A chemically defined medium was optimised for the biomass production of Clostridium acetobutylicum in the fermentor using rice husk as the carbon source.

1. Optimization of ABE Fermentation
from Rice husk medium
using Clostridium acetobutylicum
Mentor: Dr.Aradhana Srivastava
By: Pranav Dadhich
USCT-IV Year
03116101411

2. Outline of the Presentation
• Introduction
• Pathway for ABE fermentation
• Selection and Preparation of Substrate
• Microorganism : Inoculum Preparation and
Culture Maintenance
• Culture Transfer
• Design of the Experiment
• Results and Conclusions

3. ABE Fermentation – An Introduction
• Uses bacterial fermentation to form Acetone, Butanol and
Ethanol from sugar.
• Developed by Chaim Weizmann during WW I.
• Anaerobic process.
• It produces solvents in 3:6:1 ratio where 3 parts acetone,
6 part butanol and 1 part ethanol are produced.
• Uses the strain of bacteria genus Clostridia. Clostridium
acetobutylicum is generally used.
• Potential renewable source of energy with Butanol as
biofuel.

4. Pathway for the reaction

5. Metabolic stages
• It can be divided into two major and distinct
phases [1]:
– Acidogenesis
• Occurs during exponential growth phase.
• Formation of acetate and butyrate from acetyl – CoA
and Butyryl – CoA.
– Solventogenesis
• Occurs as cell growth slows down
• Solvent production such as butanol, ethanol and
acetone.

6. Substrate : Rice Husk
• One of the most widely produced agricultural waste.
• Contains 75-90% organic matter viz. cellulose, lignin etc.
• Rice husk consists of 36–40% cellulose, and 12–19%
hemicelluloses[2].
Fig 1: Rice Husk (Powdered Form)

7. Why Rice Husk?
• Approximately, 24 million tonnes of rice husk
produced in India annually.
• Most of it is burnt or dumped in land.
• Using rice husk for fermentation will ensure
less harm to the environment which is caused
due to burning or dumping.

8. Preparation of Rice Husk
• Rice Husk was sieved using a sieve tray so as to get a
evenly distributed sample of biomass.
• The smaller and equal size of the biomass will
confirm a better digestion of rice husk (or uniform
hydrolysis).
Fig 2 : Sieve tray
• 307 grams of biomass is weighed
from the collected sample.
•This weighed quantity is then
mixed with 1200 ml of water and
incubated overnight in a shaking
incubator at 40 rpm for better
mixing and softening.

9. Acid Pre-treatment
• Hydrolysis involves cleaving the polymers of cellulose and
hemicellulose into their monomers. Complete hydrolysis of
cellulose results in glucose, whereas the hemicellulose gives
rise to several pentoses and hexoses.
• Concentrated Sulphuric acid is used for Acid pre treatment of
the rice husk.
• Concentrated acid Pre-treatment process
ensures higher glucose yield and operation
at lower temperatures.
•100 ml of concentrated Sulphuric Acid was
used for the process.
•The solution was incubated at 50°C and 20
rpm in a shaking Incubator for 3 days.Fig 3 : Sample of Acid
pretreated biomass

10. Filtration of the
Pre – treated biomass
• The Pre-treated biomass was filtered using a
filter paper and the filtrate was collected in a
beaker.
• Final volume obtained after the filtration of
the pre-treated biomass was 660 ml.
• This solution is finally used as the sugar
solution.
Fig 4 : Filtered and
pretreated sugar
solution

11. Sugar Estimation
• The methods to estimate the amount of sugar
in a sample are:
1.) Phenol Sulphuric Acid test- Gives us the total
amount of sugar present in the sample[3].
2.) DNS Acid test– Gives us the total fermentable
amount of sugars present in the sample[4].
(Reducing Sugars)

12. Microorganism : Clostridium acetobutylicum
• There are several wild strains of ABE – producing bacteria, majorly of
Clostridia family, which are gram positive, spore forming obligate
anaerobes.
• There are 4 particular species of clostridia – Clostridium
acetobutylicum, C. beijirinckii, C.saccharobutylicum and C.
saccharoperbutylacetonium.
• They possess wide substrate utilization ability and can use many types
of carbon sources ranging from glucose, sucrose, lactose, xylose, xylan,
starch and glycerol.
• They are not pathogenic or toxicogenic to humans, animals or plants.
Out of all the four strains, C. acetobutylicum has the greatest yield in the
ABE fermentation in normal conditions[5].
Fig 5 : Clostridium
acetobutylicum
• Culture was obtained from Microbial Type
Culture Collection, Institute of Microbial
Technology, Chandigarh, India; was Clostridium
acetobutylicum MTCC 11274.

13. Inoculum Formation
• The growth medium (Reinforced Clostridium Medium) for the inoculum
consist of:
– Beef Extract 10.0g
– Yeast Extract 3.0g
– Peptone 10.0g
– Dextrose 5.0g
– Soluble Starch 1.0g
– Sodium Acetate 3.0g
– Cysteine Hydrochloride 0.5g
– NaCl 5.0g
– Distilled water 1.0L
– Adjust pH to 6.8
• Divide the 1L RCM into 750 ml and 250 ml respectively.
• Add 15 g of Agar to the 250 ml RCM for solid medium.
• Autoclave both the media.
Fig 6 : Liquid and Solid RCM
medium respectively

14. Culture Transfer
• Lyophilised form of the culture is transferred into a liquid broth which
is incubated for 1-2 days at 37 °C in Laminar Flow. This is our master
culture.
• From the liquid broth the culture can be grown in two forms:
– Solid Agar medium :
• Plates are made using the RCM containing agar. Agar plates were prepared
aseptically.
• 4-5 plates are filled with the medium and a drop of culture from master culture
is spread with the help of spreader aseptically.
• Finally the plates are kept for incubation at 37 °C.
– Liquid Broth :
• A drop of culture is transferred into the liquid RCM under laminar hood.
• Culture from plates can also be used to make the same with the help of loop.
• The liquid broth is kept for incubation at 37 °C and 40 rpm in a shaking incubator.

15. Fig 7 : Clostridium
acetobutylicum
MTCC 11274
Fig 8 : Master Culture
Fig 9 : Liquid Broth

16. Preparation of CBS Fermentation
medium
• Central Bureau Seer (CBS) medium is synthetically designed for anaerobic
growth of the bacteria by TU Delft, Netherlands. [6]
• CBS medium comprises of 4 solutions:
– Saline Solution :
• (NH4)2SO4
• KH2PO4
• MgSO4·7H2O
• Autoclave and store at room temperature.
– Trace Metal Solution
• EDTA
• Calcium Chloride
• Zinc Sulphate
• Ferrous Sulphate
• Boric Acid
• Manganese Chloride
• Sodium Molybdate
• Cupric Chloride
• Copper Sulphate
• Pottasium Iodide
• Adjust pH at 4.00 with NaOH, autoclave and store at 4 °C

17. Preparation of CBS Fermentation
medium
– Vitamin Solution :
• Dissolve 25mg d – Biotin in 0.1 M NaOH.
• Add 400ml water and adjust the ph to 6.5.
• Add the following:
– P-Amino Benzoic Acid
– Nicotinic acid
– Ca- panthanoate
– Pyrodixine, HCl
– Thiamine, HCl
– Adjust pH to 6.5 and add m-Inositol.
– Adjust pH to 6.5 and transfer it into an autoclave reagent bottle at
4 °C.
– Tween 80 Solution:
• Add Tween 80 to pure ethanol for making solution.4

18. Design of the experiment
• Design of the experiment for the optimization of ABE is done using
Design Expert 9.0, Statease Inc, USA.
• The chemically defined CBS medium was optimized for biomass
production of Clostridium acetobutylicum by using rice husk as the
carbon source and (NH4)2SO4 as the nitrogen source.
• Box – Behnken Method was used for the experiment design.
• 4 factors were varied and hence varying concentrations for different
factors were obtained.
• The concentrations of saline solution (KH2PO4 and MgSO4·7H2O),
Ammonium Sulphate solution, vitamin solution and trace metal
solution in the medium were optimized by response surface
methodology using a four factor, three-level Box–Behnken design[7].
• A total of 20 experimental runs with different combinations of four
factors were obtained and carried out.

19. Implementation Of Design
• Applying Box-Behnken method, 20 batches of the synthetic CBS
medium were prepared.
• Batches were divided into 2 equal halves for better estimation.
• 3 ml sugar solution was introduced in every batch and the volume was
build up till 10 ml by distilled water.
• The pH of every batch was maintained at 6.8 for optimum growth.
• A drop of Tween 80 solution was added in every batch for segregation.
• The batches were autoclaved and 0.6 mL Clostridium acetobutylicum
was added to every batch.
• The batches were left for incubation in a shaking incubator at 37°C
and 40 RPM for 2 days.
• After optimum growth and total consumption of sugar, the biomass
build up was measured by taking the OD in spectrophotometer at 600
nm.

20. Concentration of factors and response
from Design Expert
Run
Trace Metal Solution
(ml)
Vitamin
Solution
(ml)
Saline
Solution (ml)
Ammonia Solution
(ml)
OD
(at 600 nm)
1 0.10 0.01 0.33 0.35 1.72
2 0.10 0.02 0.43 0.35 2.65
3 0.10 0.02 0.43 0.65 2
4 0.07 0.02 0.23 0.50 1.62
5 0.13 0.02 0.23 0.50 1.9
6 0.07 0.02 0.33 0.65 1.78
7 0.07 0.02 0.33 0.35 2.44
8 0.13 0.02 0.33 0.35 1.79
9 0.13 0.02 0.33 0.65 1.69
10 0.10 0.02 0.23 0.35 1.42
11 0.10 0.02 0.23 0.65 1.98
12 0.13 0.02 0.43 0.50 2.17
13 0.07 0.02 0.43 0.50 1.98
14 0.10 0.03 0.33 0.35 1.87
15 0.10 0.03 0.43 0.50 1.76
16 0.10 0.03 0.23 0.50 1.76
17 0.10 0.03 0.33 0.65 1.57
18 0.13 0.03 0.33 0.50 2.13
19 0.07 0.03 0.33 0.50 2.22
20 0.10 0.03 0.33 0.50 2.27

21. Response Curves
• The Optical Density of every run was
submitted to Design Expert as a response for
developing the response curves.
• The response curves were studied for
optimized values of all the four factors with
respect to the biomass buildup and the
optimized conditions for fermenter run were
obtained.

22. Response Curves
• The experimental results were fitted to a full quadratic
second order polynomial equation by applying multiple
regression analysis.
• OD = 3.07 - 0.18*A - 0.045*B + 0.31*C - 0.023*D + 0.14*AB
-0.022*AC + 0.14*AD - 0.31*BC - 0.13*BD - 0.30*CD -
0.62*A2 - 0.76*B2 - 0.53*C2 - 0.53*D2 + 0.53*A2B - 0.15*A2C
- 0.17*A2D + 0.30*AC2
• When the values of A-D were substituted in the above
equation, the predicted biomass production (Y) was
obtained.
• The predicted values were compared with the
experimentally obtained values, indicating that these data
were in reasonably close agreement.

23. Experimental v/s Predicted Values

24. Results
• The co-efficient of multiple determinations, R
squared was found to be 0.9992, which means
that model could explain 99.92% of the total
variations in the system.
• The relatively high value of R-squared
indicated that second order polynomial
equation is capable of representing the system
under the given experimental domain.

25. P-value
• The p value of a model
suggests that the
experimental design is
statistically significant and
not just a sampling error if
p<0.05.
• The p – value suggests the
significant models in the
experiment.

26. Curve between Trace metal solution and Vitamin solution
The optimum value for the graph was obtained by drawing perpendicular from the
top most point and extending them till the Vitamins and Trace Metal axis.
The optimized values for vitamin and trace metal according to graph are 0.02 mL
and 0.1 mL, respectively

27. Curve between trace metal solution and saline solution
The optimized condition for the graph was obtained by drawing perpendicular
from the top most point and extending them till the Saline and Trace Metal axis.
The optimized values for Saline and Trace Metal according to graph are 0.35 mL
and 0.1 mL, respectively.

28. Curve between trace metal solution and ammonium sulphate solution
The optimized condition for the graph was obtained by drawing perpendicular from
the top most point and extending them till the Ammonium sulphate and Trace
Metal axis.
The optimized values for Ammonium sulphate and Trace Metal according to graph
are 0.48 mL and 0.1 mL, respectively.

29. Curve between ammonium sulphate solution and vitamin solution
The optimized condition for graph was obtained by drawing perpendicular from the
top most point and extending them till the Vitamin and Ammonium sulphate axis.
The optimized values for Vitamin and Ammonia according to graph are 0.02 mL and
0.48 mL, respectively.

30. Curve between saline solution and ammonium sulphate solution
The optimized condition for graph was obtained by drawing perpendicular from the
top most point and extending them till the Saline and ammonium sulphate axis.
The optimized values for Saline and ammonium sulphate according to graph are 0.36
mL and 0.48 mL, respectively.

31. Curve between saline solution and vitamin solution
The optimized condition for graph was obtained by drawing perpendicular from the
top most point and extending them till the Saline and Vitamin axis.
The optimized values for Saline and vitamin solutions according to graph are 0.02
mL and 0.36 mL, respectively.

32. Conclusions
• A rice husk medium supplemented with other components
(chemically defined medium) was optimized for maximum
biomass production of C. acetobutylicum using statistical
methods.
• The optimized medium composition for maximum biomass
production was found to be 150 mL/L rice husk sugar
solution, 7.2 g/L ammonium sulfate, 0.84 g/L Magnesium
Sulphate, 3.8 g/L Pottasium Phosphate, 2 mL/L vitamin
solution and 10 mL/L trace metal solution.
• The software Design Expert was used for the response
surface methodology to obtain response curves and obtain
optimized values of the rice husk medium components

33. Optimized Composition
•For a bio fermenter run under
controlled conditions with a volume of
the tank equivalent to 2.2 L, 4.4 mL of
vitamin solution, 105.6 mL of Ammonia
solution, 79.2 mL Saline Solution and 22
mL of trace metal solution will provide
the best result of ABE Fermentation.

34. References
• [1] Yukihiro Tashiro & Kenji Sonomoto. Advances in butanol production by clostridia. Current Research,
Technology and Education Topics in Applied Microbiology and Microbial Biotechnology (Microbiology Book
Series, Volume 2), Antonio Mendez Vilas (ed.), ISBN (13): 978-84-614-6195-0, Formatex Research Center
(Badajoz, Spain), p. 1383-1394 (2010.12).
• [2] Ajay Kumar, Kalyani Mohanta, Devendra Kumar and Om Parkash, Properties and Industrial Applications
of Rice Husk: A review, International Journal of Emerging Technology and Advanced Engineering, ISSN
2250-2459, Volume 2, Issue 10, October 2012.
• [3] M. Dubois, K.A. Gilles, J.K. Hamilton, P.T. Rebers, F. Smith, Phenol Sulphuric Acid Carbohydrate
Assay, Anal. Chem. 28:(1956) 350-356.
• [4] Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal.
Chem., 31, 426, 1959.
• [5] Victor Ujor, Ashok Kumar Bharathidasan, Katrina Cornish and Thaddeus Chukwuemeka Ezeji ,
Evaluation of industrial dairy waste (milk dust powder) for acetone-butanol-ethanol production by
solventogenic Clostridium species, SpringerPlus, 2014.
• [6] Cornelis Verdyun, “Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat”,
journal of general microbiology,1989
• [7] A. Ghosalkar, V. Sahai, A. Srivastava “ Optimization of chemically defined medium for recombinant
Pichia pastoris for biomass production” , Bioresource Technology, 2008

35. THANK YOU!