Clostridium Beijerinckii BA101 Research Articles

Acetone-Butanol-Ethanol (ABE) Production in Fermentation of Enzymatically Hydrolyzed Cassava Flour by Clostridium beijerinckii BA101 and Solvent Separation

Cassava constitutes an abundant substrate in tropical regions. The production of butanol in ABE fermentation by Clostridium beijerinckii BA101 using cassava flour (CF) was scaled-up to bioreactor level (5 L). Optimized fermentation conditions were applied; that is, 40℃, 60 g/l CF, and enzymatic pretreatment of the substrate. The batch fermentation profile presented an acidogenic phase for the first 24 h and a solventogenic phase afterwards. An average of 37.01 g/l ABE was produced after 83 h, with a productivity of 0.446 g/l/h. Butanol production was 25.71 g/l with a productivity of 0.310 g/l/h, high or similar to analogous batch processes described for other substrates. Solvent separation by different combinations of fractioned and azeotropic distillation and liquid-liquid separation were assessed to evaluate energetic and economic costs in downstream processing. Results suggest that the use of cassava as a substrate in ABE fermentation could be a cost-effective way of producing butanol in tropical regions.

Comparison of energy efficiency and economics of process designs for biobutanol production from sugarcane molasses

Development of technologies for biobutanol production by fermentation has resulted in higher final butanol concentrations, less fermentation by-products and higher volumetric productivities during fermentation, together with less energy intensive separation and purification techniques. These new technology developments have the potential to provide a production process for butanol from sugarcane molasses that is economically viable in comparison to the petrochemical pathway for butanol production. This objective was investigated by developing process models to compare three different possible process designs for biobutanol production from sugarcane molasses. The first two process routes incorporate well established industrial technologies: Process Route 1 consisted of batch fermentation and steam stripping distillation, while in Process Route 2, some of the distillation columns were replaced with a liquid–liquid extraction column. Some of the best production strains in these process routes, which include Clostridium Acetobutylicum PCSIR-10 and Clostridium Beijerinckii BA101, can produce total solvent concentrations up to 24g/L. Process Route 3 incorporated fed-batch fermentation and gas-stripping with CO2, an unproven technology on industrial scale. Process modeling in ASPEN PLUS® and economic analyses in ASPEN Icarus® were performed to determine the economic feasibility of these biobutanol production process designs. Process Route 3 proved to be the only profitable design in current economic conditions in South Africa. Improved fermentation strains currently available are not sufficient to attain a profitable process design without implementation of advanced processing techniques. Gas stripping is shown to be the single most effective process step of those evaluated in this study, which can be employed on an industrial scale to improve process economics of biobutanol production.

Microbial production of a biofuel (acetone–butanol–ethanol) in a continuous bioreactor: impact of bleed and simultaneous product removal

Acetone butanol ethanol (ABE) was produced in an integrated continuous one-stage fermentation and gas stripping product recovery system using Clostridium beijerinckii BA101 and fermentation gases (CO(2) and H(2)). In this system, the bioreactor was fed with a concentrated sugar solution (250-500 g L(-1) glucose). The bioreactor was bled semi-continuously to avoid accumulation of inhibitory chemicals and products. The continuous system was operated for 504 h (21 days) after which the fermentation was intentionally terminated. The bioreactor produced 461.3 g ABE from 1,125.0 g total sugar in 1 L culture volume as compared to a control batch process in which 18.4 g ABE was produced from 47.3 g sugar. These results demonstrate that ABE fermentation can be operated in an integrated continuous one-stage fermentation and product recovery system for a long period of time, if butanol and other microbial metabolites in the bioreactor are kept below threshold of toxicity.

Butanol production by Clostridium beijerinckii BA101 using cassava flour as fermentation substrate: enzymatic versus chemical pretreatments

Cassava flour (CF), a cost-effective source of starch, was employed as a substrate for successful acetone-butanol-ethanol (ABE) production by batch-fermentation with Clostridium beijerinckii. The effect of temperature, initial concentration of CF and chemical/enzymatic hydrolysis were studied in a 23 factorial design. Results revealed that temperature and initial concentration of substrate exert a significant effect on ABE production, as well as interactions of temperature with the other variables. Solvent production was maximized when working at 40°C, 60 g l−1 CF and enzymatic pretreatment. An average of 31.38 g l−1 ABE was produced after 96 h, with a productivity of 0.33 g l−1 h−1. A posterior randomized block design (3 × 2) showed that enzymatic hydrolysis (with saccharification periods of 6 h at 60°C) enhances both reducing sugar and solvent production if compared to chemical pretreatments. Average ABE production in this case was 27.28 g l−1, with a productivity of 0.28 g l−1 h−1. Results suggest that CF may be a suitable substrate for industrial ABE production.

Butanol production by Clostridium beijerinckii. Part I: Use of acid and enzyme hydrolyzed corn fiber

Fermentation of sulfuric acid treated corn fiber hydrolysate (SACFH) inhibited cell growth and butanol production (1.7 ± 0.2 g/L acetone butanol ethanol or ABE) by Clostridium beijerinckii BA101. Treatment of SACFH with XAD-4 resin removed some of the inhibitors resulting in the production of 9.3 ± 0.5 g/L ABE and a yield of 0.39 ± 0.015. Fermentation of enzyme treated corn fiber hydrolysate (ETCFH) did not reveal any cell inhibition and resulted in the production of 8.6 ± 1.0 g/L ABE and used 24.6 g/L total sugars. ABE production from fermentation of 25 g/L glucose and 25 g/L xylose was 9.9 ± 0.4 and 9.6 ± 0.4 g/L, respectively, suggesting that the culture was able to utilize xylose as efficiently as glucose. Production of only 9.3 ± 0.5 g/L ABE (compared with 17.7 g/L ABE from fermentation of 55 g/L glucose-control) from the XAD-4 treated SACFH suggested that some fermentation inhibitors may still be present following treatment. It is suggested that inhibitory components be completely removed from the SACFH prior to fermentation with C. beijerinckii BA101. In our fermentations, an ABE yield ranging from 0.35 to 0.39 was obtained, which is higher than reported by the other investigators.

Fermentation of dried distillers’ grains and solubles (DDGS) hydrolysates to solvents and value-added products by solventogenic clostridia

Pretreatment and hydrolysis of lignocellulosic biomass using either dilute acid, liquid hot water (LHW), or ammonium fiber expansion (AFEX) results in a complex mixture of sugars such as hexoses (glucose, galactose, mannose), and pentoses (xylose, arabinose). A detailed description of the utilization of representative mixed sugar streams (pentoses and hexoses) and their sugar preferences by the solventogenic clostridia ( Clostridium beijerinckii BA101, C. acetobutylicum 260, C. acetobutylicum 824, Clostridium saccharobutylicum 262, and C. butylicum 592) is presented. In these experiments, all the sugars were utilized concurrently throughout the fermentation, although the rate of sugar utilization was sugar specific. For all clostridia tested, the rate of glucose utilization was higher than for the other sugars in the mixture. In addition, the availability of excess fermentable sugars in the bioreactor is necessary for both the onset and the maintenance of solvent production otherwise the fermentation will become acidogenic leading to premature termination of the fermentation process. During an investigation on the effect of some of the known lignocellulosic hydrolysate inhibitors on the growth and ABE production by clostridia, ferulic and p-coumaric acids were found to be potent inhibitors of growth and ABE production. Interestingly, furfural and HMF were not inhibitory to the solventogenic clostridia; rather they had a stimulatory effect on growth and ABE production at concentrations up to 2.0 g/L.

Butanol production from agricultural residues: Impact of degradation products on Clostridium beijerinckii growth and butanol fermentation

During pretreatment and hydrolysis of fiber-rich agricultural biomass, compounds such as salts, furfural, hydroxymethyl furfural (HMF), acetic, ferulic, glucuronic, rho-coumaric acids, and phenolic compounds are produced. Clostridium beijerinckii BA101 can utilize the individual sugars present in lignocellulosic [e.g., corn fiber, distillers dry grain solubles (DDGS), etc] hydrolysates such as cellobiose, glucose, mannose, arabinose, and xylose. In these studies we investigated the effect of some of the lignocellulosic hydrolysate inhibitors associated with C. beijerinckii BA101 growth and acetone-butanol-ethanol (ABE) production. When 0.3 g/L rho-coumaric and ferulic acids were introduced into the fermentation medium, growth and ABE production by C. beijerinckii BA101 decreased significantly. Furfural and HMF are not inhibitory to C. beijerinckii BA101; rather they have stimulatory effect on the growth of the microorganism and ABE production.

Continuous butanol fermentation and feed starch retrogradation: butanol fermentation sustainability using Clostridium beijerinckii BA101

Use of starch solution as feed for butanol bioconversion processes employing Clostridium beijerinckii BA101 may have added economic advantage over the use of glucose. Acetone butanol ethanol (ABE) was produced from 30 g L −1 starch solution using a continuous process. The bioreactor was fed at a dilution rate of 0.02 h −1 and starch solution/feed volume (3 L) was replaced every 72 h. The continuous reactor fed with cornstarch solution (feed temperature 19 °C) produced approximately 6.0 g L −1 total ABE. Increasing the feed storage temperature to 37 °C improved ABE production to 7.2 g L −1 suggesting that retrogradation was occurring more rapidly at 19 °C. In both these cases the fermentation drifted toward acid production after approximately 260 h, consistent with the retrogradation of starch overtime. The use of soluble starch, which is less prone to retrogradation, resulted in the production of 9.9 g L −1 ABE at 37 °C feed storage temperature, as compared to 7.2 g L −1 ABE when cornstarch was used. It should be noted that gelatinized starch retrogradation takes place after sterilization and prior to use of the feed medium, and does not occur during long-term storage of the raw corn material in the months leading up to processing. The degree of hydrolysis of gelatinized starch decreased from 68.8 to 56.2% in 3 days when stored at 37 °C. Soluble starch which does not retrograde demonstrated no change in the degree of hydrolysis.

Scale-Up of a High Productivity Continuous Biofilm Reactor to Produce Butanol by Adsorbed Cells of Clostridium Beijerinckii

An immobilized cell reactor for producing acetone butanol ethanol (ABE or solvents) when using Clostridium beijerinckii BA101 demonstrated reactor productivities of 3.49, 5.99 and 16.13 gl−1h−1 at dilution rates of 0.29, 1.00 and 2.00 h−1, respectively. These dilution rates are based on the total volume of the reactor. The reactor was scaled up successfully as these productivities are close to those achieved in small reactor. Dilution rate based on the void volume increased reactor productivity from 16.13 to 34.76 g I−1 h−1. The packed bed reactor blocked after a period of 2302 h of continuous operation due to excessive cell growth. Nutrient limitation was applied as a means of reducing cell growth while keeping the reactor productive; however, this approach was not successful. It was found that most of the cell growth in adsorbed cell bioreactors occurs on the surface of the cell support. The free cell continuous reactor demonstrated much lower reactor productivity. In the immobilized cell reactor only a fraction of the biomass was in the solventogenic state. A significant amount of biomass was present as inactive biomass (spores). It is suggested that sporulation be blocked in C. beijerinckii BA101 in order for reactors to be more productive. It is postulated that once such a culture is developed nutrient limitation should be attempted to avoid reactor blockage due to excessive cell growth.

High-productivity continuous biofilm reactor for butanol production: effect of acetate, butyrate, and corn steep liquor on bioreactor performance.

Corn steep liquor (CSL), a byproduct of the corn wet-milling process, was used in an immobilized cell continuous biofilm reactor to replace the expensive P2 medium ingredients. The use of CSL resulted in the production of 6.29 g/L of total acetone-butanol-ethanol (ABE) as compared with 6.86 g/L in a control experiment. These studies were performed at a dilution rate of 0.32 h-1. The productivities in the control and CSL experiment were 2.19 and 2.01 g/(L.h), respectively. Although the use of CSL resulted in a 10% decrease in productivity, it is viewed that its application would be economical compared to P2 medium. Hence, CSL may be used to replace the P2 medium. It was also demonstrated that inclusion of butyrate into the feed was beneficial to the butanol fermentation. A control experiment produced 4.77 g/L of total ABE, and the experiment with supplemented sodium butyrate produced 5.70 g/L of total ABE. The butanol concentration increased from 3.14 to 4.04 g/L. Inclusion of acetate in the feed medium of the immobilized cell biofilm reactor was not found to be beneficial for the ABE fermentation, as reported for the batch ABE fermentation.

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping.

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H(2) and CO(2) as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l(-1) and 60 g l(-1), respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l(-1) and produced 17.6 g total solvents l(-1) (yield 0.39 g g(-1), productivity 0.29 g l(-1) h(-1)). Using the integrated fermentation-gas stripping product-recovery system with CO(2) and H(2) as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l(-1)) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g(-1) and 1.16 g l(-1) h(-1), respectively.

Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping

Summary We examined the effect of gas-stripping on the in situ removal of acetone, butanol, and ethanol (ABE) from batch reactor fermentation broth. The mutant strain (Clostridium beijerinckii BA101) was not affected adversely by gas stripping. The presence of cells in the fermentation broth affected the selectivities of ABE. A considerable improvement in the productivity and yield was recorded in this work in comparison with the non-integrated process. In an integrated process of ABE fermentation-recovery using C. beijerinckii BA101, ABE productivities and yield were improved up to 200 and 118%, respectively, as compared to control batch fermentation data. In a batch reactor C. beijerinckii BA101 utilized 45.4 g glucose l )1 and produced 17.7 g total ABE l )1 , while in the integrated process it utilized 161.7 g glucose l )1 and produced total ABE of 75.9 g l )1 . In the integrated process, acids were completely converted to solvents when compared to the non-integrated process (batch fermentation) which contained residual acids at the end of fermentation. In situ removal of ABE by gas stripping has been reported to be one of the most important techniques of solvent removal. During these studies we were able to maintain the ABE concentration in the fermentation broth below toxic levels.

Continuous production of butanol from starch-based packing peanuts.

Acetone, butanol, ethanol (ABE, or solvents) were produced from starch-based packing peanuts in batch and continuous reactors. In a batch reactor, 18.9 g/L of total ABE was produced from 80 g/L packing peanuts in 110 h of fermentation. The initial and final starch concentrations were 69.6 and 11.1 g/L, respectively. In this fermentation, ABE yield and productivity of 0.32 and 0.17 g/(L h) were obtained, respectively. Compared to the batch fermentation, continuous fermentation of 40 g/L of starchbased packing peanuts in P2 medium resulted in a maximum solvent production of 8.4 g/L at a dilution rate of 0.033 h-1. This resulted in a productivity of 0.27 g/(L h). However, the reactor was not stable and fermentation deteriorated with time. Continuous fermentation of 35 g/L of starch solution resulted in a similar performance. These studies were performed in a vertical column reactor using Clostridium beijerinckii BA101 and P2 medium. It is anticipated that prolonged exposure of culture to acrylamide, which is formed during boiling/autoclaving of starch, affects the fermentation negatively.

Production of butanol from starch-based waste packing peanuts and agricultural waste.

We examined the fermentation of starch-based packing peanuts and agricultural wastes as a source of fermentable carbohydrates using Clostridium beijerinckii BA101. Using semidefined P2 medium containing packing peanuts and agricultural wastes, instead of glucose as a carbohydrate source, we measured characteristics of the fermentation including solvent production, productivity, and yield. With starch as substrate (control), the culture produced 24.7 g l(-1) acetone-butanol-ethanol (ABE), while with packing peanuts it produced 21.7 g l(-1) total ABE with a productivity of 0.20 g l(-1) h(-1) and a solvent (ABE) yield of 0.37. Cell growth in starch, packing peanuts, and agricultural wastes medium was different, possibly due to the different nature of these substrates. Using model agricultural waste, 20.3g l(-1) ABE was produced; when using actual waste, 14.8 g l(-1) ABE was produced. The use of inexpensive substrates will increase the economic viability of the conversion of biomass to butanol, and can provide new markets for these waste streams.

Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor: increase in sugar utilization.

Acetone butanol ethanol was produced in a continuous immobilized cell (biofilm) plug-flow reactor inoculated with Clostridium beijerinckii BA101. To achieve high reactor productivity, C. beijerinckii BA101 cells were immobilized by adsorption onto clay brick. The continuous plug-flow reactor offers high productivities owing to reduced butanol inhibition and increased cell concentration. Although high productivity was achieved, it was at the expense of low sugar utilization (30.3%). To increase sugar utilization, the reactor effluent was recycled. However, this approach is complicated by butanol toxicity. The effluent was recycled after removal of butanol by pervaporation to reduce butanol toxicity in the reactor. Recycling of butanol-free effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2 g/(L x h) at a dilution rate of 1.5 h(-1). A dilution rate of 2.0 h(-1) resulted in a reactor productivity of 16.2 g/(L x h) and sugar utilization of 101.4%. It is anticipated that this reactor-recovery system would be economical for butanol production when using C. beijerinckii BA101.

Production of acetone butanol ethanol from degermed corn using Clostridium beijerinckii BA101.

In this article we report on acetone butanol ethanol (ABE) fermentation characteristics of degermed corn when using Clostridium beijerinckii BA101. Recent economic studies suggested that recovery of germ from corn and hence corn oil would help to make the ABE fermentation process more economical. C. beijerinckii BA101 ferments corn mash efficiently to produce ABE under appropriate nutritional and environmental conditions. Corn mash contains germ/corn oil that is, possibly, ancillary to the production of butanol during the ABE fermentation process. Since the presence of corn oil is not a critical factor in solvent fermentation, it can be removed and this will allow for byproduct credit. Batch fermentation of degermed corn resulted in 8.93 g/L of total ABE production as compared with 24.80 g/L of total ABE when supplemented with P2 medium nutrients. During the course of the germ separation process, corn steeping is required prior to grinding and removing the germ. It is likely that some nutrients from the corn are leached out during the steeping process. This may reduce the rate of fermentation and impact the final concentration of butanol/ABE that can be achieved. Fermentation of degermed corn with corn steep liquor resulted in the production of 19.28 g/L of ABE.

Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101

This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone-butanol-ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27-29 g x l(-1). C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g x l(-1). The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g x l(-1) has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g x l(-1) x h(-1). Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20-0.25 x lb(-1) by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices.

ABE production from corn: a recent economic evaluation

This article details an economic assessment of butanol production from corn using the newly developed hyper-butanol-producing strain of Clostridium beijerinckii BA101. Butanol is produced in batch reactors and recovered by distillation. For a plant with 153,000 metric tons of acetone, butanol, and ethanol (ABE) production capacity, the production equipment cost and total working capital cost is US$33.47x10(6) and US$110.46x10(6), respectively. Based on a corn price (C(p)) of US$79.23 x ton(-1) (US$2.01 x bushel(-1)), an ABE yield of 0.42 (g ABE/g glucose) butanol price is projected to be US$0.34 x kg(-1). An improved yield of 0.50 will reduce this price to US$0.29 x kg(-1). Assumptions, such as by-product credit for gases and complete conversion of corn steep liquor (CSL) to fermentation by-products, have been taken into consideration. An increased price of corn to US$197.10 x ton(-1) would result in a butanol price of US$0.47 x kg(-1). A grass-rooted plant would result in a butanol price of US$0.73 x kg(-1) (C(p) US$79.23 x ton(-1)). In a worst case scenario, the price of butanol would increase to US$1.07 x kg(-1) (C(p) 197.10 x ton(-1) for a grass-rooted plant and assuming no credit for gases). This is based on the assumption that corn price would not increase to more than US$197.10 x ton(-1).

Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101.

Spray-dried soy molasses (SDSM) contains the sugars dextrose, sucrose, fructose, pinitol, raffinose, verbascose, melibiose, and stachyose. Of the 746 g kg(-1) total sugars in SDSM, 434 g kg(-1) is fermentable using Clostridium beijerinckii BA101. SDSM was used to produce acetone, butanol, and ethanol (ABE) by C. beijerinckii BA101 in batch cultures. Using 80 g l(-1) SDSM, 10.7 g l(-1) ABE was produced in P2 medium. Higher concentrations of SDSM resulted in poor solvent production due to the presence of excessive salt and inhibitory components. C. beijerinckii BA101 in SDSM at 80 g l(-1) concentration produced 22.8 g l(-1) ABE when supplemented with 25.3 g l(-1) glucose. SDSM contains 57.4 g kg(-1) mineral ash and 2% tri-calcium phosphate. Tri-calcium phosphate up to 43.1 g l(-1) was not inhibitory and at a tri-calcium phosphate concentration of 28.8 g l(-1), the culture produced more solvents (30.1 g l(-1)) than the control experiment (23.8 g l(-1)). In contrast, sodium chloride was a strong inhibitor of C. beijerinckii BA101 cell growth. At a concentration of 10 g l(-1) sodium chloride, a maximum cell concentration of 0.6 g l(-1) was achieved compared to 1.7 g l(-1) in the control experiment. The effects of two salts on specific growth rate constant (mu) and specific rate of ABE production (nu) for C. beijerinckii BA101 were examined.

Economics of Butanol Fermentation using Hyper-Butanol Producing Clostridium Beijerinckii BA101

An economic assessment is presented to produce butanol from corn using the hyperbutanol producing strain of Clostridium beijerinckii BA101. Butanol is produced in batch reactors and recovered by distillation with a total acetone, butanol, and ethanol (ABE) productivity of 0.38 g L−1 h−1. For a plant with 150,000 metric tons of ABE production capacity per year, the production equipment cost and total working capital cost is $33.2× 106 and $109.56× 106, respectively. This is based on a lang factor of 3 for a plant annexed to an already existing corn milling plant in the mid-west region of the United States of America. Based on an ABE yield of 0.42, and a corn price of $71 ton−1, butanol production cost is projected to be $0.55 kg−1. Further improvements, such as increased yield of 0.45 and savings in electricity and steam will reduce this price to $0.44 kg−1. Various other alternatives such as a lang factor of 4.5 and increased corn price have also been considered in this assessment.