The document summarizes a study on the bioconversion of whey to 2,3-butanediol using Klebsiella oxytoca NRRL-13-199. It describes how K. oxytoca was able to effectively utilize lactose in whey permeate to produce 2,3-butanediol, achieving a yield of 0.259 g/g lactose utilized. The addition of acetate at 50 mM concentration increased butanediol yield 1.5-fold to 0.365 g/g lactose utilized. The process also led to 88% and 92% reductions in COD and BOD, respectively, indicating it can help reduce environmental pollution from whey waste.

1. “Bioconversion of whey to 2,3-butanediol using Klebsiella oxytoca
NRRL-13-199”
Submitted to
The Principal ,
Mahatma Gandhi Mission
College of Agricultural Biotechnology , Aurangabad
(Affiliated to Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani)
(ISO 9001: 2008 and ISO 14001: 2004 Certified Certified)
Bachelor of Science
In
Agricultural Biotechnology
Submitted by
INGLE SNEHAL DEVIDAS
Reg.No.- MGM/CABT/10/30 Semester -VII (New)
Exam Seat No.- 3274 Course No.- CS-4713
Guided by
Mr. G. V. KORE
Assistant Professor
Department of Biochemistry and molecular biology

3.  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.

4.  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.

7. 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 and
3.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 300
C for 24 h. This
culture was used as Inoculum (1% v/v)

8.  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 40
C 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

9. 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.

10. 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 200o
C, respectively.

13. 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

14. 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.


15. 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

16. 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.

17. 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.

18. 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.

19.  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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