Alcoholic Fermentation from Sweet Sorghum : Some Operating Problems
Page 1 of 6
Alcoholic Fermentation from Sweet
Sorghum : Some Operating Problems
Phowchinda 0*., Delia-Dupuy M.L.** and Strehaiano P.**
*King Mongkut's Institute of Technology North Bangkok, Bangkok 10800, Thailand
**Laboratoire de Genie Chimique, UMR-CNRS 5503 ENSIGC-INP, 18 Chemin de la Loge, 31078 Toulouse Cedex 4,
France
Abstract
In Thailand, ethanol production from sweet sorghum may be a new option
to solve the serious problem of using fossil oil as energy resource, to improve
agricultural economy and also to reduce the pollution in environment.
Kinetic studies on synthetic media (single and mixed sugars) showed that
the proportion between sugars did not influence the behaviour of selected yeast,
Saccharomyces cerevisiae. The initial concentration of inoculum from five levels
(3.106, 5.106 , 10.106, 1.5.106 and 20.106 cells/mL) were experimented to find a good
economical compromise. The 3.10 6 and 5.106 cells/mL levels seemed to be suitable
concentration for industrial process. In the same context, the kinetics of sugar
consumption and ethanol production from different cycles (run with 3.106 and 15.106
cells/mL initial concentration) showed the stability of the selected strain.
In order to use sweet sorghum juice as a fermentation substrate, the
contamination problem in non sterile media was studied. Mixed cultures with
different concentrations between pure culture of S.cerevisiae and a contaminous
yeast, Brettanomyces bruxellensis, were tested on glucose 50 g/L. S. cerevisiae
always succeeded in implantation more than 50% but B. bruxellensis has never
disappeared from the medium (remained 5 to 10 %). Then 150 g/L glucose was
used and S. cerevisiae was inoculated during the lag period of the contaminant, this
time S. cerevisiae could not develop more than 10.10 6 cells/mL. To avoid
contamination, the different growth kinetics of both strains should be considered.
The experiments showed that operating methods and process control may greatly
influence the implantation of contaminant.
Key words: Alcoholic fermentation, Sweet sorghum, lnoculum concentration, Saccharomyces
cerevisiae, Brettanomyces bruxellensis
Note : This paper was presented at the 9th Annual Meeting of the Thai Society for Biotechnology and
the 2nd JSPS-NRCT-DOST- LIPP-VCC Seminar on Biotechnology : An Essential Tool for Future
Development?, Suranaree University of Technology, 19-22 November 1997, Thailand.
Introduction
Sweet sorghum is an interesting annual plant that can be cultivated in wildspread areas
from tropical to temperate climates. In Thailand it can grow in almost every geographic part. This plant
mainly composed of sugar (saccharose, glucose, fructose), represents an interesting renewable
potential of raw material. With currently available cultivars, yields of 3,500 to 4,000 L of ethanol per
hectare can be obtained from fermentable sugars (1). Moreover, its solid components are mainly
lignocellulose, cellulose and fibers that can be used as raw material in paper pulp manufacturing (2).
An overall study (on the potential and interest of this crop for energy and industry purpose) was
initiated recently. The preliminary programme of research dealed with complete sweet sorghum
valorization (genetic improvement, ethanol production and paper pulp industry).
Three main reasons led us to convert sorghum biomass into ethanol:
1. Thailand has to face the significant problem of oil lacking. This energy resource is very
expensive and increases too much the cost of all industrial transformations. Ethanol should be an
alternative fuel or an additive.
http://202.28.17.1/article/atc42/atc00258.html
31/7/2549
Alcoholic Fermentation from Sweet Sorghum : Some Operating Problems
Page 2 of 6
2. An industrial outlet of this crop should open a new field for this culture and encourage
agriculturists. By this way agricultural economy should be improved.
3. The environmental point of view has to be considered. Thailand is concerned by pollution
and future industrial developments have to take care with this problem. In fact, ethanol is less
pollutant than petroleum.
Actually, beverage ethanol manufactures (whisky, beer and wine) utilize barley and some
fruits as fermentation substrates. But ethanol for chemical products, pharmaceuticals and some fuel
resource is produced from molasses. Indeed, sweet sorghum wastes will be more attractive because
the fibers should be used in paper pulp manufacturing beside ethanol is produced from its juice.
This work is aimed to solve the problem of industrial practices in order to use sweet
sorghum juice as fermentation substrate by studying of kinetics on single and mixed sugars, the
optimum level of initial inoculum concentration and also the relations of mixed cultures between S.
cerevisiae and B. bruxellensis (as a contaminant).
Materials and Methods
Microorganisms :
The yeast strain was Saccharomyces cerevisiae K1 produced by Lallemand Inc. (Canada).
The Breftanomyces strain was isolated from a fermentation plant and was identified as Brettanomyces
bruxeliensis by the I.H.E.M. (Belgium).
Culture media :
The synthetic medium was made of : KH2 PO4 5g/L, ( NH4) 2 SO4 2g/L, Mg SO4 . 7H 2 0 0.4
g/L, yeast extract 1 g/L, proposed by (3). The different sugars in pure solutions were: glucose,
fructose and saccharose at a concentrations of 50 g/L. When mixed, different concentrations were
prepared. The initial pH was adjusted to 4 by using orthophophoric acid.
Culture conditions :
The experiments were performed in 2 liters fermentor. Temperature was 30 ํ C. No aeration
was provided for the cultures. lnocula were incubated on the medium 150 mL at 30 ํC for 15 hours.
Analytical determinations :
Biomass was measured turbidimetrically at 620 nm, and was calibrated to cell dry weight
determination. In case of mixed cultures, cell counting was made by using the hematimeter. Viable cell
was estimated according to (4).
For sugar determination, glucose was analysed by DNS method (5). The anthrone method
(6) was used for the total sugar measurement. For the specific determination of the different sugars
when mixed, the HPLC method was used .
Ethanol was analysed by gas chromatography by using propanol-1 (1%) as internal
standard.
Results
1. Influence of substrate composition
The interest is focused on nature of sweet sorghum juice composed of saccharose, glucose
and fructose. (7) reported that sweet sorghum juice is composed of 60% saccharose , 33% glucose
and 7% fructose , (8) showed the juice from stems is 53% saccharose, 28% glucose and 19%
fructose, while (9) showed 55% saccharose, 24% glucose and 21 % fructose. The proportions of the
different sugars varied depending on the variety and the maturity of the crop. So it is necessary to
study the influence of substrate composition upon the kinetics of fermentation.
Firstly, each single sugar was used at the concentration of 50 g/L, the amount of produced
ethanol was the same one. The yield of ethanol reached to 0.48 - 0.49 g/g. The kinetics of glucose
consumption, growth and ethanol production were shown in Figure 1.
http://202.28.17.1/article/atc42/atc00258.html
31/7/2549
Alcoholic Fermentation from Sweet Sorghum : Some Operating Problems
Page 3 of 6
Figure 1. Fermentation kinetics on glucose 50 g/L by Saccharomyces cerevisiae K 1
Secondly, mixed of 3 sugars at the same and different proportions were experimented.
Total sugars were varied (60, 100 and 150 g/L). The results of ethanol production were similar to
those obtained from single sugar. Then, a mixture of sugars based on the composition of sweet
sorghum juice (56% saccharose, 30% glucose, 14% fructose) was tested. The experiments showed
any difference upon kinetics of fermentation depending on the substrate composition.
From the practical point of view, single and mixed sugars did not influence the behaviour of
S . cerevisiae. Indeed, using a high concentration of mixed sugars (150 g/L) seemed to be not
efficient, leading to the utilization of sweet sorghum juice as a fermentation substrate.
2. Influence of inoculum concentration
At the first step of industrial fermentation process, preparing an inoculum with a suitable
concentration is very important. Dry yeast utilization may represent an alternative to this operating
mode by direct inoculation of the seed tank.
The objective is to find a good economical compromise between the level of inoculum
concentration and the reaction productivity. Indeed, the purchase price of yeasts is rather expensive,
in order to limit the cost of microbial material, the minimum inoculation rate should be considered.
Firstly, different inoculum concentrations (3.106, 5.10 6, 10.106, 15.10 6 and 20.106 cells/mL)
were tested. The results showed the biomass and ethanol production yields were not modified. It was
similar to the observation of (10) when the initial levels of inoculum were varied (from 0.007 to
0.20%). However (11) studied on different inoculum levels that were much more concentrated (from 2
to 60% V/V).
In the same context when the initial inoculum level was increased, decreasing of the
viability of yeast population at the end of fermenta- tion was remarkable (from 93 to 85%.) It showed
that increasing the concentration from 3.10 6 to 20.16 6cells/mL could not develop the biomass and
ethanol production. On the contrary, using too high concentration of inoculum may cause to reduce
the viability of selected strain that will be not interesting for cell recycling. The results were
summarized as shown in Table 1.
Table 1. Yield of biomass, ethanol and cell viability depending on different inoculum concentrations
lnoculum
(cells/mL)
Biomass yield Ethanol yield
(g/g)
(g/g)
Cell viability(end of
fermentation, %)
3.106
0.06
0.50
93
5.106
0.06
0.50
89
10.106
0.06
0.49
80
15.106
0.06
0.46
83
http://202.28.17.1/article/atc42/atc00258.html
31/7/2549
Alcoholic Fermentation from Sweet Sorghum : Some Operating Problems
20.106
0.06
0.46
Page 4 of 6
85
At this moment, the 3.106 and 5.106 cells/ mL initial inoculum seeding seemed to be a good
economical way for industrial processes.
Secondly, the stability of the yeasts should be studied. In order to test the capacity of
strain, successive cultivations were run on glucose 50 g/L. Biomass at the end of fermentation of
previous batch was used to inoculate to the next one at the same concentration. Two levels of
inoculum, (3.10 6 and 15.106 cells/mL) were tested for 4 and 3 cycles respectively. The results were
similar. Growth, glucose consumption and ethanol production rates remained constant through out the
cycles. Kinetics of glucose consumption for 4 cycles with an initial 3.10 6 cells/mL were shown in Figure
2.
Figure 2. Glucose consumption for 4 cycles of fermentation with an initial 3.10 6 cells/mL inoculum by S.ceresisire K1
The rates of fermentation are linked to the initial concentration of inoculum. This parameter
is one of the main key of process control, standardization of the inoculum step should be necessary.
3. Contamination problem
Non sterile media utilization is widespread in industrial processes due to the economy.
When a fermentation is stepped up to the industrial scale, the size of plant and the volume of raw
material generally make the sterilization difficult and the cost is always expensive. Moreover many
kinds of waste products from other processes are utilized as fermentation substrates, corn steep liquor,
soya meal, molasses of sugar beet and sugar cane. This kind of each media does not stand sterilization
cycle (browning, precipitation of some compounds ... ). We intend to use sweet sorghum juice as
fermentation substrate to produce ethanol. Because of low value added of ethanol, sweet sorghum
juice cannot be sterilized, by this way it will be easily contaminated. This problem induced us to study
the development of a contaminant strain during fermentation. On fermentation plants, seeded yeasts
and indigenous population may compete and indigenous strains sometimes succeed in implanting.
Such problem may involve a decrease of the yield and rate of the process. A best understanding of the
behaviour of mixed cultures should allow a best control of the process to avoid fermentation
contamination.
The tested strain of S. cerevisiae K1 was already used on french fermentation plants. The
chosen contaminant, Brettanomyces bruxellensis was often found to be a contaminant in alcoholic
fermentation. Pure cultivation was run on glucose 50 g/L for each strain. Under aerobic conditions (0.1
vvm), B. bruxellensis showed a significant lag period (about 15h), and sugar depletion was complete
after 37 h. The production yield reached to 0.42 g/g but global rate was only 0.6 g/L/h. Without
aeration S. cerevisiae, showed 0.47 g/g and a 1.26 g/L/h productivity.
Then the two yeasts were cultivated together. The percentage of each strain was varied
from 90 to 60% for B. bruxellensis and from I0 to 40% for S. cerevisiae. Even initial S. cerevisiae level
http://202.28.17.1/article/atc42/atc00258.html
31/7/2549
Alcoholic Fermentation from Sweet Sorghum : Some Operating Problems
Page 5 of 6
was 10%, the balance between the two strains happened quickly after 5h, the part of S. cerevisiae
reached to 75%. With different proportions S. cerevisiae always succeeded in implanting more than
50%. Nevertheless B. bruxellensis had never disappeared from the medium, remained 5 to 10% of the
global population. As initial sugar contant was limited at 50 g/L, it was completely consumed after 20h.
It corresponded to the start of B. bruxellensis growth phase, S. cerevisiae seemed responsible for
glucose consumption. May be the lack of substrate limited B. bruxeliensis development, then 150 g/L
sugar concentration was tested. It was found that when B. bruxellensis succeeded well in implantation,
S. cerevisiae could slightly develop and remained at low population level. The kinetics of fermentation
on glucose 150 g/L was shown in Figure 3.
Figure 3. Fermentation kinetics on glucose 150 g/L when initial mixture was 10% Saccharomyces cerevisiae and 90%
Brettanomyces bruxellensis
In order to avoid B. bruxellensis implantation, it is necessary to consider the difference of
growth kinetics of both strains. As S. cerevisiae developed more quickly than B. bruxellensis, promoting
growth was a good way to enhance the faster growth of S. cerevisiae and limit the slower implantation
of B. bruxellensis.
Conclusion and Discussion
1. Industrial exploitation of renewable raw materials has become of greater interest. Sweet
sorghum transformation should open a new field for Thai agriculture and industry; in this way different
investigations have been started. This work is especially focused on problems connected with industrial
operating of alcoholic fermentation. As sweet sorghum juice composition may vary depending on the
crop, the influence of the nature and the sugar proportion should be examined. Any effect upon yeast
behaviour was detected. Process control should be independent of raw material arrivals.
2. Controlling microbial population is a key of biological process command :
Standardization of inoculum should be solved by dry selected yeast seeds. Inoculation with
6
3.10 or 5.106 cells/mL may offer a good compromise between cost and productivity.
Cell recycling is a widespread practice in alcoholic fermentation plants. Nevertheless this
method associated with non sterile media use increased the risks of contamination. A study of
contaminant implantation was presented. The example showed that promoting S. cerevisiae growth
could favour against indigenous strain competition. Partial recycling may be an answer to economical
management of selected strain utilization (high rates, lower cost).
The variety of possible contaminant strains in sweet sorghum juice may involve a more
complete evaluation of the contamination problem. Moreover, this study was run using synthetic
medium; testing the yeast on sweet sorghum juice should be one of the next step of the
investigations.
References
1. Kosaric N., Wieczorek A., Cosentino G., Magee R. and Prenosil J., Biotechnal, 3, 257-385, (1983).
2. Mardon C. and Gardside G., Proc. Nat. Conf. Fuels from Crops, 28 - 29 Sep., Melbourne, Australia.,
1-6, (1981).
3. Strehaiano P., These Dr. es Sciences., I.N.P. Toulouse, France (1984).
http://202.28.17.1/article/atc42/atc00258.html
31/7/2549
Alcoholic Fermentation from Sweet Sorghum : Some Operating Problems
Page 6 of 6
4. Lange H., Bavouzet J.M., Taillandier P. and Delorme C., Biotechnol. Techniques, 7, 223-228,
(1993).
5. Miller G.L., Anal Chem., 31, 426-428, (1959).
6. Weiner J., J. lnst. Brew., 84, 222-223, (1978).
7. Mohite U. and Sivaraman H., Biotechnol. and Bioeng., 26, 1126-1127, (1984).
8. El Bassam N., Commission of the European Communities, (1990).
9. Curt M.D., Martinez M. and Fernandez J., 7 th European Conference on Biomass for Energy and
Environment, Italy, 5-9 Oct., (1992).
10. Bryan W.L., Enzyme Microb. Technol., 12, 437-442, (1990).
11. Strehaiano P., Mota M. and Goma G., Biotechnol. Lett., 5, 135-140, (1983).
Last modified :
http://202.28.17.1/article/atc42/atc00258.html
31/7/2549