Optimization of ABE Fermentation from Rice husk medium

using Clostridium acetobutylicumMentor: Dr.Aradhana Srivastava

By: Pranav DadhichUSCT-IV Year 03116101411

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

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.

Pathway for the reaction

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.

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)

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.

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.

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

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

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)

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.

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

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.

Fig 7 : Clostridium acetobutylicum

MTCC 11274

Fig 8 : Master Culture

Fig 9 : Liquid Broth

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

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

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.

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.

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

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.

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.

Experimental v/s Predicted Values

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.

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.

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

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.

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.

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.

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.

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.

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

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.

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

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