ABE F ERMENTATION
4.2. Material and Methods
4.2.1. Culture maintenance and growth
Lyophilized cells of Clostridium acetobutylicum MTCC 481 were procured from MTCC (Microbial Type Culture Collection), Institute of Microbial Technology (Chandigarh, India).
These cells were maintained as spore suspension in sterile water. Three strains of Clostridium acetobutylicum NCIM 2337, 2877 and 2878 in sand have been procured from NCIM Pune (India ) in dried spore form. The cells were revived anaerobically inside an anaerobic culture bag system (Himedia) in RCA (Reinforced Clostridial Agar), and RCM (Reinforced Clostridial Medium: Broth) culture media at 37oC (Fig. 4.2). The inoculums were prepared in RCM containing following components (with concentration mentioned in g L–1): glucose, 5.0; yeast extract, 3.0; starch, 1.0; beef extract, 10.0; peptone, 10.0; sodium chloride, 5.0;
sodium acetate, 3.0; Agar, 0.5; cysteine hydrochloride, 0.5. The pH of medium was 6.8 ± 0.2.
100 mL of media was autoclaved at 121oC, 15 lb pressure and inoculated in a custom fabricated 250 mL screw capped Erlenmeyer flask (Fig. 4.3). In addition, CMM (Cooked Meat Medium) was also used for the maintenance of clostridia. Anaerobic condition in broth culture was maintained by adding 0.05% of cysteine hydrochloride, and regular sparging of
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(a) (b) (c)
(d) (e) (f)
Figure 4.4: Maintenance and culturing of clostridial species. (a) RCM Broth, (b) Cultured RCM broth, (c) clostridial slant (d) bacterial lawn (spread plate). (e) streak plate of clostridia,
(f) clostridial stab culture
through fermentation broth. All chemicals were of analytical grade procured either from Merck (Germany), Sigma Aldrich (Germany) or Himedia (India). The revived cells were maintained on RCM broth, RCA plates (bacterial lawn and streaks) and slants at 4oC, and were used as a stock (Fig. 4.4). The cells were sub–cultured every month.
Microscopic staining of clostridias via Grams stain and Malachite green (spore staining) Sterilized microscopic slides, crystal violet dye, distilled water, Lugols iodine, ethanol, acetone tissues, safranin, oil immersion, microscope (Carl Fischer, Germany) and fresh Clostridial cultures were required for staining. All the dyes and chemicals were procured from Himedia, Germany. There are four basic steps of the Gram staining as follows: (1) application of a primary stain (crystal violet) to a heat–fixed (death by heat) smear of a
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bacterial culture, (2) addition of a trapping agent (Gram's iodine), (3) rapid decolorization with alcohol or acetone, and (4) counter–staining with safranin.
Malachite staining requires sterilized microscopic slides, saturated solution of Malachite green dye, distilled water, hot plate/oven/water bath, tissues, safranin, oil immersion, microscope and old clostridial cultures (10 days +). Staining initiates with making a smear of the C. acetobutylicum species by air–drying and heat–fixing. Smear was then steamed, and flooded with the primary dye, malachite green, and left for 15 min. Extra moisture was removed with help of paper towel. The slide was steamed over boiling water for some time and again washed thoroughly with water and finally counter–stained with safranine.
4.2.2. Preparation of biomass hydrolyzate
Four biomasses, viz. fruit waste, bagasse, rice straw and rice husk were procured from local areas of Guwahati. All four biomasses were initially washed with water to remove the impurities, and then were allowed to dry at 50oC for 48 h in a hot air oven (JSJW, India).
Dried biomass was further chopped and grinded into small sizes using a mixer grinder (Sumeet, India). 3% w/v solutions of all four processed biomass were hydrolyzed using 0.5%
v/v of sulfuric acid, and was agitated at 150 rpm at 60oC using a shaker incubator (Sciegenics, India). Resulting hydrolyzate was filtered using a sterile muslin cloth, and filtrate was used as feedstock for fermentation processes.
4.2.3. Preparation of fermentation broth for selection of Clostridial strain
Reinforced Clostridial Agar medium (broth) was prepared as stated in section 5.2.1.
Before autoclaving, broth was supplemented with 2.0% of glucose. All four strains of C.
acetobutylicum were allowed to undergo fermentation in this glucose supplemented synthetic medium. Culture was selected on the basis of its ability to yield high amount of solvents, with
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4.2.4. Preparation of fermentation broth with varied glucose concentration
Experiments were performed in 4 set of flasks, each containing 2, 4, 6 and 8% w/v of glucose solution. All reagents were of analytical grade. The concentrations of all other components of RCM medium as mentioned in section 4.2.1 (except glucose) were kept constant, and the glucose concentration was varied as 2, 4, 6 and 8% w/v. Experiments were conducted in this medium with MTCC 481 strain incubated at 37oC at 120 rpm for 12 days.
4.2.5. Preparation of fermentation broth with varied xylose concentration
Experiments were performed in 4 set of flasks, each containing 2.5, 4, 5, and 6% w/v xylose. All reagents were of analytical grade. The concentrations of all other components of RCM medium as mentioned in section 4.2.1 (except xylose) were kept constant, and the xylose concentration was varied as 2.5, 4, 5 and 6% w/v. Experiments were conducted in this medium with MTCC 481 strain incubated at 37oC at 120 rpm for 12 days.
4.2.6. Fermentation
Batch fermentation experiments were carried out with 100 mL of working volume of fermentation broth in custom fabricated 250 mL screw–capped Erlenmeyer flasks (Fig. 4.4).
These flasks had a bottom port for sample withdrawal and nitrogen sparging in order to minimize oxygen contamination and maintain strict anaerobic conditions. Anaerobic condition in the flask was generated by addition of 0.5% w/v cysteine hydrochloride to the fermentation medium. An initial sample (0 h) was taken immediately after pretreatment for sugar analysis. Regular samples were withdrawn from the broth to study the growth, sugar release and utilization and solvent production by Clostridium acetobutylicum. All flasks were sparged with nitrogen at the start, and after every 24 h of fermentation to maintain anaerobic conditions. The samples of fermentation broth were withdrawn at constant intervals upto a period of 12 days. Each experiment was conducted in duplicate to assess the reproducibility of the results.
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Figure 4.5: Anthrone test for quantitative analysis of total sugar
Figure 4.6: Glucose oxidase assay for quantitative estimation of glucose
4.2.7. Analysis
The optical density of the cells in the fermentation broth was measured using UV–Vis spectrophotometer (Thermo Fischer) with absorbance at 600 nm after appropriate dilution in water. Quantification of total sugar was done by Anthrone test (Fig. 4.5) as directed by Hedge and Hofreiter [9]. Glucose was analysed using Glucose (GO) assay kit procured from Sigma Aldrich, USA (GAGO20–1KT) (Fig. 4.6). All the samples were filtered with 0.2 μm filter and diluted appropriately for the qualitative and quantitative determination of the
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fermentation products, viz. acetone, butanol and ethanol. Solvent production in the fermentation broth was monitored on a gas chromatograph (Varian) using a CP Wax 52CB (250 mm × 0.25 mm × 0.39 mm) capillary column, and a Flame Ionization Detector. The injector and detector temperatures were 230 and 250oC, respectively. The oven temperature was programmed from 45 to 100oC with an increment of 3oC/min, and after 100oC, an increment of 5oC/min up to 200oC.
Standard curve plot for quantitative estimation of acetone, butanol and ethanol using gas chromatograph
Standard curve for quantitative estimation of acetone, butanol and ethanol solvent was plotted using GC grade standard acetone, butanol and ethanol solvents from Sigma Aldrich. Standards plots achieved for all the three solvents are mentioned in Fig 4.7 to 4.9.
Figure 4.7: Standard plot for quantitative estimation of acetone
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y = 1E+06x R² = 0.9469
0 500000 1000000 1500000 2000000 2500000 3000000
0 0.5 1 1.5 2 2.5
A re a u n d er gas c h rom at ogr am
Standard butanol concentration (in %)
Figure 4.8: Standard plot for quantitative estimation of butanol
Figure 4.9: Standard plot for quantitative estimation of butanol
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C. acetobutylicum MTCC 481 (400×) C. acetobutylicum NCIM 2337 (400×)
C. acetobutylicum NCIM 2877 (400×) C. acetobutylicum NCIM 2878 (1000×)
Figure 4.10: Gram staining of four clostridial strains
4.2.8. Estimation of yield and selectivity
Solvent yield was calculated as gram per liter of solvent produced per gram litre of total sugar added (g/g). Butanol selectivity was calculated as mol of butanol produced per mol of total solvent (ABE) production.