Seminar On

“Bioconversion of whey to 2,3-butanediol using Klebsiella oxytocaNRRL-13-199”

  

Submitted to The Principal ,

Mahatma Gandhi MissionCollege of Agricultural Biotechnology , Aurangabad

(Affiliated to Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani)

(ISO 9001: 2008 and ISO 14001: 2004 Certified Certified) Bachelor of Science

InAgricultural Biotechnology

Submitted byINGLE SNEHAL DEVIDAS

Reg.No.- MGM/CABT/10/30 Semester -VII (New) Exam Seat No.- 3274 Course No.- CS-4713

Guided byMr. G. V. KORE Assistant Professor

Department of Biochemistry and molecular biology

The BOD is mainly due to lactose, which is present at concentration between 4-5%.

The cost –effective disposal or utilization has become important for dairy industries as they contribute high organic pollution load.

The most recent approach for whey utilization is ultra-filtration to separate proteins from permeate or deproteinated whey (DPW).

Options has been proposed to convert permeate to value added products as lactic acid, acetic acid, propionic acid, acetone and butanol.

Structure It is a chemical compound composed of carbon, hydrogen, and oxygen. It is one of the constitutional isomers of butanediol. The production of bio-based product bulk chemicals from renewable

resources has recently attracted increasing attention as the price of petrochemical feedstocks continuously increases and availability diminishes.

Interest in microbial production of 2,3-butanediol has been increasing recently due to the extensive industrial application.

This is colourless and odourless liquid with high boiling point and low freezing point is a potential valuable fuel additive.

Its heating value is 27.198 kJg － 1.

The medium used for the culture of K. oxytoca was described by Pirt and Callow and known as PC medium.The pH of the medium was adjusted to 6.5 using phosphoric acid. Glucose or lactose was used as the carbon source. The medium was prepared and autoclaved in three separate solutions, viz.,1.solution of the nutrients,2.solution of ammonium salts and3.sugar solution

Glucose or lactose solution (0.1%) in 100 mL portion were autoclaved at 0.7 kg/cm2 pressure for 30 min. Ammonium salt solution and nutrient medium, which were previously sterilized, were added to the flask followed by inoculation from a 24-h-old slant culture of K. oxytoca and incubated on a shaker (60 rpm) at 300C for 24 h. This culture was used as Inoculum (1% v/v)

Whey was obtained from a local dairy. The pH of whey was adjusted to 7.0 by using 1 N NaOH and then steamed for 30 min to precipitate protein. It was then cooled, kept at 40C and filtered through ordinary filter paper. The filtrate known as deproteinated whey (DPW) was used for further studies. Various constituents of DPW were estimated. Reducing sugar was estimated by dinitrosalicylic acid method lactic acid by titrimatric method protein according to the modified method of Lowery et al nitrogen by macro Kjeldahl method calcium and magnesium by complexometric titration method and sulphates by turbidity method. Total dissolved solids, ash content, COD and BOD were estimated by standard methods.

Glucose or lactose solutions (3%) in 100 mL portion were autoclaved in 250 mL flasks. PC minerals were added and then inoculated with K. oxytoca. When DPW was used for fermentation,PC minerals were not added. Autoclaved whey was directly inoculated with K.oxytoca inoculum. Agitation of 60 rpm was provided throughout the experimentation. Samples were withdrawn after every 24 h and then incubated, centrifuged and analyzed for residual sugar, BD, acetoin, acetic acid and ethanol.

Sodium acetate stock solution (5 M) was prepared and 0.2, 0.5, 1.0, 1.5, and 2.0 mL of it were added to 100 mL DPW, so as to get final concentration of 10, 25, 50, 75, and 100 mM of acetate. The pH of all the solutions was adjusted to 6.5. Then they were autoclaved, inoculated and samples were withdrawn at 24 h interval to analyze the content of BD, acetoin, acetic acid and ethanol.The fermentation broth was first centrifuged and the supernatant after proper dilution was injected into GC (Perkin Elmer Sigma 3B) equipped with flame ionization detector.The injector, detector and oven temperatures were maintained at 210, 230 and 200oC, respectively.

Trace elements DPW (ppm)(Present Day)

Permeate powder(Hargrove)

Copper 0.685-0.977 1.2-2.9

Lead 0.103-0.200

Manganese 0.105-0.234 0.5-0.6

Iron 1.544-2.452 3.1-11.3

Nickel 0.05-0.13

Cadmium 0.003-0.006

Zinc 1.506-2.280

Chromium 0.033-0.035 2.2-3.4

Results indicate that the utilization of lactose by K. oxytoca was very slow as compared to that of glucose. BD concentration of 2.1 g/L was achieved with lactose after 168 h incubation period. No BD production was observed with glucose at 48 h, whereas 6.8 g/L BD was obtained after 72 h of incubation. The lower rate of lactose consumption could be due to difference in the uptake mechanism, which is energy demanding for lactose but not for glucose.A BD concentration of 6.1 g/L was achieved form 2.35% lactose utilized after 96 h of incubation period. This corresponds to BD production of 0.259 g/g lactose utilized. The yield of BD achieved by them was 0.06 and 0.15 g/g lactose utilized after 72 and 168 h, respectively. Ethanol 0.43%, acetic acid 0.09%, and acetoin 0.02% were also produced as byproducts during fermentation..

Time(h) COD (kg/m3)

% COD reduction

BOD (kg/m3)

BOD Reduction

0 80 106

24 40 50 55.1 48

48 25.3 65 21.2 80

96 10.1 87.5 10.6 90

120 9.9 88 8.48 92

It has been reported that acetate induces the production of acetoin and BD by Aerobacter aerogenes Acetate at a concentration of 50 mM was found to be the optimum and produced 8.4 g/L of BD from 2.3% lactose. It corresponds to a BD yield of 0.365 g/g lactose utilized, which amounts to a 1.5-fold increase in the production of BD without appreciable increase in other byproducts. The concentrations above 50 mM acetate resulted in the production of decreased BD. Stromer noted the key role of acetate in the pathway of BD production. Acetate induces 3 enzymes, viz., acetoacetate forming enzyme, acetolactate decarboxlylase and dicetyl reductase, which are involved in the conversion of pyruvate to BD. It also activates the acetoacetate forming enzyme and regulates the balance between acetoin and BD. It was reported that acetate increased the production of BD by increasing the rate of carbohydrate utilization.

Characterization of DPW shows that it can be used as a substrate for the production of BD. Lactose present in the DPW was found to be effectively utilized by K. oxytoca, producing BD 0.259 g/g of lactose utilized. Addition of acetate at concentration of 50 mM was found to increase BD yield by 1.5-fold, resulting in BD 0.365 g/g lactose utilized. Further, 88 and 92% reduction in COD and BOD, respectively clearly indicates that this process of utilization of whey not only helps in the production of value added chemical BD from the waste, but it also helps in reducing the environmental problems faced due to disposal of untreated or unfermented whey directly into river.

Recently, microbial 2,3-BD production has attracted great attention worldwide, especially in China, as renewable biomass is a promising route for developing a low carbon economy.As a bio-polyol project with the annual output of 200,000 tons has been put into production by Changchun Dacheng Group, a new route for the production. By the independent innovation and the use of biomass as raw materials, Changchun Dacheng Group has successfully developed the catalyst technology.This catalyst has a high selectivity to the unique hydroxyl structure of biomass molecules and can turn the biomass materials like corn starch into glucose by hydrolysis.

Changchun Dacheng Group is planning to expand a corn industrial park with annual production of 1 million tons is under preparation, which can produce 60,000 tons 2,3-butanediol annually.

LanzaTech expects to commercialize carbon monoxide-based ethanol by 2013 and 2,3-butanediol by the end of 2014

The microorganism used is a bacterium in the clostridium family. The technology would allow for the production of up to 50% of 2,3

BDO by volumeand the rest in ethanol, said Holmgren.LanzaTech. It has been producing CO-based 2,3 BDO at a 15,000 gal/ year

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