1.12. GOALS & OBJECTIVES - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/18972/9/09_chapter...
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1.12. GOALS & OBJECTIVES
Isolation, screening and selection of microbes, capable of
producing amylase enzyme, from soil
Collection of microbes from local soil and NCIM, which are
responsible for producing α-amylase enzyme and ethanol
production
α - amylase production from isolated and collected cultures by
solid state (S.S.F) and submerged fermentations (SmF)
Optimization of α-amylase production by Response Surface
Methodology(R.S.M)
Development of neural network model and neuro fuzzy model
for enzyme production and comparison
Optimization of total protein and its activity by Genetic
Algorithm and Particle Swarm Optimization
Optimization of Enzyme mediated Saccharification of starch by
using microbial amylases
Conversion of sugars into Ethanol by employing Saccharomyces
cerevisiae and Zymomonas mobilis.
2.1. ETHANOL PRODUCTON
From 2007 to 2010, the share of ethanol in global gasoline type
fuel use increased in 2010, from 3.7% to 5.4%. In 2010, worldwide
ethanol fuel production reached 86.9 billion liters. Environmental
efficiency for agro-industry has been reviewed recently in relation to
analysis of the ecological, economical and social aspects (Borrero et.
al. 2003) and characteristics of anhydrous ethanol used as an
automotive fuel mixed with gasoline, have been described (Vilar et. al.
2003). The largest ethanol consuming region other than South
America (38%) is Asia, accounting for 20% of world consumption. In
fact, biomass plays an important role as a source of energy in Asia.
On the other hand, very little, if any, information is available on the
source of the biomass used as a source of energy.
The main exporters of ethanol are U.S.A., Brazil, France, South
Africa, and the United Kingdom. Sugarcane is produced in several
countries Africa (Zimbabwe, Kenya, Egypt, Zaire, Zambia, Sudan,
Swaziland, and Mauritius), and the ethanol production in this
continent accounts for the worlds production. The production of fuel
ethanol from biomass has been considered a laudable goal because
plant biomass is the only sustainable source of organic fuels,
chemicals, and materials available to humanity. Moreover, the
renewable energy sources are much more environmentally friendly
than fossil fuels. Following burning they are lost forever and pose no
problem of pollution comparable to fossil fuels. Thus, the extensive
use of renewable resources will slow down continued deterioration of
the environment (Wu et. al. I998; Wu et. al. 1999; Zafar et. al. 2005).
A model of ethanol fermentation considering the effect of
temperature was developed and validated. Experiments were
performed in a temperature range from 28 to 40oC in continuous
mode with total cell recycling using a tangential microfiltration
system. This work is reported by Atala, D., A. Costa, et al. (2001).
2.2. BIOLOGICAL ENERGY PRODUCTION
Lignocellulosic materials containing cellulose, hemicellulose, and
lignin are the most abundant renewable organic resource on earth.
The utilization of renewable resources for energy and chemicals is
expected to increase in the near future (Aristidou, A et.al., 2000).
Various plant biomass residues can also be converted to ethanol
rather than burning (Kadam et. al. 2000, Schugeri, 2000). These
include agricultural residues for example, bagasse from sugarcane,
corn fiber, rice straw and hulls, nutshells and farm silages (Driehuis
and Wikselaar, 2000) wood waste (sawdust, timber slash, and mill
scrap), the paper trash and urban yard clippings in municipal waste,
energy crops (fast growing trees like poplars, willows, and grasses like
switch grass or elephant grass) (Anderson et. al. 2005), the methane
captured from landfills, municipal waste water treatment, manure
from cattle or poultry and even from spent sulfite pulping liquor (SSL)
which is a high-organic content byproduct of acid bisulfite pulp
manufacture. Because of its moisture content sugar cane bagasse has
a low value fuel; therefore its conversion to ethanol has also been
considered (Teixeira et. al. 1999; Duchuis and van Wikselaar, 2000;
Gnansounon et. al.2005).
2.3. ETHANOL PRODUCTION BACTERIA VS. YEAST
According to P. Gunasekaran (1999), Zymomonas mobilis and
Saccharomyces cerevisiae and are better candidates for industrial
alcohol production. Zymomonous mobilis is better than Saccharomyces
cerevisiae with respect to ethanol productivity and tolerance.
Observed theoretical yield was 92%, if Zymomonous mobilis is
cultivated in batch fermentation process for 70hrs. But in a
continuous fermentation using mixed cultures of Zymomonous mobilis
and Saccharomyces cerevisiae, production of 54.3 g/L of ethanol was
observed with 98% theoretical yield within 3 days.
2.4. ETHANOL TOLERANCE
A fundamental problem limiting higher yields is the low ethanol
tolerance of yeasts. The adverse effects of ethanol on yeast growth,
viability, and metabolism are caused primarily by ethanol leakage of
the plasma membrane i.e., the increased membrane fluidity at higher
ethanol concentrations (Ingram, 1990; Lloyd et. al. 1993). Medium
composition, and many other surrounding environmental factors and
number of genes affect ethanol concentrations of yeasts (Chi et. al.
1991).
Traditionally, ethanol was produced in batch fermentation with
Saccharomyces cerevisiae strain that cannot tolerate high
concentrations of ethanol. Ethanol tolerance in yeast is a fundamental
problem in fermentation industry especially at higher temperatures. It
is not necessary that ethanol kills the cells, but rather temporarily
inactivate them in a reversible manner. Saccharomyces cerevisiae
cannot tolerate more than about 10-12% by volume of ethanol
(Taylor et. al. 1995; Taylor et. al. 1998) Ethanol tolerance in free and
immobilized Saccharomyces cerevisiae was measured , and the study
indicated that immobilized yeast was far less sensitive to the ethanol
(Desimone et. al. 2002).
Boyle, M., N. Barron, et al. (1997) have reported a thermotolerant,
ethanol-producing yeast strain Kluyveromyces marxianus IMB3 that
would grow at 45°C on media containing 2, 4 and 6 % (w/v) pulverized
barley straw supplemented with 2% (v/v) cellulase and produce
ethanol.
2.5. ETHANOL FERMENTATION BY IMMOBILIZATION OF CELLS
Converti, A., C. Sommariva, et al. (1994) have published results a
study carried out mathematical modelling of fermentation with
immobilized cells in a large-pore matrix.
Immobilization resulted in increased levels of ethanol tolerance
Ciesarová, Z., Z. Domeny, et al. (1998).
Immobilization has also been reported with Z. mobilis in fluidized
bed reactors with increased productivity(Davison, B. and C.
Scott.,1988).
Ethanol fermentation using immobilized cells of yeast is one the
widely has also been reported by others (Wendhausen et. al. 2001;
Shen et. al. 2003b). Most of the methods involve the use of chemicals
for pretreatment of carriers. Some of such chemicals proved toxic to
beverages; therefore, biodegradable cellulose sponge (loofa sponge) has
been opted as a safe and alternative carrier for cell immobilization
(Ogbonna et. al. 1994). Many matrices have been used for
immobilization such as flexible spongelike material (Scott and O’Reilly,
1995), agar gel, calcium alginate beads (Ogbonna et. al. 1989; Li,
1996; Sree et. al. 2000; Najafpour et. al. 2004), Kissiris (mineral glass
foam derived from lava) (Nigam et. al. 1997), carrageenan, in ethanol
fermentation.
2.6. RAW MATERIALS FOR ETHANOL PRODUCTION
Fermentation processes from any material that contains sucrose
can derive ethanol. Many and different raw materials used in the
production of ethanol through fermentation. They are starches, sugar
and cellulose materials. Sugars can be converted to ethanol directly.
Starches must first be hydrolyzed to fermentable sugars by the action
of enzymes. Once simple sugars are formed, enzymes from yeast can
readily ferment them to ethanol. Important promising raw materials
used for ethanol production are sugar, starch and cellulose materials.
Ethanol production has been reported from several substrates
(Table.2.1).
Table.2.1 Substrates for alcohol production
S.No Substrates Name of the mocrobe Proposed
method Author Year
1 sweet
potato
S.fibulyer &
Zymomonas mobilis SSF Sevendsby 1981
2 raw sweet
potato Rhizopus species
one step
process Matsuoka 1987
5 uncooked
starch Rhizopus strain SSF
Fang and
Wang 1989
6 raw starch Corticum rolfsii &
Schizosaccharomyces SSF Hariantono 1991
7 Beet
Molasses
Saccharomyces
cerevisiae fermentation
Yekta
G.KSUNGU
R
2001
8 ozonized
raw starch recombinant yeast SSF
Nakamura
& Sawada 2002
9
Molasses
Saccharomyces
cerevisiae fermentation
Ranulfo
Monte
Alegre
2003
10 soybean
molasses
Saccharomyces
cerevisiae fermentation
Paula F.
Siqueira 2008
2.7. AMYLASE PRODUCTION BY SOLID STATE FERMENTATION
Amylases used in breadmaking and to break down complex
sugars, such as starch, into simple sugars. Alpha and beta amylases
are important in brewing beer and liquor. Amylase production has
been reported from several substrates (Table.2.2 & Table.2.3)
Table 2.2. AMYLASE PRODUCTION
S.No Substrate Organism Author Year
1 Bacterial
bran B. licheniformis Ramesh 1989
2 Wheat bran B. coagulans Babu and
Satyanarayana 1995
3 banana fruit
stalk Bacillus sp. Krishna 1995
4 Wheat bran Bacillus sp Soni 2003
5 Tapioca/
rice husk B. subtilis Baysal 2003
6 Wheat bran Thermomyces
lanuginosus
Kunamneni,
A., K 2005
7 Wheat bran B. subtilis Singh, S. K 2008
8 Wheat bran Aspergillus niger Manikandan 2009
9 waste
Loquat Penicillium expansum Serkan Erdal 2010
Table 2.3. AMYLASE PRODUCTION BY SUBMERGED FERMENTATION
S.No Substrate Organism Author Year
1 wheat bran Aspergillus oryzae A. K. Kundu 1970
2 Cassava
pulp
Rhizomucor pusillus Tony M. Silva 2005
3 wheat bran Bacillus sp Sujata d 2010
4 rice bran Aspergillus sp.MK07 Murali
Krishna
Chimata
2010
5 various
starches,
flours
Bacillus subtilis Özdemir S 2011
6 wheat bran Bacillus
sp. KR-8104
Maryam
Hashem
2011
7 wheat bran Bacillus sp V. P. Zambare 2011
2.8. RESPONSE SURFACE METHODOLOGY (RSM)
Many scientists have worked on response surface optimization to
maximize enzymes production. Enzymes production processes have
been reported from several substrates and different microbes
(Table.2.4 & Table.2.5).
Table 2.4: RESPONSE SURFACE METHODOLOGY (RSM)
S.No Substrate Organism Author Year
1 Wheat
bran Aspergillus oryzae
Shah, N. K., V.
Ramamurthy 1991
2 wheat
bran Aspergillus niger M.P. NANDAKUMAR 1999
3 Wheat
bran
Bacillus
licheniformis Chen, Q.-h., H. Ruan 2007
4 wheat
bran
Bacillus
amyloliquefaciens Dhanya Gangadharan 2008
5 groundnut
oil cake
Bacillus
amyloliquefaciens
Dhanya Gangadharan
2008
6 agro-
residue Bacillus subtilis p.sudarshan 2009
7 agro-
residues B. subtilis KCC103 Gobinath Rajagopalan 2009
8 cassava Bacillus
brevis MTCC 752 Ramesh C 2009
9 starch and
glycerol Streptomyces4 Mihaela Cotârleţ 2011
10 Wheat
bran green microalgae Azma, M 2011
10 starch Saccharomyces
cerevisiae Yingling, B 2011
2.9. MODELING AND OPTIMIZATION
Koutinas, A. A., R. Wang, et al. (2004) have worked on
reestructuring and optimization of the conventional fermentation
industry for fuel and chemical production is necessary to replace
petrochemical production routes
ANN linked with genetic algorithm were used to find the optimum
concentration of L-asparaginase production by Aspergillus terreus
MTCC 1782 in submerged fermentation (Baskar, G. and S.
Renganathan (2010).
optimization strategy for design of experiments and back propagation
algorithm of. Components such as L-proline, sodium nitrate, L-
asparagine and glucose were identified as significant fermentation
media components using Plackett–Burman design
Gurunathan, B. and R. Sahadevan (2011) reported the sequential
optimization strategy for design of an experimental and artificial
neural network (ANN) linked genetic algorithm (GA) were applied to
evaluate and optimize media component for L-asparaginase
production by Aspergillus terreus MTCC 1782 in submerged
fermentation
Hashemi, M., S. M. Mousavi, et al. (2011) have done modeling for
different phases of the bacterial growth and the production of α-
amylase by Bacillus sp. in a solid-state fermentation process.
2.10. NEURAL NETWORK MODELING
A.J.G. da Cruz, C.O. Hokka and R.C. Giordano et al.(1997) worked
on the production of penicillin G by Penicillium chrysogenum and it
was simulated by employing a feed forward neural network.
Backpropagation and random search methods were used in this
method. It was observed that the nonlinear behavior of the process
was able to describe accurately.
Nikhil, Bestamin Özkaya, Ari Visa, Chiu-Yue Lin, Jaakko A.
Puhakka, and Olli Yli-Harja et al.(2008) have reported the
performance of a sucrose-based H2 production in a completely stirred
tank reactor (CSTR) was modeled by neural network back-propagation
(BP) algorithm. The H2 production was monitored over a period of 450
days at 35±1 ºC. The proposed model predicts H2 production rates
based on hydraulic retention time (HRT), recycle ratio, sucrose
concentration and degradation, biomass concentrations, pH,
alkalinity, oxidation-reduction potential (ORP), acids and alcohols
concentrations. Artificial neural networks (ANNs) have an ability to
capture non-linear information very efficiently. In this study, a
predictive controller was proposed for management and operation of
large scale H2-fermenting systems. The relevant control strategies can
be activated by this method. BP based ANNs modeling results was
very successful and an excellent match was obtained between the
measured and the predicted rates. The efficient H2 production and
system control can be provided by predictive control method combined
with the robust BP based ANN modeling tool.
Charef Chabbi, M. Taibi and B. Khier et al.(2008) described two
modeling techniwues.They ANN and the hybrid artificial neural
network. The ANN’s fail the justification, but the HANN is good in
interpolation even for fewer training data.
2.11. FUZZY AND NEURO FUZZY MODELING
Bioprocess parameters are highly nonlinear and it is difficult to
control. Good modeling and control of these variables facilitates real-
time identification of these regimes. A fuzzy neural network model for
a bioprocess was developed by Hamrita,T.K and ShuWang et al (2000)
based on decomposition of the process into its different regimes. The
method is illustrated through the development of a real-time product
estimation model for simulated gluconic acid batch fermentation.
Baruch, I.; Galvan-Guerra, R.; Mariaca-Gaspar, C.-R.; Castillo, O
et al(2008) have proposed a new recurrent fuzzy-neural multi-model
(FNMM) identifier applied for identification of a distributed parameter.
2.12. MULTI OBJECTIVE OPTIMIZATION
If more than one response is present, then multiobjective
optimization need to be used for optimal solution. Adrian Dietz,
Catherine Azzaro Pantel, Luc Guy Pibouleau, and Serge Domenech et
al(2007) worked on the case study on multiproduct batch plant for the
production of four recombinant proteins. A Genetic Algorithm with a
Discrete Event Simulator was used in this method. Genetic Algorithm
was developed with a Pareto optimal ranking method.
Sendin, J.O.H. Exler, O. Banga, J.R et al (2010) have reported
multi-criteria optimization in the field of systems biology. The authors
present a novel solution scheme based on a global optimization. The
authors shown how the set of optimal solutions successfully and
efficiently obtained, providing further insight into the systems under
consideration regarding their optimal solution.
Sandu TARA and Alexandru Woinaroschy et al (2011) have dealt
with economical and ecological multi-criteria optimization of the
bioprocess of pyruvic acid production from glucose using a mutant
Escherichia coli strain. The optimization is possible due to an
automatic and repeated exchange of variables between Matlab
optimization algorithms.
2.13. MULTI OBJECTVE OPTIMIZATION BT GENETIC ALGORITHM
M.V.R.K. Sarma, Vikram Sahai and V.S. Bisaria et al(2009) have
worked on Genetic Algorithm optimization. Genetic algorithm was
applied for the optimization of siderophore and biomass production in
shake flask experiments. This method also used successfully to
estimate the kinetic parameters of the mathematical models of the
batch fermentation
Zhang, h and zhang, z et al (2010) have done optimization of
penicillin using genetic algorithm. The proposed approaches can be
productively applied to optimization problems of fed-batch
fermentation to develop the operation of such processes.
2.14. MULTI OBJECTIVEOPTIMIZATION BY PSO
Carlos A. Coello Coello et al (2004) have presented an approach in
which Pareto optimal is incorporated into particle swarm optimization.
Other current proposals to extend PSO to solve multi objective
optimization problems,. Results specify that the approach is extremely
competitive and that can be measured a viable alternative to solve
multi objective optimization problems.
Nor Azlina Ab. Aziz et al (2011) worked on Particle swarm
optimization (PSO) is an optimization method that belongs to the
swarm intelligence family. It was initially introduced as continuous
problem optimization tool. It has evolved to being applied to more
complex multi objective and constrained problem. This paper presents
a systematic literature review of PSO for constrained and multi
objective optimization problems.
Leticia Cagnina, Susana Esquivel et al (2005) this paper proposes
a hybrid particle swarm optimization. The results are compared with
those obtained with the Pareto Archived Evolution Strategy and the
Multi-Objective Genetic Algorithm.
DESIGN OF THE PROJECT WORK
Fig: 3.1 Flow diagram for design of project work
Collection & Processing of the
Vegetable Wastes
Cutting& Grinding + Water----- Sterilization
α- amylase production modelling for protein prediction
α- amylase production optimization with GA & PSO
Enzyme mediated saccharification
optimization
Alcohol fermentation
Separation of alcohol by fractional
distillation
Ethyl Alcohol
Enzyme
Purification
Isolation& collection of microbes
from MTCC for
α- amylase production
Collection of microbes
from MTCC for ethanol
fermentation
1
Fig.3.2. Proposed flow diagram for α-amylase and ethanol production