The earliest and leading liquid energy carriers are ethanol and a mixture of acetone, butanol and ethanol. If these solvents are produced using a practical fermentative processes, it will decrease environmental pollution and reduce dependency on fossil fuels (Ezeji et al., 2007). There are two major biological processes that can convert biomass to liquid energy carriers via anaerobic breakdown of organic matter: ethanol fermentation and mixed acetone, butanol, ethanol (ABE) fermentation.
Ethanol is the most widely used liquid energy carrier produced biologically from three types of biomass - (i) starch and (ii)sucrose containing energy crops such as sugarcane, corn, etc (1st generation bioethanol technology) and (iii) lignocellulosic residues/wastes (2nd generation bioethanol technology). First generation bioethanol technology is conventional and well-established with majority of the ethanol is produced by this technology. Nearly all fuel ethanol is produced by fermentation of either corn glucose (in the USA 5 billion liters of ethanol are produced annually) or sucrose (in Brazil 12.5 billion liters of ethanol are produced annually). 2nd generation biofuels are gaining impetus recently (Karakashev et al., 2007).
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Butanol, ethanol and acetone are major basic commodity chemicals consumed in bulk in a variety of ways, e.g., as fuels, fuel additives and solvents. Before the 1950s, ABE fermentation ranked second to ethanol in its importance and scale of production; but this declined due to increasing substrate costs and the availability of the much cheaper, petrochemically-derived butanol. Currently, the ABE fermentation process is operated commercially only in China (Karakashev et al., 2007).
Butanol is a fuel that can be produced from agricultural crops such as corn, molasses, etc. using Clostridium acetobutylicum or C. beijerinckii. The advantage of using these and some other butanol-producing bacteria is that they can utilize both ligno-cellulosic hydrolysate sugars (hexoses and pentoses) as opposed to traditional ethanol-producing yeast strains that cannot use pentses. Butanol has properties that other fermentation-derived fuels do not have, including ethanol (Qureshi & Ezeji, 2008).
Butanol has similar characteristics to gasoline to be used directly in any gasoline engine without modification and/or substitution. Butanol is superior to ethanol as a fuel additive in many regards: higher energy content, lower volatility, less hydroscopic and less corrosive (Karakashev et al., 2007). The energy content of butanol is 30% more than ethanol and is closer to gasoline, its low vapor pressure facilitates its application in existing gasoline supply channels, it is not sensitive to water, less hazardous to handle, less flammable and can be mixed with gasoline in any proportion (Qureshi & Ezeji, 2008).
Butanol (ABE) production by fermentation is one of the oldest fermentation processes employed for commercial production of a chemical to benefit mankind. In ABE fermentation the carbohydrate substrate is converted to a mixture of solvents: acetone, butanol and ethanol, in the approximate ratio 3:6:1, at a total solvent concentration of around 20 g /L (Karakashev et al., 2007).
A typical feature of the clostridial solvent production is biphasic fermentation. The first phase is the acidogenic phase, during which the acids forming pathways are activated, and acetate, butyrate, hydrogen, and carbon dioxide are produced as major products. This acidogenic phase usually occurs during the exponential growth phase. The second phase is the solventogenic phase during which acids are reassimilated and used in the production of acetone, butanol and ethanol. The transition from acidogenic to solventogenic phase is the result of a striking change in gene expression pattern (Lee et al., 2008).
Solventogenesis is closely coupled to sporulation. The transcription factor responsible for initiation of sporulation (Spo0A) also initiates solvent production in C. acetobutylicum by activating transcription of acetoacetate decarboxylase (adc), alcohol dehydrogenase (adhE), and CoA transferase (ctfAB) genes. Spo0A deletion mutants are severely deficient in solvent production and fails to septate, while strains with amplified Spo0A overexpress solventogenic genes but fail to produce more solvent due to an accelerated sporulation process (Lee et al., 2008).
The three major factors that impede the growth of ABE fermentation are: (i) high costs of the substrate (e. g. molasses), (ii) low product yield (23% of total carbon consumed and solvent toxicity) and (iii) high product recovery costs (e.g. distillation). In order to for the process to be economically competitive and be reintroduced for large scale production of butanol, these drawbacks need to be overcome. The 2nd generation biofuels utilize low cost non-feed substrates like LBM and agro-wastes (Lee et al., 2008). Also there has been a lot of research on development of new processes in fermentation technology and recovery methods since 1980 (Ezeji et al., 2007). With the new tools of genetic engineering and molecular biology, organisms can be developed that will have better yield and higher tolerance towards solvents (Lee et al., 2008). Thus, put all together into an integrated process, ABE fermentation can allow establishment of an economically feasible and environmentally friendly industrial process for butanol production.
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In the past 20 years, there have been numerous engineering attempts to improve butanol production in ABE fermentation which includes the use of cell recycle and cell immobilization to increase cell density and reactor productivity respectively and using extractive fermentation to minimize product inhibition. Despite all these efforts, the best results ever obtained for ABE fermentations to date are still less than 15 g/L butanol concentration corresponding to 4.5 g/L/h productivity, and yield of less than 25 % (w/w) on glucose (Ramey & Yang, 2004).
In Chapter 2, there is a thorough review on the various aspects of ABE fermentation and there are many methods through which butanol production can be made economically competitive. Keeping that in mind the present work was initiated to isolate a butanol producing strain that can be grown on a variety of substrates with minimization of costs involved in the fermentation media. The objective of thtion e present work is as follows:
To identify (through literature survey and in silico analyses) solvent producing strains of bacteria or fungi species for the production of butanol.
To optimize the fermentation parametersfor maximum production of butanol using substrate like abc xyz mnp concentration, pH, temperature, etc. To increase the solvent formation and / or solvent resistance using mutagenesis, leading to strain improvement.
To analyze various fermentation types like fed-batch, continuous system integrated with continuous product removal so as to maximize the yield as compared to original batch fermentation.
Analytical method for quantification of solvents
The important operation in butanol fermentation is the isolation and quantification of solvents. The commonly available methods for quantification of solvents are HPLC (high- performance liquid chromatography) and GC (gas chromatography). Both these methods are extended applications of column chromatography. Column chromatography is the classical procedure developed by Tswett in 1906 (when researching on plant pigments) called as open column chromatography, is a type of liquid chromatography in which the mobile phase is allowed to flow through the packed column under the influence of gravity or at most, low pressure. The mode of separation depends primarily on nature of the stationary phase. The five modes available are adsorption, partition, ion exchange, size exclusion and affinity.
High performance liquid chromatography (HPLC): This is a type of liquid chromatography in which mobile phase is forced through the packed column under influence of high pressure (1000 to 3000 psi). In HPLC, particle diameter is typically 10 Î¼m or less and as a result columns are packed more tightly. The stationary phase may be solid or a liquid coated on an inert material. The most widely used stationary phase is silica based. Reversed phase packing material is produced by bonding of octadecylsilyl groups to silica gel. The elution of components may be isocratic or gradient. Recent introduction of chiral stationary phases, allows separation of enantiomers from racemic mixtures. It has become the most versatile, safest and sensitive chromatographic technique for quality control of various drug components.
Basic components of HPLC system are:
Reservoirs: These are glass or stainless steel containers capable of holding up to 1l of mobile phase.
Pump: Pump can be either mechanical or pneumatic.
Gradient Controller: essential for gradient analysis.
Solvent conditioning column.
Injector: Rotary valve and loop injector capable of applying predetermined volumes are used.
Pre-column / Guard column: It is used mainly to protect main column by trapping particulate matter and retaining substances.
Analytical column: It is a stainless steel tube, usually of 5-25 cm in length and 2 to 4.6 mm in internal diameter, packed with stationary phase.
Detectors: The most commonly used detector is UV- visible spectrometer. A refractive index detector is used for analysis of solvents and sugars. Photodiode detectors, light scattering detectors, and detectors based on electrochemical methods such, as amperometry, coulometry and polarography are also available.
Gas chromatography (GC): GC is divided into two classes depending upon the nature of stationary phase. These are -
Gas-solid chromatography (GSC) - in which, the stationary phase is a solid adsorptive material and solute particles are removed from the mobile phase by electrostatic forces.
Gas-liquid chromatography (GLC) - in which, stationary phase is a thin layer of a liquid, usually as a coating on the surface of inert particles. In this method, solute molecules are retained in the liquid phase based on their partition coefficients between it and the gaseous mobile phase.
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GC finds its main application with volatile components, fatty acids, mono and sesquiterpenes, hydrocarbon and sulphur compounds. The developments in stationary phase synthesis and capillary column technology have opened new perspectives in the analysis of high molecular weight compounds and thermo labile organic compounds by high temperature - high resolution gas chromatography (HT-HR GC). This branch deals with analysis performed up to 390°C oven temperature.
GC can be used for both quantitative as well as qualitative analysis. Qualitative analysis can be accomplished in either of two ways; by comparing the retention parameters of the unknown with known compounds or by subjecting the column effluents to classical chemical or spectrometric methods. The parameter, which is proportional to the concentration of a compound in the GC effluent, is the area under the elution peak, which is the integral of the elution curve from the point where it leaves the baseline to the point where it returns.
The present work was undertaken to focus on the callus induction and generation of biomass and understanding the correlation between the levels of the PGRs and the production levels of gymnemic acids at callus stage. Hence, in order to fully understand the optimization profile in terms of PGR levels, a sequential approach was required.
Materials and methods
After the complete literature review it was determined that Clostridium acetobutylicum were to be used for butanol fermentation. Work done on bio-butanol project was initiated when the strains of Clostridium acetobutylicum were procured. in form of stabs from National Collection of Industrial Microorganisms (NCIM), Pune, India
C. acetobutylicum NCIM 2337
C. acetobutylicum NCIM 2877
C. acetobutylicum NCIM 2878
While these strains exhibited substantial growth in the complex media, it failed to grow in the synthetic medium. Also, growth was not correlated with a concomitant production of butanol. Hence, work on these strains was discontinued and fresh strains were procured from Northern Regional Research Laboratory (NRRL), USA. They were:
C. acetobutylicum NRRL B527
C. acetobutylicum NRRL B530
C. bejerinckii NRRL B594
C. saccharobutylicum NRRL B643 [butyric acid producing strain]
C. butyricum NRRL B1024 [butyric acid producing strain]
Initial work began on only the solvent producing strains. Strains of C. acetobutylicum NRRL B527 & NRRL B530 were isolated from soil by McCoy et al. in 1920 and NRRL B594 was isolated from soil by Donker et al. in 1926. NRRL B527 & NRRL B530 were designated as ATCC 824 & ATCC 4259 respectively while NRRL B594 was designated as ATCC 10132. The NRRL strains arrived as lyophilized spores.
The cultures were revived at 37°C under anaerobic condition using cooked meat medium (CMM). Stock cultures were maintained as spores in 20% glycerol in reinforced clostridial broth (RCB) at -80°C. To start the cultures, spores (1 mL aliquots) were heat-shocked in 80 °C water bath for 10mins, and cultures were grown anaerobically at 37°C in CMM.
All the inorganic salts, sucrose, glucose, fructose, xylose, starch, ammonium acetate and ammonium sulfate were analytical grade and were procured from Sisco Research Laboratories (SRL) Pvt. Ltd., India. Vitamins, agar and other fine chemicals were S.D. Fine-Chem Ltd., Mumbai, India. Yeast extract, medium components and dehydrated media were bought from Hi-media Laboratories Pvt. Ltd., India. Membrane filters were procured from Rankem (formerly RanBaxy Fine Chemicals Ltd.) as syringe filters.
Media compositions This is not a place to discuss media composition this should be added in last Appendix section!! Here you can add significance of the media wrt clostridia one has been worked as an example for you.
Reinforced Clostridial Broth (RCB): Reinforced Clostridial Broth is formulated by Hirsch and Grinsted (1954) for selective isolation of Clostridia, Casein enzymic hydrolysate, yeast extract, beef extract, starch, L-cysteine and sodium acetate provide all the necessary nutrients for the growth of Clostridia . Dextrose is a fermentable carbohydrate in the medium while sodium chloride maintains osmotic equilibrium. This media can be made selective by addition of 15-20 mg Polymyxin B per litre of media. (Barnes and Ingram 1956)
Cooked Meat Medium (CMM):
Semi-defined synthetic medium
Maintenance of anaerobic conditions:
The initial anaerobic conditions were maintained by layering filter sterilized paraffin oil over the cuture medium and then placing the medium in a desiccator. This desiccator was closed tightly with a burning candle inside. It resulted in % CO2 environment. The method was useful for growing stabs of Clostridia. Cultures failed to grow in broth by these anaerobic conditions.
To obtain good growing clostridia anaerobic jars and anaerobic packs were procured from Hi-media Lab. Pvt. Ltd. code # LE013 and LE002F respectively. Anaerobic conditions were maintained by withdrawing air out of the jar using vacuum pump and then flushing in nitrogen. These jars were placed into incubators at desired temperatures. It was an effective method for providing anaerobic conditions for both liquid broths and solid agar plates. (Photograph with plates inside)
Anaerobic work station procured from Ruskinn Technology Ltd., (UK) sold as Anaerobic Bug-Box. It is small air-tight cabinet attached to nitrogen and mixed gas (80% N2, 10% H2 and 10% CO2) cylinder. It is a bench-top station which has a thermostat and an anaerobic compartment for working and incubating the culture. (Photograph????)
Preparation of inoculum:
Clostridial spores were grown in CMM for 6 days under anaerobic conditions at 37Â°C. After there is enumeration of the culture, this culture broth is heated in water bath at 80Â°C for 10mins and rapidly cooled to room temperature. Then 10 ml is added to RCB to make final volume of 100ml and incubated for 48hrs under anaerobic conditions at 30Â°C. This is used as inoculum for further experiments. Inoculum transfer in each case was 10% (v/v).
Spore staining and Gram staining:
Clostridial spore staining was done to ensure the presence of sporulation and any changes in morphology of the bacterium due to experimentation. Spore staining was done using 5% (w/v) Malachite green and 0.5% (w/v) safranine. The smear prepared from the culture is heat-fixed on the slide and malachite green (primary stain) was flooded onto the slide which was kept at 100Â°C for 5 min; malachite green was reapplied if the stain dries out. After the heat treatment the slide was washed with water till water runs clear. Then the slide was flooded with safranin for 30 secs and rinsed with water. The sample was dried and observed under oil immersion (1000x TM) of compound light microscope. Results not here!!
Gram staining kit was procured from Hi-media Lab. Pvt. Ltd., code # K001. Grams crystal violet (primary stain) is flooded on the smear for 30sec and excess is removed. Grams iodine is then flooded onto the slide for 30sec and excess is removed. Grams decoloriser (95% ethanol) is used for rinsing and then safranin is added as a counter stain for 30sec. The slide is rinsed with water and dried gently and then observed under oil immersion (1000x TM) of compound light microscope.
Aliquot removal and preparation of sample for analysis:
The cultures maintained under anaerobic conditions are temporarily removed from the jar or bug-box and aliquots of _____ ml were removed in laminar air flow under sterile conditions. The cultures were then quickly replaced back to anaerobic jar or bug-box so as to provide minimum oxygen exposure. For pH and cell concentration measurements, minimum of 3ml broth was required and for fermentation product analysis 1ml broth was removed. The collected aliquots were centrifuged at 10,000 rpm at 4Â°C for 10mins. The supernatant was analyzed for pH and fermentation product analysis. Samples for GC analysis were filtered through 0.2 micron membrane. Before analysis .
Analysis of fermentation broth
Determination of pH
The aliquot of the fermentation were first analyzed for its pH, using a portable hand-held pH meter with resolution of 0.01 pH. The pH meter was calibrated with standard pH buffer capsules before use (pH 7 and 4.2). A minimum of 3ml sample was required for appropriate analysis. The pH meter was Hanna Ins.
Estimation of cell concentration
The biomass studies were done using a spectrophotometer (Labtronics Model LT-12) at a wavelength of 660 nm. . A standard curve was plotted for the culture and a factor of 0.1435 was obtained from the straight line. which was correlated to give cell concentration. .
Estimation of fermentation products
HPLC - High performance liquid chromatography
The analysis of fermentation products was done by injecting 10Âµl of the aliquot in to the HPLC column of Bio-Rad Aminex 87H Ion exclusion column with 5mM H2SO4 as the mobile phase and refractive index (RI) detector. The flow rate was 0.6 ml/min and the column was maintained at temperature of 50Â°C. The run time was 50 min and retention time for glucose, acetic acid, butyric acid and butanol was 9.2 min, 15.8 min, 23.4 min and 37min, respectively. Acetone was detected at 23.8 min in the above system. Thus the fermentation broth samples with higher concentration of acetone or butyric acids would lead to inter-merging of peaks. to avoid this GC was used as it provided better resolution. The initial results are reported from HPLC analysis. A standard curve for each of the compounds was plotted and was used for determination of concentrations of the fermentation products. Xylose was detected using the same method and had retention time of 9.1min.
GC - gas chromatography
The concentrations of solvents (ethanol, acetone, and butanol) and acids (acetic and butyric) were determined by injecting centrifuged samples into Agilent 7890A GC System equipped with a flame ionization detector (FID, equipped with capillary colum DB-624. with 30m length, internal diameter of 0.53 mm and matrix film thickness of 3 microns. The analysis of products was carried out at injector temperature, 150Â°C; detector temperature, 210Â°C; N2 (carrier gas) flow rate, 1 ml/min; column temperature, was 0-4 min 40Â°C then a gradient of 10Â°C/min to reach maximum temperature of 190Â°C and maintained at 190Â°C for 2min. The fermentation products were resolved and had retention time of 3.5 min, 4.1min, 9.2min, 10.4min and 13.6 min ethanol, acetone, butanol, acetic acid and butyric acid, respectively. Iso-propanol was used as internal standard.
Results and discussion
Now whatever you are writing in materials and method results of which should come here in same sequence!!
Strain selection for optimization:
NCIM strains were able to grow only under strict anaerobic conditions and in complex medium. The culture failed to grow in synthetic medium supplied with yeast extract and glucose. Only NCIM 2877 as it was able to grow in synthetic medium which was supplemented with beef extract and could produce 1.8 g/L butanol. Thus comparisons between strains were done using complex medium. The parameter for strain selection was butanol yields and productivity. NCIM 2877 and 2377 were able to grow in RCB and could produce butanol up to 2 g/L. NCIM 2878 was able to grow in complex medium but failed to form solvents. Spore staining showed the presence of growth.
Of the three solvent producing strains from NRRL (B527, B530 and B594), only two could be revived (B527 and B594) from the procured lyophilized spores. NRRL B594 failed to produce solvents. These were studied for butanol production along with NRRL strains. The comparison is given in table 1.
Cell Conc. (g/L)
Glucose consumed (g/L)
Butanol yield (g/g glucose)
Table 1: Comparison of performance on day 6 of different strains on Clostridium acetobutylicum
These are the solvent concentrations obtained after 6 days (fermentation cycle) of incubation under anaerobic conditions at 30Â°C in RCB supplemented with glucose. NRRL B527 evidently produced higher concentrations of butanol and hence was selected for further studies.
Effect of temperature on growth:
The different temperatures studied were 20, 25, 30, 37 and 42Â°C. The cultures were grown in semi-defined synthetic medium for 96hrs under anaerobic conditions. Butanol yield and productivity were measured after 96hrs and given in graph 1. At lower temperatures there was reduced butanol production (< 4 g/L) while at 42 Â°C there was reduced cell biomass (< 3 g/L) and consequently no butanol ormation. Temperatures between 30-37 Â°C are optimum for growth and butanol production. Productivities were higher in 37 Â°C (0.082 g/L/h) compared to 30Â°C (0.079 g/L/h). While the yields were comparable, final butanol concentrations were higher at 30 Â°C (7.7 g/L as compared to 7.2 g/L at 37 Â°C). Hence 30 Â°C was used for experimentation for further studies.
Graph 3.1: Effect of temperature on butanol concentration
Effect of pH on growth:
The cultures were grown in semi-defined synthetic medium at varying initial pH. Ceteris paribus all the cultures were grown under anaerobic conditions for 96hr at 30 Â°C. Butanol yields and productivity were observed at the end of 96 hr are given in graph 3.2. NRRL B527 fails to grow at alkaline pH range; while the butanol production is drastically reduced when initial pH was neutral (3.26 g/L as compared to 7.3 g/L). Also as the initial pH goes on reducing the amount of butanol produced is reduced. This could be because as the culture grows it produces acids and thus leading to acid crash. Maximum butanol productivity of (0.09 g/L/h) is obtained when the initial pH of semi-defined synthetic medium was not changed. The optimum pH for butanol production range lies from 6.2 to 6.5.
Graph 2: effect of pH on butanol concentration of ----- species
Effect of inoculum on growth and butanol production:
The effect of source of the inoculum and the volume of the inoculum on growth and solvent production was studied. four types of inoculum development were carried out. In this, inoculum was transferred to the semi-defined synthetic medium from:
a) RCB to synthetic medium (5% v/v)
b) RCB to synthetic medium (10% v/v)
c) Semi-defined synthetic medium to semi-defined synthetic medium (5% v/v)
d) Semi-defined synthetic medium to semi-defined synthetic medium (10% v/v)
These were incubated for 96hr under anaerobic conditions at 30 Â°C. Fermentation yields observed at the end of 72 hr are given in graph 3.3. It was observed that, even though maximum butanol was observed in inoculum from RCB to semi-defined medium (9.8 g/L) in all the types of inoculum there was almost complete consumption of glucose and butanol yield of 0.23 g/g glucose consumed. It shows that any of the above inoculation type can be used for fermentation. Inoculums of higher volumes were also studied. Addition of 15% and 20% inoculum showed that the yield decreased to 0.129 and 0.115 g/g glucose consumed, respectively (compared to 0.167 g/g glucose consumed of 10% inoculum) indicating negative effect on butanol production, which was also observed by â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦..!!!!. RCB (10% v/v) was used for experimentation for further experimentation.
effect of the medium added along with inoculum was also studied. 10% v/v transfer from RCB to semi-defined medium there could be chance that it could affect the butanol yields, especially due to the presence of acids produced during growth in RCB. Hence inoculum was added to the semi-defined medium as follows:
a) Control (from RCB to synthetic medium (10% v/v))
b) Cell pellet (from RCB pellet is collected and transferred into synthetic medium)
These were incubated for 96hr under anaerobic conditions at 30 Â°C. Butanol yields were observed at the end of 72 hr is given in graph 4. It can be observed that butanol concentrations in RCB are higher in volumetric transfer (9.95 g/L) as compared to cell pellet transfer (6.09 g/L) and also the productivity is higher with volumetric transfer of inoculum (0.13 g/L/h as compared to 0.09 g/L/h). This lower productivity could also be attributed due to the aerobic shock the culture gets during centrifugation.
From graph 5 it is evident that, even though when cell pellet was used as inoculum it did not affect the acetic acid or butyric acid profile and acid concentrations resumed to normal within 6 hrs of fermentation. The amount of acid produced remains almost constant after 24hr of inoculation. This phenomenon was observed in all results obtained in the study. Also there was no uptake of butyric acid during solventogenesis observed. In all studies, henceforth, acid concentrations are noted but it was observed that there was no quantifiable effect of acids produced during acidogenesis on butanol production. This result was comparable withâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦..!!!
Graph 3: Effect of inoculum source and inoculum volume on butanol production
Graph 4: Effect of inoculum (cell) pellet on butanol production
Graph 5: Effect of inoculum (cell) pellet on acid fermentation profile
Effect of butanol on growth:
Butanol is toxic to Clostridia and therefore the inhibition concentration of butanol has to be determined. A range of initial butanol concentration was used. Butanol was added to the semi-defined medium and the culture was inoculated under anaerobic conditions at 30 Â°C and observed after 72hr. Growth observed after 72hr of incubation is given in graph 3.6. It can be concluded that there is complete inhibition for growth above 1.25%.
Graph 6: Effect of butanol on growth
Effect of butyric acid on growth and its effect on butanol production:
Butyric acid is an important intermediate to Clostridia metabolism and therefore its effect on butanol production was studied. Butyric acid was added to the semi-defined medium and the culture was inoculated under anaerobic conditions at 30 and observed for 72hr. Growth observed after 72hr of incubation id given in graph 7 there is complete inhibition for growth above 50mM butyric acid. Above 30mM butyric acid there was a drastic decrease in butanol concentration. There has been no uptake of butyric acid observed during fermentation. Thus if butyric acid or sodium butyrate is supplied in the medium, it will not result in increase in butanol production with NRRL B527 strain.this is in comparison with It was also observed that addition of butyric acid after 48 hr of growth in semi-defined medium restricted butanol yield to 0.17 g/g glucose consumed.
Graph 7: Effect of butyric acid on butanol production and growth
Effect of glucose concentration on growth and determination of optimal glucose concentration:
It is reported that, usually glucose concentrations of 6% is used in butanol fermentation . Similar results were obtained using NRRL B527. At lower glucose concentrations (<2%) the butanol yields dropped to 0.08 g/g glucose consumed and a productivity of 0.03 g/L/h (at 0.5% glucose). The inhibition concentration of glucose was 20% glucose, i.e. at a glucose concentration of 20% and above there is no growth observed. A drastic decrease in yield is observed above 10% glucose concentrations. Thus glucose from the range of 5 g/L (0.5%) to 75 g/L (7.5%) was observed and an optimum range of was identified between 3% to 6%. The fermentation yields after 72 hrs of incubation is given in graph 8a. Results o of effect of glucose in the optimum range is given in graph 8b.
Graph 8a: Effect of glucose on fermentation yields
Graph 8b: Effect of glucose on fermentation yields
It is observed that complete consumption of glucose in semi-defined medium occurs up to 40 g/L in 72hr at 30 Â°C by NRRL B527. The butanol yields are within the range of 0.23-0.25 g/g glucose consumed in the optimum glucose concentration range (3% to 6%). Any higher concentrations the butanol yields decreased concomitant with increased butyric acid production, while any lower butanol concentrations had lower butanol yields and decreased cell biomass. The glucose concentration with maximum butanol yield and productivity is 30 g/L with a yield of 0.24 g/g glucose consumed.
Effect of nitrogen on growth:
There are two nitrogen sources in semi-defined medium: yeast extract and ammonium acetate. On deletion of these components from the medium it was observed that on removal of yeast extract there was a marginal effect on fermentation yield while removal of ammonium acetate had a drastic effect on growth and butanol production. The biomass and butanol concentration had reduced by 50% as compared to normally achieved levels. The fermentation yield of deletion of yeast extract and ammonium extract from semi-defined medium at 72 hr is given in table 2. Ammonium acetate is essential component in semi-defined medium for butanol production by C. acetobutylicum NRRL B527.
Cell conc. (g/L)
Butyric acid (g/L)
Butanol yield (g/g glucose consumed)
Control (complete semi-defined medium)
Semi-defined medium without yeast extract
Semi-defined medium without ammonium acetate
Table 2: Effect of yeast extract and ammonium acetate on fermentation balance
Effect of acetate on growth:
Chen and Blaschek (1999) reported that acetate is key element for butanol production in C. beijerinckii BA 101. Hence the effect of acetate from ammonium acetate was studied on C. acetobutylicum NRRL B527. The experiments included substitution of ammonium acetate with equivalent amounts sodium acetate or ammonium salts. The experiments were as follows:
a) Control (semi-defined synthetic medium with NH4-acetate)
b) Semi-defined medium without NH4-acetate; but equivalent amount of NH4Cl
c) Semi-defined medium without NH4-acetate; but equivalent amount of (NH4)2SO4
d) Semi-defined medium without NH4-acetate; but equivalent amount of Na-acetate
The result proved that acetate and not ammonium is essential for butanol production in NRRL B527. Cultures showed butanol production in presence ammonium acetate and sodium acetate; while in presence of only ammonium salts there was reduced growth. The results on fermentation yields after 72hr incubation is given in table 3.
Cell conc. (g/L)
Butyric acid (g/L)
Butanol yield (g/g glucose consumed)
Semi-defined medium (control)
Medium with NH4Cl (without acetate)
Medium with (NH4)2SO4 (without acetate)
Medium with sodium acetate as substitute
Table 3: Effect of acetate on fermentation yield
Effect of other media components on growth:
The results of elimination of single component at time showed that without FeSO4, phosphate and NaCl the culture failed to grow. Of the other components MgSO4 had a marked effect on butanol production and glucose uptake. All other components had marginal effect on the butanol yields. The fermentation balance after 72 hr of incubation under anaerobic conditions at 30 °C is given in table 4. In absence of MgSO4, even though there was growth comparable observed but there was a drastic drop in butanol production. There was a higher amount of butyric acid production (> 60%). The butyric acid yield increased from 0.07 g/g to 0.22 g/g glucose consumed. Therefore, for butanol production by NRRL B527 MgSO4 is required, while minor components can be removed from semi-defined medium without affecting the growth or butanol production.
Cell conc. (g/L)
Glucose consumed (g/L)
Butyric acid (g/L)
Butanol yield (g/g glucose consumed)
Semi-defined synthetic media (control)
Semi-defined media without MgSO4
Semi-defined media without thiamine
Semi-defined media without biotin
Semi-defined media without pABA
Table 4: Effect of other components on fermentation yields
Effect of various sugars:
There different sugar substitutes considered for fermentation were sucrose, fructose, xylose and starch. Xylose and starch were primarily considered because of their importance. Xylose is the major sugar produced during breakdown of lignin while presence of amylolytic strain would help in effective butanol fermentation. The initial total sugar concentration was in all experiments was at 3%. The results observed on substitution of different sugars of glucose in semi-defined synthetic medium, after 96hr of incubation under anaerobic conditions at 30 °C is given in table 5. It is observed that the best results obtained using glucose or glucose xylose mixtures. C. acetobutylicum NRRL B527 was able to consume xylose and starch as a substrate for fermentation in semi-defined synthetic medium. The butanol yields and productivities for xylose and starch were comparable to glucose and they can be used for further studies of NRRL B527.
(g/g sugar consumed)
Glucose + Fructose (1:1)
Glucose + Sucrose (1:1)
Glucose + Xylose (1:1)
Fructose + Sucrose (1:1)
Table 5: Effect of different sugars on butanol production
Effect of sub-optimal concentrations of media components on butanol production:
It was observed during the study that the strain was being adapted to the semi-defined synthetic medium. Due to optimization and adaptation, the fermentation cycle was reduced from 120hr to 96hr; thus increasing the productivity from 0.07g/L/h to 0.13 g/L/h. Hence in parallel all the components of the medium were reduced together; thus obtaining a semi-defined medium which had 10% lower concentrations of all the components barring glucose and phosphate. The results after 72hr of incubation are compared in table 6.
Reduced medium semi-defined medium were also studied. They contained only the essential components for growth and the rest of the components of the semi-defined medium were not added. The different types of sub-optimal concentrations of media are:
a) Semi-defined synthetic medium (optimized)
b) Sub-optimal semi-defined media (concentrations of all media components reduced by 10%, except glucose)
c) Medium containing 3% glucose + 0.15% yeast extract only [glc + ye]
d) Medium containing 3% glucose + 0.15% yeast extract + 0.25% ammonium acetate [glc + ye + ace]
The results after 72hr of incubation are compared in table 6. It was observed that, the NRRL B527 was able to produce butanol in the sub-optimal semi-defined medium with equal amount of yield (0.25 g/g glucose consumed) as compared to the optimal semi-defined synthetic medium. The medium consisting of only 3% glucose + 0.15% yeast extract + 0.25% ammonium acetate was able to produce butanol without addition of any vitamins of salts. The butanol yields were reduced by 50% but NRRL B527 was able to survive and produce butanol in a reduced semi-defined medium.
Cell conc. (g/L)
Glucose consumed (g/L)
Butyric acid (g/L)
Butanol yield (g/g glucose consumed)
Semi-defined synthetic medium
Sub-optimum semi-defined medium
Reduced medium [glc + ye]
Reduced medium [glc + ye + ace]
Table 6: Fermentation balance in different reduced synthetic medium
Clostridium acetobutylicum NRRL B527 was the strain selected for ABE fermentation and a medium was designed and optimized for maximum growth and butanol production for the following strain. The conclusions of the study can be given as:
Semi-defined synthetic medium was designed and optimized.
The optimum temperature and pH of C. acetobutylicum NRRL B527 with semi-defined medium is 30 °C and 6.23 respectively.
The optimum glucose concentrations are from 3% to 4.5% (w/v) which produced upto 11 g/L of butanol in batch studies.
98% theoretical yields achieved frequently. (0.25 g/g glucose consumed)
Butanol productivities of 0.1-0.15 g/L/h were reached; which is typical to batch fermentations.
Acetate is essential for butanol production in C. acetobutylicum NRRL B527.
Pentose (xylose) can be used as a substrate for fermentation using C. acetobutylicum NRRL B527 with yields up to (0.24 g/g xylose consumed)
C. acetobutylicum NRRL B527 is a amylolytic strain able to produce butanol from starch with a yield of 0.2 g/g starch consumed.
Thus ABE fermentation was effectively established with C. acetobutylicum NRRL B527. The optimization process achieved 98% theoretical yields with wild-type strain and further strain improvement or process improvement can provide higher yield and productivities.