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20 Φεβ 2013 (πριν από 4 χρόνια και 5 μήνες)

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Business, Administrative & Contact Information




Business name: Eureka Powers



Business type: Private Enterprise



Company registration number: xxxxx



Tax reference number: xxxxx



VAT number: self explanatory



Tel: 0314
-
4134331



E
-
mail:
eurekapowers@hotmail.com



Physical address: New Lahore Road Nishatabad, Faisalabad, Pakistan



Website:
www.eurekapowers.wordpress.com



Banking Details: Habib M
etropolitan Bank



Account No: DHL
-
126410



Members:

1.

M. Adeel

2.

Maria Maqsood

3.

Adnan Iqbal

4.

Sania Munir

5.

Sayyada Azka

6.

Adeela Hussain

7.

Awais Afzal

8.

Manya Sarmad

9.

Ambash Riaz

10.

Amna Shakrullah

11.

Zon Yousaf

12.

Ayesha Qadir

13.

Sumia Khan



Eureka Powers

Aim:

This is an
entrepreneurial venture, aiming to achieve clean products with the use of industrial
biotechnology. This includes the production of four enzymes:

1.

Amylase

2.

Phytase

3.

Cellulase

4.

Xylanase

The other goal of the industry is the production of power alcohol from mola
sses.

Eureka Powers is an entrepreneurial venture in the domain of industrial biotechnology.
Innovative use of Biotechnology and effective use of marketing are two pillars of our industry.
Our industrial products include oft used enzymes (Phytase, Xylanase
, Amylase and Cellulase)
and Power Alcohol. Using novel expression systems (Multiplex Automated Genome
Engineering) and advanced Solid State Fermentation Technique we are able to produce effective
enzymes. Eureka Powers is able to create efficient industr
ial products in the form of enzymes.
Our company specializes in optimizing enzymes for replacement of harsh chemicals and
processes in commercial applications. Our second domain of production is Power
Fuel/Bioethanol. Fossil Fuel is a dying resource, and p
rices are sky high. Usage of Power
Alcohol not only provides environmentally friendly fuel but also a cheaper alternative. We have
means in place to achieve profitable and sustainable growth in both the short and long term. Our
business plan is backed by a

strong dependence on optimization of our production process,
which would be able to churn out hefty returns each year. The result is higher quality, lower
costs, lower CO2 emissions, and a better environment. Our primary domains of business are two
pronge
d: Enzyme Business and Bio Fuel Business.

Industrial Biotechnology primarily involves the usage of biotechnology for industrial purposes.
This would also include the production of alternate energy and biomaterials as well. We aim to
use cells and their com
ponents (enzymes) to produce industrially benefitting products.
The
premise behind power alcohol is the reduction of dependence on fossil fuels.
Industrial
Biotechnology has been labeled as the “White Biotechnology”, and one of the three waves of
biotechnology.

One of the major benefits of Industrial Biotechnology is the production of
components that are environmentally friendly and do not cause h
arm.
On one side, they are cost
effective and on the other they are quite efficient as well.
The premise behind our entrepreneurial
venture is that, we would be able to offer cost effective items that do not harm the nature, and are
powerful in their mode
of action.

















Production of Enzymes:

Before going into detail about the production of enzymes commercially, we would like to
elaborate the fermentation technique that we would be employing. Our company intends to excel
in the fact that it intends to use the current developments in biotech for

the consumer.
Solid state
fermentation (SSF) holds tremendous potential for the production of enzymes. It can be of
special interest in those processes where the crude fermented products may be used directl
y as
enzyme sources. This following section

focus
es on the production of various industrial enzymes
by SSF processes.

Solid State Fermentor:

Solid
-
state fermentation is the cultivation of microorganisms, and hence enzymes on a

solid
substrate. Carbon containing compounds in or on the substrate are broken

down

by the micro
organisms, which produce the enzymes either intracellularly or

extracellularly. The enzymes are
recovered by methods such as centrifugation, for

extracellularly produced enzymes and lysing of
cells for intracellular enzymes.

SSF processe
s generally employ a natural raw material as carbon and energy source. SSF can
also employ an inert material as solid matrix, which requires supplementing a nutrient solution
containing necessary nutrients as well as a carbon source. Solid substrate (matri
x), however,
must contain enough moisture. Depending upon the nature of the substrate, the amount of water
absorbed could be one or several times more than its dry weight, which leads relatively high
water activity (a w) on the solid/gas interface in order

to allow higher rate of biochemical
process. Low diffusion of nutrients and metabolites takes place in lower water activity conditions
whereas compaction of substrate occurs at higher water activity. Hence, maintenance of adequate
moisture level in the so
lid matrix along with suitable water activity is essential elements for SSF
processes. Solid substrates should have generally large surface area per unit volume (say in the
range of 10 3
-
10 6 m 2/cm 3 for the ready growth on the solid/gas interface). Small
er substrate
particles provide larger surface area for microbial attack but pose difficulty in
aeration/respiration due to limitation in inter
-
particle space availability. Larger particles provide
better aeration/respiration opportunities but provide lesse
r surface area. In bioprocess
optimization, sometimes it may be necessary to use a compromised size of particles (usually a
mixed range) for the reason of cost effectiveness. For example, wheat bran, which is the most
commonly used substrate in SSF, is obt
ained in two forms, fine and coarse. Former contains
particles of smaller size (mostly smaller than 500
-
600 µ) and the latter mostly larger than these.
Most of SSF processes use a mix of these two forms at different

ratios for optimal production.

Solid sub
strates generally provide a good dwelling environment to the microbial flora
comprising bacteria, yeast and fungi. Among these, filamentous fungi are the best studied for
SSF due to their hyphal growth, which have the capability to not only grow on the sur
face of the
substrate particles but also penetrate through them. Several agro crops such as cassava, barley,
etc. and agro
-
industrial residues such as wheat bran, rice bran, sugarcane bagasse, cassava
bagasse, various oil cakes (e.g. coconut oil cake, palm

kernel cake, soybean cake, ground nut oil
cake, etc), fruit pulps (e.g. apple pomace), corn cobs, saw dust, seeds (e.g. tamarind, jack fruit),
coffee husk and coffee pulp, tea waste, spent brewing grains, etc are the most often and
commonly used substrate
s for SSF processes. During the growth on such substrates hydrolytic
exo
-
enzymes are synthesised by the micro
-
organisms and excreted outside the cells, which create
and help in accessing simple products (carbon source and nutrients) by the cells. This in t
urn
promotes biosynthesis and microbial activities.

The following image would elaborate it further:


The figure above is quite self explanatory and would be able to shed light on the working of a
standard solid state fermentor. We intend to show how the e
ntire process takes place.
As
you can
see from the figure,
the solid state fermentation can be carried out inside the horizontal drum
bioreactor with any amount of dry substrate.

The treated and inoculated substrate is placed inside
the fermentor. This par
ticular model shown above is a horizontal drum bioreactor that consists of
a shovel coupled to a motor axis and is then rotated at controlled speed.
The material then is
revolved as per our requirement. Following the warranted time for fermentation, the sa
turated air
is inserted continuously into the drum in order to control the substrate temperature and moisture.
The fermentation can be carried out at room temperature and the moisture is controlled as per our
optimization requirement.

Advantages of Solid S
tate Fermentation over Sub
-
Merged Fermentation:

Our company would employ the
use of Solid state fermentation, which is more effective than
submerged owing to the following factors:

?
Higher volumetric productivity

?
Usually simpler with lower energy
requirements

?
Might be easier to meet aeration requirements

?
Resembles the natural habitat of some fungi and bacteria

?
Easier downstream processing

The following table would be able to enhance the process of our solid state fermentor in a better
way:

Ty
pical process characteristics

Downstream processing


None or centrifugation for many applications

Duration




1 to 5 days

Aeration





0.5 to 5 vessel volumes per hour

Agitation





Intermittent or constant rotation

Moisture content



50 to 85%

Temperature



5 to 95 C

Inoculation




1 to 20% (w/w) of fungi or bacteria suspension

Liquid Medium



Basic Mineral Medium

Solid Matrix



Rice bran, sawdust, sugar beet pulp, etc.


Production of Enzymes:

1.

EP
-
Xyl (Xylanase)

EP
-
Xyl

is our product, which is the industrially important Xylanase.

The enzyme named xylanase used to down regulate plant structural material by breaking down
hemicellulose, a major component of the plant cell wall. Plant cell walls are necessary to prevent
deh
ydration and maintain physical integrity. They also act as a physical barrier to attack by plant
pathogens. In nature, some plant consumers or pathogens use xylanase to digest or attack plants.
Many microorganisms produce xylanase, but mammals do not. Some

herbivorous insects and
crustaceans also produce xylanase.

The Xylanase enzyme (Endo
-
1,4
-
Beta
-
xylanase) is produced through bacterial and fungal
cultures. Xylanase consists of 190 amino acids and has a molecular weight of 21 kD. Xylanases
belong to the
glucanase enzyme family and are characterized by their ability to break down
various xylans to produce short
-
chain xylo
-
oligosaccharides. Xylanase readily crystallizes in
ammonium sulfate and sodium/potassium phosphate across pH 3.5 to 9.0. Xylanase can al
so be
crystallized with other salts, polymers, and organic solvents. Xylanase solubility increases with
increasing temperature in moderate concentrations of ammonium sulfate. Xylanase solubility in
phosphate pH 9 decreases in the temperature range of 0 to
10 degrees Celsius but remains
constant in the range of 10 through 37 degrees Celsius. Xylanase has been extracted from many
different fungi and bacteria. It is commonly used in animal feeds, paper production, and food
production.

Specifications of xylanas
e:


Systematic Name
:
1,4
-
β
-
D
-
xylanohydrolase


Recommended Name
:
endo
-
1,4
-
β
-
xylanase


Type
:
Hydrolase







Substrate
:
Xylan


Product
:
Xylose


Sources
:
Bacterial, Funga
l



pH Range
:
Acidic (3.5


7.0)


Optimum
pH: 5.3



Temperature Range
:
35


60 °C



Optimum Temperature
:
45 °C

Applications of Xylanase:

a)
Kraft Pulp Bleaching:

Xylanase is used in paper industry to improve
the strength of cellulose fibers in bleached Kraft
Pulp (from bamboo, eucalyptus etc.).

It improves pulp fibrillation and water retention, reduces beating times in virgin pulps, restores
bonding, increases freeness in recycled fibers and selectively remov
es xylan from dissolving
pulps.

b) De
-
inking of Newsprint:

Cellulase blended with xylanase in specific ratios is used for deinking newsprint. At optimum
ratio (50
-
50), brightness of deinked pulp is higher, ink removal ratio is lower and breaking
length,
bursting index and tearing index are higher than for chemically deinked pulp. This
process also gives a higher yield than chemical deinking.


c) In Poultry Industry:

Xylanase is added to poultry feed material such as rice/wheat bran along with various other
enzymes to improve absorption of nutrients by the birds and to reduce the quantity and nutrient
concentration of the birds’ droppings.

d) In Wine Production:

It i
s used to break down xylans, pectin and hemicellulose present in fruits into simpler molecules
such as xylose and glucose. By breaking down cell walls, it helps extract more juice from the
fruit.

e) In Baking:

Xylanase breaks down glycosidic linkages in
arabinoxylans (in endo or exo fashion), producing
smaller fragments. This improves the handling properties of dough, the over
-
spring and the bread
volume.

f) In Forage Digestions:

Xylanase along with other fibrolytic enzymes is sprayed onto forages in sp
ecific total mixed
ratios and fed to lactating dairy cows to enhance FCM and milk production.

g) Agricultural Waste Degradation

h) Yielding Cellulose from Rayon

Industrial Scale Production of Xylanase:

Xylanase enzyme can be produced by different method
s and by using different strains. It can be
produced by using fungal strain or by using bacterial strain. From a commercial viewpoint,
xylanases are an important group of carbohydrolases and have a worldwide market of around
$200 million each year. Xylanas
es have been widely applied in food, animal feed, bioconversion,
textile, and in paper and pulp industries. However, high cost and low yields of xylanase have
been the main problems for its industrial production. Therefore, there is a great need to develop

a
new fermentation medium with inexpensive substrates that provides a high xylanase yield.

Among existing technologies in the fermentation industry, solid
-
state fermentation

(SSF) shows
many advantages over fermentation with submerged culture.

Usage of
Fungal Strain and Solid State Fermentation:

We are going to use fungal strain (
Thermomyces lanuginosus

) with the wheat bran inoculation
(using solid state fermentation) for the following reasons:

1.

Thermomyces lanuginosus 195 is easy and fast to grow on
potato dextrose
-
agar.

2.

The wheat bran is easily available in Pakistan
.

3.

Wheat bran is cheap to use for the production of xylanase.

4.

It is easy to optimize the conditions using
Thermomyces lanuginosus
195 and wheat bran.

5.

By optimizing the condition
s
Thermomyces lanuginosus
195 can produce 21.7% more
enzyme.

Methods and materials:

The stain of
Thermomyces lanuginosus
195 is grown on potato dextrane
-
agar (PDA) at 45°C for
40hrs and stored at 4°C. The spores are then collected from PDA slants with 0.1%

v/v Tween
-
20
washed, and diluted in sterile water. Liquid second seed medium was inoculated with1000000
spore/ml and incubated for 30 hrs at 40°C, 200rpm. Liquid seed growth medium is consisted of
(g/l of deionised water): corn starch, 60; peptone: 18; gl
ucose, 5; megnisium sulphate: 1.5;
potassium phosphate, 1; potassium chloride, 0.5 and is sterilized at 105°C for 30min. Before
optimization and for the purpose of an initial determination of xylanase activity, 10g of sterilized
wheat bran in 250
-
ml Erlenm
eyer flask was inoculated with 8 ml of liquid seed culture, diluted
1:4 with sterile water. Flasks prepared in triplicate, are then fermented for 40hrs, at 40°C at 80%
relative humidity in the chamber.

Detection of xylanase activity:

Fermented media were t
reated with 90ml of deionized water for 90min. The crude enzyme is
filtered through muslin cloths and centrifuge at 10,000x g for 10min at 4°C. Enzyme preparation
is examined for total xylanase activity. Sample absorbance was calculated at 540nm.

Optimizat
ion of growth parameter in solid state fermentation (SSF):

The influence of the fermentation temperature on xylanase production was investigated by
incubating the inoculated wheat bran at the temperature ranging from 25 to 45°C increments.
The effect of fe
rmentation duration at each individual temperature is also assessed by analyzing
SSF cultures for xylanase production at various time points. These optimized parameters can also
be employed to investigate the effect of inoculums volume on xylanase producti
on. This can be
assessed by combining 10g of sterilized wheat bran with varying volumes of inoculums, ranging
from 4ml to 10ml.



References for Xylanase Production:

1.
Timell, T.E. (1967) Recent

progress in the chemistry of wood hemicelluloses. Wood Sci.
Technol. 1, 45
-
70.

2.
Goheen, D.W. (1981) Chemicals from wood and other biomass. Part I : Future

supply of organic chemicals. J. Chem. Educ. 58, 465
-
468.

3.
Dekker, R.F.
H. and Richards, G.N. (1976) Hemicellulases: Their occurrence,

purification,
properties and mode of action. Adv. Carbohydr. Chem. Biochem. 32, 277
-
352.

4.
Prade, R.A. (1995) Xylanases: from biology to biotechnology. Biotechnol. Genet. Eng. Rev.
13, 101
-
13
1.

5.
Gilbert, H.J. and Hazlewood, G.P. (1993) Bacterial cellulases and xylanases. J.

Gen.
Microbiol. 139, 187
-
194.

6.
Biely, P. (1993) Biochemical aspects of the production of microbial hemicellulases.

In:
Hemicellulose and Hemicellulases (Coughlan, M.P.
and Hazlewood, G.P.Eds.), pp. 29
-
51.
Portland Press, Cambridge.

7.
Wong, K.K.Y., Tan, L.U.L. and Saddler, J.N. (1988) Multiplicity of L
-
1,4
-
xylanase in
microorganism: Functions and applications. Microbiol. Rev. 52, 305
-
317.

8. Biely, P. (1985) Microbial

xylanolytic systems. Trends Biotechnol. 3, 288
-
290.

9.
Warren, R.A.J. (1996) Microbial hydrolysis of polysaccharides. Annu. Rev.

Microbiol.50,
183
-
212.

10. Sunna, A. and Antranikian, G. (1997) Xylanolytic enzymes from fungi and
bacteria.Crit. Rev. Biotechnol. 17, 39
-
67.



2. EP
-
Amy (Amylase)

Amylase is a digestive enzyme classified as a saccharidase (an enzyme that cleaves
polysaccharides). It is mainly a constituent of pancreatic juice and saliva, needed for the
breakdown of lon
g
-
chain carbohydrates (such as starch) into smaller units. It is a digestive
enzyme made primarily by the pancreas and salivary glands. The primary function of the
amylase enzyme is to break down starches in food so that they can be used by the body. Amyla
se
is also synthesized in the fruit of plants during ripening, causing them to become sweeter.

Amylases are among the most important enzymes and are of great significance for
biotechnology; constituting a class of industrial enzymes having approximately 25
% of the world
enzyme market .They can be obtained from several sources, such as plants, animals and
microorganisms. Today a large number of microbial amylases are available commercially and
they have almost completely replaced chemical hydrolysis of starc
h in starch processing
industry. The amylases of microorganisms have a broad spectrum of industrial applications as
they are more stable than when prepared with plant and animal
-

amylases. The major advantage
of using microorganisms for the production of
amylases is the economical bulk production
capacity and the fact that microbes are easy to manipulate to obtain enzymes of desired
characteristics.
-
Amylase has been derived from several fungi, yeasts and bacteria. However,
enzymes from fungal and bacteria
l sources have dominated applications in industrial sector.


Types:

a.

α
-
Amylase

The α
-
amylases are calcium metalloenzymes
, completely unable to function in the absence of
calcium., α
-
amylase breaks down long
-
chain carbohydrates by acting at random locations along
the starch chain, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose
and "limit dextri
n" from amylopectin. α
-
amylase tends to be faster
-
acting than β
-
amylase
because it can act anywhere on the substrate. In human physiology, both the salivary and
pancreatic amylases are α
-
Amylases. Also found in plants (adequately), fungi (ascomycetes and
b
asidiomycetes) and bacteria (Bacillus)

b. β
-
Amylase

β
-
amylase is another form ofamylase synthesized by bacteria, fungi, and Plants. β
-
amylase
catalyzes the hydrolysis of the second α
-
1,4 glycosidic bond, working from the non
-
reducing
end, cleaving off two
glucose units (maltose) at a time. During the ripening of fruit, β
-
amylase
breaks starch into maltose, resulting in the sweet flavor of ripe fruit. Both α
-
amylase and β
-
amylase are present in seeds; β
-
amylase is present in an inactive form prior to germina
tion,
whereas α
-
amylase and proteases appear once germination has begun. Animal tissues do not
contain β
-
amylase.

c. γ
-
Amylase

γ
-
amylase cleaves α 1, 6
-
glycosidic linkages, in addition to cleaving the last α(1
-
4)glycosidic
linkages at the nonreducing end o
f amylose and amylopectin, yielding glucose. Unlike the other
forms of amylase, γ
-

amylase is most efficient in acidic environmentsand has an optimum pH 3.

Industrial Importance:

The industrial enzyme producers sell enzymes

for a wide variety of applicatio
ns. The estimated

value of world market is presently about US$ 2.7

billion and is estimated to increase by 4%

annually through 2012. Detergents (37%),

textiles (12%), starch (11%), baking (8%) and

animal
feed (6%) are the main industries, which

use about 7
5% of industrially produced

enzymes.
Amylases constitute a class of

industrial enzymes having approximately 25% of

the enzyme
market.
An extra
-
cellular amylase,

specifically raw starch digesting amylase has

found important
application in bioconversion of

s
tarc
hes and starch
-
based substrates.

Uses of amylase:

Amylases have potential application in a number

of industrial processes such as in the food,

textiles, paper industries, bread making
,

glucose and fructose syrups, detergents, fuel

ethanol

from starches, fruit juices
, alcoholic

beverages
, sweeteners (Peppler and Periman,

1978),
digestive aid and spot remover in dry

cleaning
. Bacterial α
-
amylases are now also

used in areas
of clinical, medicinal, and

analytical chemistry
. The most widely use
d

thermostable enzymes are
the amylases in the

starch industry
. Some of the applications are

discussed in detail as follows:

1.

Bread Making

Modern bread making techniques have

included amylase enzymes (often in the

form of malted
barley) into bread improver

thereby making the bread making process

faster and more practical
for commercial

use.

2.

Sugar Industry


In sugar processing industry, the first step

includes gelatinization of the starch slurry

which is
achieved by heating starch with

water at temperature ar
ound 100
C, due to

insolubility of starch
at lower temperatures.

This step involves dissolution of starch

granules, thereby forming a
viscous

suspension. Because of this high viscosity it

poses serious problem with mixing and

pumping. To overcome this visco
sity

associated problems, geltinization is

coupled with
liquefaction which involves

partial hydrolysis and loss in viscosity. This

action is brought about
by thermostable

alpha amylase, which can act at

temperatures around 70
-
100oC depending

upon
the
temperature profile of alpha

amylase
.

3.

Diet Aid


An inhibitor of alpha
-
amylase called

phaseolamin has been tested as a potential

diet aid.

4.

Medicinal Applications


A higher than normal concentration of

amylases may reflect one of several

medical conditions,
including acute

inflammation of the pancreas (concurrently

with the more specific lipase), but
also

perforated peptic ulcer, torsion of an

ovarian cyst, strangulation ileus,

macroamylasemia and
mumps. Amylase

may be measured in other body fluids,

including

urine and peritoneal fluid.

5.

Molecular Applications


In molecular biology, the presence of

amylase can serve as an additional method

of selecting for
successful integration of a

reporter construct in addition to antibiotic

resistance. As reporter
genes are

flanked

by homologous regions of the structural

gene for amylase, successful
integration

will disrupt the amylase gene and prevent

starch degradation, which is easily
detectible

through iodine staining.

Production:

Effectiveness of Solid
State
Fermentation and Usage of Fungal Strain:

It

has been reported that SSF is the most appropriate

process in developing countries due to the
advantages it

offers. The hyphen mode of

growth and good tolerance to low water activity (aw)
and

high osmotic pressur
e conditions make fungi most

efficient for bioconversion of solid
substrates. The objective of this study were selection of a suitable

strain for the production of
glucoamylase, screening

of different agricultural byproducts as substrates for maximum

enzym
e
production, application of different combinations

of these substrates for enzyme production, and

optimization of cultural conditions for the production of

glucoamylase.

Research has shown that the
Thermomyces lanuginosus

can be used for the production of

amylase as well. The good thing about the strain is that our cost of production would be lowered
since we are already using this strain for the production of xylanase.


Substrates:

The substrates that can be used in this process include wheat bran, molass
es bran, rice bran,
maize meal, millet cereal, wheat flakes, barley bran, crushed maize, corn cobs and crushed
wheat. All of them are easily available from the local market.

Optimum Conditions:

In optimum conditions the substrate used is wheat bran with su
pplements of soluble starch and
peptones. The operable pH is 6.0, and a moisture content of 90 percent is good enough. The
incubation temperature is 50 C and the incubation period is 120 hours.

Yield

Using this method
, an enzyme titer of 534 U/g can be ob
tained. This can be only obtained if the
moisture level is kept at 90 %. Higher inoculums levels are inhibitory in nature. There is a great
deal of influence from supplements, so we would prefer using them for greater yield. Further
yield can be enhanced i
f the salt solution concentration is increased.

Increased Yield through mtutations:

As would be cited later on, the company is also focused on getting the best possible yield of the
enzyme. For this we would go for mutations and recombination for effective yield.
Mutagenesis
and genetic recombination

techniques using protoplast fusion and

transformation have been used
widely by

several companies

as a tool of protein

engineering to achieve strains with higher

enzyme productivity or desired characters
. In our case Protoplast fusion can

b
e achieved using
filamentus fungi, yeast,

streptomycete
s and bacteria. Amylase hyperproducing,

catabolite
-
repression
-
resistant,

recombinant strains are produced by

intraspecific protoplast fusion of
thermophilic

fungus Thermomyces lanuginosus strains, using

well
-
characterized, morphological,
and 2
-
deoxy
-
D
-
gluc
ose resistant markers
.

References

1. Sidhu GS, Sharma P, Chakrabarti T and

Gupta JK, Strain improvement for the

production of a
thermostable alpha amylase

by
Bacillus
species. Enz. Microbial Technol,

21: 525
-
530, (1997).

2. Rao MB, Tanksale AM, Gathe MS,

Deshpande VV, Molecular and

Biotechnological aspects
of microbial

proteases. Microbial. Microbial. Rev, 62:

597
-
635, (1998).

3. M.W. Fogarty. Microbial Amylases. In: W.M.

Fogarty (eds.),
Microbial Enzymes and

Biotechnology
, Applied Science Publishers

Ltd,
London, UK, 1983, pp. 1

92.

4. Song SY. et al., Med. J, 44: 74
-
75, (2004).

5. Shigemura M. et al., Clin Chem Lab Med,

42(6): 677
-
80, (2004).

6. Abou
-
Sheif Ma. et al., Clin Chem Acta
.,

346(2):161
-
70, (2004).

7. Lin LL, Biotechnol. Appl. Biochem, 28:61
-

68,
1998

8. Lin LL, Hsu WH, Chu WS, A gene encoding

for α
-
amylase from thermophilic
Bacillus

sp.,
strain TS
-
23 and its expression in

Escherichia coli.,
J. Appl. Microbiol, 82:

325
-
334, (1997).

9. Pandey A, Soccol CR and Mitchell D, New

developments in solid st
ate fermentation.

Process
Biochem, 35: 1153
-
1169, (2000a)


3. ep
-

Celu (
Cellulase
)

Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that
catalyze cellulolysis (i.e. the hydrolysis of cellulose). However, there are
also cellulases
produced by a few other types of organisms, such some termites and the microbial intestinal
symbionts of other termites. Several different kinds of cellulases are known, which differ
structurally and mechanistically. Cellulase is used for c
ommercial food processing in coffee. It
performs hydrolysis of ce
llulose during drying of beans.

Furthermore, cellulases are widely used in textile industry and in laundry detergents. They have
also been used in the pulp and paper industry for various
purposes, and they are even used for
pharmaceutical applications. Cellulase is used in the fermentation of biomass into biofuels,
although this process is relatively experimental at present. Cellulase is used as a treatment for
phytobezoars, a form of cell
ulose bezoar found in the human stomach.

Production of Cellulase

The organism that we would be using for the production of cellulase would be
Trichoderma
reesei

RUT C30
.

Substrate

Sugar cane bagasse is a low
-
cost
and abundant biomass material
containing ab
out

27

54%
cellulose
, which can serve as a potent substrate for cellulase production.

Production

The following protocol would explain how we are going to produce our product. A fixed amount
of the substrate is

weighed into 250
-
ml Erlenmeyer flasks and was

moistened with mineral salt
medium to attain 70% initial moisture content. The basal

mineral salts solution used for the
experiment had the following composition (g/l):

KH2PO4, 5; NH4NO3, 5; MgSO4.7H2O, 21; urea, 2; CaCl2, 1; Peptone, 5; NaCl, 5; Tween
-

80
,
0.5; and trace elements: FeSO4.7H2O, 0.005; MnSO4.7H2O, 0.001; ZnSO4.7H2O,

0.001; and
CoCl2, 0.0002. The ini
tial pH of the salt solution is

adjusted to 5. The flasks

are

sterilized by
autoclaving at 121 °C for 15 min at 15 lbs pressure, and after cooling
,

filter sterilized crude
inducer prepa
ration was added. The medium is

inoculated with 1 ml

of the seed inoculum (150
mg/ml t
otal protein). The contents are

mixed thoroughly and

are

incubated under controlled
conditions of temperature and humidity.
Incubat
ion is
continued for the duration indicated in the
experimental designs, and at the end of

incubation
period;

the enzyme is

recovered by extraction
with 0.05 N citrate
buffers

(pH 4.8). The extract is

centrifuged to remove debris at 6,000 rpm for
10 min at 4 °C, and

the supernatant i
s used as the crude enzyme sample

continued for the
duration indicated in the experimental
designs

and at the end of

incubation period, the enzyme
was recovered by extracti
on with 0.05 N citrate buffer

(pH 4.8). The extract was centrifuged to
remove debris at 6,000 rpm for 10 min at 4 °C, and

the supernatant was used as the crude
enzyme sample.

The optimal conditions include an optimal temperature of 32
-
33
o
C and an
incubation time of 66
hours. The moisture level is to be maintained at 80 percent.

References:

1. Lynd, L. R., Wyman, C. E., & Gerngross, T. U. (1999). Biocommodity engineering.
Biotechnology

Progress, 15, 777

793.

2. Reith, J. H., den Uil, H., van Veen, H
., de Laat WTAM, Niessen, J. J., de Jong, E., et al.
(2002). Coproduction

of bioethanol, electricity and heat from biomass residues. 12th European
Conference and

Technology Exhibition on Biomass from Energy, Industry and Climate
Protection, Amsterdam, The

Netherlands, 17

21 June.

3. Wen, Z., Liao, W., & Chen, S. (2005). Production of cellulase/b
-
glucosidase by the mixed
fungi culture

Trichoderma reesei and Aspergillus phoenicis on dairy manure. Process
Biochemistry, 40, 3087

3094.

4. Chahal, D. S. (1985). S
olid
-
state fermentation with Trichoderma reesei for cellulase
production. Applied

Environmental Microbiology, 56, 554

557.

5. Pandey, A., Soccol, C. R., Nigam, P., & Soccol, V. T. (2000). Biotechnological potential of
agroindustrial

residues I: sugarcane b
agasse. Bioresource Technology, 74, 69

80.

6. Du Toit, P. J., Olivier, S. P., & van Bijon, P. L. (1984). Sugarcane bagasse as a possible source
of

fermentable carbohydrates. I. Characterization of bagasse with regard to monosaccharide,
hemicellulose,

and a
nimoacid composition. Biotechnology and Bioengineering, 26, 1071

1078.

7. Aiello, C., Ferrer, A., & Ledesma, A. (1996). Effect of alkaline treatments at various
temperatures on

cellulase and biomass production using submerged sugarcane bagasse
fermentation

with Trichoderma

reesei QM9414. Bioresource Technology, 57, 13

18.

8. Gutierrez
-
Correa, M., & Tengerdy, R. P. (1997). Production of cellulase on sugar cane
bagasse by fungal

mixed culture solid substrate fermentation. Biotechnology Letters, 19(7), 665

667
.

9. De
-
Paula, E. H., Ramos, L. P., & Azevedo, M. O. (1999). The potential of Humicola grisea
var

thermoida for bioconversion of sugar cane bagasse. Bioresource Technology, 68, 35

41.















4.

EP Phy
-
Phytase

Background:

Phosphorus is an essential
element for the growth and development of all animals,
playing key roles in skeletal structure and in vital metabolic pathways. All animal diets must
contain adequate amounts of this element.

The principle storage form of phosphorus in feedstuffs of plant
origin is the phytic acid
(inositol hexa phosphate). Phytates are enclosed in all kinds of nuts, cereals, beans, seeds, spores
and pollens in an amount of 1
-
3% and up to 80% of the phosphorus in grains and seeds.

Phytase is the only recognized enzyme that

can used as an animal feed supplement to
enhance the nutritive value of plant material by liberation of inorganic phosphate from phytic
acid (myo
-
inositol hexakisphosphate) and, thereby, to reduce environmental phosphorus
pollution. Phytase is a natural e
nzyme used to improve the nutritional quality of phytic acid rich
feed components. The supplementation with Phytase decreases the need for calcium phosphate
addition and has a positive effect on environment (less manure to be spread in fields). For feed
ap
plication a thermostable Phytase is of general interest to circumvent problems that may occur
during the formulation and feed pelleting process where temporarily high temperatures (80
-
100
°C) and shear stress may affect protein structure and lead to activi
ty loss.

In disparity, only 0
-
40% of phytate phosphorous is available to mono gastric animals such as
pigs and poultry as lack Phytase
-
producing microorganisms in their gut. Hence

1.


Due to this inability of mono gastric animals to utilize phytate, i
t is therefore necessary to
supplement the diets with expensive sources of available inorganic phosphate.

2.


Phytate phosphorus passes through the intestinal tract and ends up in the feces causing
environmental problems in areas of intensive livestock

production.

3.


In addition, phytate is considered to be anti
-
nutritional factors. Phytate forms complexes
with essential minerals such as calcium, zinc, magnesium, iron, and may also react with
proteins, thereby decreasing the bio
-
availability of pro
tein and nutritionally important factors.



Phytase is important to a greater extent for its accessibility to the animals and to protect the
environment. The addition of microbial Phytase to the feedstuff of mono gastric animals was
described as long as 25

years ago. Addition of this Phytase to feed significantly improved as:

1.


Increase in phosphorous availability by 60%.

2.


Reducing or even eliminating the need for supplemental dietary inorganic phosphorous.

3.


Decrease in the excretion of

phosphorous in the feces by over 30%.

4.


Moreover, such treatments can also introduce that works against the anti
-
nutritional
properties coupled with unhydrolyzed phytate.

Phytin:


Most of the stored phosphorus in plants is institute in seeds mainly as a component of a
molecule called phytin. Phytin phosphorus within a given feedstuff is variable, but typically
averages 72 and 60 percent of total seed phosphorus in corn and soybean m
eal respectively, the
two prime feed ingredients in poultry and swine diet. Phytic acid is highly reactive and readily
forms complexes with Ca, Fe, Mg, Cu, Zn, carbohydrates, and proteins. These complexes are
substantially less soluble in the small intesti
ne and, therefore, less likely to interact with Phytase.


Phytin is often considered to be an anti
-
nutrient because of its ability to bind with other nutrients
rendering those nutrients as well as the phosphorus contained in the phytin

molecule partially or
completely unavailable to the animal. Phytin in feedstuffs is relatively heat stable. The location
of phytin within seeds differs among different plants. Ninety percent of the phytin in corn is
found in the germ portion of the kernel
, while in wheat and rice most of the phytin is in the
aleurone layers of the kernel.

Phytase:


Phytin
-
phosphorus is poorly available to poultry and swine, and this availability varies
both within and among ingredients. The enzyme Phytase releases phosphat
e groups from phytin
potentially making this released phosphorus available to the animal. Phytase is the only
recognized enzyme that can commence the release of phosphate from phytin.

The International Union of Biochemistry (1979) recognizes two general cl
asses of Phytases,
3
-

Phytase and 6 Phytase based on the location of the phosphate group, within the phytin
molecule which can be hydrolyzed first. Microbial or fungal Phytase typically hydrolyze the
phosphate at the
3rd

position and plant Phytase the phos
phate at the six position of the phytin
molecule. After releasing the first phosphate group, the five remaining phosphate groups can be
successively released from phytin by Phytase and some non
-

specific, acid phosphatases.

Enzyme Activity:


One unit of P
hytase is defined as the amount of enzyme required to liberate one µmol of
orthophosphate from phytin per minute at pH 5.5 and 37° C. As enzyme characteristics differ
among Phytases, therefore, a unit of activity in the above conditions does not necessaril
y
transform into the same amount of phosphorus released within the animal. The last point is a key,
because it is not the commercially misused term “effectiveness” that is important when
considering commercial Phytases, but the amount of phosphorus liberat
ed by the Phytase at the
manufacturers recommended inclusion level for the specific dietary ingredients and nutrient
levels being used. This ultimately would translate to a cost of the Phytase per unit of phosphorus
made available.


Even though an
extensive assortment of microorganisms can produce Phytase, the expression
levels of Phytase in them are too low for economic considerations. Due to the high cost of
Phytase production, application of Phytase has not been prevalent. For economic considerat
ions,
inorganic phosphorous supplement of mono gastric animal feedstuffs is still the method of
choice in many cases. For commercially viable procedure for the production of a large quantity
of Phytase would be of tremendous value both from the feed
-
conver
sion and environment point
of view.

Towards the eventual objective of improving the production of Phytase and reducing its
production cost, here we have developed recombinant yeast, which can greatly improve Phytase
production levels.



Advantages in Phyta
se Production by Recombinant Yeast:

Yeast
Pichia pastoris

have been effectively engaged as host for the industrial production of
extra cellular Phytase. The advantages of the technique include:

1.


The expression level of Phytase in recombinant
Pichia
pastoris

is very high, and the amount
of expressed Phytase reaches to 5gram/liter medium (equal to 15000IU/mL) in industrial
fermentation.

2.


Large scale, high
-
density fermentation technology for production of Phytase has well
customary, and fermentat
ion method is easy and trouble
-
free.

3.


Large scale downstream processing for Phytase production has soundly established, and
processing methods is very simple and uncomplicated.

4.


The fermentation medium, a defined mixture of glucose, salts and

trace elements that is easy
to get, is economical and easy on the pocket and free of toxins.

5.


The microorganism selected for the purpose of producing Phytase is
Pichia pastoris

due to
its approval as safe for human use through experience with the microorganism.

6.


The overall production cost of Phytase products is very low. The production cost of 1 liter of
liquid Phytase products (5000IU/mL) or 1 kg of solid Phytase produc
ts (5000IU/g) is about 1
US dollar, which can be added in 10,000kg of feedstuff.

7.


The equipment of producing Phytase by recombinant yeast is simple. The equipment in
general fermentation factory can meet the needs for Phytase production.


Production of Phytase:

Strain → Inoculum Seed Flask Preparation → Seed Fermentor Preparation →
Fermentation of Producing Phytase → Phytase Secreted Into the Growth Medium → Harvesting
Supernatant Without Cells → Liquid Phytase Products → Mixing With Stuffi
ng → Drying →
Solid Phytase Products.


Construction of Recombinant
Pichia pastoris

producing Phytase:

High
-
level expression of Phytase with high specific activity is an effectual way to
improve Phytase fermentation strength and reduce its production cost.
The gene appA encoding
Escherchia coli

Phytase AppA with high specific activity was modified and artificially
synthesized according to the bias in codon choice of the high expression gene in
Pichia pastoris

without changing the amino acid sequence of the A
ppA. The modified gene, appA
-
m, was
inserted in the
Pichia pastoris

is expression vector pPIC9, and then introduced into the Pichia
Pastoris by electroporation. The
Pichia pastoris

recombinants for Phytase over expression were
screened by enzyme activity a
nalysis and SDS
-
PAGE. The result of southern blotting analysis of
the recombinant yeast indicated that only one copy of the appA
-
m gene was integrated into the
genome of
Pichia pastoris
. The result of Northern analysis of the recombinant yeast showed that
the modified gene was effectively transcribed. SDS
-
PAGE analysis of the Phytase expressed in
Pichia pastoris

exposed that the Phytase was over expressed and secreted into the medium
supernatant. There are three Phytase with apparent molecular in approximat
ely 50kD, 52kD and
54kD respectively in the media, which are larger in the size than the native Phytase from E.coli.
The results of N
-
terminal sequencing and deglycosylation of the expressed Phytase in
Pichia
pastoris
proved that the articulated Phytase we
re glycosylated protein with different
glycosylation degree. The expressed Phytase
Pichia pastoris

shared similar pH and temperature
optima to those of the natural from E.coli and had highly resistant to pepsin digestion. In 30000
-
L Fermentor, after induce
d by 0.5% methanol, the expression level of Phytase protein was
5mg/mL, and the Phytase activity (fermentation potency) exceeded 15000IU/mL, which was the
highest among those of all kinds of recombinant strains reported now.


References:

1.

Yin, Q.Q., Q.H. Zh
eng and X.T. Kang, 2007.

Biochemical characteristics of phytase from
fungi

and transformed microorganism. Ani. Feed Sci.

Technol., 341
-
350.

2.


Youssef, K.A., M. Ghareib and M.M. Nour
-
el
-

Dein, 1987. Purification and general
properties of

extracellular
phytase from Aspergillus flavipes.

Zentralbl. Mikrobiol.,
142(5): 397
-
402.

3.

Zimmermann, B., H.J. Lantzsch, R. Mosenthin, F.J. Schoener, H.K. Biesalski and W.
Drochner,

2002. Comparative evaluation of the efficacy of

cereal and microbial phytases
in growing
pigs fed

diets with marginal phosphorus supply, J. Sci.

Agric., 82: 1298
-
1304.

4.

. Zyta, K. and D. Gogol, 2002. In vitro efficacies

of phosphorolytic enzymes synthesized
in
mycelia
cells of Aspergillus niger AbZ4 grown by a liquid

surface fermentation. J. of

Agric. and Food Chem.

50: 899
-
905.




Production of Power Alcohol:

When ethyl alcohol is used as fuel in internal combustion engine, it is called as "power alcohol".
Generally ethyl alcohol is used as

its 5
-
25% mixture with petrol.
Alcohol produced by
fermenting and then distilling sugars from sugar
-
rich plants (e.g. sugarcane, maize, sugar beet,
cassava, wheat and sorghum). The alcohol is then purified to remove water. Both anhydrous
bioethanol (less than 1 per cent water) and hydrous bioethanol (1

5 p
er cent water) can be used
in a pure form as fuel, but they are usually blended with petrol before use. Blends of 5

10 per
cent of bioethanol in gasoline (named E5 and E10 respectively) do not require any modification
to the vehicle engine either.

When eth
anol is obtained from biological materials and by biological processes, e.g.
fermentation they are known as bio alcohols (e.g. bio ethanol). There is no chemical difference
between biologically produced and chemically produced alcohols. Ethanol and methano
l are two
types of alcohol fuels. The use of pure alcohols in internal combustion engines is only possible if
the engine is designed or modified for that purpose. However, in their anhydrous or pure forms,
they can be mixed with gasoline /petrol in various

ratios for use in unmodified gasoline engines,
and with minor modifications can also be used with a higher content of ethanol. Normally, only
ethanol is used widely in this manner, particularly since methanol is more corrosive to standard
engine component
s than ethanol.

Different types of Mixes:

Ethanol fuel mixtures have "E" numbers which describe the percentage of ethanol in the mixture
by volume, for example, E85 is 85% anhydrous ethanol and 15% gasoline. Gasoline is the usual
fuel mixed with ethanol bu
t there are other fuel additives that can be mixed, such as an ignition
improver used in the E95 Swedish blend. Low ethanol blends, from E5 to E25, are also known as
‘’gasohol’’ though internationally the most common use of the term gasohol refers to the E
10
blend. Ethanol fuel is an alternative to gasoline. It can be combined with gasoline in any
concentration up to pure ethanol (E100). Anhydrous ethanol, that is, ethanol with at most 1%
water, can be blended with gasoline in varying quantities to reduce c
onsumption of petroleum
fuels and in attempts to reduce air pollution. Worldwide automotive, ethanol capabilities vary
widely and most spark
-
ignited gasoline style engines will operate well with mixtures of 10%
ethanol (E10).


Schema of Production:


Project Description

The proposed project envisages setting up of a molasses based Fuel ethanol (Bio
-
Fuel) plant
either as an additional value added processing facilities with a Sugar mill or as a standalone unit
based on molasses available from cooperative

Sugar mills in
Jhang
. This distillery unit will be
manufacturing Ethanol and absolute alcohol to be used as Bio
-
fuel for blending with petrol or
diesel.
All the s
ugar mills have molasses as a by product of Sugar manufacturing
.

Manufacturing Process

Ethano
l is produced by yeast fermentation of the sugar extracted from sugarcane or sugar beets.
Sugarcane requires a tropical climate to grow productively. Sugarcane returns about 8 units of
energy for each unit expended compared to corn which only returns about

1.34 units of fuel
energy for each unit of energy expended. Thus sugarcane gives 20 times more energy as corn.
Carbon dioxide, a potentially harmful greenhouse gas, is emitted during fermentation. Yet, the
net effect is offset by the uptake of carbon gase
s by the plants grown to produce ethanol. As
compared to gasoline, ethanol releases less greenhouse gases.

Molasses is the main raw material used for production of Ethanol. It contains about 50% of the
total sugar; of which 30 to 33% is cane sugar and rest

is reducing sugar. The molasses is diluted
to mash containing 10

20 wt% sugar. After the pH of the mash is adjusted to about 4


5 with
mineral acid, it is inoculated with the yeast, and the fermentation is carried out non
-
aseptically at
20


32°C for ab
out 1


3days.During the fermentation, yeast strains to the species
Saccharomyces Cerevisiae, a living microorganism of class fungi it converts sugar present in the
molasses such as sucrose and glucose to alcohol. This conversion took place in large tank t
ype
Fomenters. The fermented beer, which typically contains ca 6


10 wt% ethanol, is then set to the
product recovery in purification section of the plant. The machinery used for separation of this
ethanol from aqueous solution is low temperature distill
ation using low pressure steam for
heating. This ethanol is further purified using multiple distillation process and forming azeo
-
tropic mixture (a mixture of two or more liquids in such a ratio that its composition cannot be
changed by simple distillation
, because, when an azeotrope is boiled, the resulting vapour has the
same ratio of constituents as the original mixture) with Benzene to get total anhydrous alcohol,
which is used for blending with petrol or diesel. In modern Fuel Ethanol plants, molecular

sieve
technology is used for removing water molecules from alcohol and manufacture alcohol, which
gives significant savings in terms of energy consumption. The Ethanol manufacturing process
from Molasses is shown in following Flow chart:


Water removal

F
or the ethanol to be usable as a fuel, water must be removed. Most of the water is removed by
distillation, but the purity is limited to 95
-
96% due to the formation of a low
-
boiling water
-
ethanol azeotrope. The 96% m/m (93% v/v) ethanol, 4% m/m (7% v/v) wa
ter mixture may be
used as a fuel, and it's called hydrated ethyl alcohol fuel. In 2006/2007, an estimated 17 billion
litters (4,5 billion gallons) of hydrated ethyl alcohol fuel will be produced, to be used in ethanol
powered vehicles.

For blending with
gasoline, purity of 99.5 to 99.9% is required, depending on temperature, to
avoid separation. Currently, the most widely used purification method is a physical absorption
process using molecular sieves. Another method, azeotropic distillation, is achieved
by adding
the hydrocarbon benzene which also denatures the ethanol (so no extra methanol/petrol/etc. is
needed to render it undrinkable for duty purposes). However, benzene is a powerful carcinogen
and so will probably be illegal for this purpose soon. Eth
anol is not typically transported by
pipeline for three reasons. Current production levels will not support a dedicated pipeline. The
costs of building and maintaining a pipeline from Midwestern United States to either coast are
prohibitive. Any water whic
h penetrates the pipeline will be absorbed by the ethanol, diluting the
mixture.


The schema above shows the main process of ethanol production, and the one below shows the
separation unit:





Equipment Used:

The following equipment would be used:


Fermentation Plant for Ethanol manufacturing including Molasses storage, Molasses

weighing,
preparatory and yeast preparatory,
Fermentor
, and yeast settling tanks etc


Distillation plant for Ethanol separation and yeast recycling


Rectified spirit, Indus
trial alcohol manufacturing plant


Anhydrous alcohol manufacturing multiple distillation facilities, using low pressure steam

for
heating process or Molecular sieve plant.

Usage of Molecular Sieve Technology:

The rectified spirit before sending in for deh
ydration process, it is heated and portion of water is
stripped in the pre concentration column through a pre
-
heater. The mixture of alcohol and the
remaining portion of water are sent to molecul
ar sieve in the form of
vapor
.

The molecular sieves are hard

granular substances, cylindrical extrudates manufactured from
clay like silica gel material such as potassium alumino silicates. They are grated according to the
nominal diameter of the myriad of internal pores, which make up the interstitial free volume

found within their structure. A typical sieve used in ethanol dehydration is Type 3A. This
designation means that the average diameter of the interstitial passageways is 3 Angstroms (One
Angstroms: 1A is unit of measures equivalent to 10
-
8 centi meters)
. Thus the passageways in
the structure have a diameter that is molecular scale. The water molecule has a mean diameter
less than 3A, while the ethanol molecule has a mean diameter greater than 3A.

In addition, the water molecules can be absorbed on the
internal surface offered by the
passageways within the molecular sieve structure. It is the physical property of the sieves, which
make them useful for the separation of mixture of ethanol and water.

Water molecules can invade the inner structure of the m
olecular sieve beeds and be absorbed
thereon, while the ethanol molecules are too large and pass out of the vessel leaving the water
behind, Thus, dehydration of ethanol takes placer in the molecular sieve technology.

In this technology, the re
-
generation of beads is done by one bed under vacuum; while the other
bed is producing anhydrous ethanol under pressure with an automated operation, the feed of
vapour to the molecular sieve system can be taken directly from the p
re concentration column
passing through a super heater. The condensed anhydrous alcohol vapour are then cooled and
passed to storage through product cooler. The recovered ethanol and water from the
regeneration phase is fed back to the pre
-
concentration
column for recovery. Thus, there is no
generation of effluent except the condensate water from steam is cooled and taken back process.