b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 403–409

http://www.bjmicrobiol.com.br/

Biotechnology and Industrial Microbiology

Enhanced ethanol production at commercial scale

from molasses using high gravity technology by
mutant S. cerevisiae

Muhammad Arshad a , Tariq Hussain a , Munawar Iqbal b,∗ , Mazhar Abbas c

a Department of Basic Sciences, Biochemistry Section, College of Veterinary and Animal Sciences, Jhang Campus 35200, Pakistan
b The University of Lahore, Department of Chemistry, Lahore, Pakistan
c The University of Lahore, Institute of Molecular Biology and Biotechnology, Lahore, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Very high gravity (VHG) technology was employed on industrial scale to produce ethanol
Received 5 June 2015 from molasses (fermented) as well as by-products formation estimation. The effect of dif-
Accepted 29 November 2015 ferent Brix◦ (32, 36 and 40) air-flow rates (0.00, 0.20, 0.40, and 0.60 vvm) was studied on
Available online 16 February 2017 ethanol production. The maximum ethanol production was recorded to be 12.2% (v/v) at 40
Associate Editor: Solange Ines Brix◦ with 0.2 vvm air-flow rate. At optimum level aeration and 40 Brix◦ VHG, the residual
Mussatto sugar level was recorded in the range of 12.5–18.5 g/L, whereas the viable cell count remained
constant up to 50 h of fermentation and dry matter production increased with fermentation

Keywords: time. Both water and steam consumption reduced significantly under optimum conditions

Very high gravity technology of Brix◦ and aeration rate with compromising the ethanol production. Results revealed VHG
Ethanol with continuous air flow is viable technique to reduce the ethanol production cost form
Molasses molasses at commercial scale.
© 2017 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de
Aeration
Microbiologia. This is an open access article under the CC BY-NC-ND license (http://
Brix
creativecommons.org/licenses/by-nc-nd/4.0/).

(v/v) ethanol production from diluted molasses (15–16%).2,3

Introduction
However, during ethanol production, huge amount of efflu-
ent (∼12 liter effluent/liter absolute alcohol) with very high
Due to higher petroleum product prices and air pollution biological oxygen demand BOD (45,000–60,000 mg/L) and COD
issues, researchers are focusing to explore alternative renew- (80,000–120,000 mg/L) is produced, which is discharged into
able sources. In this connection, ethanol has been emerged as the environment without proper treatment.4 So for, the
a potential alternative candidate as it is eco-friendly and use- ethanol production using current distilleries is a potential
able as blending with gasoline in current combustion engine source of environmental pollution in Pakistan and ethanol
systems.1 Sugar cane molasses (high fermentable sugars) is production process needs to be improved and optimized for an
the major feedstock for distilleries in Pakistan for ethanol pro- environment friendly, fast and cheap ethanol production. The
duction. The industrial process is well established with 7–8% sugar cane molasses (sugar production by-product) is a good

∗
Corresponding author.
E-mail: bosalvee@yahoo.com (M. Iqbal).
http://dx.doi.org/10.1016/j.bjm.2017.02.003
1517-8382/© 2017 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Microbiologia. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
404 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 403–409

option to produce ethanol.5,6 Biomasses are efficient source of Saccharomyces cerevisiae UAF-1 and used for ethanol produc-
energy and are eco-friendly in nature.7–11 tion). The selected strain was cultured in medium containing
The application of VHG fermentation technology (the (g/L): sucrose (10.0), yeast extract (3.0), (NH4 )2 SO4 (2.0), and
preparation and fermentation of mashes containing 27 g or MgSO4 (0.5). We found that gene creAdelta4 was significantly
more dissolved solids per 100 g mash) has been proposed mutated and levels of mRNA of CreA protein in the wild strain
for efficient ethanol production and to reduce stillage vol- were more in quantity than those of the mutant derivative
ume. Among different ethanol production methods, the VHG (creA delta4 gene is useful in enhancing the production of
fermentation technology is very efficient at industrial scale extracellular invertase (protein) level in the medium as well
since it offers higher yield (12–15%), low waste generation as take part in carbon catabolite repression. The Sko1 gene
and can be operated at low cost. Higher ethanol level can has role in activation and repression of protein transcription
reduce the distillation cost as compared to the traditional involved in osmotic and oxidative stress and also suppression
fermentation, where maximum 7–8% ethanol is produced.2,12 off kinase overexpression is controlled by Sko1 gene). More-
The ethanol percentage production can be enhanced through over, copy number of sko1 gene in the mutant derivative was
VHG technology using strains tolerable to high sugar contents. sufficiently decreased to cause repression of transcription
Yeasts are known to tolerate higher sugar contents (up of CreA motif as done in wild strain. Good quality molasses
to 40%), however, at higher sugar concentration, the yeast having 89 ◦ Bx, total reducing sugars (TRS) (50% w/v) with
growth is affected, which grows very slowly.13 Yeast growth addition of nitrogen and phosphorus contents was used for
is regulated by metabolites generated during ethanol produc- the inoculate preparation and very high gravity technique
tion. Generally, industrial ethanol production is performed was used for fermentation of molasses.
at low carbohydrate concentration in fermentable sugar.2,14
Previous reports indicated that yeast can grow up to 50% Molasses pre-treatment
sucrose in fermenting media15 and batch fermentation with
23.8% (v/v) ethanol at laboratory scale was achieved in a For molasses pretreatment, diluted molasses was settled
simultaneous saccharification and fermentation mode.16 Lab- in tanks of conical bottom type, especially designed for
scale multistage continuous cascade, chemostat fermentation the removal of sludge, ash contents and other particulates.
system have been constructed to investigate its applica- Sodium hexameta phosphate was added and pH was adjusted
bility toward ethanol production under increased gravity at 4.0–4.5 with commercial sulfuric acid which converted Ca2+
conditions.17 Ethanol production using VHG technology can be into calcium sulfate.22 Brix◦ of the substrate was adjusted at
enhanced applying various strategies, i.e., vitamin feeding,12 32, 36 and 40 and sugar contents were 21%, 24%, and 27% (w/v)
nutrient feeding,13 media supplementations,18 temperature in pre-treatment molasses, respectively.
optimization19 and aeration rate12 have been studied well to
improve the ethanol percentage yield. Among these strate- Inoculum preparation
gies, aeration improved the viability of yeast during high-level
ethanol production.20 Initially, the propagation of yeast was started in 1 m3 vessel
The VHG fermentation technology in alcohol industry and cell count reached to 3.00 × 106 CUF/mL; it was transferred
(operated in Pakistan) is of great interest, as it saves consider- to 60 m3 vessel to maintain appropriate cell count2 and this
able amount of water with higher alcohol yield, which needs inoculum was used for fermentation of molasses.
low energy input, less capital cost, more efficient and is also
free from bacterial contamination.21 Therefore, this study was Fermentation
aimed to evaluate the ethanol production using Saccharomyces
cerevisiae (mutant strain). Moreover, the effects of aeration rate Fed-batch experiments were performed in fermenters of
and Brix’s on ethanol percentage yield along with by-products 300 m3 capacity. The temperature was maintained at 32 ± 1 ◦ C
estimation in fed-batch mode fermentation at industrial scale and stirred by circulation of mash with the help of elec-
(using current distilleries system in Pakistan) was also inves- tric pump at 300 rpm. To control the foaming, silica based
tigated. antifoaming agent was used. Air was supplied by the air blow-
ers. The aeration rates used were 0.0, 0.1, 0.3, 0.6 and 0.9 (vvm).
The fermenter was filled in 16 h after the transfer of inocu-
Materials and methods lum (25%) with appropriately diluted molasses. Level rise of
the fermenter was kept at 5%/h (15 m3 molasses were fed to
Microorganism and culture media fermenter/h (Fed-batch mode), which is 5% of total fermenter
molasses volume used for fermentation). After transferring of
The Saccharomyces cerevisiae parental strain was obtained from inoculum (100 m3 ), the substrate (200 m3 ) was supplied contin-
Shakarganj distillery (Instant-France). The mutant strain of uously. Five fermenters were run for each treatment in order
Saccharomyces cerevisiae UAF-1 was used for fermentation to check reproducibility.
(S. cerevisiae SAF-INSTANT strain were exposed to gamma
radiation (500 Krad using Co-60 gamma radiation source) and Distillation
resultantly, 1135 mutant strains were recorded. All survivors
were tested for sugar tolerance and ethanol production. Fermented mash was distilled in double effect distilla-
The survivor (GAMMA-11) furnished higher tolerance to tion plant (Firlli, Italy) having 40,000 liters processing
sugar as well as ethanol production, which was named as capacity/day.2 Fermented mash was transferred to the mash
 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 403–409 405

column and steam was applied from bottom in such a way

Table 1 – Fermentation kinetic parameters at different
that temperature maintained in the range of 78–80 ◦ C. Vac- Brix◦ .
uum was applied through condensers. Vapors traveled from
Parameters 32◦ 36◦ 40◦
column to condensers were condensed and cooled. The resul-
tant liquid was re-boiled in depuration column for removing Fermentation time (h) 60 60 60
impurities and finally, fed to rectification column. Rectifica- Residual sugars (g/L) max 35.4 39.7 60.4
Residual sugars (g/L) min 12.5 14.5 18.7
tion of ethanol was done in the range of 96.0–96.4% and this
max (h−1 ) 0.38 0.34 0.35
column was operated at 1.5 kg m/s2 pressure.
min (h−1 ) 0.31 0.42 0.43
Xmax (g/L) 20.3 22.2 22.7
Determination of cell count/viability Xmin (g/L) 16.1 18.4 19.0
Ethanol max (g/L) 8.2 8.9 9.6
For cell count determination hematocytometer was used, fer- Ethanol (min g/L) 7.18 7.30 7.50

menter broth sample was serially diluted with a sterile saline

solution (0.89% (w/v) NaCl) to a point where reasonable num-
ber of cells could be counted (3.00 × 106 CUF/mL). The cell
viability was determined by the Methylene Blue technique.12 million/mL at 0.60 vvm. High oxygen concentration exerted
strong influence on yeast growth during fermentation and this
Brix, reducing sugars, ethanol and by-products trend was in line with previous findings that oxygen supple-
determination ment has significant effect on ethanol production as well as
cell viability.26 The viable cell count increased to 16%, 20% and
Concentration of TRS in diluted molasses and fermented 22% at 0.2, 0.4 and 0.6 vvm, respectively. The final biomass
mash (after sucrose inversion using HCl) was measured by (Fig. 1D) and ethanol production was recorded considerably
Fehling-Soxhlet method.23 Brix was measured with the help of at higher sugar level, which indicates that increasing level
ATAGO densimeter (model 2312; ATAGO Co. Ltd., Tokyo, Japan). of sugar, the yeast cope the osmotic stress due in response
Ethanol and by-products in fermented samples were deter- of ethanol production in fermenter. The studied parameters
mined using GC (Shimadzu GC-17A, 3.0) equipped with flame (Table 1) during ethanol fermentation clearly showed that the
ionization detector (FID) as reported earlier.2 The aldehyde aeration affected the process at higher sugar level.
content was estimated through ASTM-D-1363 method. Ethanol production (%) was recorded to be 9.3% (v/v) at 36◦
Brix without aeration and reached to 11.3% (v/v) with aeration
(Fig. 2A). Aeration rates of 0.20, 0.40 and 0.60 vvm increased the
Results ethanol production by 20%, 16% and 14% over the non-aerated
fermentation, respectively. The residual sugar increased up
The VHG technology with and without aeration was evaluated to 30 h of fermentation and then, decreased sharply up to
on industrial scale to improve the traditional fermentation for 60 h (Fig. 2B). Maximum viable cell count was enhanced with
ethanol production. VHG technology resulted in higher alcohol increasing sugar level and 499 million/mL cell count was
production (11–12%, v/v) as compare to 7–8% (v/v) in conven- recorded at 36◦ Brix (Fig. 2C). Similarly, the biomass increasing
tional process and overall, variation among replicates was in trend was observed as the time of fermentation is increased at
the range of ±2–3% throughout the experimentation. High different VHG levels (Fig. 2D). The reduction is residual sugar
gravity molasses media containing different concentration of and production of biomass is a good indication of ethanol
dissolved solid, ranging from 32 to 40◦ Brix were prepared and production and Brix level significantly affected the ethanol
fermented for 60 h in 300 m3 vessel. Maximum ethanol was production. The maximum ethanol production of 12.2% (v/v)
produced (12.2%) from 27% TRS at 0.2 vvm aeration rate. The was recorded at 40◦ Brix with 0.2 vvm aeration rate (Fig. 3A).
higher sugar content results in higher ethanol production and The residual sugar levels were 18.7 g/L, 24.8 g/L and 21.2 g/L
these findings are in line with reported finding, i.e., pilot-scale at 0.2, 0.4 and 0.6 vvm, respectively (Fig. 3B), whereas in non-
ethanol production from rice straw hydrolysates using xylose- aerated fermenter, the residual sugar was recorded to be
fermented with Pichia stipites was studied24 and influence of 60.4 g/L, which indicates that aeration along with Brix◦ level
aeration on bioethanol production from ozonized wheat straw is also important factor in ethanol production. The cell count
hydrolysates using Pichia stipitis.25 was recorded to be 514 million/mL at 40◦ Brix (Fig. 3C), which
Viable cell count, ethanol formation, residual sugar con- was maximum cell count as compared to other Brix levels.
tent and dry matter generation were determined during the The biomass production trend at 40◦ Brix was recorded sim-
fermentation period. By-products, i.e., methanol, acetic acid, ilar to other Brix levels, which increased with the passage of
higher alcohols (1-propanol, 1-butanol, 2-butanol) production time.
and aldehyde appearance time were recorded and results are Different by-products, i.e., methanol, acetic acid, higher
depicted in Table 2. Ethanol (%, v/v) and residual sugar (g/L) alcohols (1-propanol, 1-butanol, 2-butanol) were recorded
at 32◦ Brix with different aeration rates are shown in Fig. 1A and by-products production was recorded to be minimum at
and B, respectively. Residual sugar showed increasing trend 0.2 vvm air flow-rate (Table 2) as compared to control (0.00 vvm
up to the completion of batch. Without aeration, the ethanol aeration rate). The aldehyde appeared after longer time of fer-
production was 8.9%, with 35.41 g/L residual sugar contents. mentation when aeration rate was 0.2 vvm versus all other
Fig. 1C shows the viable cell count at 32◦ Brix with differ- air flow-rates. Higher alcohols, i.e., 1-propanol, 1-butanol, 2-
ent aeration rates. Maximum viable cell count remained 417 butanol productions were recorded to be 2.4, 2.8 and 3.6 g/h L
406 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 403–409

A 11
B 140

120
10

Residual sugar (g/L)

Ethanol, %
9 100

8 80

7
60

6
40
5
20
4
0
10 20 30 40 50 60 0 10 20 30 40 50 60

Time (h) Time (h)

C D
420 20.0

400 17.5
Cell count (x 106 /mL)

380
15.0
360 Dry matter (g) 12.5
340
10.0
320
7.5
300
5.0
280
2.5
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (h) Time (h)

0 VHG 0.02 VHG 0.04 VHG 0.06 VHG

Fig. 1 – (A) Ethanol production, (B) residual sugar contents, (C) cell count and (D) dry matter at 32◦ Brix at different very high
gravity levels.

Table 2 – Effect of aeration on by-products formations at different Brix◦ levels.

Brix◦ Air level Methanol (g/h L) Acetic acid (g/h L) Higher alcohols (g/h L) APT* (min)

32◦ 0.00 vvm 2.3 3.4 1.2 28

0.20 vvm 1.8 2.1 1.3 40
0.40 vvm 3.1 4.1 2.2 24
0.60 vvm 3.5 5.0 2.4 16

36◦ None 3.7 3.5 1.3 9

0.20 vvm 2.7 2.1 1.3 30
0.40 vvm 4.7 4.1 2.6 18
0.60 vvm 5.2 5.0 2.8 12

40◦ None 4.4 5.2 1.5 10

0.20 vvm 3.2 3.1 1.7 26
0.40 vvm 5.6 6.1 3.3 19
0.60 vvm 6.2 7.4 3.6 14

∗
Aldehydes appearance time and higher alcohols include 1-propanol, 1-butanol, 2-butanol.

in high aerated (0.6 vvm) fermenter in comparison with non- 0.2 vvm, however, the ethanol production decreased at higher
aeration medium (1.2, 1.3 and 1.5 g/h L), respectively. It was aeration rates. Sugar fermentation by S. cerevisiae is gener-
observed that increased ethanol production inhibited the cell ally inhibited in the presence of oxygen, but small amount
growth. Cell count viability declined up to 25% in non-aerated of dissolved oxygen enhances ethanol production compared
culture at 32◦ Brix. Overall, at Brix◦ three levels used, resid- to highly aerobic condition.27 Residual sugar recorded to be
ual sugars were very high in non-aerated fermenters. Aeration 39.7% in non-aerated fermenter and only 14.5% in aerated fer-
improved the ethanol production by 18% at aeration level of menter. Maximum residual sugar during feeding was reached
 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 403–409 407

A 12
B 140

11
120
10

Residual sugar (g/L)

100
9
Ethanol, %
80
8

7 60

6 40

5
20
4
0
10 20 30 40 50 60 0 10 20 30 40 50 60
Time (h) Time (h)

20.0
C 500 D
480 17.5
460
15.0
Cell count (x 106 /mL)

440

Dry matter (g)

420
12.5
400
380 10.0
360
340 7.5

320
5.0
300
280 2.5
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (h) Time (h)

0 VHG 0.02 VHG 0.04 VHG 0.06 VHG

Fig. 2 – (A) Ethanol production, (B) residual sugar contents, (C) cell count and (D) dry matter at 36◦ Brix at different very high
gravity levels.

to 130.5 g/L. The viability of cells maintained 80% even after stage and ethanol induced oxidative stress at the end of fer-
48 h of fermentation as compared to control (60%). Higher alco- mentation. These findings are in line with.29 Authors reported
hols production along with other by-products was higher in that aerated fed batch process on glucose medium is useful in
aerated fermentation and acidity was low at 0.2 vvm as com- ethanol production. Similarly, Maemura30 also found increase
pared to non-aerated process and high aerated cultures at all in viable cell count with the aeration level. The total num-
Brix◦ levels. The formation of by-products took place under ber of cells reached maximum level after incubation for about
certain unfavorable fermenting conditions. Therefore, signif- 24 h and cell growth cultivation was found to be dependent
icant reduction in the by-products was observed at 0.2 vvm on the aeration. More aeration enhanced the dissolved oxy-
controlled aeration at all high gravity fermenting medium. The gen and at higher Brix◦ , the viable cell count decreased and
production of oxidized metabolites (acetaldehyde, acetate, this is in agreement with observations reported previously.31
and acetoin) is always favored,2 however, under aerobic con- It has been reported that the cell viability decreased as the
ditions and acetic acid production was recorded to be high. concentration of ethanol increased using S. cerevisiae during
fermentation.16 In present investigation, ethanol production
was maximum at the lowest aeration rate and Seo32 also
Discussion observed similar trend. Moreover, they reported that the
growth and ethanol production may decrease at later stages
The inhibitory effect of by-products in ethanol production of fermentation, when the ethanol concentration reached
is main factor in ethanol production.28 It was observed that ∼100 g/L. Overall, ethanol production was higher in the aer-
under aerated conditions, decline in cell count viability was ated fermenters and these findings are in line with Alfenore
much lower as compared to non-aerated culture at all sugar et al.12 and in another study, production of higher alcohols was
levels. Residual sugar under non aerated condition and aer- markedly enhanced under oxidative conditions maintained by
ated process as well as the viability of cells after 48 h of agitation or sparging with air.2 So for, based on current find-
fermentation clearly indicates that the yeast cells need some ing, it is concluded that the ethanol production using VHG
aeration in order to overcome the osmotic stress at initial technology along with aeration is efficient method since at
408 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 403–409

13
A B
12 140

11 120

Residual sugar (g/L)

10
100
Ethanol, %

9
80
8

7 60

6 40

5
20
4
0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (h) Time (h)

C 520 D 25.0

500 22.5
480
20.0
Cell count (x 106 /mL)

460
17.5
440

Dry matter (g)

15.0
420
400 12.5

380 10.0
360 7.5
340
5.0
320
2.5
300
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (h) Time (h)

0 VHG 0.02 VHG 0.04 VHG 0.06 VHG

Fig. 3 – (A) Ethanol production, (B) residual sugar contents, (C) cell count and (D) dry matter at 40◦ Brix at different very high
gravity levels.

optimized condition a significantly higher ethanol production

was achieved along with minimum by-products production.
Acknowledgements

The Jhang sugar mill Authorities are highly acknowledged for

providing research facilities at Jhang sugar mill laboratories
Conclusions

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