Influence of Bacillus Subtillis for Bioethanol Production From Agro Bio Waste

8/13/2019 Influence of Bacillus Subtillis for Bioethanol Production From Agro Bio Waste

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Influence of Bacillus subtillis for Bioethanol production from

Agricultural waste

1*Karthikeyan, R.,

2Sivasubramanian, C.,

1Research scholar, Department of Environmental and Herbal science, Tamil university, Thanjavur, Tamil Nadu, India.

2Assistant 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

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

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: [email protected]

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

J. Biochem. 2013;148(6):332338 doi:10.1091/jb/mvq112

Received September 21, 2013; accepted October 31, 2013; published online December 17, 2013

332The 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

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).

Traditionally, ethanol has been produced in

batch fermentation with fungal strains such as, Scizo

saccharomyces pombe and Saccharomyces cerevisiae,

which cant 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

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,

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 usingB.subtilis.

MATERIALS AND METHODSCollection and processing of samplesPaddy 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).

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Isolation and characterization of

microorganismsB.subtilis was isolated from sugar cane molasses that

obtained from the Arignar Anna sugar industry,

kurunkulam, Thanjavur and identified in the

environmental and herbal science laboratory of the

Tamil University Thanjavur, Tamilnadu, India

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

media were used as such or were solidified with 1 %

agar (Davis Gelatine Ltd, Warwick, Warwickshire).

The microorganisms were grown with shaking at 37C

in a medium containing hydrolysed casein and inorganic

ions (Sterlini & Mandelstam, 1969). When the cells

were growing exponentially and the bacterial

concentration had reached 0.25 mg dry wt/ml, the

Oligosporogenous mutants of Bacillus subtilis3 culture

was centrifuged and the cells transferred at the same

density to a re suspension medium containing L-

glutamate and inorganic ions with 0.04 M-Mg2+

(Sterlini

& Mandel- stam, 1969). Cells shaking in this medium a

T37C gave a yield in the wild-type of about 80

retractile spores in 7 to 8 h.

Bioethanol productionMethods used for production of bioethanol include

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

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

121C. The Flasks were allowed to cool, filtered

through No. 1 What man filter paper and the pH was

adjusted to 4.5 with 0.4 M NaOH.

FermentationThe fermentation was carried out along with

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 30C.

Insignificant distillationThe 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

78C was used to heat the round-bottomed flask

containing the fermented broth.

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Fortitude of quantity of ethanol producedThe distillate collected over a slow heat at 78C 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 78C by the density

of ethanol (0.8033 g/l) is equivalent to the yield of 100 g

of dried substrate (Humphrey and Okafoagu, 2007).

Ethanol produced from paddy husk using B.

subtillis

Fortitude of percentage ethanol

concentrationA 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.

RESULTSCultural, morphological and biochemical

characteristics of isolatesThe 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

lot of interest in b. subtillisis its ability to differentiate

and formendospores. b. subtillisforms 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

fermentation, followed by 15.01 g/l at 96h. The lowest

volume (2.01 g/l) was produced at 24h.

DISCUSSIONPaddy 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.subtiliswas capable

of producing starch / carbohydrate hydrolases from

scarified paddy husk. B.subtilis is able to produced

335


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Influence of Bacillus Subtillis for Bioethanol Production From Agro Bio Waste

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