ISSN: 0970-020 X

ORIENTAL JOURNAL OF CHEMISTRY CODEN: OJCHEG

An International Open Access, Peer Reviewed Research Journal
2019, Vol. 35, No.(2):
Pg. 744-750
www.orientjchem.org

A rapid spectrophotometric method for quantitative

determination of ethanol in fermentation products
Malinee Sriariyanun1, Parita Mutrakulcharoen1, Surapun Tepaamorndech2,
KraiPAT CHEENKACHORN3 and KITTIPONG RATTANAPORN4*,

1
The Sirindhorn International Thai-German Graduate School of Engineering (TGGS), KMUTNB,
Bangkok, 10800 Thailand.
2
Food Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology,
Pathumthani, 12120 Thailand.
3
Department of Chemical Engineering, Faculty of Engineering, KMUTNB, Bangkok, 10800 Thailand.
4
Department of Biotechnology, Faculty of Agro-industry, Kasetsart University, Bangkok, 10900 Thailand.
*Corresponding author E-mail: [email protected]

http://dx.doi.org/10.13005/ojc/350234

(Received: January 08, 2019; Accepted: March 26, 2019)

Abstract

A rapid and accurate assay for monitoring ethanol production is required to control the
progress of fermentation in various industrial-related research and processes. In this study,
a spectrophotometric assay to measure ethanol concentration in fermentative samples was
developed. Ethanol in unknown aqueous solution was extracted using tri-n-butyl phosphate (TBP)
and subsequently oxidized by dichromate reagent. The oxidation product of ethanol with dichromate
reagent could be visualized as blue green-color. The A595 values detected by spectrophotometer
and ethanol concentration between 0.7%-8.0% were plotted in linear regression with high correlation
coefficiency (R2). In addition, The concentrations of methanol, propanol and butanol were determined
as did in ethanol suggesting the broad application of this assay. Our established method was applied
to commercial wine and fermentative products from yeast culture broths and the results were
compared with Gas Chromatography Mass Spectrophotometry (GCMS) method. In this study, this
assay was demonstrated as a cheap, rapid, and high accuracy method for determination of ethanol
concentration in unknown solutions.

Keywords: Ethanol, Alcohol, Quantitative determination, Colorimetric method, Fermentation.

Introduction produced and utilized as an alternative biofuel with

production capacity of 1, 200 billion liters in 2016.
Ethanol is biologically produced through the According to the government policy, the production
fermentation of sugars by various types of microbes of ethanol is targeted to be 3, 240 billion liters in
or chemically produced via chemical reaction or 2021, remarking the potential market growth of
petro-chemical processes. In Thailand, ethanol was ethanol1. Currently, ethanol is mainly obtained from

This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC- BY).
Published by Oriental Scientific Publishing Company © 2018
 RATTANAPORN et al., Orient. J. Chem., Vol. 35(2), 744-750 (2019) 745

fermentation of agricultural by-products, for example, to determine ethanol concentrations in different

molasses and cassava pulp in Thailand. In addition, solutions as listed in Table 1. An ideal method for
lignocellulosic wastes obtained after harvesting this purpose should be rapid, cheap, simple and
seasons could be raw materials for bioethanol high accuracy, high sensitivity, high repeatability.
production in industrial scale2. Furthermore, the method that does not require
expensive instrument and well-trained personnel
In general, ethanol fer mentation is is primarily preferred. Gas chromatography (GC) is
considered as a slow process that normally takes one of the common methods for determination of
48-72 h per batch in industrial scale production. The ethanol concentration in alcoholic beverages due to
fermentation process sometimes fails due to many its accuracy, and sensitivity4. However, this method
reasons, for example, contamination, inconsistent requires expensive instrument and skilled worker for
quality of raw materials, failure of operational analysis. In addition, one instrument could analyze
controllers, presence of fermentation inhibitors3. Due one sample at a time of analysis. Distillation is one
to large scale of production in industries, one-time of the selected choices for factory’s lab, although
failure leads to big loss of expenses. Therefore, the it is time-consuming, and laborious 5,6. During
rapid quantitative measurement method to monitor fermentation, the product not only contains ethanol
the progress of ethanol production is necessary but also other by-products for example, butanol,
to promptly adjust the on-going and real-time or volatile fatty acid that could be unspecifically
fermentation to be proper schemes. detected by many methods7. Enzymatic assay offers
high specificity and sensitivity to ethanol8, although it
Various techniques have been developed is relatively expensive compared to other methods.

Table 1: Comparison of different quantitative measurement methods for determination of ethanol

concentration in samples

Methods Sensitivity Assay time/sample Minimum volume Remark References

of sample

Gas chromatography 100 ppm Approx. 5 min 1 µL High accuracy, Expensive equipment, [4]
per sample required high cost of sample analyses
and skilled analysts
Gravimetric method Rough 5 min 100 mL Required large amount of samples [5,6]
(i.e. alcohol and must use at reference standard
meter, hydrometer) condition at 20oC
Distillation method 2 g/L 15 min 50 mL Required large amount of samples and [5,6]
(i.e. Ebulliometer) may be interfered with the volatile acids
for the result interpretration. The total
suspended solid is limited
Solvent extraction- 0.25- 8% (v/v) 10 min 1.2 mL Taking long time for [7]
dichromate oxidation extraction (1 hours)

Enzymatic assay 0.093 mg/L-0.1 g/L 5 min 10 µL High accuracy, high cost of sample [8]
analyses, limited range of ethanol
concentration
Cerric Ammonium 0.3-3.85 % (w/v) 10 min 1 mL Does not work for fermentation [9]
Nitrate broths due to residual sugars

In most biological solutions, including to reduce cross-reactivity and other interferences.

fermentation broth, contains a variety of soluble In this study, a detection assay to quantitatively
and insoluble compounds. In ethanol production, determine the ethanol concentration using the
the final fermented supernatant contains remaining modified solvent extraction and dichromate oxidation
raw materials, i.e. molasses, starch hydrolysate, and method which was developed and tested for its
other by-products that may cross react with the assay sensitivity to fermentation broth. GCMS analysis
due to their strong color10,11. Thus, the extraction of ethanol was also conducted as a benchmark
of ethanol from unknown samples is suggested of this dichromate oxidation colorimetric method.
 RATTANAPORN et al., Orient. J. Chem., Vol. 35(2), 744-750 (2019) 746

Our results demonstrated that the modified solvent 180oC at 20oC/min and held for 5 min12. The mass
extraction and dichromate oxidation method was spectra operating parameters were, ionization
able to determine the ethanol concentration in many potential, 70 eV; ion source temperature; 250oC,
numbers of fermentation samples simultaneously solvent delay 1.5 min, scan range 60-350 amu, EV
which is suitable for research and process control voltage 3,000 volts.
applications.
Yeast fermentation for ethanol production from
lignocellulose hydrolysates
Materials and methods
In this study, ethanol samples were prepared
from yeast cultures grown in Modified Lignocellulose
Spectrophotometric method for determination
Hydrolysate (MLH) media and Yeast Malt (YM)
of ethanol concentration
media. To prepare MLH media, 1 g of dried rice straw
In spectrophotometric method, ethanol
was hydrolyzed in 40 mL of 50 mM sodium citrate
concentration was determined by solvent extraction
buffer (pH 4.7) containing cellulase mixtures (20 FPU
and dichromate oxidation reaction7 with protocol
of Celluclast 1.5L (Sigma-Aldrich, USA) and 100
modification described as follows. Firstly, ethanol in
CBU of Novozyme 188 (Sigma-Aldrich, USA)13-17].
liquid sample was extracted by Tri-n-butyl phosphate
The hydrolysis reaction was conducted at 45oC for 72
(TBP, Sigma Aldrich, USA). One mL of TBP and
h in a 200 rpm shaking incubator. The supernatant of
1 mL of aqueous sample was mixed in a microtube
hydrolysate was collected by centrifugation at 3,420
and then vortex vigorously for 1 minutes. The mixture
xg for 10 minutes. The reducing sugars released from
was centrifuged at 3,420 xg for 5 min to separate
rice straw biomass was quantitively measured using DNS
into two phases. Upper phase, TBP layer, was clear
assay18. The hydrolysate supernatant was mixed with 6
and transparent, while lower phase, water later, was
g/L yeast extract, 1 g/L KH2PO4, 0.5 g/L MgSO4.7H2O and
turbid. Then, 500 µL of upper phase was transferred
1 g/L glucose to be MLH media and pH was adjusted
to a new microtube and mixed with 500 µL of
to pH 5.512.
dichromate reagent (containing 10% w/v of K2Cr2O7
in 5 M of H2SO4), and vortex vigorously for 1 min. The To grow yeast culture, Saccharomyces
mixture was set still for 10 min at room temperature cerevisiae TISTR 5339 (Sc 5339) and Saccharomyces
to allow oxidation product in lower phase developed cerevisiae TISTR 5596 (Sc 5596), obtained from
its color to blue green. One hundred microliters Thailand Institute of Scientific and Technological
of the oxidation products were diluted with 900 Research (TISTR), were inoculated in 20 mL of
µL of deionized water. The optical density at 595 YM broth media (containing 5 g/L peptone, 3 g/L
nm (A595) of tested sample was measured in yeast extract, 3 g/L malt extract and 10 g/L glucose,
spectrophotometer (T80+ UV/Vis Spectrometer, PG pH 5.5) and MLH media. The yeast cultures were
Instrument Ltd., USA). The ethanol concentration in placed in shaker incubator at 30oC, 150 rpm for
sample was estimated from the ethanol standard 48 hours. Ethanol samples were collected from
curve representing the relationship between A595 supernatant fractions by centrifugation at 3,420 xg
and the concentrations of ethanol. for 10 min, and kept in -20oC until it was analyzed by
spectrophotometric assay and GCMS. Each analysis
GCMS analysis of ethanol
was repeated at least 4 replicates.
Standard ethanol diluted with deionized
water were prepared to 0.01% - 0.5% (v/v) and 1 μl Results and discussion
was injected into the injector port of GCMS apparatus
(GCMS-QP2020, Shimadzu, Japan) equipped with Sensitivity of spectrophotometric method
autoinjector (AOC-20i, Shimadzu, Japan) and In this study, an assay to determine ethanol
DB-wax capillary column (30 m x 0.25 mm x 0.25 concentrations in unknown sample was conducted
μm, Agilent, USA). The samples were analyzed in in two step reactions. Firstly, ethanol in aqueous
GCMS using helium as carrier gas and the flow rate solution was extracted using non-alcoholic solvent,
was 1.5 mL/minuted. The injector temperature was tri-n-butyl phosphate (TBP)19. Secondly, ethanol was
220 o C with the split ratio of 40:1. The oven oxidized by chromium ions, and chromium ions were
temperature was programmed as follows: the initial reduced from +6 oxidation state, orange color, to +3
temperature was 60oC for 5 min, then ramped to oxidation state, blue-green color7.
 RATTANAPORN et al., Orient. J. Chem., Vol. 35(2), 744-750 (2019) 747

To estimate the appropriate range of were not enough to oxidize remaining ethanol in
ethanol concentration for this assay, ethanol the sample. Furthermore, when the sample volume
solutions diluted with deionized water were prepared used in this assay was reduced from 1 mL to
to concentration of 0.2% to 10% ethanol. Each 0.5 mL, the linear correlation was also observed
sample was subjected to the assay and A595 values similarly. This results suggested that this assay is
were recorded by spectrophotometer (Fig. 1). The applicable to small volume sample and suitable
correlation plot between ethanol concentrations and for screening research. Previously, the solvent
A595 was generated, and the linear regression with extraction and dichromate oxidation method was
high correlation efficiency (R2 = 0.9948) could be tested in yeast culture in both microtube and a
observed when the ethanol concentration ranging 96-deep well microplate7. It was found that different
from 0.8% – 7%. The plateau of A595 could be composition of yeast culture showed different ranges
observed when ethanol concentration was 8% or of linear regressions of ethanol concentration, and
higher. This could be the result of TBP extractability the maximum concentration that could be estimated
reached its limitation, or chromium ion supplies by this assay was 8%7.

Fig. 1. Correlation of optical density values at 595 nm and concentrations of each ethanol sample varied between (A) 0.2% – 10%
and (B) 0.8% – 7%. The color of dichromate oxidation of ethanol with different concentration ranged from 1% - 7%
(from left hand to right hand). Standard deviations were calculated from four replicates of each ethanol concentration

Specificity of spectrophotometric method

During ethanol fermentation by microbes,
other by-products, including other alcohols, could
be produced simultaneously and could interfere the
color signal in the assay11. In this section, methanol,
ethanol, propanol and butanol with different
concentration ranging from 0.8% - 6% were tested
to evaluate the specificity of the assay. Based on
the results, it indicated that our assay is compatible
to different types of alcohols. Thus, this assay could
Fig. 2. Correlation plot of the optical density values at 595 nm
be further applied to various purposes, not only for
and concentrations of alcohol samples, including methanol,
ethanol concentration determination. The correlation ethanol, propanol and butanol. Standard deviations were
plots between alcohol concentration and A595 calculated from four replicates of each alcohol concentration
values were drawn and the linear regression trend
lines were generated with high correlation coefficient Interestingly, the slopes of each alcohol
(R2 > 0.9950) (Figure 2). were different, which may be due to the redox
 RATTANAPORN et al., Orient. J. Chem., Vol. 35(2), 744-750 (2019) 748

potential of each alcohol. Wang et al.,20, studied the Comparison of spectrophotometric method and
oxidation activity of methanol, ethanol and propanol on Pd GCMS
electrode in alkaline medium.The alcohol electrooxidation The accuracy of spectrophotometric
activity of ethanol and propanol is higher than that of assay based on solvent extraction and dichromate
methanol suggesting that ethanol and propanol is more oxidation was evaluated by comparing with GCMS
easily oxidized than methanol. The results in our study analysis. Two yeast isolates, Sc 5339 and Sc 5596,
also agreed well to this report that the slopes of ethanol were cultured in two types of media, MLH and
and propanol were less than methanol. YM, for ethanol production. Sc 5339 was shown
to grow in pineapple core and peel hydrolysates22
Effect of pH in assay accuracy and produce high ethanol yield23. Sc 5596 was
One of the target goal of this assay is
previously demonstrated to grow in sorghum straw
for determination of ethanol concentrations in
hydrolysate with presence of lignin and produce
fermentation broths. Practically, during the progress
of ethanol fermentation, pH of broth decreases ethanol in the strength condition 24. Therefore,
gradually until the end of process21. Besides, the these two yeast strains could utilize sugars from
oxidation-reduction reaction, which is the key of this lignocellulose hydrolysates. In this study, MLH media
assay, may be affected by pH changes. To investigate is composed of rice straw hydrolysate as a main
the effect of pH to the consistency of the assay, pH carbon source. After unpretreated rice straw was
of 4% (v/v) ethanol solutions were variant adjusted enzymatic hydrolyzed, the reducing sugar released
between pH 1.0 – 13.0. Each sample was subjected from the reaction was quantitatively measured by
to the assay and the A595 value was recorded by DNS assay to be 4.28 mg/mL. Glucose was added
using spectrophotometer. The ethanol concentration into MLH media to obtain final sugar concentration
was calculated as shown in Fig. 3 using standard of 4.75 mg/mL.
curve in Fig. 1. The results showed that calculated
ethanol concentration of all samples was 4.00%- After 48 h fermentation, the supernatant
4.24% suggesting the broad compatibility of this fractions of Sc 5339 and Sc 5596 cultures were
assay at various pH of samples. The determined collected and ethanol concentrations were analyzed
ethanol concentration at each pH was analyzed by by spectrophotometric assay and GCMS analysis.
ANOVA analysis to assess whether pH has effect on The calculated ethanol concentrations of 4 yeast
the assay. The obtained P-value at < 0.001 (F-value cultures, Sc 5339 and Sc 5596 grown in YM and
= 7.12) indicated that pH resulted in deviation of MLH media, obtained from GCMS analysis were
determined ethanol concentration. Also, to verify the less that that obtained from spectrophotometric
application of this assay to fermentation aqueous assay for 0.034%-0.063% (Fig.4). This result
products, commercial wine products with pH 3.02 suggested that spectrophotometric assay could be
(Haut-Medoc, France) containing 12% ethanol was used to quantitative determine ethanol concentration
subjected to this assay, and the calculated ethanol with minor difference from GCMS analysis. This
concentration obtained from A595 was 12.00% ± differences of the determined ethanol concentration
0.08%. These results suggested that this assay also obtained from two methods were evaluated by using
suitable for application in alcoholic beverages. two-tailed T-test. The results showed that P-values
of Sc 5596, Sc 5339, H-Sc 5596, H-Sc 5339 were
0.0159, 0.0657, 0.0042, 0.0285, respectively. Thus,
with criteria of P-value at 0.05, the determined
ethanol concentrations of Sc 5339 samples that
obtained from spectrophotometric assay and GCMS
analysis were not statistically different.

The ethanol concentration measured by

using spectrophotometric assay of Sc 5339 and Sc
5596 grown in YM were 0.704% and 0.788%, and
Sc 5339 and Sc 5596 grown in MLH were 0.666%
Fig. 3. Effect of pH on the determined concentration of and 0.727%, respectively. Comparing between two
ethanol using spectrophotometric method types of media, both yeast strains showed that they
 RATTANAPORN et al., Orient. J. Chem., Vol. 35(2), 744-750 (2019) 749

produced ethanol with little higher yield in YM media intrinsic colors that cause interference in final color
than MLH. For example, ethanol yield in Sc 5339 that of oxidation reaction detected by spectrophotometer.
was grown in YM was higher than in MLH for 0.038%. Therefore, it suggested that the extraction step by
While, the carbon source concentration in YM (13 TBP was a crucial step for this assay to remove false
g/L) is much higher than MLH (4.75 g/L) suggesting signal of the tested samples.
that these two yeast strains have good ability to grow
and to produce ethanol in lignocellulose hydrolysate Conclusion
media.
In this study, the solvent extraction and
Interestingly, all of ethanol concentrations dichromate oxidation method to quantitative
obtained from spectrophotometric assay were higher determine ethanol concentration in fermentation
than that obtained from GCMS analysis. Based on broth was developed and tested for its sensitivity
previous studies, the dichromate oxidation reaction and specificity. In addition, this method required
could cross-react with several types of alcohols, and small volume of samples (0.5-1.0 mL) with less
the results in Fig. 2, also agreed well with it. However, than 20 min/sample and many samples could be
GCMS analysis of these fermentation broths could analyzed at the same time in one batch. It did not
detect only ethanol, suggesting that the difference of require expensive equipment, chemicals and skilled
both methods was not due to cross-reactivity. Yeast analysts. It is useful for process monitoring and
fermentation broth of YM and MLH media contained optimization for improvement of ethanol fermentation
yeast extract, peptone, glucose, malt extract, because it can handle many samples in the same
lignin, and unknown components in lignocellulose time in one batch. Here, this method was validated
hydrolysates. These chemicals have their own to show its applications in alcoholic beverages
and fermentation broths containing lignocellulosic
hydrolysates.

Acknowledgement

The authors would like to thank King

Mongkut's University of Technology, North Bangkok
(Research Grant Contract No. KMUTNB-61-
GOV-03-47) and Kasetsart University Research
and Development Institute (KURDI) at Kasetsart
University for financial supports of this work.
Fig. 4. Determined ethanol concentration in different
fermentative yeast cultures of Saccharomyces.cerevisiae
5596 and Saccharomyces.cerevisiae 5339 grown in YM media
Conflict of interest
(Sc 5596 and Sc 5339) and MLH media (H-Sc 5596 and H-Sc The authors declare that there are conflicts
5339) using spectrophotometric assay and GCMS method of interest.

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