Butanol production using Clostridium beijerinckii BA101 hyper-butanol producing mutant strain and recovery by pervaporation

Clostridium beijerinckii BA101 (mutant strain) and C. beijerinckii 8052 (wild type) were compared for substrate and butanol inhibition. The wild-type strain is more strongly inhibited by added butanol than is the mutant strain. Acetone and butanol were removed from a fed-batch reactor inoculated with C. beijcrinckii BA101 by pervaporation using a silicone membrane. In the batch reactor, C. beijerinckii BA101 produced 25.3 g/L of total solvents, whereas in the fermentation-recovery experiment it produced 165.1 g/L of total solvents. Solvent productivity increased from 0.35 (batch reactor) to 0.98 g/L·h (fed-batch reactor). The fed-batch reactor wasfed with 500 g/L of glucose-based P2 medium. Acetone selectivities ranged from 2 to 10 whereas butanol selectivities ranged from 7 to 19. Total flux varied from 26 to 31 g/m²·h.     

Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor

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 butanolfree effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2g/(L·h) at a dilution rate of 1.5 h⁻¹. A dilution rate of 2.0h⁻¹ resulted in a reactor productivity of 16.2g/(L·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.     

Ultrasound-Enhanced Recovery of Butanol/ABE by Pervaporation

The search for renewable sources of energy has led to renewed interests on the biochemical route for the production of butanol. Butanol production suffers from several drawbacks, mainly caused by butanol inhibition to the butanol-producing microorganism which makes it economically uncompetitive against the chemical process. One possible solution proposed is the in situ recovery of acetone–butanol–ethanol (ABE). Among the in situ recovery options, membrane processes like pervaporation have a great potential. Thus, the effects of temperature, feed concentration, and ultrasound irradiation on permeate concentration and permeation flux for the recovery of butanol/ABE by pervaporation from aqueous solutions were investigated in this study. In the butanol–water system, permeate butanol concentration as well as flux increased with an increase in temperature and butanol feed concentration. When pervaporation studies with ABE–water mixture were carried out at 60 °C for 2, 4, 8, 16, and 24 h, pervaporation profile revealed an optimal permeate concentration as well as permeation flux. Applications of ultrasound irradiation on pervaporation improved permeate concentration by about 23 g/L for both butanol and ABE. Ultrasound irradiation also improved butanol and ABE mass permeation flux by about 13 and 11 %, respectively.     

The Escherichia coli adenylate cyclase complex: activation by phosphoenolpyruvate.

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.     

Modeling and simulation of butanol separation from aqueous solutions using pervaporation

A study was conducted to separate butanol from an aqueous solution using pervaporation. A specially designed and manufactured cell was used to separate the butanol from butanol/water solutions of different butanol concentrations (6-8-11-16-20-50) g/l. A 250 cm³ butanol mixture at 33 °C was used to feed the cell, while the pressure of permeation side was about 0 bar. Results revealed that butanol concentration changes non-linearly during the first 3 h, and then proceeds linearly. The percentage of butanol removal increases with increasing feed concentration. The permeability of the used membrane was determined experimentally. A resistance in series model was used to simulate the pervaporation step. The butanol concentration in the feed during the pervaporation step was predicted by using the developed model. There is a fair agreement between butanol concentration in feeding tank of pervaporation cell both experimentally and predicted from the developed model.     

The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS): an interface between energy and signal transduction

The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates, and is involved in signal transduction. It comprises two general phosphotransferase proteins (EI and HPr) and a—species dependent—variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC). EI and HPr transfer phosphoryl groups from PEP to the IIA units. IIA and IIB sequentially transfer phosphates to the sugar, which is translocated by the IIC unit. The ratio of phosphorylated to non-phosphorylated IIA and IIB varies with transport activity, and the phosphorylation state of some of the IIA and IIB serves as signal input for regulation of catabolite repression, intermediate metabolism, gene expression and chemotaxis in response to the availability of carbohydrates and PEP (glycolytic activity). PTS occur in about one-third of all eubacteria and in a few archaebacteria but not in animals and plants. Uniqueness and pleiotropic function make the PTS a potential target for anti-infectives. The PTS transporter for mannose is utilized as a gate for the penetration of bacteriophage lambda DNA across, and insertion of certain bacteriocins (small antimicrobial peptides) into the inner membrane. The PTS of Escherichia coli is in the focus of this review, but occasionally comparisons with other species are made. The topics are: History; Modular design of the E. coli PTS; Structure function and catalytic mechanism of the protein modules; Regulation of and by the PTS; The PTS in pathogenicity and virulence; Computational models; Metabolic engineering.     

The mechanism of sugar-dependent repression of synthesis of catabolic enzymes in Escherichia coli.

Previous studies have indicated that the Escherichia coli adenylate cyclase (AC) activity is controlled by an interaction with the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS). A model for the regulation of AC involving the phosphorylation state of the PTS is described. Kinectic studies support the concept that the velocity of AC is determined by the opposing contributions of PEP-dependent phosphorylation (V1) and sugar-dependent dephosphorylation (V2) of the PTS proteins according to the expression percent VAC=100/[1 + (Max V2/Max V1)]. Physiological parameters influencing the rate of the PTS are discussed in the framework of their effects on cAMP metabolism. Factors that increase cellular concentration of PEP (and stimulate V1) appear to enhance AC activity while increases in extracellular sugar concentration (which stimulate V2) or internal levels of pyruvate (which inhibit V1) inhibit the activity of this enzyme.     

Butanol Tolerance in a Selection of Microorganisms

Butanol tolerance is a critical factor affecting the ability of microorganisms to generate economically viable quantities of butanol. Current Clostridium strains are unable to tolerate greater than 2% 1-butanol thus membrane or gas stripping technologies to actively remove butanol during fermentation are advantageous. To evaluate the potential of alternative hosts for butanol production, we screened 24 different microorganisms for their tolerance to butanol. We found that in general, a barrier to growth exists between 1% and 2% butanol and few microorganisms can tolerate 2% butanol. Strains of Escherichia coli, Zymomonas mobilis, and non-Saccharomyces yeasts were unable to surmount the 2% butanol growth barrier. Several strains of Saccharomyces cerevisiae exhibit limited growth in 2% butanol, while two strains of Lactobacillus were able to tolerate and grow in up to 3% butanol.     

Production of Acetone–Butanol–Ethanol (ABE) in Direct Fermentation of Cassava by Clostridium saccharoperbutylacetonicum N1-4

In this work, acetone–butanol–ethanol (ABE) fermentation characteristics of cassava starch and cassava chips when using Clostridium saccharoperbutylacetonicum N1-4 was presented. The obtained results in batch mode using a 1-L fermenter showed that C. saccharoperbutylacetonicum N1-4 was a hyperamylolytic strain and capable of producing solvents efficiently from cassava starch and cassava chips, which was comparable to when glucose was used. Batch fermentation of cassava starch and cassava chips resulted in 21.0 and 19.4 g/L of total solvent as compared with 24.2 g/L of total solvent when using glucose. Solvent productivity in fermentation of cassava starch was from 42% to 63% higher than that obtained in fermentation using corn and sago starches in the same condition. In fermentation of cassava starch and cassava chips, maximum butanol concentration was 16.9 and 15.5 g/L, respectively. Solvent yield and butanol yield (based on potential glucose) was 0.33 and 0.41, respectively, for fermentation of cassava starch and 0.30 and 0.38, respectively for fermentation using cassava chips.     

Adsorbent screening for biobutanol separation by adsorption: kinetics, isotherms and competitive effect of other compounds

Butanol, considered as one of the best renewable alternatives for gasoline, has attracted significant attention in recent years. However, biobutanol production via fermentation is plagued by the low final product concentration due to product inhibition. It is possible to enhance productivity by selectively removing biobutanol from the fermentation broth. Adsorption is one of the most promising and energy-efficient techniques for butanol separation and recovery. In the present study, different adsorbents were tested by performing kinetic and equilibrium experiments to find the best adsorbent for butanol separation. Activated carbon (AC) F-400 showed the fastest adsorption rate and the highest adsorption capacity amongst ACs and zeolites tested. AC F-400 also showed the highest affinity toward butanol and to a lesser extent for butyric acid whereas its adsorption capacity for the other main components present in acetone–butanol–ethanol fermentation broths was very low. In addition, the butanol adsorption capacity was not affected by the presence of ethanol, glucose and xylose while the presence of acetone led to a slight decrease in adsorption capacity at low butanol concentrations. On the other hand, the presence of acids (acetic acid and butyric acid) showed a significant effect on the butanol adsorption capacity over a wide range of butanol concentration and this effect was more pronounced for butyric acid.     

Continuous production of butanol by clostridium acetobutylicum immobilized in a fibrous bed bioreactor

We explored the influence of dilution rate and pH in continuous cultures of Clostridium acetobutylicum. A 200-mL fibrous bed bioreactor was used to produce high cell density and butyrate concentrations at pH 5.4 and 35°C. By feeding glucose and butyrate as a cosubstrate, the fermentation was maintained in the solventogenesis phase, and the optimal butanol productivity of 4.6g/(L h) and a yield of 0.42 g/g were obtained at a dilution rate of 0.9h⁻¹ and pH 4.3. Compared to the conventional acetone-butanol-ethanol fermentation, the new fermentation process greatly improved butanol yield, making butanol production from corn an attractive alternative to ethanol fermentation.     

Process improvement in acetone-butanol production from hardwood by simultaneous saccharification and extractive fermentation

The acetone-butanol production by simultaneous saccharification and extractive fermentation (SSEF) was investigated. In the SSEF employing cellulase enzymes andClostridium acetobutylicum, both glucan and xylan fractions of pretreated aspen are concurrently converted into acetone and butanol. Continuous removal of the fermentation products from the bioreactor by extraction was an important factor that allowed long-term fed-batch operation. The use of membrane extraction prevented the problems of phase separation and extractant loss. Increase in substrate feeding as well as reduction of nutrient supply was found to be beneficial in suppressing the acid production, thereby improving the solvent yield. Because of prolonged low growth conditions prevalent in the fed-batch operation, the butanol-to-acetone ratio in the product was significantly higher at 2.6–2.8 compared to the typical value of two.     

Recovery of butanol from model ABE fermentation broths using MFI adsorbent: a comparison between traditional beads and a structured adsorbent in the form of a film

Butanol, a promising biofuel, can be produced by ABE (acetone, butanol and ethanol) fermentation using e.g. Clostridium acetobutylicum. However, the butanol concentration in the resulting broth is limited to only ca. 20 g/L due to the toxicity for the microorganisms. This low product concentration demands an efficient recovery process for successful commercialization of this process. In this study, a structured adsorbent in the form of steel monolith coated with a silicalite-1 film was prepared using the in situ growth method. The adsorbent was carefully characterized by SEM and XRD. The performance of the adsorbent was evaluated by performing breakthrough experiments at room temperature using model ABE fermentation broths and the performance was compared with that of traditional adsorbents in the form of beads. The structured silicalite-1 adsorbent showed less saturation loading time as compared to commercial binder free silicalite-1 beads, reflecting the different dimensions of the columns used, set by experimental constraints. Studies of the desorption process showed that by operating at appropriate conditions, butanol with high concentration i.e. up to 95.2 wt% for butanol–water model system and 88.5 wt% for ABE fermentation broth can be obtained using the structured silicalite-1 adsorbent. Commercial silicalite-1 beads also showed good selectivity but the concentration of butanol in the desorbed product was limited to 70 % for the butanol–water model system and 69 % for ABE fermentation broth, probably as a result of entrained liquid between the beads.     

Economic analysis of biobutanol recovery from the acetone-butanol-ethanol fermentation using molasses

Commercial production of biobutanol via acetone-butanol-ethanol (ABE) fermentation is challenged by the energy-intensive recovery of biobutanol from inherently diluted fermentation broth. Towards the solution of this problem, four combinations (scenarios) of separation techniques such as distillation, gas stripping (GS), liquid-liquid extraction (LLE), and pervaporation (PV) are investigated for the minimum unitary production cost of biobutanol. Cost components for the installation and operation of the plant are estimated using Matlab software and method given by Peters and Timmerhaus. Production costs with significant variables viz. biobutanol yield, price of molasses, and fermenter volume are individually estimated. Gas stripping followed by distillation is found as the most economical scenario with a unitary production cost of INR 74/kg. The optimum values of yield and fermenter volume are found as 0.4 kg ABE/kg sugars and 100 m³, respectively. The optimization of three major variables around the baseline values provides nearly 10% reductions in the cost with present fermentation technology. This economic analysis of biobutanol recovery certainly contributes to the commercial production of biobutanol from renewable raw materials. The economic feasibility of biodiesel production may offer better environment to the mankind.     

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 starch-based 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 acetone-butanol-ethanol from corn mash and molasses in batch fermentation

The production of solvents from corn mash and molasses in batch fermentation usingClostridium acetobutylicum P 262 was examined. The content of saccharose of beet molasses used in experiments is determined by using the gravimetric method (52.45% saccharose). The quantities of molasses that are used in the nutrient medium are calculated after doing the above determination. The samples of fermentation liquid are taken within a certain time, the determination of saccharose is done by using the same method, and all the saccharose is converted by the microorganism to organic end products. The quantitative and qualitative determination of acetone-butanol has been made by using gas chromatography. On the other hand, using the three isolation way, three different cultures are obtained, and with microscopic observations, the cultures obtained are of the C.acetobutylicum genus. According to the literature values, the concentration of maximum mixed solvent formed during fermentation is about 2%. This is seen in this experiment. There is only a slight difference from this value. This difference is caused by another organic product that is formed during fermentation.     

The Ultrafast and Continuous Fabrication of a Polydimethylsiloxane Membrane by Ultraviolet‐Induced Polymerization

The polydimethylsiloxane (PDMS) membrane commonly used for separation of biobutanol from fermentation broth fails to meet demand owing to its discontinuous and polluting thermal fabrication. Now, an UV‐induced polymerization strategy is proposed to realize the ultrafast and continuous fabrication of the PDMS membrane. UV‐crosslinking of synthesized methacrylate‐functionalized PDMS (MA‐PDMS) is complete within 30 s. The crosslinking rate is three orders of magnitude larger than the conventional thermal crosslinking. The MA‐PDMS membrane shows a versatile potential for liquid and gas separations, especially featuring an excellent pervaporation performance for n‐butanol. Filler aggregation, the major bottleneck for the development of high‐performance mixed matrix membranes (MMMs), is overcome, because the UV polymerization strategy demonstrates a freezing effect towards fillers in polymer, resulting in an extremely high‐loading silicalite‐1/MA‐PDMS MMM with uniform particle distribution.     

Direct Fermentation of Gelatinized Cassava Starch to Acetone,Butanol, and Ethanol Using Clostridium acetobutylicum Mutant Obtained by Atmospheric and Room Temperature Plasma

The mutant strain designated as ART18, obtained from the wild-type strain Clostridium acetobutylicum PW12 treated by atmospheric and room temperature plasma, showed higher solvent tolerance and butanol production than that of the wild-type strain. The production of butanol was 11.3?±?0.5 g/L, 31 % higher than that of the wild-type strain when it was used for acetone, butanol, and ethanol fermentation in P2 medium. Furthermore, the effects of cassava flour concentration, pH regulators, and vitamins on the ABE production were also investigated. The highest butanol production of 15.8?±?0.8 g/L and butanol yield (0.31 g/g) were achieved after the above factors were optimized. When acetone, butanol, and ethanol fermentation by ART18 was carried out in a 15-L bioreactor, the butanol production, the productivity of butanol, and the total solvent were 16.3?±?0.9, 0.19, and 0.28 g/L^/h, respectively. These results indicate that ART18 is a promising industrial producer in ABE fermentation.     

Continuous ethanol extraction by pervaporation from a membrane bioreactor

In order to obtain a high productivity of ethanol, a membrane bioreactor consisting of a fermentor and a pervaporation system was applied to the continuous alcoholic fermentation process. A microporous hydrophobic polytetrafluoroethylene membrane was used for pervaporation. Glucose medium and baker's yeast were used for the fermentation. Three types of continuous fermentation experiment were carried out: conventional free-cell fermentation as the standard process; a fermentation in which product ethanol was extracted continuously by pervaporation from the membrane bioreactor; and a fermentation in which ethanol was extracted by pervaporation and part of the culture broth was simultaneously removed from the fermentation system.

The fermented ethanol was continuously extracted, and simultaneously concentrated by pervaporation, from the membrane bioreactor, and the extracted ethanol concentration was 6 to 8 times higher than in the broth. A high concentration of microorganisms was realized by immobilizing cells in the membrane bioreactor. When the ethanol concentration in the broth was kept low by pervaporation, the specific rate of ethanol production increased. However, the fraction of viable cells decreased because of the accumulation of inorganic salts fed as a nutrient, of nonvolatile by-products and of aged cells, which were not extracted by pervaporation from the fermentation solution. In order to achieve a high ethanol productivity, part of the fermentation broth must be removed from the membrane bioreactor.