Applied Energy 87 (2010) 23522355

Contents lists available at ScienceDirect

Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Comparative study of bio-ethanol production from mahula (Madhuca latifolia L.)

owers by Saccharomyces cerevisiae and Zymomonas mobilis
Shuvashish Behera a, Rama Chandra Mohanty a, Ramesh Chandra Ray b,*
a
Department of Botany, Utkal University, Vanivihar, Bhubaneswar 751004, Orissa, India
b
Microbiology Laboratory, Central Tuber Crops Research Institute (Regional Centre), Bhubaneswar 751019, Orissa, India

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

Article history: Mahula (Madhuca latifolia L.) ower is a suitable alternative cheaper carbohydrate source for production
Received 6 August 2009 of bio-ethanol. Recent production of bio-ethanol by microbial fermentation as an alternative energy
Received in revised form 22 October 2009 source has renewed research interest because of the increase in the fuel price. Saccharomyces cerevisiae
Accepted 13 November 2009
(yeast) and Zymomonas mobilis (bacteria) are two most widely used microorganisms for ethanol produc-
Available online 28 December 2009
tion. In this study, experiments were carried out to compare the potential of the yeast S. cerevisiae (CTCRI
strain) with the bacterium Z. mobilis (MTCC 92) for ethanol fermentation from mahula owers. The eth-
Keywords:
anol production after 96 h fermentation was 149 and 122.9 g kg1 owers using free cells of S. cerevisiae
Bio-ethanol
Fermentation
and Z. mobilis, respectively. The S. cerevisiae strain showed 21.2% more nal ethanol production in com-
Madhuca latifolia parison to Z. mobilis. Ethanol yield (Yx/s), volumetric product productivity (Qp), sugar to ethanol conver-
Saccharomyces cerevisiae sion rate (%) and microbial biomass concentration (X) obtained by S. cerevisiae were found to be 5.2%,
Zymomonas mobilis 21.1%, 5.27% and 134% higher than Z. mobilis, respectively after 96 h of fermentation.
2009 Published by Elsevier Ltd.

1. Introduction
The natural energy resources such as fossil fuels (petroleum and
coal) are being utilized at a rapid rate and these fossil fuel re-
sources would last only for few more years [1]. From various alter-
native energy resources, bio-ethanol is the most promising
resource because of its biological and renewable origins, normally
derived from energy crops such as maize, sugarcane, cassava,
sweet potato, mahula ower and by-products of agriculture and
forestry [2]. Biofuels offer a number of environmental, social, and
economic advantages, lower emission of harmful pollutants and
good fuel properties for vehicles [3].
Current ethanol production processes using crops such as sugar
cane and corn are well-established. However, utilization of cheaper
substrates such as lignocelluloses and other renewable biomasses
would make bio-ethanol (ethanol from biomass) more competitive
than fossil fuels [4]. But ironically, due to the inherent complexities
in processing and utilizing these lignocellulosic and starchy bio-
mass, the cost of production increases signicantly [1], thereby
leading to a growing interest worldwide to nd alternative feed
stocks for bio-ethanol production [5]. In this context, mahula
(Madhuca latifolia L.) owers provide a great premise as an alterna-
tive bio-resource for production of ethanol through fermentation
* Corresponding author. Tel./fax: +91 674 247052.
E-mail address: rc_rayctcri@rediffmail.com (R.C. Ray).
[68]. Mahula is a forest tree found in the tropical rain forests of
Asian and Australian continents. This tree species, however, has
been domesticated by tribal people in India and Pakistan for use
as food (ower), feed (leaves and ower), wood (timber) and bev-
erages (ower) locally called mahuli.
Traditionally, the yeast, Saccharomyces cerevisiae has been used
all over the world as the major ethanol producing microorganism.
In our earlier studies, S. cerevisiae strain CTCRI was used as free and
immobilized cells for production of ethanol from mahula owers in
submerged fermentation [6,8]. Likewise, Mohanty et al. [7] re-
ported bio-ethanol production from mahula owers by solid-state
fermentation using S. cerevisiae. In recent years, however, research
is focused on processes involving the gram-negative anaerobic bac-
terium, Zymomonas mobilis, because of several better fermentation
attributes such as it converts glucose almost stoichiometrically to
ethanol and CO2, grows more rapidly and demonstrates highest
ethanol productivity during continuous fermentation [9]. Zymomo-
nas spp. grow anaerobically and unlike yeasts do not require the
controlled addition of oxygen to maintain viability at high cell con-
centrations [10,11]. Further the ethanol tolerance of Zymomonas
spp. is comparable with strains of S. cerevisiae [12] and these pro-
duce less by-products [13]. Considering the above, this study was
carried out to compare the performance of yeast, S. cerevisiae
(CTCRI strain) with bacterium Z. mobilis (MTCC 92) on ethanol pro-
duction from mahula owers in submerged fermentation. Further,
the growth and fermentation kinetics of S. cerevisiae and Z. mobilis
cells during fermentation are compared.

0306-2619/$ - see front matter 2009 Published by Elsevier Ltd.

doi:10.1016/j.apenergy.2009.11.018
 S. Behera et al. / Applied Energy 87 (2010) 23522355 2353

2. Materials and methods 2.6. Population count

2.1. Mahula owers
Fresh mahula owers were collected from the forests of the
Keonjhar District of Orissa, India, during MarchApril, 2007. The
owers were brought to the Microbiology Laboratory of CTCRI,
washed in tap water to get rid of dust and other debris and sun-
dried in the open for 7 days to reduce the moisture content to
1618.6%. The sun-dried owers collected from various locations
were mixed thoroughly before being used for ethanol fermenta-
tion. The owers have the following compositions (expressed in
g 100 g1 dry weight basis): moisture, 2425.85; starch, 0.94
0.95; total sugar (glucose, fructose, fructose, sucrose and maltose),
3638; crude protein, 67; crude ber, 10.012.5; total ash, 1.6
2.0; undetermined solids, 10.613.7; and pH 4.54.8.
2.2. Microorganisms and culture conditions
Z. mobilis MTCC 92 was procured from the Institute of Microbial
Technology, Chandigarh, for this investigation. Z. mobilis and S.
cerevisiae (CTCRI strain) was earlier used in our laboratory for eth-
anol fermentation [7,8] maintained on Z. mobilis specic medium
(ZSM) [(g l1): glucose, 100; yeast extract, 2; urea, 1; KH2PO4, 1;
MgSO47H2O, 0.5; agar, 15; pH 6.5] and the yeast (S. cerevisiae)
was maintained on malt extractyeast extractglucosepeptone
(MYGP) medium [(g l1): malt extract, 3; yeast extract, 5; peptone,
5; glucose, 20; agar, 15; pH 5.5]. Both the cultures were stored at
4 0.5 C for further use.
Yeast and bacterial populations in the fermented mash were
calculated by serially diluting the substrate (fermented mahula
slurry) in distilled water and plating suitable dilution (106108)
on Petri plates (18 mm  150 mm) containing either MYGP (for S.
cerevisiae) and ZSM (for Z. Mobilis) medium. Data were given as
mean of three replicates.
2.7. Statistical analysis
The data of ethanol production using S. cerevisiae and Z. mobilis
were analyzed using one way ANOVA. Where signicant difference
in ANOVA (p < 0.05) was detected by the Fishers Least Signicance
Difference (LSD) multiple comparison test was applied to compare
the factor level difference. The analysis was performed using
MSTAT-C (version 2.0, Michigan State University, MI, USA).
3. Results and discussion
The main fermentable sugar components of the mahula owers
were reported to be glucose and fructose [6,8]. In this study, mah-
ula owers (100 g) after cleaning were blended with water in the
ratio of 1:5 so as to dilute the bulkiness of the mash before steam-
ing and subsequent fermentation (submerged) by either yeast, S.
cerevisiae or bacterium, Z. mobilis. The comparison of sugar utiliza-
tion and ethanol production prole by these two microbial strains
are given in Fig. 1.

2.3. Preparation of starter culture A 400 400

350 350

Ethanol Concentration
The starter cultures were prepared in 100 ml of the respective
growth medium (as mentioned above) were sterilized (at 121 C 300 300
Residual sugar

(g/kg Flowers)
(g/kg Flowers)

for 20 min) in 250 ml Erlenmeyer asks. The asks were inoculated

250 250
with a loopful of the microbial cultures (Z. mobilis or S. cerevisiae)
and were incubated at 30 C for 24 h under stationary conditions. 200 200
150 150
2.4. Fermentation medium
100 100

The mahula owers were grinded (ower:water ratio, 1:5) in a 50 50

mixer-cum-grinder (TTK Prestige Ltd., Bangalore, India) to make 0 0
slurry. The slurry was cooked by steaming at 120122 C for 60 0 24 48 72 96
80 min. After cooling (NH4)2SO4 was added to the slurry (as nitro- Incubation period (h)
gen source at the concentration of 1 g l1) and pH of the medium
was adjusted to 5.5 and 6.5 for subsequent inoculation of the yeast Residual sugar Ethanol
and bacterial culture, respectively. Then the slurry was inoculated
with 10% starter culture (dry weight basis). The akes (n = 3) were B 400 400
incubated for 96 h at the room temperature (30 2 C).
Ethanol Concentration

350 350
(g/kg Flowers)
Residual sugar

300 300
(g/kg Flowers)

2.5. Analytical methods

250 250
At 24 h intervals, fermented broths (in triplicate) were removed
200 200
and the contents were analyzed for total sugar and ethanol. The
ethanol content of the fermented broth was determined by mea- 150 150
suring the specic gravity of the distillate according to the proce- 100 100
dure described by Amerine and Ough [14]. In this procedure, the
weight of a certain volume of an alcohol distillate was compared 50 50
to the weight of exactly the same volume of distilled water. The ra- 0 0
tio of the weights of the two (alcohol:water) gave the specic grav- 0 24 48 72 96
ity of the distillate [14]. The total sugar was assayed by the Incubation period (h)
Anthrone method [15]. The pH was measured using a pH meter
Residual sugar Ethanol
(Systronics, Ahmadabad, India) tted with a glass electrode. Fer-
mentation kinetics were calculated using the formulae by Bailey Fig. 1. Comparison of ethanol production between free cells of S. cerevisiae (A) and
and Ollis [16]. Z. mobilis (B).
2354 S. Behera et al. / Applied Energy 87 (2010) 23522355
In case of S. cerevisiae, the concentration of total sugar fell rap-
idly by 24 h (81.8% over the initial content) of fermentation, with
concomitant production of ethanol (64.9 g kg1 owers); thereaf-
ter, the decline was gradual (between 24 h and 96 h). At the end
of 96 h incubation period, the residual sugar concentration was
30.1 g kg1 owers and the nal ethanol production was
149.0 g kg1 owers. Earlier study by Swain et al. [6] reported
the total ethanol production from mahula owers in submerged
fermentation using free yeast (S. cerevisiae) cells were 193 and
148 g kg1 owers, from fresh and 12 month stored mahula ow-
ers, respectively. In that study, the initial concentration in mahula
owers ranged between 420 and 474 g kg1 owers; whereas in
the present study, the initial sugar concentration was 364
368 g kg1 owers. The variation in the initial sugar concentration
of mahula owers might be due to the collection from different
localities, time of ower maturation and period of harvest [6,7].
In contrast to S. cerevisiae, there was a marginal fall (5.5% over
initial content) in total sugar albeit no production of ethanol after
24 h of fermentation by Z. mobilis. The marginal decrease in sugar
reserve might be due to its utilization for growth and metabolism
by Zymomonas. After 24 h, there was a gradual increase in ethanol
concentration over the incubation period with simultaneous de-
crease in total sugar (Fig. 1B). At the end of 96 h incubation, the
ethanol production from Z. mobilis from mahula owers was
122.9 g kg1. The result shows that Z. mobilis was signicantly less
efcient than S. cerevisiae [Fishers LSD test, P < 0.05, LSD between
treatments is 1.48 (sugar utilization) and 1.3 (ethanol production)]
in converting sugar into ethanol at least in the case of mahula
owers.
A similar nding on production of ethanol from molasses using
S. cerevisiae and Z. mobilis was reported by Bansal and Singh [17].
There was no signicant difference in ethanol production from
molasses diluted to 510% (v/v), by both the cultures; however,
S. cerevisiae produced more ethanol than Z. mobilis at sugar concen-
tration above 15% (v/v). In a continuous-ow bioreactor study,
immobilized S. cerevisiae cells were found as the best alcohol pro-
ducer from 1% to 5% glucose than immobilized Z. mobilis cells [18].
However, there are reports also to demonstrate that Z. mobilis is a
better ethanol producer than S. cerevisiae because of its higher su-
gar to ethanol conversion rate [19]. Raman and Pothiraj [20] re-
ported that ethanol production from cassava (Manihat esculenta
Crantz) at pH 6.0 and 36 h (residence time) of fermentation was
the highest in the Z. mobilis (9.26 g l1) mediated fermentation
(5% more) than the ethanol production rate of S. cerevisiae
(8.73 g l1). In yet another study, co-immobilized cells of S. cerevi-
siae and Z. mobilis produced a high ethanol concentration com-
pared to immobilized cells of S. diastaticus during batch
fermentation of liqueed cassava starch [21]. The co-immobilized
cells produced 46.7 g l1 of ethanol from 150 g l1 liqueed cas-
sava starch, while immobilized cells of yeast, S. diastaticus pro-
duced only 37.5 g l1 ethanol. It seems from the literature that
there is no consistency in report which organism, S. cerevisiae or
Z. mobilis, is superior in ethanol production from sugary/starchy
biomass. According to Karsch et al. [10], for industrial fermenta-
tion, Z. mobilis seems to be inferior to S. cerevisiae, because the
advantage of the higher ethanol and the lower biomass production
of the bacterium is disadvantaged by the decrease in pH from 5.5
to 3.8 (as in the present study) during yeast fermentation thus
greatly impeding the occurrence of contaminants and making ster-
ilization of the medium not necessary; whereas, in case of Z. mobi-
lis, there might be a chance of contamination. Besides, during
ethanol fermentation, Z. mobilis encounters various environmental
stresses which adversely affect the ability of cells to perform ef-
ciently and consistently in converting sugars to ethanol. The major
stresses experienced by this microorganism are ethanol toxicity
and heat, which accumulate during metabolism of cells. Several
Table 1
Growth and fermentation kinetics of free cells of S. cerevisiae (CTCRI strain) and Z.
mobilis (MTCC 92) on mahula ower.
Free cells of Free cells of
S. cerevisiae Z. mobilis
Final ethanol (P, g l1) 24.83 20.47
Final biomass concentration (X, g l1) 4.34 1.85
Specic growth rate (l, h1) 0.098a 0.079b
Cell yield (Yx/s, g g1) 0.077 0.038
Ethanol yield (Yp/s, g g1) 0.445 0.423
Volumetric substrate uptake (Qs, g l1 h1) 0.581 0.504
Volumetric product productivity (Qp, g l1 h1) 0.258 0.213
Conversion rate (%) into ethanol 89.02 84.56
Mass of product ethanol formed
YP=S Mass of substrate glucose consumed
.
Mass of biomass
YX=S Mass of substrate
yeast cell formed
glucose consumed
.
QS = Substrate (glucose) uptake (g) per liter of hydrolysate per hour.
QP = Product formed (g) per liter of hydrolysate per hour.
a
l (h1) = Standardized value (0.098) for specic growth rate of microorganism
(yeast: S. cerevisiae) under specic substrate (glucose) consumption.
b
l (h1) = Standardized value (0.079) for specic growth rate of microorganism
(bacteria: Z. mobilis) under specic substrate (glucose) consumption.
researchers have demonstrated that ethanol and heat stresses re-
tard the specic growth rate, viability [22], fermentation ability
[23,24], modify plasma membrane uidity [24], and trigger specic
stress responses [25,26] of Z. mobilis.
The growth and fermentation kinetics of S. cerevisiae and Z.
mobilis cells were studied (Table 1). The ethanol concentration
(P) obtained with free cells of S. cerevisiae (24.83 g l1) was 21.2%
more than that of free cells of Z. mobilis (20.47 g l1), where as
the volumetric substrate uptake (Qs) was found to be 15.2% more
in case of S. cerevisiae (0.581 g l1h1) than that of Z. mobilis
(0.504 g l1h1). The ethanol yield (Yp/s = 0.445 g g1) and volu-
metric product productivity (Qp = 0.258 g g1) obtained with S.
cerevisiae was found to be 5.2 and 21.1%, respectively higher than
that of Yp/s (0.423 g g1) and Qp (0.213 g g1) of Z. mobilis cells.
Likewise, the nal sugar to ethanol conversion rate (%) with S. cere-
visiae cells was 5.27% more than that of Z. mobilis. However, the -
nal biomass concentration (X) of S. cerevisiae cells (4.34 g l1) was
134% higher than that of Z. mobilis cells (1.85 g l1), which might
be due to more tolerance to temperature and ethanol [17].
In this study S. cerevisiae (strain CTCRI) was found to be more
efcient than Z. mobilis MTCC 92 in ethanol production from mah-
ula owers. Inuganti and Bhate [27] reported that mahula ower is
associated with several natural yeasts such as S. cerevisiae and S.
ellipsoideus. Hence, mahula ower considered as a suitable natural
habitat for S. cerevisiae which might have inuenced its fermenta-
tion capacity in comparison to Z. mobilis.
4. Conclusions
The results showed that ethanol production from mahula ow-
ers by S. cerevisiae was 21.2% higher in comparison to Z. mobilis. For
both the microorganisms, the peak ethanol concentration was ob-
tained after 96 h of fermentation. Despite many efforts undertaken
world wide, Zymomonas has not yet been commercialized for
ethanol production due to its low tolerance to temperature, etha-
nol and utilization of limited substrate range, as evident from the
present study.
Acknowledgment
The authors are thankful to UGC [(Letter F. No. 32-573/
2006(SR), dated 17.07.07], New Delhi for nancial support to carry
out this work.
 S. Behera et al. / Applied Energy 87 (2010) 23522355 2355

References [13] Nowak J, Roszyk H. Co-immobilization of Aspergillus niger and Zymomonas

mobilis for ethanol production from starch. Pol J Food Nutr Sci 1997;6:
6570.
[1] Chandel AK, Chan ES, Rudravaram R, Narasu ML, Rao LV, Ravindra P. Economics
[14] Amerine MA, Ough CS. Wine and must analysis. New York (USA): Wiley; 1984.
and environmental impact of bioethanol production technologies: an
[15] Mahadevan A, Sridhar R. Methods in physiological plant pathology. 5th
appraisal. Biotechnol Mol Biol 2007;2:01432.
ed. Madras (India): Sivakami Publication; 1999.
[2] Ward OP, Singh A. Bioethanol technology: development and perspectives. Adv
[16] Bailey JE, Ollis DF. Biochemical engineering fundamentals. New
Appl Microbiol 2002;51:5380.
York: McGraw-Hill; 1986.
[3] Demirbas A. Bioethanol from cellulosic materials: a renewable motor fuel from
[17] Bansal R, Singh RS. A comparative study on ethanol production from molasses
biomass. Energy Sources 2005;27:32737.
using Saccharomyces cerevisiae and Zymomonas mobilis. Ind J Microbiol
[4] Zaldivar J, Nielson J, Olsson L. Fuel ethanol production from lignocellulose: a
2003;43:2614.
challenge for metabolic engineering and process integration. Appl Microbiol
[18] McGhee JE, Julian GSt, Detroy RW, Bothast RJ. Ethanol production by
Biotechnol 2001;56:1734.
Saccharomycces cerevisiae, Saccharomyces uvarum and Zymomonas mobilis.
[5] Ward OP, Singh A, Ray RC. Production of renewable energy from agricultural
Biotechnol Bioeng 1981;24:115563.
and horticultural substrates and wastes. In: Ray RC, Ward OP, editors.
[19] Kunduru MR, Pometto AL. Continuous ethanol production by Zymomonas
Microbial biotechnology in horticulture, vol. 1. Eneld (New
mobilis and Saccharomyces cerevisiae in biolm reactors. Ind J Microbiol
Hampshire): Science Publishers; 2006. p. 51758.
1996;16:24956.
[6] Swain MR, Kar S, Sahoo AK, Ray RC. Ethanol fermentation of mahula (Madhuca
[20] Raman N, Pothiraj C. Screening of Zymomonas mobilis and Saccharomyces
latifolia L.) owers using free and immobilized yeast Saccharomyces cerevisiae.
cerevisiae strains for ethanol production from cassava waste. Rasayan J Chem
Microbiol Res 2007;162:938.
2008;1:53741.
[7] Mohanty SK, Behera S, Swain MR, Ray RC. Bio-ethanol production from mahula
[21] Amutha R, Gunasekaran P. Production of ethanol from liqueed cassava starch
(Madhuca latifolia L.) owers by solid state fermentation. Appl Energy
using co-immobilized cells of Zymomonas mobilis and Saccharomyces
2009;86:6404.
diastaticus. J Biosci Bioeng 2001;92:5604.
[8] Behera S, Kar S, Mohanty RC, Ray RC. Comparative study of bio-ethanol
[22] Thanonkeo P, Laopaiboon P, Sootsuwan K, Yamada M. Magnesium ions
production from mahula (Madhuca latifolia L.) owers by Saccharomyces
improve growth and ethanol production of Zymomonas mobilis under heat or
cerevisiae cells immobilized in agar agar and Ca-alginate matrices. Appl Energy.
ethanol stress. Biotechnology 2007;6:1129.
doi:10.1016/j.apenergy.2009.05.030.
[23] Osman YA, Ingram LO. Mechanism of ethanol inhibition of fermentation in
[9] Queresi N, Manderson GJ. Bioconversion of renewable resources into ethanol:
Zymomonas mobilis CP4. J Bacteriol 1985;164:17380.
an economic evaluation of selected hydrolysis, fermentation, and membrane
[24] Moreau RA, Powell MJ, Fett WF, Whitaker BD. The effect of ethanol and oxygen
technologies. Energy Sources 1995;17:24165.
on the growth of Zymomonas mobilis and the levels of hopanoids and other
[10] Karsch T, Stahl U, Esser K. Ethanol production by Zymomonas and
membrane lipids. Curr Microbiol 1997;35:1248.
Saccharomyces, advantages and disadvantages. Eur J Appl Microbiol
[25] Barbosa M, De FS, Yomano LP, Ingram LO. Cloning, sequencing and expression
Biotechnol 1983;18:38791.
of stress genes from the ethanol-producing bacterium Zymomonas mobilis: the
[11] Rogers PL, Strzelecki AT, Goodman AE. Commercial potential of Zymomonas
groESL operon. Gene 1994;94:517.
process for ethanol production. Found Biotech Indus Ferment Res
[26] Thanonkeo P, Sootsuwan K, Leelavacharamas V, Yamada M. Cloning and
1986;4:6379.
transcriptional analysis of groES and groEL in ethanol-producing bacterium
[12] Busche RM, Scott CD, Davison BH, Lynd LR. Ethanol, the ultimate feedstock. A
Zymomonas mobilis TISTR548. Pak J Biol Sci 2007;10:1322.
technoeconomic evaluation of ethanol manufacture in uidazed bed
[27] Inuganti NN, Bhate SR. The conditions of fermentation of the sugars of mahua.
bioreactors operating with immobilized cells. Appl Biochem Biotechnol
<http://Journal.library.iisc.ernet.in/archives/iiscjournal/p81-118.pdf>.
1992;34/35:395415.