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Applied Energy 86 (2009) 640–644
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Applied Energy
journal homepage: www.elsevier .com/ locate/apenergy
Bioethanol production from mahula (Madhuca latifolia L.)flowers by solid-state fermentation
Sujit Kumar Mohanty a, Shuvasis Behera b, Manas Ranjan Swain c, Ramesh Chandra Ray c,*
a Division of Biotechnology, Majhighariani Institute of Technology and Science, Sriram Vihar, Bhujabal, Rayagada 765017, Indiab Department of Botany, Utkal University, Bhubaneswar 751004, Indiac Central Tuber Crops Research Institute (Regional Centre), Bhubaneswar 751019, India
a r t i c l e i n f o a b s t r a c t
Article history:Received 17 June 2008Received in revised form 19 August 2008Accepted 28 August 2008Available online 7 October 2008
Keywords:BioethanolEthanol productivityMahula (Madhuca latifolia L.) flowerSolid-state fermentationSaccharomyces cerevisiae
0306-2619/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.apenergy.2008.08.022
* Corresponding author. Tel./fax: +91 674 247052.E-mail address: [email protected] (R.C. Ray).
There is a growing interest worldwide to find out new and cheap carbohydrate sources for production ofbioethanol. In this context, the production of ethanol from mahula (Madhuca latifolia L.) flowers by Sac-charomyces cerevisiae in solid-state fermentation was investigated. The moisture level of 70%, pH of 6.0and temperature of 30 �C were found optimum for maximum ethanol concentration (225.0 ± 4.0 g/kgflower) obtained from mahula flowers after 72 h of fermentation. Concomitant with highest ethanol con-centration, the maximum ethanol productivity (3.13 g/kg flower/h), yeast biomass (18.5 � 108 CFU/gflower), the ethanol yield (58.44 g/100 g sugar consumed) and the fermentation efficiency (77.1%) werealso obtained at these parametric levels.
� 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Mahula (Madhuca latifolia L.) is a forest tree found in abundancein the tropical rain forests of Asian and Australian continents. Thistree species, however, has been domesticated by tribal people inIndia and Pakistan for its uses as food (flower), feed (leaves andflower), wood (timber) and alcoholic beverage (fermented flowers)which is locally called ‘mahuli’ in India. The annual production ofmahula (also called as ‘mahula’) flowers in India in 2005–2006was about 48,000 M tonnes [1]. Fresh mahula flowers are a cheapsource of fermentable sugars [2].
As demand for the limited global supply of non-renewable en-ergy resources increases, the price of oil and natural gas keepincreasing. As a result, production of ethanol by fermentation fromrenewable carbohydrate materials for use as an alternative liquidfuel has been attracting worldwide interest [3]. There is a growinginterest to find alternative bioresources apart from sugarcane/beetmolasses and starchy crops like cassava, sweet potato and sweetsorghum for ethanol production. Further, considerable interesthas been shown in using these agricultural crops and their prod-ucts for ethanol production using solid-state fermentation (SSF)[4–7].
SSF is defined as the cultivation of microorganisms on moist so-lid support, either on inert carriers or insoluble substrates that can
ll rights reserved.
in addition be used as carbon and energy sources. SSF takes placein absence or near absence of free water thus being close to thenatural environment to which microorganisms are adapted [8].Agricultural substrates and crop residues serve as cheap raw mate-rials for use in SSF for production of various bio-products, i.e. or-ganic acids, enzymes, amino acids, and bio-ethanol [9,10].
Mahula trees have many distinct advantages over traditionalcrops, such as its flowers have high fermentable sugars, goodgrowth in poor soil under rain fed conditions and high toleranceto various plant diseases [11]. The price of mahula flowers is verycheap, US$35–40 M tonnes; hence, can be economical for ethanolproduction. Recently, the production of ethanol from mahula flow-ers by free and immobilized Saccharomyces cerevisiae cells in sub-merged fermentation has been described [1]. The production ofethanol from mahula flowers by SSF has not been studied. There-fore, the aim of the present investigation was to examine the po-tential of mahula flowers as a source for ethanol production by S.cerevisiae cells via SSF, as well as to study the effect of various fer-mentation parameters such as incubation period, moisture, pH andtemperature on kinetic parameters of ethanol fermentation.
2. Materials and methods
2.1. Mahula flowers
Fresh mahula flowers were collected from the forests of Keonj-har district of Orissa, India, during March–April, 2006. The flowers
S.K. Mohanty et al. / Applied Energy 86 (2009) 640–644 641
were brought to the Microbiology Laboratory of CTCRI, washed intap water to get rid of dust and other debris and sun-dried in openfor 7 days to reduce the moisture content to 11.0–12.5%. The sun-dried flowers collected from various locations were mixed thor-oughly before being used for ethanol fermentation. The flowershave the following compositions (expressed in g/100 g dry weightbasis): moisture, 10–12; starch, 0.94–0.95; total sugar (glucose,fructose, sucrose and maltose), 46–48; crude protein, 6.0–6.5;crude fibre, 10.0–11.5; total ash, 1.2–1.8; pH 4.5–4.8 [2].
2.2. Yeast
Saccharomyces cerevisiae (strain CTCRI) was grown in 250 mlErlenmeyer flasks containing 100 ml sterilized medium (yeast ex-tract-nutrient broth, YENB) with sugar concentration of 12% (w/v) and the pH was adjusted to 5.5 by dilute HCl. It was culturedfor 24 h at 30 �C in an incubator. This served as the starter culturefor ethanol production.
2.3. Fermentation medium
Fifty grams of mahula flowers supplemented with 1% NH4Cl (asnitrogen source) was placed in 1000 ml Roux bottles(132 mm � 275 mm) and moistened with appropriate amount ofdistilled water in order to contain 70% moisture. The pH of the sub-strate was adjusted to 6.0 with 1 N NaOH. The content was pres-sure-cooked for 30 min at 120 �C and inoculated with yeaststarter culture [10% v/w (3 � 109 Colony Forming Unit (CFU)/ml)]. The Roux bottles, in triplicate, were incubated at 30 �C in anincubator under stationary conditions for 120 h.
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Fig. 1. Role of incubation period on ethanol concentration and sugar consumptionby Saccharomyces cerevisiae in solid-state fermentation using mahula flower.
2.4. Study of fermentation parameters
(1) Moisture content: A series of Roux bottles containing 50 gmahula flowers were moistened with an appropriateamount of distilled water in order to contain 40, 50, 60, 70,80 and 90% moisture. The flasks, in triplicate, were inocu-lated and incubated at 30 �C for 72 h.
(2) Initial pH: The substrate consisting of 50 g mahula flowerswith 70% moisture and a pH 3, 4, 5, 6, 7 and 8 was inoculatedwith 10% (v/w) yeast culture and incubated as above.
(3) Temperature: The medium (50 g mahula flowers, moisture70% and pH 6.0) was inoculated for 72 h with 10% (v/w)yeast culture and incubated at different temperatures (20–40 �C).
2.5. Analytical techniques
At appropriate time intervals, fermentation bottles were re-moved and the contents were analyzed. The number of living cellswas determined by plate counting S. cerevisiae that was cultivatedon yeast extract-nutrient agar (YENA) medium at 30 �C for 24 h.The fermented mash in each Roux bottle was mixed with 150 mldistilled water [1:3 (w/v)] and the mixture was shaken on a rotaryshaker (Remi Pvt. Ltd., Mumbai, India) at 250 rpm for 30 min at30 �C in order to extract the ethanol and the whole mash was dis-tilled to collect the ethanol [1]. Ethanol concentration of the fer-mentation liquid was determined by measuring the specificgravity of the distillate according to the procedure described byAmerine and Ough [12]. The ethanol yield was expressed as g eth-anol/100 g sugar consumed. Fermentation efficiency was calcu-lated by dividing the sugar consumed during fermentation by theinitial sugars and multiplying the results by 100.
Concentrations of the total sugar (glucose, fructose, sucrose andmaltose) in the flowers and in the fermentation broth were deter-mined as glucose equivalent by Anthrone method [13]. The otherproximate compositions like starch, crude protein, crude fibreand ash were estimated as per the standard ‘‘AOAC” procedure[14]. The pH was measured by a pH meter (Systronics, Ahmedabad,India) using glass electrode. Fermentation kinetics was studied asper the formulae given by Bailey and Ollis [15].
2.6. Population count
Yeast population on the fermented mash was calculated by seri-ally diluting the substrate in the distilled water and plating suit-able dilution (108–109) on YENA solidified on Petri plates(18 mm � 150 mm). Data were given as mean of six replicates.
3. Result and discussion
The production of ethanol from mahula flowers by S. cerevisiaein SSF is shown in Fig. 1. The concentration of ethanol increasedwith the increase of fermentation time and yeast biomass. Themaximum ethanol (195 ± 4 g/kg flowers) concentration (95%) wasobtained after 72 h of incubation. In a previous study, maximumethanol concentration of 193 and 205 g/kg flowers were obtainedwhen free and immobilized yeast cells were grown in mahulaflower slurry [mahula flower:water, 1:5 (w/v) ratio], respectivelyafter 96 h in submerged shake-flask fermentation [1]. Hang et al.[5,16] reported maximum ethanol concentration of 43 g/kg applepomace and 53.5 g/kg grape pomace for various yeast strainsgrown in SSF, whereas Roukas [6] found that maximum ethanolconcentration (160 ± 3 g/kg dry pods) was obtained when S. cerevi-siae was grown on pods of carob tree (Ceratonia siliqua) after 48 hof fermentation. Similarly, Kiran Sree et al. [7] reported highestethanol concentration of 50 g/kg substrate (sweet sorghum andsweet potato) in SSF at 37 �C using a thermotolerant strain of S.cerevisiae. There are some possible reasons for these differences,including the strain of S. cerevisiae used, biochemical compositionof the substrate, fermentation system and the condition underwhich the fermentation took place [17,18]. The viable cell numbersincreased from 3 � 108 CFU/g substrate (0 h) to 18.5 � 109 CFU/gsubstrate (72 h) after which it decreased drastically at 96 h(1 � 108 CFU/g substrate). The decline in biomass concentrationcould be due to reduced substrate availability and the inhibitoryeffect of ethanol on yeast cells [3,19].
Further, the concentration of residual sugars decreased duringthe fermentation coinciding with an increase in biomass and etha-nol production (Fig. 1). The concentration of residual sugars fell
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rapidly and consistently during the first 72 h of fermentation, afterwhich it decreased slowly. This was due to rapid increase in bio-mass and ethanol concentration, observed at the same time. Atthe time (72 h) when the maximum concentration of ethanolwas achieved, 78% of sugar consumed was converted to ethanol.This finding differs from our previous study of mahula slurry fer-mentation by S. cerevisiae that showed maximum sugar conversion(80–82%) and ethanol concentration (193–205 g/kg flowers) wereachieved at 96 h of submerged fermentation [1].
3.1. Effect of moisture content
Moisture content is one of the important factors that affect theperformance of SSF. As shown in Fig. 2A and B, the ethanol concen-tration, ethanol productivity, ethanol yield and fermentation effi-ciency were increased significantly with the increase in moisturecontent. The highest value of fermentation parameters wereachieved at a moisture level of 70%. Roukas [6] reported a moisturelevel of 70% was the best to achieve ethanol concentration(160 ± 3 g/kg) from carob pod in SSF. Similarly, Kargi et al. [20]and Ngadi and Correia [21] reported that the maximum ethanolproduction was obtained from sweet sorghum and apple pomacein SSF at a moisture level of 70% and 85%, respectively. Decreasingthe moisture level from 70% to 40% resulted in a decrease in all ki-netic parameters (ethanol concentration, ethanol productivity, eth-anol yield and fermentation efficiency). This is because anoptimum moisture level (70% in our study) is essential for sustain-ing optimum growth of microorganisms and thereby ethanol pro-duction. The decrease in moisture level is to a certain extentadvantageous since the chance of contamination of fermentationis reduced. However, there is a lower limit of moisture content be-low which yeast cells may not function to produce ethanol [22].
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Fig. 2. Effect of moisture content (%) on (A) ethanol concentration and ethanolyield, (B) fermentation efficiency and ethanol productivity by Saccharomycescerevisiae in solid-state fermentation of mahula flowers.
Likewise, above 70% moisture content in SSF, there was a decreasein ethanol accumulation. This might be due to decrease in porosity,lower oxygen transfer and poor aeration inside the substrate mass(mahula flower) under the stationary fermentation condition [23].
3.2. Effect of initial pH
The effect of initial pH on kinetic parameters of mahula flowerfermentation is shown in Fig. 3A and B. The fermentation parame-ters increased drastically with the increase in pH up to 6.0 and de-creased beyond this value. On the other hand, ethanol yield andethanol productivity remained more or less same over the pHrange of 5.0–6.0, and decreased marginally above 6.0. The maxi-mum ethanol concentration (225.0 ± 4 g/kg mahula flower), etha-nol productivity (3.13 g/kg/h), ethanol yield (58.44 g/100 g sugarconsumed) and fermentation efficiency (77.1%) were obtained incultures grown at 6.0. Roukas [6] studied the effect of pH on etha-nol production from carob pod by S. cerevisiae and found that themaximum ethanol concentration, ethanol yield, and fermentationefficiency were obtained at pH 4.5. Yeasts have a pH optimum be-tween 4.0 and 6.0, and can grow in a large pH range of 2.5–8.5 [24].
3.3. Effect of temperature
As shown in Fig. 4A and B, increasing the fermentation tempera-ture from 20 �C to 40 �C significantly affected the ethanol concentra-tion, ethanol productivity and fermentation efficiency. The ethanolyield decreased at temperature values lower or higher than 25–30 �C. The ethanol concentration, ethanol productivity and fermen-tation efficiency increased with the increase in fermentationtemperature from 20 to 30 �C and decreased gradually between30 and 35 �C and drastically above 35 �C. This was probably due to
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Fig. 3. Effect of initial pH on (A) ethanol concentration and ethanol yield, (B)fermentation efficiency and ethanol productivity by Saccharomyces cerevisiae insolid-state fermentation of mahula flowers.
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Fig. 4. Effect of initial temperature (�C) on (A) ethanol concentration and ethanolyield, (B) fermentation efficiency and ethanol productivity by Saccharomycescerevisiae in solid-state fermentation of mahula flowers.
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the decrease in viable cell number above 30 �C. Temperature in therange of 25–30 �C is commonly found optimum for mesophillic S.cerevisiae strain for production of ethanol in SSF of various sub-strates, i.e. apple pomace [5], carob pod [6], sweet sorghum [25], etc.
Average production (M tonne/ha) of different bio-ethanol cropsunder irrigated condition in India is as follows: sugarcane (8–12),sweet sorghum (2–3), cassava (12–18), sweet potato (8–10) andmahula (1.7–2.0) [1,3]. All these crops except mahula are culti-vated in fertile (sugar cane) and marginal (sweet sorghum, cassavaand sweet potato) agricultural lands with substantial inputs of fer-tilizers and provision for irrigation. In contrast, mahula is a self-pollinated tree grown naturally in tropical rain forest and needno extra cost for its production and growth. The sugar rich ediblefleshy parts (corolla) of mahula flowers fall naturally on groundafter maturity [2] and only costs that involve are for collection,transportation and storage. Further, the cost (US$) per M tonneof these substrates from which ethanol is obtained are tentativelyas follows: sugarcane (53.0–60.0), sweet sorghum (110.0–125.0),cassava (50.0–60.0), sweet potato (58.0–70.0) and mahula (35.0–40.0) [3,26]. The cost (US$) of ethanol production/l has been esti-mated (US$1 � INR 44.80): sugar cane (0.27), sweet sorghum(0.29), cassava (0.55), sweet potato (0.71) and mahula (0.24). In In-dia, total production of mahula in the year 2006 was 48,000 M ton-nes [1]. Therefore, mahula flower can serve as another potentialfeedstock for bioethanol in tropical countries like India, Pakistan,Indonesia and Australian continent [1,27].
4. Conclusion
The results showed that ethanol production from mahula flow-ers in SSF was as per with the data obtained from submerged
fermentation from our previous study. Moreover, the peak ethanolconcentration was obtained at 72 h in SSF in comparison to sub-merged fermentation in which the same concentration wasachieved at 96 h. This spares considerable time and energy besidesease in operation and recovery process that are advantageous char-acteristics of SSF. Mahula flowers are available in plenty in theAsia-Pacific regions but its commercial potential for fuel ethanolhas not been fully explored. This is mainly because collection, stor-age and marketing of mahula flowers are not well organized unlikemarketing of sugarcane/beet molasses in Asian Sub-continent.Nevertheless, as evident from this study, mahula flowers are acheap fermentable carbohydrate bio-resource for production offuel ethanol. Further studies are needed to find out the compara-tive economics of fuel ethanol production from mahula flowersand sugarcane/beet molasses.
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