Cells Of Clostridium Acetobutylicum Research Articles

Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

A method is presented for the introduction of plasmids into Clostridium acetobutylicum ATCC 8052 by electroporation. A plasmid shuttle vector, pMTL500E, which contains the erythromycin resistance gene and replication machinery of plasmid pAMβ1, was constructed and introduced into C. acetobutylicum by electroporation. The vector was then used to introduce a 2.2 kb ClaI/SphI chromosomal fragment from C. pasteurianum into a leucine requiring mutant of C. acetobutylicum, SBA9, where complementation of auxotrophy was observed. Plasmid DNA indistinguishable from that introduced, on the basis of agarose gel electrophoresis, was observed in transformants containing either plasmid.

Model for continuous production of solvents from whey permeate in a packed bed reactor using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar

A mathematical model has been developed to describe the operation of a packed bed reactor for the continuous production of solvents from whey permeate. The model has been used to quantitate the amounts of different physiological/ morphological types of biomass present in the reactor. The majority of biomass is inert, i.e. it neither grows nor produces solvent. Only relatively small amounts of biomass actively grow (vegetative, non-solvent-producing cells), while even smaller amounts are responsible for solvent production (clostridial, solvent-producing cells).

In situ extractive fermentation of acetone and butanol

The productivity of the acetone-butanol fermentation was increased by continuously removing acetone and butanol from the fermentation broth during fed-batch culture. Whole broth containing viable cells of Clostridium acetobutylicum was cycled to a Karr reciprocating plate extraction column in which acetone and butanol were extracted into oleyl alcohol flowing counter-currently through the column. By continuously removing these toxic metabolites from the broth, end product inhibition was reduced, and a concentrated feed solution containing 300 g/L glucose was fermented at an overall butanol productivity of 1.0 g/L h, 70% higher than the productivity of normal batch fermentation. The continuous extraction process provides flexible operation and lends itself to process scale-up.

Reactor design for the ABE fermentation using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar

Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar. and used for the production of solvents (ABE fermentation) from whey permeate. When the process was performed in packed bed reactors operated in a vertical or inclined mode, solvent productivities approximating 6 kg/(m3h) were obtained. However, the systems suffered from blockage due to excess biomass production and gas hold-up. These problems were less apparent when a partially-packed bed reactor was operated in the horizontal mode. A fluidized bed reactor proved to be the most stable of the systems investigated, and a productivity of 4.8 kg/(m3h) was maintained over a period of 2000 h of operation. The results demonstrate that this type of reactor may have a useful future role in the ABE fermentation.

In-line toxic product removal during solvent production by continuous fermentation using immobilized Clostridium acetobutylicum

Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar, and used in a two-stage continuous reactor for solvent production from whey permeate. Gas-stripping (N 2 gas), an adsorbent resin (XAD-16) and a molecular sieve (silicalite) were evaluated for their use in between-stages solvent removal. All three techniques removed significant quantities of solvents, but not lactose, and allowed increases in sugar utilization and solvent productivity in the second stage. Gas stripping was the most successful technique, possibly because it removed only volatile solvents and not essential nutrients.

Continuous solvent production from whey permeate using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar

Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar and used in a packed bed reactor for the continuous production of solvents from whey permeate. A maximum solvent productivity of 4.1 g l −1 h −1, representing a yield of 0.23 g solvent/g lactose utilized, was observed at a dilution rate of 1.0 h −1. The reactor was operated under stable conditions for 61 days. High concentrations of lactose in the whey permeate favored solventogenesis, while low concentrations favored acidogenesis.

L(+)-lactate dehydrogenase ofClostridium acetobutylicumis activated by fructose-1,6-bisphosphate

Cells of Clostridium acetobutylicum contained an NADH-dependent L(+)-lactate dehydrogenase which was activated specifically by fructose-1,6-bisphosphate (F-1,6-P2), with calcium or magnesium ions as positive effectors. During the purification steps the enzyme was very unstable. The purified enzyme existed in a tetrameric structure (apparent Mr of about 159 kDa) and had its pH optimum at pH 5.8. Little activity was left at pH values below 5.0. The enzyme was unidirectional, catalysing only the reduction of pyruvate. The half maximal activation of the reaction velocity with F-1,6-P2 depended on the pyruvate concentration.

Phosphotransferase Activity in Clostridium acetobutylicum from Acidogenic and Solventogenic Phases of Growth

Clostridium acetobutylicum cells, when energized with fructose, transported and phosphorylated the glucose analog 2-deoxyglucose by a phosphoenolpyruvate-dependent phosphotransferase (PT) system. Butanol up to 2% did not inhibit PT activity, although its chaotropic effect on the cell membrane caused cellular phosphoenolpyruvate and the 2-deoxyglucose-6-phosphate to leak out. Cells harvested from the solventogenic phase of batch growth had a significantly lower PT activity than did cells from the acidogenic phase.

Rôle de l'acétate et du butyrate dans l'induction de la NADH: rubrédoxine oxydoréductase chez Clostridium acetobutylicum

Study of the biosynthesis of NADH: rubredoxin oxidoreductase in resting cells of Clostridium acetobutylicum shows that this enzyme is synthesized at a maximal rate in the presence of acetic acid at a concentration of 3 g . l-1 and at pH 4.8. Protons do not play any role in this biosynthesis since no induction is observed in a medium without acetate for the same values of pH. Butyric acid at a concentration of 0.5 g . l-1 gives 50% induction and formic acid, isobutyric acid and propionic acid have no inductive action on NADH: rubredoxin oxidoreductase. These results are confirmed by studies using a dialysis bag. Only a culture against acetic acid at an initial concentration of 2 g . l-1 gives maximal biosynthesis of the enzyme, whereas a culture in which all products of metabolism are eliminated gives an activity which is 80% lower.

Induction of acetoacetate decarboxylase in Clostridium acetobutylicum

Factors that may initiate the biosynthesis of acetoacetate decarboxylase were investigated in resting cells of Clostridium acetobutylicum. Linear acids from C1 to C4 were inducers, whereas branched acids and linear acids from C5 to C7 were not inducers of acetoacetate decarboxylase biosynthesis. Induction of acetoacetate decarboxylase was maximal at pH 4.8 in the presence of acid concentrations comparable with those found during fermentation. In growth conditions repression of acetoacetate decarboxylase biosynthesis was found. This fact explains that acetone production by Clostridium acetobutylicum occurs when growth slows down.

Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum

Clostridium acetobutylicum cells were collected from chemostats which were run at pH 4.3 or 6.0 and which produced either acetone-butanol or acetate-butyrate; they were used to determine the level of enzymes involved either in solvent or in acid formation. The highest activity of phosphotransacetylase, phosphotransbutyrylase, acetate kinase, and butyrate kinase was found in cells which carried out an acetate-butyrate fermentation; these enzymes were present in solvent-producing cells at a level of about 10–50% as compared to acid-producing cells. Hydrogenase activity was detectable in approximately the same amounts in both cell types; however, in solvent-producing cells it was only measurable following a lag-period. Butyraldehyde and butanol dehydrogenases were found in small amounts exclusively in solvent-producing cells. It was demonstrated that the formation of acetone was initiated by the action of a coenzyme A-transferase which transferred coenzyme A from acetoacetyl-CoA to either acetate or butyrate. This coenzyme A-transferase as well as acetoacetate decarboxylase were hardly detectable in acid-producing cells, but reached high levels in solvent producing cells. Similar changes of the activity of the enzymes mentioned were observed when a batch culture was shifted from acid to solvent formation.

Continuous production of butanol with immobilized cells ofClostridium acetobutylicum

Spores ofClostridium acetobutylicum were immobilized in calcium alginate. An active gel preparation was obtained after outgrowth of the spores to vegetative cells within the gel matrix. A 100 mL column containing the immobilized cells was used for continuous production. At steady-state conditions the productivity of butanol was 67 g/L reactor volume/day.

Pentose Metabolism in Clostridium acetobutylicum

L-Arabinose ketol-isomerase (EC 5. 3. 1. 4) was extracted from the L-arabinose-grown cells of Clostridium acetobutylicum and partially purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and the gel filtration on Sephadex G-200. Optimum pH and temperature for the activity of the enzyme were 7.5 and 37°C, respectively. The enzyme was inhibited by EDTA, Na-pyrophosphate and KCN, and its activity was restored by the addition of divalent metal ions such as Mn2+ and Co2+. The Michaelis-Menten's constants for L-arabinose, Mn2+ and Co2+ were 1.08×10-2M, 0.83×10-4M and 0.38×10-4M, respectively. The equilibrium constant (K=ribulose/arabinose) was 3.14 (37◊C, pH 7.5). The thermodynamic quantities, ΔH, ΔG (37°C), ΔS (37°C) and E were +7354cal/mole, -710.7cal/mole, +26.0cal/(deg•mole) and +7471cal/mole, respectively.

Triosephosphates and pyruvic acid intermediates in fermentations of pentoses by living cells of Clostridium acetobutylicum.

Da fermentazioni dello xilosio e dell'arabinosio con cellule vive diClostridium acetobutylicum e stato possibile isolare i triosofosfati e l'acido piruvico. Il risultato depone a favore dell'ipotesi che la meta a C-3 dei pentosi venga demolita secondo lo schema di Meyerhof.

An improved means of giving increased strength to paper

Production of acetone–butanol–ethanol (ABE) from potato waste starch using free and immobilized cells of Clostridium acetobutylicum was investigated. The starch was directly fermented to ABE by C. acetobutylicum without hydrolysis, and the results were compared with those obtained from glucose as a reference. Maximum butanol yields from waste starch and glucose were 0.21 and 0.26 g/g of initial carbon source (20 g/L), respectively. Batch fermentation of 60 g/L of waste starch in a 5 L bioreactor with free cells resulted in production of 9.9 g/L butanol. In a repeated batch fermentation, using immobilized cells of the bacterium in calcium alginate–polyvinyl alcohol (PVA)–boric acid beads and 60 g/L of the waste starch, a final butanol concentration of 15.3 g/L was achieved.