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ST-P-038

Burapha University International Conference 2015

“Moving Forward to a Prosperous and Sustainable Community”

Nutrients and ions optimization for ethanol fermentation from

cane molasses
Pongpannee Poomikhata,*, Ancharida Akaracharanyaa, Vasana Toliengband
Somboon Tanasupawatc
a
Departmentof Microbiology, Faculty of Science, Chulalongkorn University, Thailand, bInstitute of Biotechnology and Genetic
Engineering, Chulalongkorn University, Thailand, cDepartment of Microbiology and Biochemistry, Faculty of Pharmaceutical
Sciences,Chulalongkorn University, Thailand.

Abstract

Though molasses is enriched with fermentable sugars, proteins, vitamins and trace elements required for growth and
ethanol fermentation of Saccharomyces cerevisiae. It sometimes found that molasses gave ethanol yield lower than
sucrose containing medium which had the same sucrose concentration. This phenomenon caused by Ca2+, metal ions
and/or sugar derivatives contaminated in the molasses was previously discussed. Metal ions, for example Cu 2+, Mn2+ and
Zn2+ enhance ethanol fermentation at low concentration but inhibit at high concentration. In this study, ethanol
fermentation from molasses was maximized through nutrients and ions supplemented optimization.

© 2015 Published by Burapha University.

Keywords: nutrients; ions; Saccharomyces cerevisiae

* Corresponding author. Tel.: +6-683-954-9589

E-mail address: namfon_pink@hotmail.com (P. Poomikhat).

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1. Introduction

Molasses, a by-product from sugarcane production, is a cheap and abundant source of fermentable sugars,
one hundred grams contain approximately 30-40 g sucrose , 4-9 g glucose and 5-12 g fructose (Chen and
Chou, 1993). Apart from the fermentable sugars, abundant nutrients including proteins, vitamins (biotin, folic
acid, riboflavin, thiamine and niacin) and minerals (potassium, calcium, sodium, magnesium, copper, iron,
manganese, zinc, chloride and sulfur etc.) are also present in the molasses (Chotineeranat et al., 2010).
Molasses is interesting raw material for microbial industrial fermentation e.g. baker’s yeast, amino acid and
ethanol production (Sirianuntapiboon and Prasertsong, 2008). However, its quality (chemical composition) is
not consistant. Ethanol yield of molasses is varied among production lot. Ethanol yield from molasses by
yeast is lower than ethanol yield from equal fermentable sugar because molasses has been contaminated such
as 1) Calcium ion has about 10-20% of all carbonate ash from using lime or calciumoxide for juice
purification. Calcium ion can inhibit invertase activity of Saccharomyces cerevisiae cause ethanol yield from
molasses fermentation have low efficiency 2) Hydroxymethyl furfural (HMF) which is a product of Maillard
reaction significantly inhibited metabolisms of S. cerevisiae (Modig et al., 2002). 3) Copper ion and
potassium ion which are dialyzable molecules and stable to heating at 90C were reported as inhibitors for
ethanol production presented in molasses.
Based on these characteristics, metal ions are candidate of the inhibitors. Removal of metal ions from
molasses enhanced ethanol yield due to release of invertase activity of S. cerevisiae which is suppressed by
metal ions. Copper, potassium and calcium were strong inhibitors compare to other metal ions. Concentration
required for 50% inhibition of these metal ions in molasses was 10 times less than in buffer solution
(Takeshige and Ouchi, 1995). It is interesting that the copper ion at optimal amount (1-10 M) in medium
enhances yeast growth and fermentation activity. Because it acts as cofactor of some enzymes such as
cytochrome c oxidase, Cu,Zn-superoxide dismutase etc. Though, molasses contains microelements required
for yeast metabolisms. Zinc, copper and manganese are presented in molasses is very small concentration.
Because they are bound to organic carriers. Addition of these microelements into molasses improved ethanol
fermentation activity of yeast (Stehlik-Tomas et al., 2004). During ethanol fermentation, yeast has found
inappropriate condition for growth and ethanol production such as osmotic pressure, high temperature and
inappropriate carbondioxide (Laopaiboon et al., 2008). Objective of this work is to analyse for strong ethanol
production - inhibitors found in molasses of Thailand and to improve ethanol yield of the molasses by optimal
nutrients and trace elements concentration.

2. Methodology

2.1. Molasses samples

Molasses sample collected from Angvien industry, NakhonRatchasima province was used in this study.
The molasses was kept at 4C until use. It was diluted to 20 % (w/v) total sugar by adding 100 g into 42 ml
distilled water, then centrifuged at 5,000 rpm, 4C for 5 min. The obtained supernatant was used for medium
preparation.

2.2. Microorganism

Saccharomyces cerevisiaeTISTR 5596 was obtained from Thailand Institute of Science and Technology
Research. It gave ethanol (83.47 g/l) after 96 h (ethanol productivity 0.87 g/L/h) from 15 % (w/v) sucrose
medium at 30C.

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2.3. Determination of molasses characteristics

Chemical composition of the diluted molasses was analysed. Total sugar and reducing sugar contents of
the diluted molasses were analysed by phenol sulfuric method (Dubois et al., 1956) and Somogyi-Nelson
method (Somogyi, 1952), respectively.

2.4. Preparation of molasses medium

Fermentation medium was prepared by adding 0.2%(w/v) (NH4)2SO4, 0.2%(w/v) KH2PO4, 0.075%(w/v)
MgSO4.7H2O, 1%(w/v) yeast extract (w/v) into the diluted molasses, pH adjusting to 4.5, and autoclaving
(120C, 15 lb/inc2, 3 min). Then sterile stock solution of ZnSO4, CuSO4 and MnSO4 were added to each final
concentration of 0.01% (w/v).

2.5. Preparation of inoculum

Single colony of S. cerevisiae TISTR 5596 which was grown on modified YPD agar (10% (w/v) sucrose,
0.3% (w/v) yeast extract, 0.3% (w/v) bactopeptone, 2% (w/v) agar, pH 5) at 30C for 48 h was inoculated into
50 ml of modified YPD broth in 250 ml flask and incubated at 30C, 200 rpm for 24 h. The obtained culture
was transferred at 1% (v/v) into fresh modified YPD broth and incubated at the same above condition

2.6. Ethanol production

The inoculum was centrifuged to separate S. cerevisiae cells by centrifugation (8000 rpm, 4C, 10 min). S.
cerevisiae cells were resuspended in fermentation medium (50 ml in 100 ml Duran) to final cell number of
1.6x106 cells/ml. The inoculated fermentation medium was incubated at 30C under oxygen limited condition
(using rubber stopper coverd with parafilm, the stopper connected to glass trap filled with copper sulfate) with
130 rpm mixing. Samples taken every 24 h were centrifuged (8000 rpm, 4C, 10 min) and analysed for
ethanol and reducing sugar in supernatant by gas chromatography (Jutakanoke et al., 2012), and Somogyi-
Nelson method, respectively.

3. Results and Discussion

3.1. Molasses composition

Chemical composition of the diluted molasses is shown in Table 1. Previous study, one hundred grams of
molasses contain approximately 30-40 g sucrose , 4-9 g glucose and 5-12 g fructose (Chen and Chou, 1993).
However, its quality (chemical composition) is not consistant so glucose analysis of this study could not
detect.

Table 1. Chemical composition of the diluted molasses

Composition (g/L) Method Limit of detection (g/L)

Calcium (Ca) 2.8
Copper (Cu) NB 3.6x10-4
Zinc (Zn) 2.1 mg/L Inhouse method based on
Manganese (Mn) 1.9 mg/L AOAC(2010),984.27,975.03
Potassium (K) 9.8
Phosphorus (P) 210 mg/L

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Nitrogen (N) 2.8 Inhouse method based on AOAC(2012),991.20

Magnesium (Mg) 1.2 Inhouse method based on
AOAC(2010),984.27,975.03
Volatile acid 5.5
(Acetic acid)
Non-Volatile 8.2 AOAC(2010),935.57,942.15
(Lactic acid)
Sucrose 112.8
Glucose NB Asean Manual of Food Analysis (2011) p.27-32 1
Fructose 96.5
*NB : Not detectable

3.2. Ethanol production from molasses

Molasses, which is the main substrate for the yeast and ethanol production, have most of the necessary
microelements, but not in the optimal concentrations. Jones and Gadd (Jones and Gadd, 1990) have suggested
concentrations of many microelements which can be optimal for the yeast growth and the ethanol production.
In Table 2, Molasses which were not added microelements gave maximum ethanol by S. cerevisiae TISTR
5596 was 64.53 g/L after 48h

Table 2 Ethanol production in molasses medium

Time (h) Ethanol (g/L)

24 55.89±2.44
48 64.53±1.85
72 61.07±0.55
96 58.37±2.41
120 61.23±0.89

Experiment design of each nutrients concentration as shown in Table 3A. When (NH 4)2SO4, KH2PO4,
MgSO4 or yeast extracts was added into molasses medium without trace elements, it can be seen that ethanol
concentration increase more than previous experiment.

In the absence of trace elements medium No.13 (without yeast extract) gave maximum ethanol (85.21)
(Table 3B). (NH4)2SO4, KH2PO4 and MgSO4 modified molasses medium which trace elements and yeast
extract were not added was use in next experiment. Concentration of (NH4)2SO4, KH2PO4 and MgSO4.7H2O
added were varied (Table 4A).

Table 3A Modification of molasses medium (w/o trace elements)

Nutrients Medium No.

1 2 3 4 5 6 7 8
(NH4)2SO4 - + + - - - + -
(2g/L)
KH2PO4 - + - + - - + +
(2 g/L)
MgSO4 - + - - + - - +
(0.75 g/L)
Yeast - + - - - + - -
extract
(10 g/L)

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Nutrients Medium No.

9 10 11 12 13 14 15 16
(NH4)2SO4 - + + - + + + -
(2g/L)
KH2PO4 - - - + + + - +
(2 g/L)
MgSO4 + - + - + - + +
(0.75 g/L)
Yeast + + - + - + + +
extract
(10 g/L)
+with, - without

Table 3B Ethanol production in various modified molasses medium (w/o trace elements)

Medium No. Ethanol (g/L)

1 76.92±0.93
2 85.03±0.36
3 80.25±0.48
4 78.02±1.16
5 79.18±0.62
6 79.31±0.25
7 80.03±0.44
8 79.45±0.06
9 81.05±0.42
10 79.16±0.32
11 81.18±0.07
12 80.27±0.30
13 85.21±0.38
14 82.12±0.57
15 82.48±0.15
16 84.15±1.22
Inoculums 9.6x108 cells/ml, 72 h fermentation

Table 4A Concentration of nutrients added into modified molasses medium (w/o trace elements and yeast extract)
Medium No. (NH4)2SO4 (g/L) KH2PO4 (g/L) MgSO4 (g/L)
17 1 0.25
18 1 1
19 4 0.25
20 1
21 1 0.25
22 4 1
23 4 0.25
24 1
13 2 2 0.75

Table 4B Optimalconcentration of nutrients on ethanol production in modified molasses medium (w/o trace elements and
yeast extract)
Medium No. Ethanol (g/L)
17 83.38±2.53
18 80.68±1.20
19 86.77±1.61
20 83.88±1.59
21 75.84±0.85
22 74.70±0.79
23 80.51±0.79
24 77.80±1.43
13 85.21±0.38
8
Inoculums 9.6x10 cells/ml, 72 h fermentation

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As shown in Table 4B, The maximum ethanol (85.21 g/L) produced in medium No.13 was increased to
86.77 g/L in medium No.19 which (NH4)2SO4 1 g/L, KH2PO4 4 g/L and MgSO4.7H2O 0.25 g/L were added.

Molasses medium supplemented with (NH4)2SO4 2 g/L, KH2PO4 2 g/L and MgSO4 0.75 g/L was used in
this experiment. Various concentration of CuSO4, MnSO4 and ZnSO4 were added into the above molasses
medium (Table 5A).

Table 5A Various concentration of trace elements supplementedin molasses medium (with (NH 4)2SO4, KH2PO4 and MgSO4.7H2O)

Medium No. CuSO4 (g/L) MnSO4 (g/L) ZnSO4 (g/L)

25 0.5
26 1.0 0.1 0.1
27 2.0
28 3.0
29 0.5
30 0.1 1.0 0.1
31 2.0
32 3.0
33 1.0
34 0.1 0.1 2.0
35 4.0
36 0.1 0.1 0.1

Table 5B Effect of Cu2+, Mn2+ or Zn2+concentration on ethanol production

Medium No. Ethanol (g/L)

25 84.19±0.22
26 84.56±0.55
27 85.91±0.42
28 79.73±0.18
29 84.92±0.44
30 85.38±0.52
31 82.83±0.30
32 80.65±0.19
33 85.58±0.33
34 82.02±0.25
35 79.53±0.35
36 84.75±0.46
Inoculums 9.6x108 cells/ml, 72 h fermentation

Optimal concentration of CuSO4, MnSO4 and ZnSO4 were 2.0, 1.0 and 1.0 g/L, respectively. At this
condition, maximum ethanol produced were 85.91, 85.38 and 85.58 g/L, respectively. Copper is cofactor of
cytochrome c oxidase. Manganese is cofactor of pyruvate decarboxylase. Zinc is co-factor of alcohol
dehydrogenasewhich is a key step enzyme for ethanol production.

4. Conclusion

Optimal concentration of N, P, Mg in molasses for ethanol production of S. cerevisiae TISTR 5596 were
3,800, 4,210, 1,450 mg/L and Cu2+, Mn2+ and Zn2+ ions in molasses should not higher than 2,000, 119 and
102 mg/L, respectively.

Acknowledgements

The author thank Angvien industry, NakhonRatchasima province, Thailand for providing molasses and
Thailand Institute of Science and TechnologyResearch for providing the TISTR 5596 strain.

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