Solid carriers for a Clostridium acetobutylicum that produces acetone and butanol

Several high strength solids have been tested as carriers for acetone-butanol production by Clostridium acetobutylicum ATCC 824. In batch fermentation, coke, kaolinite and Gel White (a montmorillonite clay) appeared to have a beneficial effect on this fermentation, although the effectiveness appeared to be dependent on the medium used. One of the least expensive materials, coke, was found to be suitable for use in continuous culture. Steady state conditions could be maintained for more than 30 days with total solvent production, productivity and yield of 12 g/l, 1.12 g l⁻¹h⁻¹and 0.3 g TS/g glucose used, respectively.     

Evaluation of solvents for extractive butanol fermentation with Clostridium acetobutylicum and the use of poly(propylene glycol) 1200

Summary In an effort to improve the viability of acetone-butanol-ethanol fermentation by extractive fermentation, 63 organic solvents, including alkanes, alcohols, aldehydes, acids, and esters, were experimentally evaluated for biocompatibility with Clostridium acetobutylicum by observing gas evolution from cultures in contact with candidate solvents. Thirty-one of these solvents were further tested to determine their partition coefficient for butanol in fermentation medium. The biocompatible solvent with the highest partition coefficient for butanol (4.8), was poly(propylene glycol) 1200, which was selected for fermentation experiments. This is the highest partition coefficient reported to date for a biocompatible solvent. Extractive fermentations using concentrated feeds were observed to produce up to 58.6 g·l^–1 acetone and butanol in 202 h, the equivalent of three control fermentations in a single run. Product yields (based on total solvent products and glucose consumed) of 0.234 g·g^–1 to 0.311 g·g^–1 and within run solvent productivities of 0.174 g·l^–1·h^–1 to 0.290 g·l^–1·h^–1 were consistentwith conventional fermentations reported in the literature. The extended run-time of the fermentation resulted in an overall improvement in productivity by reducing the fraction of between-run down-time for fermentor cleaning and sterilization.     

Production of ethanol from molasses at 45 °C using alginate-immobilized Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain, Kluyveromyces marxianus IMB3, has been immobilized in calcium alginate matrices. The ability of the biocatalyst to produce ethanol from cane molasses originating in Guatemala, Honduras, Senegal, Guyana and the Philippines was examined. In each case the molasses was diluted to yield a sugar concentration of 140?g/l and fermentations were carried out in batch-fed mode at 45?°C. During the first 24 hours, the maximum ethanol concentrations obtained ranged from 43–57?g/l with optimum production on the molasses from Honduras. Ethanol production during subsequent re-feeding of the fermentations at 24-hour intervals over a 120-hour period, decreased steadily to concentrations ranging from 20–36?g/l and it was found that ethanol productivity remained highest in fermentations containing the molasses from Guyana. When each set of fermentations was re-fed at 120?h and allowed to continue for 48?h, ethanol production again increased to a maximum with concentrations ranging from 25–52?g/l. It was also found however, that increasing the time between re-feeding at this stage in fermentation had a detrimental effect on the functionality of the biocatalyst.     

Improvement of mannitol production by Lactobacillus brevis mutant 3-A5 based on dual-stage pH control and fed-batch fermentations

Lactobacillus brevis 3-A5 was isolated and expected to produce mannitol efficiently by regulating pH in batch and fed-batch fermentations. In 48 h batch fermentations with free and constant pH, the optimal pH for cell growth and mannitol production in the first 24 h of incubation was 5.5, whereas that for mannitol production in the second 24 h of incubation was 4.5. To achieve high cell density and mannitol yield simultaneously, a dual-stage pH control strategy was proposed based on the kinetic analysis of mannitol production. The pH value was controlled at 5.5 for the first 12 h of fermentation and subsequently shifted to 4.5 until the fermentation was completed. Under dual-stage pH control fermentation, a 103 g/L yield of mannitol with a volumetric production rate of 3.7 g/L/h was achieved after 28 h. The dual-stage pH control fed-batch fermentation strategy was further developed to improve mannitol yield, wherein the yield increased by 109 % to 215 g/L after 98 h of fermentation. This value is the highest yield of mannitol ever reported using L. brevis.     

System Development for Linked-Fermentation Production of Solvents from Algal Biomass

Five species of the genus Dunaliella (D. tertiolecta, D. primolecta, D. parva, D. bardawil, and D. salina) were examined for glycerol accumulation, growth rate, cell density, and protein and chlorophyll content. The suitability of each algal species for use as a fermentation substrate was judged according to glycerol accumulation and quantities of neutral solvents produced after sequential bacterial fermentations. When grown in 2 M NaCl, with 24 mM NaHCO₃ or 3% CO₂ at 28°C and with 10,000 to 15,000 lx of incident light on two sides of a glass aquarium, four of the five species tested produced ca. 10 to 20 mg of glycerol per liter of culture. Clostridium pasteurianum was found to convert an algal biomass mixture supplemented with 4% glycerol to ca. 16 g of mixed solvents (n-butanol, 1,3-propanediol, and ethanol) per liter. Acetone was not detected. Additionally, it has been demonstrated that Dunaliella concentrates of up to 300-fold can be directly fermented to an identical pattern of mixed solvents. Overall solvent yields were reduced by >50% when fermentations were performed in the presence of 2% NaCl. These results are discussed in terms of practical application in tropical coastal zones.     

Production of acetone and butanol by Clostridium acetobutylicum in continuous culture with cell recycle

Summary To increase the solvent productivity of the acetone-butanol fermentation, a continuous culture of Clostridium acetobytylicum with cell recycling was used. At a dry cell mass concentration of 8 g l^-1 and a dilution rate of D=0.64 h^-1, a solvent productivity of 5.4 g l^-1 h^-1 was attained. To prevent degeneration of the culture, which occurs with high concentrations of solvents (acetone, butanol and ethanol), different reactor cascades were used. A two-stage cascade with cell recycling and turbidostatic cell concentration control turned out to be the best solution, the first stage of which was kept at relatively low cell and product concentrations. A solvent productivity of 3 and 2.3 g l^-1 h^-1, respectively, was achieved at solvent concentrations of 12 and 15 g l^-1.Symbols D Dilution rate (h^-1) - r _p solvent productivity (g l^-1 h^-1) - s residual glucose concentration (g l^-1) - V _R reactor volume (l) - V _O overall volume (l) - x (dry) cell mass concentration (g l^-1) - Y _P/S solvent yield (g g^-1)     

In-situ recovery of butanol during fermentation

End product inhibition can be reduced by the in situ removal of inhibitory fermentation products as they form. Extractive fermentation, in which an immiscible organic solvent is added to the fermentor in order to extract inhibitory products, was applied to the acetone-butanol fermentation. Six solvents or solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetradecanol in kerosene, 50 wt% dodecanol in kerosene, oleyl alcohol, 50 wt% oleyl alcohol in a decane fraction and 50 wt% oleyl alcohol in benzyl benzoate. The best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of Clostridium acetobutylicum, glucose consumption is limited to about 80 kg/m³ due to the accumulation of butanol in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, over 100 kg/m³ of glucose can be fermented. Removal of butanol from the broth as it formed also increased the rate of butanol production. Maximum volumetric butanol productivity was increased by as much as 60% in extractive fermentation compared to batch fermentation. Butanol productivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations.     

Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici

Cells of the propionate-tolerant strain Propionibacterium acidipropionici P200910, immobilized in calcium alginate beads, were tested for propionic and acetic acid production both in a semidefined laboratory medium and in corn steep liquor in batch, fed-batch, and continuous fermentation. Cell density was about 9.8 × 10⁹ cells/g (wet weight) of beads, and beads were added to the medium at 0.1 g (wet weight) beads/ml. Beads could be reused for several consecutive batch fermentations; propionic acid production in the tenth cycle was about 50%–70% of that in the first cycle. In batch culture complete substrate consumption (glucose in semidefined medium, lactate in corn steep liquor) and maximum acid production were seen within 36 h, and acid yields from the substrate were higher than in free-cell fermentations. Fed-batch fermentations were incubated up to 250 h. Maximum propionic acid concentrations obtained were 45.6 g/l in corn steep liquor and 57 g/l in semidefined medium; this is the highest concentration achieved to date in our laboratory. Maximum acetic acid concentrations were 17 g/l and 12 g/l, respectively. In continuous fermentation of semidefined medium, dilution rates up to 0.31 h^–1 could be used, which gave higher volumetric productivities (0.96 g l^–1 h^–1 for propionic acid and 0.26 g l^–1 h^–1 for acetic acid) than we have obtained with free cells. Corn steep liquor shows promise as an inexpensive medium for production of both acids by immobilized cells of propionibacteria.Journal paper no. J- 15614 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project no. 3122     

Immobilized Clostridium acetobutylicum P262 Mutants for Solvent Production

The production of acetone, butanol, and ethanol by two immobilized, sporulation-deficient (spo) Clostridium acetobutylicum P262 mutants which were held in the solventogenic phase was investigated. The spoA2 mutant, which was an early-sporulation mutant and did not form a forespore septum, produced higher solvent yields than did the spoB mutant which was a late-sporulation mutant and was blocked at a stage after forespore septum formation. The spoA2 mutant was also granulose and capsule negative. In a conventional batch fermentation, the wild-type strain produced 15.44 g of solvents per liter after 50 h at a productivity of 7.41 g of solvents per liter per day. The spoA2 mutant produced 15.42 g of solvents per liter at a productivity of 72.4 g of solvents per liter per day, with a retention time of 2.4 h in a continuous immobilized cell system employing a fluidized bed reactor. This represents a major advance, since the immobilization of wild-type cells showed similar increases in productivity but a ca. fivefold reduction in final product concentrations.     

Inhibitor effect of products of metabolism on growth of Clostridium acetobutylicum

Summary The growth of Clostridium acetobutylicum was studied by three ways. 1. In batch fermentation, referred to as the control. 2. Fermentation in dialysis which permits elimination of all the products of metabolism: acids, solvents and gases. In order to test the toxic effect of acids, cultures were dialysed against 2 g l^-1 acetic acid or 2 g l^-1 butyric acid. 3. To test the toxic effect of gases only, batch fermentations were carried out under vacuum or with a continuous bubbling of nitrogen. The first method resulted in a productivity of 1.2 g l^-1 dry cell weight and a maximal specific growth rate of 0.2 h^-1; the second, 20 g l^-1 dry cell weight and a constant maximum specific growth rate (μ=0.39 h^-1) between 14 and 20 h. The toxic effect of acetic and butyric acids, starts at low concentrations and about 4 g l^-1 of both acids results in a decrease of 50% of maximal specific growth rate. The third series of experiments showed that gases produced by the bacteria have a high toxic effect, comparable to that of 5 g l^-1 of acid.     

Isolation and Characterization of Microorganisms Associated with the Traditional Sorghum Fermentation for Production of Sudanese Kisra

Sorghum flour obtained from Sudan was mixed with water in a 1:2 (wt/vol) ratio and fermented at 30°C for 24 h. The bacterial populations increased with fermentation time and reached a plateau at approximately 18 h. At the end of 24 h, sorghum batter pH had dropped from 5.95 to 3.95 and the batter had a lactic acid content of 0.80%. The microbial population during the 24 h of fermentation consisted of bacteria (Pediococcus pentosaceus, Lactobacillus confusus, Lactobacillus brevis, Lactobacillus sp., Erwinia ananas, Klebsiella pneumoniae, and Enterobacter cloacae), yeasts (Candida intermedia and Debaryomyces hansenii), and molds (Aspergillus sp., Penicillium sp., Fusarium sp., and Rhizopus sp.). P. pentosaceus was the dominant microorganism at the end of the 24-h fermentation. When three consecutive fermentations using an inoculum from the previous fermentation were carried out, the bacterial population increase plateaued at 9 h. The microbial populations in these fermentations were dominated by P. pentosaceus.     

Kissiris: A mineral support for the promotion of ethanol fermentation by Saccharomyces cerevisiae

Ethanol production using Saccharomyces cerevisiae, promoted by the mineral kissiris, is reported on. A three-fold increase of ethanol productivity in the fermentation of molasses was achieved. An ethanol yield factor 0.43 g/g and conversion of 93.3% at an initial sugar concentration (ISC) 208.5 g/l were obtained in the presence of this mineral in molasses fermentation, compared to 0.21 g/g and 44.2% in its absence. It is also shown that the fermentation of molasses takes place even at relatively higher ethanol levels, with kissiris contributing to a 35% reduction of the energy demand in grade-fuel and potable ethanol production. The proposed mineral was shown to have a smaller effect in fermentations carried out in synthetic media containing glucose or saccharose.     

Lactic Acid Production in a Mixed-Culture Biofilm Reactor

Novel solid supports, consisting of polypropylene blended with various agricultural materials (pp composite), were evaluated as supports for pure- and mixed-culture continuous lactic acid fermentations in biofilm reactors. Streptomyces viridosporus T7A (ATCC 39115) was used to form a biofilm, and Lactobacillus casei subsp. rhamnosus (ATCC 11443) was used for lactic acid production. For mixed-culture fermentations, a 15-day continuous fermentation of S. viridosporus was performed initially to establish the biofilm. The culture medium was then inoculated with L. casei subsp. rhamnosus. For pure-culture fermentation, L. casei subsp. rhamnosus was inoculated directly into the reactors containing sterile pp composite chips. The biofilm reactors containing various pp composite chips were compared with a biofilm reactor containing pure polypropylene chips and with a reactor containing a suspension culture. Continuous fermentation was started, and each flow rate (0.06 to 1.92 ml/min) was held constant for 24 h; steady state was achieved after 10 h. Lactic acid production was determined throughout the 24-h period by high-performance liquid chromatography. Production rates that were two to five times faster than those of the suspension culture (control) were observed for the pure- and mixed-culture bioreactors. Both lactic acid production rates and lactic acid concentrations in the culture medium were consistently higher in mixed-culture than in pure-culture fermentations. Biofilm formation on the chips was detected at harvest by chip clumping and Gram staining.     

The fermentative production of acetone-butanol by Clostridium acetobutylicum.

Fourteen different media were used in the fermentative production of acetone-butanol. The highest total yields were achieved in medium I. Potato starch and soluble starch were suitable as carbon sources. The best concentrations of potato starch and soluble starch were 500.0 and 10.0 g/l, respectively. Peptone was the most favourable nitrogen source. The best concentration of peptone was 4.0 g/l. Calcium carbonate in 3.6 g/l acted as buffering agent in the fermentation process. The best initial pH value of the fermentation medium was 6.0. The optimum temperature was 32--33degreesC. The fermentation process required 120 h to obtain maximum yields of acetone-butanol.     

Production of volatile fatty acids from bagasse by rumen bacteria

Summary Conditions are described for converting bagasse lignocellulose to volatile fatty acids (VFA) by anaerobic fermentation. Although yields of VFA were as high as 74% by weight of digestible organic matter (or 54% of dry bagasse), limitations were imposed by both fermenter design and fibre digestibility. All fermentations were substrate-limited up to the maximum initial concentration examined of 50 g bagasse · l^-1 and no product inhibition was evident (up to 260 mM VFA produced). Maximum VFA productivities of 0.25 to 0.65 g · l^-1 · h^-1 were obtained in batch fermentations and this is greater than those previously reported using lignocellulosic substrates. Batch fermentations neared completion after 66 h.     

A general inhibition kinetics model for ethanol production using a novel carbon source: sodium gluconate

In this study, sodium gluconate was applied as a novel carbon source for the fuel ethanol production using an engineered Escherichia coli strain KO11 in batch fermentations. Ethanol and acetic acid were produced as two major products as well as small amount of lactic acid during the fermentation. Compared to the conventional carbon source glucose, the bioconversion of sodium gluconate possessed two distinct advantages: faster utilization rate of sodium gluconate (1.66 g/L per h) compared to glucose (0.996 g/L per h) and no requirement for pH control during fermentation. A general inhibition model including both substrate and products inhibitory effects was proposed, which adequately simulated batch fermentation kinetics at various concentrations of sodium gluconate. All of the products showed inhibitory effects on cell growth. The order of the inhibitory strength of all products and substrate was for the first time clarified in this study. Acetic acid was the most inhibitory product mitigating the cell growth, followed by ethanol and lactic acid. Sodium gluconate stimulated cell growth when its concentration was below 16 g/L, while it inhibited the cell growth when the concentration was above this concentration. It completely inhibited the cell growth when the concentration was 325 g/L. The high value of both the coefficient of determination (R ²) and the adjusted R ² verified the good fit of the model. This paper provides key insights into further engineering these strains to improve ethanol production.     

Non-porous magnetic supports for cell immobilization

A new method for covering magnetic particles with a stable non-porous layer of a material like zeolite or activated carbon was used for the preparation of support materials with good properties for the immobilization of yeast Saccharomyces cerevisiae cells. The immobilized cells can be used in batch and continuous alcoholic fermentation. A productivity of 35.6 g ethanol/l · h was reached. The adsorption isotherms of the immobilized yeast cells were determined. Yeast cell immobilization on non-porous magnetic supports obeyed the Langmuir isotherm equation. Satisfactory results were obtained also from repeated batch fermentations with fixed cells on supports additionally treated with glutaraldehyde or by simple adsorption.     

Uptake and activation of acetate and butyrate in Clostridium acetobutylicum

Summary The pathway for uptake of acids during the solvent formation phase of an acetone-butanol fermentation by Clostridium acetobutylicum ATCC 824 was studied. ¹³C NMR investigations on actively metabolizing cells showed that butyrate can be taken up from the medium and quantitatively converted to butanol without accumulation of intermediates. The activities of acetate phosphotransacetylase, acetate kinase and phosphate butyryltransferase rapidly decreased to very low levels when the organism began to form solvents. This indicates that the uptake of acids does not occur via a reversal of these acid forming enzymes. No short-chain acyl-CoA synthetase activity or butyryl phosphate reducing activity could be detected. Based on our results and a critical analysis of literature data on acetone-butanol fermentations, it is suggested that an acetoacetyl-CoA: acetate (butyrate) CoA-transferase is solely responsible for uptake and activation of acetate and butyrate in C. acetobutylicum. The transferase exhibits a broad carboxylic acid specificity. The key enzyme in the uptake is acetoacetate decarboxylase, which is induced late in the fermentation and pulls the transferase reaction towards formation of acetoacetate. The major implication is that it is not feasible to obtain a batch-wise butanol fermentation without acetone formation and retention of a good yield of butanol.     

Factors in acid treated bagasse inhibiting ethanol production from d-xylose by Pachysolen tannophilus

The fermentation of d-xylose, the major sugar-cane bagasse hemicellulose component, to ethanol by Pachysolen tannophilus is inhibited by various factors produced or released during the acid hydrolysis of the bagasse or during the fermentation process. These include ethanol, iron, chromium, copper, nickel, acetic acid and furfural. Ethanol production by P. tannophilus is inhibited by ethanol fconcentrations >24 g l^?1. Furfural and acetic acid concentrations as low as 0.3 and 7 g l^?1, respectively, and iron, chromium, nickel and copper at concentrations of 0.07, 0.01, 0.01 and 0.004 g l^?1, respectively. Similar concentrations may be found in acid-hydrolysed bagasse. The removal of these factors by treatment with ion-exchange resin resulted in the fermentation of the sugars to ethanol. The d-glucose was used rapidly and completely whereas d-xylose utilization was slow and incomplete. An ethanol concentration of 4.1 g l^?1 was produced and an ethanol yield of 0.32 was obtained. Xylitol in significant amounts was produced.     

Fed-batch sorbose fermentation using pulse and multiple feeding strategies for productivity improvement

Microbial oxidation of D-sorbitol tol-sorbose byAcetobacter suboxydans is of commercial importance since it is the only biochemical process in vitamin C synthesis. The main bottleneck in the batch oxidation of sorbitol to sorbose is that the process is severely inhibited by sorbitol. Suitable fed-batch fermentation designs can eliminate the inherent substrate inhibition and improve sorbose productivity. Fed-batch sorbose fermentations were conducted by using two nutrient feeding strategies. For fed-batch fermentation with pulse feeding highly concentrated sorbitol (600 g/L) along with other nutrients were fed intermittently in four pulses of 0.5 liter in response to the increased DO signal. The fed-batch fermentation was over in 24 h with a sorbose productivity of 13.40 g/L/h and a final sorbose concentration of 320.48 g/L. On the other hand, in fed-batch fermentation with multiple feeds, two pulse feeds of 0.5 liter nutrient medium containing 600 g/L sorbitol was followed by the addition of 1.5 liter nutrient medium containing 600 g/L sorbitol at a constant feed rate of 0.36 L/h till the full working capacity of the reactor. The fermentation was completed in 24 h with an enhanced sorbose productivity of 15.09 g/L/h and a sorbose concentration of 332.60 g/L. The sorbose concentration and productivity obtained by multiple feeding of nutrients was found to be higher than that obtained by pulse feeding and was therefore a better strategy for fed-batch sorbose fermentation.