The Manufacture of Ethanol From Casein Whey a Two-fold Solution to the Dilemmas of Waste Disposal and Energy Crunch

8/3/2019 The Manufacture of Ethanol From Casein Whey a Two-fold Solution to the Dilemmas of Waste Disposal and Energy Crunch

1/19

The manufacture of ethanol from casein whey: A two-fold solution

to the dilemmas of Waste Disposal and Energy Crunch

Document by:BharadwajVisit my website

www.engineeringpapers.blogspot.comMore papers and Presentations available on above site

Abstract:

Casein whey is treated biologically to yield ethanol. Casein Whey is a major by product of the sweetmeat

industry. The sweetmeat industry being quite a lucrative industry in India produces a gigantic amount of

Casein Whey. Thus Casein Whey disposal becomes a major problem because of the very high BOD and

COD values of Whey. The entire world is facing an Energy crunch. Energy Economy is now the primary

concern of the developed and developing countries alike. Substantial research on alternate economic

sources of energy is being undertaken. Among various options tried so far, gaseous and liquid fuels based

on natural gas and biomasses respectively, have emerged as best options for the transport sector. The

petroleum industry now looks very committed to the use of ethanol as fuel, where sugarcane dominates

the scene as the raw material. The blend of ethanol to unleaded gasoline is a major factor in the cyclic

variability and emissions of spark ignited engines. The present attempt has been made to focus ethanol

production from casein whey by a two step fermentation technology, i.e., hydrolysis of lactose to glucose

by -galactosidase, secreted by aspergillus oryzae, a common -galactosidase producing fungal source,

which naturally grows in the whey. Simultaneously saccharomyces cerevisiae, a trivially used fungal

strain in beverage industries, has been used to convert the glucose to ethanol. It is important to note that

the fungi used in the fermentation process have been isolated from the whey itself. Thus the treatment of

whey using fungal growth from itself has not only helped in treating the waste which has a high BOD

and COD value, but an important fuel in the form of ethanol has also been obtained from it. The present

work thus addresses not only the waste disposal problem, but it also provides an alternative source of fuel

for future use, thus opening new frontiers to solve the energy crisis and fuel crunch which the world is

facing.
http://www.engineeringpapers.blogspot.com/http://www.engineeringpapers.blogspot.com/


2/19


3/19

is the main carbohydrate source of whey, a disaccharide which is composed of two

monosaccharides namely, glucose and galactose. -galactosidase is a well known lactase trivially

used in dairy industries for lactose hydrolysis, but due to its high cost it is not a viable option for

dairy industries. India has practiced basic biotechnological approaches in several decades, like,

isolation of the microbes, enzyme production, pharmaceutical applications etc (6). The present

attempt has been made to focus ethanol production from casein whey by two step fermentation

technology, i.e., hydrolysis of lactose to glucose by -galactosidase, secreted by Aspergillus

niger(Figure 1), a common -galactosidase producing fungal source, which naturally grows in

the whey. Simultaneously saccharomyces cerevisiae (Figure 2), a trivially used fungal strain in

beverage industries, has been used to convert the glucose to ethanol. The important fact is that

both Aspergillus nigerand saccharomyces cerevisiae have been isolated from the casein whey

and they have used to obtain ethanol from the same. The work not only helps in the treatment of

whey as per the environmental norms but also is a technological development since the

production cost is minimized for ethanol.

Experiment :

Materials:

Casein whey is collected from a local confectionary house, Hindusthan Sweets, Kolkata-700032,

West Bengal, India. All chemicals used in the experiment were procured from Hi Media

(Mumbai, India), E.Merck (Mumbai, India) and SigmaAldrich (St. Louis, USA). HPLC grade

methanol and water purchased from Spectrochem PVT. LTD., Mumbai, India. The deionized

water used in all the experiments was obtained from Arium 611DI, ultrapure water system

(Sartorius AG, Gttingen, Germany). HPLC grade methanol was purchased from Spectrochem

PVT. LTD. Mumbai, India.

Instruments :

UV Spectoscopy (Hitachi U2000, Germany), Atomic absorption Spectroscopy (Perkinelmer,

Japan), weighing machine (Sartorius BS 223 S, Gttingen, Germany), laminar flow (Kenzflow,

India), water bath and autoclave (Gurpreet Engineering Works, Kanpur, U.P., India), Remi make

Centrifuge, microoven and mixy (Vediocon, Mumbai, India), were used during the experiment.


4/19

Digital pH meter and all other instruments were procured from (Sartorius AG, Gttingen,

Germany).

Method:

The flowchart of the process is shown in Figure 3.

Isolation ofAspergillus nigerandSachharomyces cerevisiae:

Aspergillus niger and Saccharomyces cerevisiae were isolated from casein whey by serial

dilution method at pH 5.5 and both two cultures were maintained at Sabouraud dextrose agar.

Microbial consortium were identified by their morphology, sporulation type and growth

characteristics. Final identifications were performed by 16S rRNA technique.

3.4. Lactose hydrolysis:

The lactose hydrolysis reaction was performed in a fermentor by a lab prepared lactase enzyme

at different pH and temperatures.

Enzyme production:

The modified Sabouraud dextrose broth, containing lactose 20gram and neopeptone 10 gm and

water 1000ml with pH maintained around 5.5, was treated by three days old inoculum of

Aspergillus nigerfor five days.

Microfiltration:

Microfiltration was carried out by Whatman filter paper 1 for proper removal of fungi i.e.,

Aspergillus nigerfrom the harvested broth.

Enzyme purification:

Two stage ultrafiltration process was performed using membrane modules of 100kDa MWCO

(Molecular Weight Cut Off) followed by 150 kDa MWCO. The whole operation was performed

at chilled condition ( 100C). The enzyme was dried by lypholized for further experimental works.


5/19

Enzyme dosing:

The fermentation media, viz. casein whey was treated by the lactase enzyme (0.4 mg/ml) for the

hydrolysis of lactose. The fixed dosage of enzyme was maintained and studies were performed

on the lactose hydrolysis with repect to different pH levels, temperature variations and different

salt concentrations, thus arriving at the optimum condition for carrying out the hydrolysis of

lactose.

Fermentation media preparation and fermentation:

The pH of fermentation media was maintained around 7 by 10N potassium hydroxide for the

optimum growth of Sachharomyces cerevisiae. The 18 hour old inoculum of Sachharomyces

cerevisiae from the Sabouraud dextrose broth was considered for ethanol production. The 96

hour anaerobic fermentation was performed in conical flasks.

Analysis:

Total protein concentration was measured by UV Spectrophotometer following the standard

procedure of Bradford protein assay. Individual protein concentrations were measured using

Waters HPLC system, [Waters (India) Pvt. Ltd] consisting of an waters 1525 Binary HPLC

pump, symmetry C18 reversed phase column (4.6x150 mm) and waters 2487 dual absorbance

detector. 40% Methanol (v/v) was used as mobile phase in HPLC with a flow rate of 0.5mL/min

for 20 min. Column temperature was maintained at 250C.

Alcohol Measurement :

Ethanol concentration was estimated by Alcohol Dehydrogenase (ADH) method.

Glucose Estimation :

Glucose concentration of fermentation was estimated by 2,4-Dinitro salicylic (DNS) method.

Protein Estimation :

Total protein concentration in food sample was measured by UV Spectrophotometer following

the standard procedure of Bradford protein assay.


6/19

pH Measurement :

pH of fermented broth was measured by pH meter.

Results and discussions:

Isolation of microbes: The microbes Aspergillus nigerand Saccharomyces cerevisiae were

successfully isolated from the microbial consortium present in the casein whey and it was grown

in the Sabouraud dextrose agar as a pure culture. The microbes were identified using the

confirmatory 16 S rRNA technique.

Enzyme purification: For lactase production the fungal Aspergillus nigeris extremely useful in

industry since it produces extracellular -galactosidase enzyme and is very efficient in this which

can be attributed to the presence of lac operon. The advantage of this production is due to the

fact that no sonication or liquid nitrogen purging is used for cell disruption for obtaining the

enzyme, the only associated cost is the purification of it just by two stage ultrafiltration where

the enzyme obtained is almost 90% pure which is enough for industrial applications.

Lactose hydrolysis: The enzyme dosing was kept constant at 0.4 mg/ml (dry basis) and the set of

experiments were performed at different pH levels and temperatures. The enzyme produced can

operate at a pH as low as 5.5 which as shown in the experiment (Figure 4). The optimum

temperature obtained was 600C (Figure 5) as per the experimental observations. The important

observation is that 2% glucose is obtained from 4% lactose due to some product inhibitions, like

galactose which inhibits the hydrolysis process. The hydrolysis process is also affected by the

presence of magnesium and manganese sulphates, the activity increases in their presence thereby

increasing the glucose conversion from around 2% to 3%.On the other hand in the presence of

magnesium chloride and manganese chloride, the conversion rate drops appreciably. The same

results are also obtained in the case of potassium phosphate and potassium sulphate where the

conversion increases but decreases in the case of potassium chloride. Calcium sulphate and

calcium chloride also brings down the conversion of lactose to glucose and the behavior is

replicated in the case of sodium sulphate and sodium chloride.


7/19

Ethanol production and growth characteristics of Saccharomyces cerevisiae: The fungi do not

have lac operon in their genomic DNA, so they are not able to metabolise lactose. The simplest

monosaccharide glucose can be uptaken by the fungi and thus can produce ethanol by the

glycolysis pathway. Whey itself is a combination of different carbohydrates and proteins, thus

Saccharomyces cerevisiae grows in it naturally and produces ethanol even in that state. Thus if

lactose is hydrolysed to form monosaccharide, then Saccharomyces cerevisiae growth is

enhanced, simultaneously ethanol production is also more pronounced (Figure 6). It has been

deduced from the experiments that ethanol production is highly substrate dependent (Figure 7).

The important observation is that the substrate utilization is directly proportional to

Saccharomyces cerevisiae growth and which is directly proportional to ethanol production, thus

it can be safely stated that substrate utilization is directly proportional to ethanol production

(Figure 9). The growth of Saccharomyces cerevisiae not only depends on substrate utilization

but also depends on temperature variations. Experimentally it has been established that 370 C to

400C is the most favourable temperature domain for the growth of the fungi, but the ethanol

production has been found to be highest at around 370C, so it has been taken as the optimum

temperature. The low supplement of carbohydrate sources helps in the growth of Saccharomyces

cerevisiae but it approaches saturation at 3% glucose addition after 72 hours (Figure 6). But on

the other hand protein supplement does not produce any sensible results in the growth of the

fungi (an increase of 1%-1.5%), but has no effects whatsoever on the production of ethanol. This

is because the growth primarily depends on carbohydrate to protein ratio which if kept at 2:1,

produces the highest amount of ethanol. The ethanol yield is around 2%-2.5% as observed from

the experiment. Different chlorides of magnesium, manganese, zinc and sodium have a positive

effect on ethanol production, where the production can be increased form 2% to 3% -3.5% at

optimum temperature (370C) in proper anaerobic conditions (Figure 10). It is a low cost process

since only casein whey, which is an industrial effluent, is used in this and the strain is an isolated

one, so the yield obtained from it is quite satisfactory. The strain, if modified further genetically,

then higher yield can be expected in the current set up.

Conclusion

With the ever rising population growth and depleting resources, the world is racing against time

to cope up with the demand posed by us. Pollution and its abatement is one of the primary issues


8/19


9/19

feasible tradeoff between energy invested and product recovered. However, biological treatment

can yield in better yield of ethanol if more pure and better strains of microbes are used.

The future scope of the work lies in the better production and yield of ethanol and a possible

suggestion of a tradeoff between conventional membrane separation techniques and biological

treatment. After all, the present scenario presents only conducive atmosphere towards nothing

but the end of life on planet earth. Not far will be the day when we meet our Armageddon if we

continue in this vein of consuming what Mother Nature has dished out for us.

References:

1. Zhang Bo, Fu Weibiao, Gong Jingsong. Study offuelconsumption when introducing DME

orethanolinto diesel engine. Fuel, Volume 85, Issues 5-6, Pages 778-782.

2. Lorena Capezio, Diana Romanini, Guillermo A. Pic, Bibiana Nerli

Partition ofwheymilkproteinsin aqueous two-phase systems of olyethylene glycolphosphate as

a starting point to isolate proteinsexpressed in transgenic milk

Journal of Chromatography B, Volume 819, Issue 1, 5. Pages 25-31

3. Svetlana Butylina, Susana Luque, Marianne Nystrm . Fractionation ofwhey-derived peptides

using a combination of ultrafiltration and nanofiltration. Journal of Membrane Science, Volume

280, Issues 1-2, Pages 418-426

4. P. Czermak, M. Ebrahimi, K. Grau, S. Netz, G. Sawatzki, P. H. Pfromm Membrane-assisted

enzymaticproductionof galactosyl-oligosaccharides from lactose in a continuous process

Journal of Membrane Science, Volume 232, Issues 1-2, Pages 85-91

5. M.J. Gonzlez-Muoz, H. Domnguez, J.C. Paraj. Depolymerization of xylan-derived products

in an enzymatic membrane reactor.Journal of Membrane Science, Volume 320, Issues 1-2, Pages

224-231.

6. Serpil Ozmihci, Fikret Kargi. Ethanolproduction from cheesewheypowder solution in a packed

column bioreactor at different hydraulic residence times. Biochemical Engineering

Journal, Volume 42, Issue 2, Pages 180-185
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4JHMY0S-1&_user=10&_coverDate=09%2F01%2F2006&_alid=1060391462&_rdoc=4&_fmt=high&_orig=search&_cdi=5257&_sort=r&_docanchor=&view=c&_ct=38&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a7d651f012317193edf281681dff9595http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4JHMY0S-1&_user=10&_coverDate=09%2F01%2F2006&_alid=1060391462&_rdoc=4&_fmt=high&_orig=search&_cdi=5257&_sort=r&_docanchor=&view=c&_ct=38&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a7d651f012317193edf281681dff9595http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4JHMY0S-1&_user=10&_coverDate=09%2F01%2F2006&_alid=1060391462&_rdoc=4&_fmt=high&_orig=search&_cdi=5257&_sort=r&_docanchor=&view=c&_ct=38&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a7d651f012317193edf281681dff9595http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4JHMY0S-1&_user=10&_coverDate=09%2F01%2F2006&_alid=1060391462&_rdoc=4&_fmt=high&_orig=search&_cdi=5257&_sort=r&_docanchor=&view=c&_ct=38&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a7d651f012317193edf281681dff9595http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4BKPSYY-6&_user=10&_coverDate=03%2F15%2F2004&_alid=1060391208&_rdoc=2&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1bedeb9409d68f5c2b46371fb828b7e6http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4BKPSYY-6&_user=10&_coverDate=03%2F15%2F2004&_alid=1060391208&_rdoc=2&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1bedeb9409d68f5c2b46371fb828b7e6http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4BKPSYY-6&_user=10&_coverDate=03%2F15%2F2004&_alid=1060391208&_rdoc=2&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1bedeb9409d68f5c2b46371fb828b7e6http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4BKPSYY-6&_user=10&_coverDate=03%2F15%2F2004&_alid=1060391208&_rdoc=2&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1bedeb9409d68f5c2b46371fb828b7e6http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4BKPSYY-6&_user=10&_coverDate=03%2F15%2F2004&_alid=1060391208&_rdoc=2&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1bedeb9409d68f5c2b46371fb828b7e6http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4BKPSYY-6&_user=10&_coverDate=03%2F15%2F2004&_alid=1060391208&_rdoc=2&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1bedeb9409d68f5c2b46371fb828b7e6http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4S80XG3-3&_user=10&_coverDate=07%2F15%2F2008&_alid=1060391208&_rdoc=1&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=de19622262f24d6b605ce647307aec99http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4S80XG3-3&_user=10&_coverDate=07%2F15%2F2008&_alid=1060391208&_rdoc=1&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=de19622262f24d6b605ce647307aec99http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V3B-4H6GW6Y-2&_user=10&_coverDate=04%2F30%2F2006&_alid=1060379111&_rdoc=3&_fmt=high&_orig=search&_cdi=5726&_sort=r&_docanchor=&view=c&_ct=28511&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=522d2653e0f59a8504ced55512530a1fhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6X0P-4FJ5XHM-1&_user=10&_coverDate=05%2F05%2F2005&_alid=1060386673&_rdoc=6&_fmt=high&_orig=search&_cdi=7220&_sort=r&_docanchor=&view=c&_ct=3905&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=07e705b53558d737fb503cf1a1c1b495http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4JHMY0S-1&_user=10&_coverDate=09%2F01%2F2006&_alid=1060391462&_rdoc=4&_fmt=high&_orig=search&_cdi=5257&_sort=r&_docanchor=&view=c&_ct=38&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a7d651f012317193edf281681dff9595http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4JHMY0S-1&_user=10&_coverDate=09%2F01%2F2006&_alid=1060391462&_rdoc=4&_fmt=high&_orig=search&_cdi=5257&_sort=r&_docanchor=&view=c&_ct=38&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a7d651f012317193edf281681dff9595http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4BKPSYY-6&_user=10&_coverDate=03%2F15%2F2004&_alid=1060391208&_rdoc=2&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1bedeb9409d68f5c2b46371fb828b7e6http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4BKPSYY-6&_user=10&_coverDate=03%2F15%2F2004&_alid=1060391208&_rdoc=2&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1bedeb9409d68f5c2b46371fb828b7e6http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4S80XG3-3&_user=10&_coverDate=07%2F15%2F2008&_alid=1060391208&_rdoc=1&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=de19622262f24d6b605ce647307aec99http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGK-4S80XG3-3&_user=10&_coverDate=07%2F15%2F2008&_alid=1060391208&_rdoc=1&_fmt=high&_orig=search&_cdi=5257&_docanchor=&view=c&_ct=2&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=de19622262f24d6b605ce647307aec99http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4SWFNX8-3&_user=10&_coverDate=11%2F01%2F2008&_alid=1060393050&_rdoc=2&_fmt=high&_orig=search&_cdi=5791&_sort=r&_docanchor=&view=c&_ct=3701&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f9296ba1b40b4460fec081c6d1487103


10/19

Figure 1: Scanning Electron Microscope picture ofAspergillus niger.


11/19

Figure 2: Scanning Electron Microscope picture of, Saccharomyces cerevisiae


12/19

Raw Casein WheyF

igure 3: Schematic diagram to show the experimental steps carried out in ethanol production.

Isolation ofAspergillus niger

Production of -galactosidase by

Aspergillus niger

Isolation ofSachharomyces

cerevisiae

RawCasein whey

Hydrolysis

Ethanol productionfrom glucose

Ethanol

Addition ofSachharomyces

cerevisiae .

Glucose andGalactose formedby hydrolysis by -galactosidase

Purification ofenzyme usingultrafiltrationtechnique

Purified Enzyme


13/19

Figure 4: Rate of hydrolysis of lactose by lactase (0.4 mg/ml) fromA. nigerin whey containing4% lactose at different pH at 500C.

0 1 2 3 4 5

0

2 0

4 0

6 0

8 0

1 0 0

%

L

actose

hydrolysis

T i m e ( h o u r s )

H y d r o l y s i s a t p H 7 . 0 H y d r o l y s i s a t p H 5 . 5 H y d r o l y s i s a t p H 4 . 5


14/19

0 1 2 3 4 5

0

2 0

4 0

6 0

8 0

1 0 0

%

L

act

ose

hydrolysis

T i m e ( H o u r )

h y d r o l y s i s a t 5 00

C


C


C


C

Figure 5: Rate of hydrolysis of lactose by lactase (0.4 mg/ml) fromA. nigerin whey containing4% lactose at different temperatures and pH 5.5.


15/19

0 2 0 4 0 6 0 8 0 1 0 0

0 . 0

0 . 1

0 . 2

0 . 3

0 . 4

0 . 5

OpticalDensityat600nm

I n c u b a t io n t im e ( h o u r s )

Biom ass g row th w i thou t l ac tose hydro lys i

Biom ass g row th af ter l ac tose h ydro lys is

B i o m as s g ro w t h a t 2 % g l u cos e s u p p l em e

B i o m as s g ro w t h a t 3 % g l u cos e s u p p l em e

Figure 6: Biomass growth with different concentration of glucose supplements.


16/19

0 2 0 4 0 6 0 8 0 1 0 0

2 . 0

2 . 5

3 . 0

3 . 5

4 . 0

4 . 5

5 . 0

5 . 5

6 . 0

Resid

ualsubstrateconcentration(mg/ml)

I n c u b a t io n t im e (h o u r s )

1 % g lu c o s e

2 % g lu c o s e3 % g lu c o s e

4 % g lu c o s e

Figure 7: Substrate concentration variation with incubation time (in hours).


17/19

0 2 0 4 0 6 0 8 0 1 0 0

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

OpticalDensity(340nm)

I nc uba t ion t im e ( hou r )

B i o m a s s g r o w t h a t 3 00C




Figure 8: Growth of biomass (Sachcharomyces cerevisiae) at different temperatures.


18/19

Figure 9: Ethanol production with various concentrations of glucose supplement.

0 2 0 4 0 6 0 8 0 1 0 0

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

%E

thanolproduction

Incubation t ime (hours)

Ethanol production with no lactose hydrolysis

Ethanol production after lactose hydrolysis

Ethanol production with 1% glucose suppleme n

Ethanol production with 2% glucose suppleme n


19/19

0 5 10 15 20

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Ethanolconcentration(V/V)

Trace metal Concentration (g/ml)

Ethanol Concentration at presence of Fe++

Ethanol Concentration at presence of Co++

Ethanol Concentration at presence of Mo+6

Figure 10: Ethanol concentration variation with different concentrations of metal ions.

Documents

The Manufacture of Ethanol From Casein Whey a Two-fold Solution to the Dilemmas of Waste Disposal and Energy Crunch