemical pretreatment shown in
the Figure are as follows: Dilution: 100 g molasses was diluted with water to obtain
solution. Centrifugation: Diluted or insoluble impurity containing molasses
was centrifuged for 20 min at 6000 g and
conditions. Hydrolysis: Sucrose
hydrolysis of molasses was accomplished either with a liquid acid, i.e.
and
or a solid acid, i.e. Amberlite IR-120P. Liquid acid hydrolysis was made at
temperature,
rpm agitation rate, and
h conditions with 6M

acid. In some of the pretreatments further hydrolysis with liquid at

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was made in

Pretreatment processes of molasses for the utilization in fermentation processes

order to hydrolyse sucrose completely. Solid hydrolysis reaction was established in

packed column including strongly acidic cation exchange resin, Amberlite IR-120P, at
temperature and
flow rate conditions by using molasses solution
containing
sucrose. Precipitation: Metal ions in molasses were precipitated
with
and
solutions,
and

precipitation was carried out either at

temperature conditions with 1.5 M base.

or

and
h,
rpm,
precipitation was similar to

In the precipitation with

and
and
were added separately to molasses solution to adjust pH to 3 and
8, respectively. The reaction was carried out for 1 h at
temperature. To separate
ions from the medium
was added.
precipitation was
performed with

solution. EDTA Entrapment: 5 % (w/v),

EDTA was used in order to entrap metal ions from the solution. SACE
Pretreatment: In one of the pretreatment processes metal ions were removed with a
strongly acidic cation exchange (SACE) resin, Amberlite IR-120P. For this purpose,
molasses solution was fed to the ion exchange column at
flow rate. SBAE
Pretreatment: Organic acids were removed with a strongly basic anion exchange
(SBAE) resin, Amberlite IRA-400. After the pH of
molasses solution had
adjusted to 7, it was fed to the ion exchange column at
flow rate. Filtration:
The solution was filtered with Whatman No:l filter paper after the hydrolysis and
precipitation steps for the separation of the solid impurities.
2.2. BIOPROCESSES

2.2.1. Glutamic acid fermentation

Corynebacterium glutamicum (NRRL B-2784) was grown, inoculated and cultivated as
described elsewhere (Berk, 1995).
urea was added to molasses solution
involving
reduced sugar. pH was adjusted with 25 %
to 7.3. Batch
experiments were conducted in agitation and heating rate controlled orbital shakers,
using microbial air filtered
flasks having
working volume capacities at
temperature and
rpm agitation rate conditions.
penicillin G was added to the bioconversion medium at
h cultivation time.
2.2.2. Alkaline protease fermentation

Bacillus licheniformis (DSM 1969) was grown, inoculated and cultivated as described
elsewhere (alik et al., 1998). The reference medium for the investigation of
pretreatment processes in alkaline protease fermentation was

glucose, 6.0;

(alik et al., 1998). Glucose was replaced by

pretreated molasses solution PP0, PP1, and PP13 in order to investigate the effects of
the pretreatment. Batch experiments were conducted in agitation and heating rate
controlled orbital shakers, using microbial air filtered
flasks having
working volume capacities at
temperature and
rpm agitation

rate conditions.

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Gzide alik, et al

3. Results and discussions

3.1. EFFECT OF PP ON METAL ION CONCENTRATIONS

The beet molasses solution contains compounds used as substrates in bioprocesses such
as sucrose, invert sugar, amino acids, organic acids, inorganic compounds and vitamins
(Schneider,1979; Cejka,1985). However, some compounds can inhibit growth Table 2.
The efficiency of metal ion removal with the pretreatment processes for molasses of
the microorganism and product formation. Moreover, the stability of the product can be
decreased by the compounds involved in molasses. Consequently, the molasses must be
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Pretreatment processes of molasses for the utilization in fermentation processes

modified by some pretreatment processes before using as a substrate in bioprocesses.

One of the objectives of this work was to remove the metal ions,
and
by pretreatment processes and to determine
the pretreatment process effects in glutamic acid and alkaline protease fermentations.
The order of different pretreatment processes designed in this work, which were the
combination of dilution, centrifugation, acid hydrolysis of sucrose, precipitation of
metal ions, removal of organic acid, entrapment of metal ions and filtration are given in
detail in Figure 1. After the employment of the pretreatment processes from PP1 to
PP11, the efficiency of metal ion removal can be seen in Table 2. The metal ions were
removed completely by the pretreatment processes PP12 and PP14.

3.2. EFFECT OF PP ON GLUTAMIC ACID FERMENTATION

The glutamic acid fermentation was carried out by PP0, PP1, PP2, PP3, PP7, PP11 and
PP14 molasses for 37 h. The effect of the pretreatment processes on the relative
concentrations of Corynebacterium glutamicum and glutamic acid are shown in Figures
2 and 3, respectively. As it is clear in Figure 2, pretreatment processes inhibit cell

growth to some extent, where PP14 has the most detrimental effect, because all the
metal ions are removed totally. When glutamic acid concentration is considered the best
results were achieved with PP1, PP13 and PP2. The comparison of results of
fermentations carried out with untreated and pretreated molasses showed that the
pretreated molasses increased the glutamic acid yield up to 70 %. The highest glutamic
acid concentration was obtained with PP1. In PP1,
and
ions were precipitated
together with
ions as their sulphates; however, in
and
ions precipitate as their phosphates. From the results given in Figure 2 one can conclude
that phosphate ion is still a stronger inhibitor than sulphate ion for the microorganism in
glutamic acid fermentation. Therefore, its concentration in the media should be very
low. Organic acids were removed by strongly basic anion exchange resin with

PP13. PP13 was almost as effective as PP1, hence the concentration of organic acids
does not produce a negative effect in glutamic acid fermentation.

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3.3. EFFECT OF PP ON SERINE ALKALINE PROTEASE FERMENTATION

The effect of pretreatment processes on alkaline protease fermentation was investigated
with PP0, PP1, and PP13 molasses. Alkaline protease productions were carried out for
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Pretreatment processes of molasses for the utilization in fermentation processes

h. For this purpose, readily accessible carbon source glucose in the fermentation
media was replaced with PP0, PP1, and PP13 molasses corresponding to different
sucrose or reduced sugar concentrations between
The variations of cell
concentration and enzyme activity were measured throughout the bioprocess. The
activity and cell concentrations obtained with PP1 and PP13 molasses did not differ
significantly. The results showed that when sucrose/reduced sugar concentration of the
PP0 and PP1 molasses increased, the cell concentration first increased and then
decreased, giving a maximum at 45 and
respectively. With PP0 and PP1
molasses, maximum cell concentrations were 2.5 and
,
while for the reference
medium involving
glucose as the carbon source this value was
Reduced sugar concentration affected the enzyme activity in the same manner and
gave maximum relative activity with PP1 when compared with the reference
solution, as 1.1 and it was obtained at
h. However, only diluted and centrifuged
molasses PP0 was more effective in the protease production with the relative activity
value 2.1 obtained with
sucrose containing molasses (Figure 4). These results
show that besides the positive effects of amino acids, organic acids, inorganic
compounds and vitamins involved in molasses the use of sucrose instead of a readily
accessible carbon source, e.g. glucose and fructose, may cause the microorganism to
function the bioreaction network under stress that it increases the protease production.

4. Conclusions

14 pretreatment processes were developed for the utilisation of beet molasses in

fermentation process, i.e., glutamic acid and serine alkaline protease. The best results
were obtained with PP1 for glutamic acid production, however, PP0 molasses was more
beneficial for the protease enzyme production. When essential components are removed
from the media, the cell growth is strongly inhibited. This consequently causes a
decrease in the product formation. However, the presence of some ions may induce the
stability of a product, e.g. an enzyme as well. Therefore, the design of the pretreatment
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Gzide alik, et al

process to be applied to a complex carbon and energy source should be made

considering the needs of the cell and its metabolism. The investigation of different
pretreatment processes to protease enzyme production as well as other bioprocesses are
being continued.
Acknowledgements

The financial support of SPO (Turkey) Grant No. 89K120390 and 95K120290 are
gratefully acknowledged.
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