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J. Biochem.

2013;148(6):332–338

doi:10.1091/jb/mvq112

Influence of Bacillus subtillis for Bioethanol production from Agricultural waste
Received September 21, 2013; accepted October 31, 2013; published online December 17, 2013
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*Karthikeyan, R., Sivasubramanian, C.,

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Research scholar, Department of Environmental and Herbal science, Tamil university, Thanjavur, Tamil Nadu, India. Assistant professor, Department of Environmental and Herbal science, Tamil university, Thanjavur, Tamil Nadu, India.

Agricultural wastes, including wood, herbaceous plants, crops and forest residues, as well as animal wastes are potentially huge source of energy. A worldwide interest in the utilization of bioethanol as an energy source has stimulated studies on the cost and efficiency of industrial processes for ethanol production (Tanaka, 2006). The practice is usually to burn them or leave them to decompose. However, studies have shown that these residues could be processed into liquid fuel such as biogas and bioethanol, or combusted to produce electricity and heat (Soltes, 2000). Paddy husk were used to produce ethanol through acid hydrolysis,and SSF with B.subtilis. In SSF, the microorganisms behaved differently, according to their nutrient

from paddy husk in this study is in agreement with that (16.22 g/l). Key words: Bioethanol, paddy husk, B.subtilis

*Corresponding

author:

Karthikeyan,R,

Research

Scholar, Department of Environmental and Herbal Science ,Tamil University,Thanjavur-4. Email: krbiotech@gmail.com

Introduction:
Bioethanol is a renewable energy source produced mainly by the sugar fermentation process; although it can also be synthesized by chemical processes such as reacting ethylene with steam (Anujetal., 2007). Ethanol based fuel blend are broadly sold in the United States of America. The majority frequent blending is 10% ethanol and 90% petrol (E10). Motor vehicle engines need no alteration to run on E10 and vehicle warranties are not affected. Only flexible fuel vehicles can run on up to 85% ethanol and 15% Petrol blends (E85) (Tanaka, 2006). The usual energy resources like fossil fuel gasoline and coal are being utilized at a rapid rate and these resources have been estimated to last only a few

requirements, but synergistically in the degradation of organic substrate. The results revealed that ethanol could be produced from agricultural residues, such as paddy husk, using B.subtilis fermenting organisms The higher ethanol yield from fresh fruit was due to higher presence of fructose and glucose in fresh fruits, (Hanh Thi My Tran, Benjamas Cheirsilpa, 2010). The maximum volume of bioethanol (16.22 g/l) produced

The Authors 2013. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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years. Therefore, alternative energy sources such as ethanol, methane and hydrogen are being considered. Some biological processes have rendered possible routes for producing ethanol and methane in large quantities. A worldwide interest in the utilization of bioethanol as an energy source has stimulated studies on the cost and efficiency of industrial processes for ethanol production (Tanaka, 2006). Human activities generate large amounts of waste such as crop residues, solid waste from mines and municipal Waste. They may become a nuisance and sources of pollution. It is therefore important to handle them Sensibly to avoid health problems, since these wastes may harmful pathogenic microorganisms (Ledward etal., 2003). Agricultural wastes, including wood, herbaceous plants, crops and forest residues, as well as animal wastes are potentially Traditionally, ethanol has been produced in batch fermentation with fungal strains such as, Scizo saccharomyces pombe and Saccharomyces cerevisiae, which can’t tolerate high concentrations of ethanol. Therefore, improvement programmes are required in order to obtain alcohol-tolerant strains for fermentation (Hanh Thi My Tran, Benjamas Cheirsilpa, 2010). Bacillus subtillis, a Gram-positive bacterium, is

huge source of energy. In Nigeria, large quantities of these wastes are generated annually and are vastly underutilized. The practice is usually to burn them or leave them to decompose. However, studies have shown that these residues could be processed into liquid fuel such as biogas and bioethanol, or combusted to produce electricity and heat (Soltes, 2000). Ethanol production processes only use energy from renewable sources and there is no net CO2 emission to the atmosphere, thus making ethanol an environmentally beneficial energy source. In addition, ethanol derived from biomass is the only liquid transportation fuel that does not contribute to the greenhouse gas effect. This reduction of greenhouse gas emission is the main advantage of utilizing biomass conversion into ethanol (Anuj et al., 2007).

Benjamas Cheirsilpa, 2010). Several agricultural wastes have been tested for their bioethanol-producing

potential. In the present study, the utilization of some Industrial bio-waste (paddy husk) for the production of bioethanol was evaluated. The objectives of the study were to produce bioethanol from paddy husk residues through fermentation using B.subtilis.

MATERIALS AND METHODS Collection and processing of samples
Paddy husk were collected from Thanjavur Local Rice mill industries. The samples were dried and ground to a powder form using a Waring blender (OSAKA CHEMICAL).

considered an alternative organism in large scale ethanol production. Its advantages over yeasts include higher sugar uptake and ethanol yield, lower biomass production and higher ethanol tolerance glucose, fructose and sucrose. This organism can be isolated from palm wine or rotten oranges (Hanh Thi My Tran,

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Isolation

and

characterization

of

Bioethanol production
Methods used for production of bioethanol include

microorganisms
B.subtilis was isolated from sugar cane molasses that obtained from the Arignar Anna sugar industry, kurunkulam, Thanjavur and identified in the

hydrolysis, fermentation and fractional distillation.

Hydrolysis
One hundred grams (100 g) of paddy husk was weighed into eight conical flasks, and 1 l of 2 MH2SO4 was

environmental and herbal science laboratory of the Tamil University Thanjavur, Tamilnadu, India

added to each conical flask. The flasks were covered with cotton wool, wrapped in aluminum foil, heated for 2 h in a water bath and then autoclaved for 30 min at 121°C. The Flasks were allowed to cool, filtered

following standard procedures described by Sterlini & Mandelstam (1969), but without the excess Mg2+ added. When the L-glutamate in this medium was replaced by L-lactate (0.2 %) or supplemented with either L-lactate or D-glucose (0.2 %) the media are referred to as lactate minimal, lactate-glutamate minimal and glucose-glutamate minimal respectively. These

through No. 1 What man filter paper and the pH was adjusted to 4.5 with 0.4 M NaOH.

Fermentation
The fermentation was carried out along with

media were used as such or were solidified with 1 % agar (Davis Gelatine Ltd, Warwick, Warwickshire). The microorganisms were grown with shaking at 37°C in a medium containing hydrolysed casein and inorganic ions (Sterlini & Mandelstam, 1969). When the cells were growing exponentially and the bacterial

saccharification and fermentation [SSF), as described by Kroumov et al. (2006) and Oghgren et al. (2006). The flasks containing the hydrolyzed samples were

covered with cotton wool, wrapped in Aluminum foil, autoclaved for 15 min at 121° C, and allowed to cool at room temperature. B.subtilis were aseptically inoculated into each flask and incubated at 30°C.

concentration had

reached 0.25 mg dry wt/ml, the

Oligosporogenous mutants of Bacillus subtilis 3 culture was centrifuged and the cells transferred at the same density to a re suspension medium containing Lglutamate and inorganic ions with 0.04 M-Mg2+ (Sterlini & Mandel- stam, 1969). Cells shaking in this medium a T37°C gave a yield in the wild-type of about 80 retractile spores in 7 to 8 h.

Insignificant distillation
The fermented broth was dispensed into round-bottom flasks fixed to a distillation column enclosed in running tap water. A conical flask was fixed to the other end of the distillation column to collect the distillate. A

heating mantle with the temperature adjusted to 78°C was used to heat the round-bottomed flask containing the fermented broth.

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Fortitude of quantity of ethanol produced
The distillate collected over a slow heat at 78°C was measured using a measuring cylinder, and expressed as the quantity of ethanol produced in g/l by multiplying the volume of distillate collected at 78°C by the density of ethanol (0.8033 g/l) is equivalent to the yield of 100 g of dried substrate (Humphrey and Okafoagu, 2007).

RESULTS Cultural, morphological and biochemical

characteristics of isolates
The organisms used for fermentation were b.subtilis is a positive, rod-shaped and endospore-forming aerobic bacterium. It is found in soil and rotting plant material and is non-pathogenic. It is one of the most studied gram-positive bacteria. One feature that has attracted a

Ethanol produced from paddy husk using B. subtillis

lot of interest in b. subtillis is its ability to differentiate and form endospores. b. subtillis forms colonies that are dull and may be wrinkled, cream to brown in colour and when grown in broth have a coherent pellicle; usually with a single arrangement.

Ethanol produced from paddy husk using B. subtillis
Figure 1 shows the volume (g/l) of ethanol produced from paddy husk after acid hydrolysis with 2.5 MH2SO4 and SSF with both Bacillus subtillis. The highest volume (16.22 g/l) was produced at 144h of

Fortitude concentration

of

percentage

ethanol

fermentation, followed by 15.01 g/l at 96h. The lowest volume (2.01 g/l) was produced at 24h.

A standard ethanol density curve was prepared by taking series of percentage (v/v) ethanol solutions, which were prepared in volumetric flasks, and the weight was measured. The density for each of the prepared ethanol solutions was calculated and a standard curve of density against percentage ethanol was plotted. The percentage ethanol concentration of ethanol produced was obtained by comparing its density with the standard ethanol density curve.

DISCUSSION
Paddy husk were used to produce ethanol through acid hydrolysis, and SSF with B.subtilis. In SSF, the microorganisms behaved differently, according to their nutrient requirements, but synergistically in the degradation of organic substrate. B.subtilis was capable of producing starch / carbohydrate hydrolases from scarified paddy husk. B.subtilis is able to produced

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ethanol due to the presence of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), which are key enzymes in ethanol formation, as reported by (Hanh Thi My Tran, Benjamas Cheirsilpa, 2010). It was also acknowledged by the authors that the maximum ethanol yield at 120 h from fresh fruit (64.01 g/l) and waste fruits (21.14 g/l) using B. subtilis. The higher ethanol yield from fresh fruit was due to higher presence of fructose and glucose in fresh fruits, (Hanh Thi My Tran, Benjamas Cheirsilpa, 2010). The maximum volume of ethanol (16.22 g/l) produced from paddy husk in this study is in agreement with that (16.22 g/l). The results discovered that bio-ethanol could be produced from agricultural residues, such as paddy husk, using B.subtilis fermenting organisms.

beijerinckii BA101 using a low-cost fermentation medium based on corn steep water, Appl. Microbiol. Biotechnology. 51 (1999), pp. 152–157. [2] D.T. Jones and D.R. Woods, Acetone–butanol fermentation

revisited, Microbiol. Rev. 50 (1986), pp. 484–524. [3] T.W. Jesse, C.T. Ezeji, N. Qureshi and H.P. Blaschek, Production of butanol from starch-based waste packing peanuts and agricultural waste, J. Ind. Microbiol. Biotechnol. 29(2002), pp. 117–123. [4] P. Dürre, New insights and novel developments in clostridial acetone/butanol/ isopropanol

fermentation, Appl.Microbiol. pp. 639–648.

Biotechnol. 49 (1998),

[5] Y. Tashiro, K. Takeda, G. Kobayashi, K. Sonomoto, A. Ishizaki and S. Yoshino, High butanol production by Clostridium saccharoperbutylacetonicum N1-4 in

Considering the cost-effectiveness, in addition to being resources to manage environmental contamination, the use of paddy husk for ethanol production is concluded as a worthwhile venture.

fed-batch culture with pH-stat continuous butyric acid and glucose feeding method, J. Biosci. Bioeng. 98 (4) (2004), pp. 263–268. [6] J. Zaldivar, J. Nielsen and L. Olsson, Fuel ethanol production from lignocellulose: a challenge for

ACKNOWLEDGEMENT
I gratefully thankful to Prof.M.Jagadeesan, Head Department of Environmental and herbal Science, all staff members and Lab Technician Department of Environmental and herbal Science, Tamil University, Thanjavur for providing the necessary facilities for the preparation of the paper.

metabolic engineering and process integration, Appl. Microbiol. Biotechnol. 56(2001), pp. 17–34. [7] V.V. Zverlov, O. Berezina, G.A. Velikodvorskaya and W.H. Schwarz, Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery, Appl.Microbiol. Biotechnol. 71 (2006), pp. 587–597. [8] B.K. Soni, K. Das and T.K. Ghose,

References
[1] M. Parekh, J. Formanek and H.P. Blaschek, Pilotscale production of butanol by Clostridium

336

Bioconversion

of

agro-wastes

into

acetone

niger grown on native starch sources, J. Sci. Food Agric. 69 (1995), pp. 109–115. [16] M.S. Madihah, A.B. Ariff, K.M. Sahaid, A.A. Suraini and M.I.A. Karim, Direct fermentation of gelatinized sago starch to acetone–butanol–ethanol by Clostridium acetobutylicum, World J. Microbiol. Biotechnol. 17 (2001), pp. 567–576. [17] G. Coleman and W.H. Elliott, Studies on αamylase formation by Bacillus subtilis, Biochem.

butanol, Biotechnol. Lett. 4 (1982), pp. 19–22. [9] O. Fond, E. Petitdemange, H. Petitdemange and J.M. Engasser, Cellullose fermentation by a coculture of a mesophilic cellulolytic Clostridium and Clostridium acetobutylicum 13 (1983), pp. 217–224. [10] E.K.C. Yu, M.K.H. Chan and J.N. Saddler, Butanol production from cellulosic substrates by sequential coculture of Clostridium thermocellum and C.

acetobutylicum, Biotechnol. Lett. 7 (1985), pp. 509– 514. [11] D. Stevens, S. Alam and R. Bajpai, Fermentation of cheese whey by a mixed cultureof Clostridium beijerinckii and Bacillus Microbiol. 3 (1988), pp. 15–19. [12] S. Chauvatcharin, C. Siripatana, T. Seki, M. Takagi and T. Yoshida, Metabolism analysis and on-line physiological state diagnosis of acetone– cereus, J. Ind.

J. 83 (1962), pp. 256–263. [18] M.M. Nakano and F.M. Hullet, Adaptation of Bacillus subtilis to oxygen

limitation, FEMSMicrobiol. ett. 157 (1997), [19] L.D. Clements, B.S. Miller and U.N. Streips, Comparative growth analysis of the facultative

anaerobes Bacillus subtilis,Bacillus licheniformis and Escheria coli, Syst. Appl. Microbiol. 25 (2002), pp. 284–286. [20] F. Mohammad, O. El-Tayeb and M. Aboulwafa, Optimization of the industrial production of bacterial amylase in Egypt. V. Analysis of kinetic data for enzyme Analysis of kinetic data for enzyme production by two strains of Bacillus amylolique

butanolfermentation, Biotechnol. Bioeng. 58 (6) (1997), pp. 561–571. [13] J.R. Gapes, D. Nimcevic and A. Friedl, Long-term continuous cultivation of Clostridium beijerinckii in a two-stage chemostat with on-line product

removal, Appl.Environ. microbiol.62 (1996) pp. 3210– 3219. [14] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31 (1959), pp. 426–429. [15] B.N. Okolo, L.I. Ezeogu and C.N. Mba, Production of raw starch digestive amylase by Aspergillus

faciens, AfricanJ.Biotechnol7 (24) (2007), pp. 4537– 4543.Biotechnol. 7 (24) (2007), pp. 4537–4543. [21] C.N. Hipolito, E. Crabbe, C.M. Badillo, O.C. Zarrabal, M.A.M. Mora, G.P. Flores, M.A.H. Cortazar and A. Ishizaki, Bioconversion of industrial wastewater from palm oil processing to butanol by Clostridium

337

saccharoper butylacetonicum N1-4 (ATCC 13564), J. Cleaner Prod. 16 (2008), pp. 632–638. [22] F. Hillmann, R. Fischer, F. Saint-Prix, L. Girbal and H. Bahl, PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum, Mol. Microbiol. 68 (4) (2008), pp. 848–860. [23] S. Kato, S. Haruta, Z.J. Cui, M. Ishii and Y. Igarashi, Effective cellulose degradation by a mixedculture system composed aerobic of a

cellulolytic Clostridium and

non-cellulolytic

bacteria, FEMS Microbiol. Ecol. 51 (2004), pp. 133– 142.

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