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The manufacture of ethanol from casein whey: A two-fold solution to the dilemmas of Waste Disposal and Energy Crunch

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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.

Keywords: Casein Whey treatment, Ethanol production, Alternate sources of Energy, Waste disposal Introduction: For more than two centuries, the worlds energy supply has relied heavily on non renewable crude fuels derived from natural resources, out of which 90 percent is examined to be consumed for energy generation and transportation. Transport sector is likely to suffer badly because of following reasons, i.e., fuel reservoirs are limited, price of crude oil in international market are on the rising trend; it has reached US $ 100 mark per barrel in January 2008. Moreover number of vehicles based on petroleum fuels is on increase worldwide, simultaneously, petroleum is monopoly of some countries and rest of the world depends on them. Looking into this scenario, there is sizeable increase of import budget for petroleum of all oil importing countries. This situation has forced such countries to search and do research and development on alternate fuels. Among various options tried so far, gaseous and liquid fuels based on natural gas and biomasses respectively, have emerged as best options for 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 (1). The policy set by the Government of India has lead to the usage of 10% ethanol blended petrol as compared to the previous ratio of 5%. Dairy industries are major economical sources of tropical and subtropical countries creating a vast amount of dairy waste, mainly whey, a heterogeneous mixture of several valuable proteins and carbohydrate which has a lot of unique values associated with them. Due to stricter environmental legislations, research is now underway to promote successful disposal and recycling of dairy wastes without much damage being caused to the environment. Although, many attempts have been performed in the fields of recovery and utilization of whey in terms of protein isolation (2,3) and GOS production (4,5) but, unfortunately no such venture has been made into the production of ethanol from casein whey(6). Whey disposal is one of the main problems which India faces and the reason being two fold. One is the fact that dairy industry in India is a flourishing area, the economic importance of which cannot be overlooked. Thus, waste disposal is a primary cause of concern since the BOD of whey is in order of thousands and COD is in the order of lakhs. Second is the lack of research work on the safe disposal of whey. The present work specifically addresses this problem .Lactose

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 niger and 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.

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 of Aspergillus niger and Sachharomyces 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 niger for five days. Microfiltration: Microfiltration was carried out by Whatman filter paper 1 for proper removal of fungi i.e., Aspergillus niger from 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.

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.

pH Measurement : pH of fermented broth was measured by pH meter. Results and discussions: Isolation of microbes: The microbes Aspergillus niger and 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 niger is 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.

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 37 0 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

which the scientific and engineering community needs to address. Todays world is the classic case of Too many consuming too much at too fast a rate. Population rise signifies the fact that more and more resources are being consumed but there is an upper limit attached to the availability of these resources. Hence looking for alternative resources is the best possible solution that comes to the mind. Energy crisis is one such aspect where conventional sources of energy are being replaced by non-conventional ones. Though more and more research and development is underway, yet there is an upper bound trend in this respect. However, resources being consumed pose another problem, i.e., the rejection of wastes from these resources. The rate of waste rejection is almost as high resources being utilized, if not higher. Hence recycling of these wastes is one of the probable solutions to the problem. A very hard-hitting exposition of this is Al Gore's film and book follow-up: 'An Inconvenient Truth'. This film depicts in graphic pictures what words fail to: the human effect on this fragile world. Most recently a ship ran aground off the Cornish coast in bad weather and alongside the images in the media of all the consumer goods that were washing up on the nearest beach, were images of the damage from the leaking oil - birds covered in oil, long slicks sat in the water, killing the life of the sea. All this for our compulsion to transport more and more goods round the world. The present work has been an effort to address this issue and take a small step towards reaching the solution. Casein whey is a rich pool of various bacteria fungi and other microorganisms which can do wonders at the right temperature, pH and other operating conditions. Isolation of these microbes and characterization of the same can deliver us with some of the most potent organisms ever witnessed. These microbes can be used up to eat the wastes generated by us. Though at present, biological treatment of wastes is indeed a slow process but future endeavors in this field can suggest some possible solutions towards fastening up the same. Recycling or treating up wastes using microbes serves a two-fold purpose. One, the process is more ecofriendly, since any mechanical or chemical methods designed by man would lead to the consumption of energy, hence more pollution and more wastes and also depletion of resources. Secondly, microbes do not give off harmful side products, on the hindsight, the by products as seen in this work is the fuel in form of ethanol. The work has been done to specifically to show the fact that membrane separation technique and biological treatment both are useful to treat industrial wastes, but membrane techniques are energy intensive. Moreover, the percentage recovery of valuable products from membrane techniques, like -lactalbumin, might not be a

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 of fuel consumption when introducing DME
or ethanol into diesel engine. Fuel, Volume 85, Issues 5-6, Pages 778-782.

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


Partition of whey milk proteins in aqueous two-phase systems of olyethylene glycolphosphate as a starting of point to isolate proteinsexpressed B, Volume 819, in Issue transgenic 1, 5. Pages milk 25-31 Journal Chromatography

3. Svetlana Butylina, Susana Luque, Marianne Nystrm . Fractionation of whey-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


enzymatic production of 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. Ethanol production from cheese whey powder solution in a packed
column bioreactor at different hydraulic residence times. Biochemical Engineering Journal, Volume 42, Issue 2, Pages 180-185

Figure 1: Scanning Electron Microscope picture of Aspergillus niger.

Figure 2: Scanning Electron Microscope picture of, Saccharomyces cerevisiae

Raw Casein Whey F

Isolation of Sachharomyces cerevisiae

Isolation of Aspergillus niger

Raw Casein whey

Production of galactosidase by Aspergillus niger

Purification of enzyme using ultrafiltration technique

Hydrolysis

Purified Enzyme
Addition of Sachharomyces cerevisiae .

Glucose and Galactose formed by hydrolysis by galactosidase

Ethanol production from glucose

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

1 0 0

L a c to s e h y d r o ly s is

8 0

6 0

4 0

H y d r o ly s is a t p H H y d r o ly s is a t p H H y d r o ly s is a t p H

7 .0 5 .5 4 .5

2 0

%
0 0 1 2 3 4 5

T im e (h o u r s )

Figure 4: Rate of hydrolysis of lactose by lactase (0.4 mg/ml) from A. niger in whey containing 4% lactose at different pH at 500C.

1 0 0

L a c to s e h y d r o ly s is

8 0

6 0

4 0

2 0

h h h h

y y y y

d d d d

r r r r

o o o o

ly ly ly ly

s s s s

is is is is

a a a a

t t t t

5 6 7 3

0 C 0 0 C 0 0 C 0 0 C

%
0 0 1 2 3 4 5

T im e (H o u r )

Figure 5: Rate of hydrolysis of lactose by lactase (0.4 mg/ml) from A. niger in whey containing 4% lactose at different temperatures and pH 5.5.

0 .5 0 .4
Optical Density at 600 nm

0 .3 0 .2 0 .1 0 .0 0 20 40 60 80 100
In cu b atio n tim e (h ou rs)
B io m ass B io m ass B io m ass B io m ass g ro w th g ro w th g ro w th g ro w th w ith ou t lacto se hyd ro lysis afte r lacto se h yd rolysis at 2 % glu cose sup p lem e n t at 3 % glu cose sup p lem e n t

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

6 .0 5 .5
Residual substrate concentration (mg/ml)

5 .0 4 .5 4 .0 3 .5 3 .0 2 .5 2 .0 0 20 40 60

1% 2% 3% 4%

g lu co se g lu co se g lu co se g lu co se

80

100

In c u b a tio n tim e (h o u r s )

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

2 .5 2 .0

Optical Density (340 nm)

1 .5 1 .0 0 .5 0 .0 0 20 40 60

B io m ass B io m ass B io m ass B io m ass


80

gro w th C at 0 gro w th C at 0 gro w th C at 0 gro w th C at


100

30 35 37 40

In cu b atio n tim e (h o u r)

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

2 .5 2 .0

% Ethanol production

1 .5 1 .0 0 .5 0 .0 0 20 40 60 80 100
E thanol production E thanol production E thanol production E thanol production w ith no lactose hydrolysis after lactose hydrolysis w ith 1% glucose supplem ent w ith 2% glucose supplem ent

Incubation tim e (hours)

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

1.8 1.6

Ethanol concentration (V/V)

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 5 10 15 20


++

Ethanol Concentration at presence of Fe ++ Ethanol Concentration at presence of Co +6 Ethanol Concentration at presence of Mo

Trace metal Concentration (g/ml)


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