Development of markers for product formation and cell cycle in batch cultivation of Clostridium acetobutylicum ATCC 824

Experiments were performed to identify relationships between morphological and physiological events during batch fermentation of Clostridium acetobutylicum ATCC 824. Differing fermentation conditions were obtained by manipulation of the culture pH value during the process. The bacterium showed marked changes in morphology during its cultivation, similar to those previously observed for other strains. However, although the acidogenic phase was characterized by the presence of rod-shaped cells, and the solventogenic phase by clostridial forms, there was no simple relationship between the proportion of clostridial forms present and the ratio of solvents to acids. Nevertheless, the shift from acidogenesis to solventogenesis was always coincident with the presence of some clostridial forms and with the accumulation of granulose within the cells. In addition, the “solvent shift” was associated with major changes in the cellular protein pattern, as analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Hence, the potential solventogenic ability of any particular culture may be recognised by its morphological appearance and/or its cellular protein pattern. Received: 19 September 1997 / Received revision: 9 February 1998 / Accepted: 9 February 1998     

Influence of acetic and butyric acid addition on polysaccharide formation byClostridium acetobutylicum

Summary The production of granulose (an intracellular reserve polygranule), capsule and exopolysaccharide was investigated in a synthetic medium in which the oxido-reduction level was modified by the addition of acetic or butyric acid. After addition of the acids, granulose synthesis increased from 150 to 300 mg glucose equivalents ·1^–1 and capsular synthesis decreased by 25%. Exopolysaccharide production was unchanged under these conditions. A hypothesis that attributes a role to the polymer in the oxido-reduction sequences is discussed.     

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.     

Solvent Production and Morphological Changes in Clostridium acetobutylicum

The morphological and cytological changes which occurred in Clostridium acetobutylicum P262 during the production of acetone, butanol, and ethanol in an industrial fermentation medium were identified and correlated with the growth and physiological changes. The swollen, cigar-shaped clostridial forms were involved in the conversion of acids to neutral solvents, and there was a correlation between the number of clostridial forms and the production of solvents. Sporulation mutants which were unable to form clostridial stages (cls mutants) did not produce solvents. Oligosporogenous mutants which showed reduced clostridial stage formation produced intermediate levels of solvents. Sporulation mutants blocked after the clostridial stage, which were unable to form mature spores (spo mutants), produced normal levels of solvents.     

Initiation of solvent production,clostridial stage and endospore formation in Clostridium acetobutylicum P262

Summary A minimal medium was used to investigate the triggers regulating the initiation of solvent production and differentiation in Clostridium acetobutylicum P262. The accumulation of acid end-products caused the inhibition of cell division and the initiation of solvent production and cell differentiation. Initiation only occurred with a narrow pH range. Glucose or ammonium limited cultures failed to achieve the necessary threshold of acid end-products and solvent production and differentiation were not initiated. The addition of acid end-products or ammonium to cultures containing suboptimal levels of glucose or nitrogen respectively, enhanced solvent production. Resuspension of cells in media containing the threshold level of acid end-products and residual glucose induced endospore formation. Glucose or ammonium limitation did not induce sporulation and there was a requirement for glucose and ammonium during solventogenesis and endospore formation. Initiation of solvent production and clostridial stage formation were essential for sporulation. The induction of endospore formation in C. acetobutylicum P262 differs from that in the aerobic endospore forming bacteria where sporulation is initiated by nutrient starvation.     

Effect of temperature upon growth rate and solvent production in batch cultures ofClostridium acetobutylicum

Summary The effect of temperature on the solvent production byClostridium acetobutylicum has been studied in the range 25 to 40°C. It was found that the solvent yield decreased with increasing temperature; seemingly because of a reduction in acetone production. It appeared that the yield of the other major solvent, butanol, was not affected by the temperature. Considering total solvent yield and productivity only, the optimum fermentation temperature is 35°C.     

Influence of temperature on solvents production from whey

Summary The influence of temperature on solvent production from whey was investigated by using strains ofClostridium acetobutylicum andbutylicum. Higher yields of solvents were observed at 37°C or at 30°C depending on the strain used.     

Improvement of operational stapility of yeast lipase by chemical treatment

Summary Treatment with dithiothreitol stabilized the esterification activity of Celite-adsorbed lipase fromCandida cylindracea in organic solvent systems. The treated lipase showed the stereoselectivity in the esterification of menthol with 5-phenylvaleric acid as in the case of the native enzyme.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

A two-layer culture method was established that uses an organic solvent to remove shikonin derivatives produced on cell surfaces during the culture of suspension cells of Lithospermum erythrorhizon. Some paraffins and a fatty acid ester made suitable solvents, whereas olefins and aromatic solvents extensively inhibited the production of shikonin derivatives. The yield of derivatives increased with an increase in the carbon chain length of the n-paraffin used as the solvent and when the oxygen supply was sufficient it reached the value found for the ordinary culture method.     

Characterization, Biosynthesis, and Regulation of Granulose in Clostridium acetobutylicum

Synthesis of granulose was investigated in 15 solvent-producing Clostridium strains. Only one of the strains did not produce granulose. The structure of granulose in Clostridium acetobutylicum P262 consisted of a high-molecular-weight polyglucan containing only (1-->4) linked d-glucopyranose units. Biosynthesis of granulose in C. acetobutylicum P262 was dependent on ADPglucose pyrophosphorylase, and granulose synthase and mutants defective in granulose accumulation lacked either one or both enzyme activities. Granulose-positive revertants exhibited both enzyme activities. ADPglucose pyrophosphorylase and granulose synthase were not subject to allosteric control by metabolites. Granulose accumulation and the biosynthetic enzyme activities were initiated immediately before the pH breakpoint and were detected in cells only at the end of the exponential growth phase. Granulose accumulation did not occur under conditions of nitrogen limitation, excess carbon, or excess energy.     

Analysis of Tn916-induced mutants ofClostridium acetobutylicum altered in solventogenesis and sporulation

Summary The conjugative transposon Tn916 was used for mutagenesis ofClostridium acetobutylicum ATCC 824. Tetracycline-resistant mutants were screened for loss of granulose synthesis and five classes of granulose mutants, that contained single transposon insertions, were identified on the basis of altered solvent production. Class 1 mutants did not make acetone or butanol, lacked activity of enzymes induced during solventogenesis, and did not sporulate, indicating that they are regulatory mutants. The class 2 mutant strains also did not produce acetone but did form small amounts of butanol and ethanol while the class 3 mutants produced low amounts of all solvents. Class 4 and 5 mutants produced essentially the same or higher amounts of solvents than the parent strain. Transposon insertions in the class 1 mutants were used as markers for in vitro synthesis of flanking chromosomal DNA using Tn916-specific primers. The DNA fragments were labeled to produce specific probes. Transposon insertion sites in the chromosomes of 13 different class 1 regulatory mutants were compared by hybridization of the specific probes to Southern blots of restriction endonuclease-digested parental chromosomal DNA. Insertions in two mutants appeared to be, in the same region of the chromosome. These results predict, that multiple regulatory elements are required to induce solvent production and sporulation.     

The bioconversion of wood hydrolyzates to butanol and butanediol

Steam-exploded aspenwood chips were acid hydrolysed to their component sugars. Near theoretical solvent yields were achieved in both the acetone-butanol-ethanol (ABE) fermentation and 2,3-butanediol fermentation of these liberated sugars. When Clostridium acetobutylicum was grown on wood hydrolysates, final butanol yields of 9.0 g/L (0.26 g of butanol per g of sugar consumed) were obtained. When Klebsiella pneumoniae was grown on the wood hydrolysates, final butanediol concentrations exceeded 20 g/L, resulting in a bioconversion efficiency approaching 0.5 g of butanediol per g of sugar utilised.     

Extraction and quantitation of astaxanthin from Phaffia rhodozyma

Summary The rapid, quantitative release of astaxanthin and other carotenoids from the yeast Phaffia rhodozyma is described. Hashed cells are ruptured with dimethylsulfoxide (DMSO) and carotenoids extracted into an organic solvent. Extraction and spectrophotometric quantitation of total carotenoids is rapid, reproducible and only small volumes (0.1–2 ml) of culture are required. HPLC analysis in normal phase silica gel column indicates that astaxanthin comprises 65–95% of the total pigmented carotenoids of P. rhodozyma.     

Isolation of Clostridium acetobutylicum strains producing butanol and acetone

Summary A system is described for the isolation of bacteria (Clostridium acetobutylicum) from broad beans, potatoes or maize. The isolates were tested in molasses medium and solvent yields up to 18–20 g/litre of butanol plus acetone were obtained.     

The metabolism of lactose byClostridium acetobutylicum

Summary Acetone and butanol biosynthesis byClostridium acetobutylicum ATCC 824 was affected by lactose concentration and by agitation during the fermentation. At 1% and 3% lactose concentrations acid production predominated, while butanol production predominated at 5% lactose concentration. Higher solvent production was observed in fermentors without agitation than in fermentors with agitation.     

Acetone-butanol fermentation of xylose and sugar mixtures

Summary Three strains ofCl. acetobutylicum and one ofCl. butyricum have been tested for their ability to ferment xylose to butanol. ATCC 824 and NRRL 527 produced 0.28 g solvents/g xylose, while ATCC 8260 and NRRL 594 produced much butyric acid. In 2-stage fermentations in which ATCC 8260 or NRRL 594 acted upon xylose for 12 to 20 h, followed by NRRL 527 for a total of 3 days, yields of solvent were better, 0.32 g/g xylose. Upon fermenting a mixture of sugars simulating sulphite waste liquor 0.36 g solvents/g sugar were obtained. Sugar consumption in both cases was about 96%.     

Solvent production by Clostridium beijerinckii NRRL B592 growing on different potato media

Very good solvent formation rates were observed when Clostridium beijerinckii NRRL B592 was cultivated on different whole potato media. The increase in whole potato concentration contributed to the increased final solvent concentrations, while the addition of yeast extract or mineral salts gave negative effects. To obtain good solvent productivities and high final solvent concentrations during batch fermentation, no enzymatic hydrolysis of the potato starch was necessary, indicating high activity of the clostridial amylases produced by the strain applied. Received: 17 April 1998 / Received revision: 22 June 1998 / Accepted: 27 June 1998     

Microbial steroidconversion in lyotropic liquid crystal

Summary The activity and stability of the 1(2)-dehydrogenation system ofPimellobacter simplex in an organic solvent/liquid crystalline phase environment has been studied.     

Production of citric acid from sugars present in wood hemicellulose usingAspergillus niger andSaccharomycopsis lipolytica

Summary One strain each of the fungus,Aspergillus niger, and the yeast,Saccharomycopsis lipolytica, were investigated for their ability to produce citric acid from the sugars present in hemicellulose hydrolysates.S. lipolytica produced citric acid as efficiently from mannose as from glucose, but failed to assimilate xylose, arabinose or galactose.A. niger readily assimilated mannose, xylose and arabinose, and produced citric acid from these sugars although the yields were lower than from glucose. A possible inhibitory effect of arabinose on citric acid production from other sugars was observed usingA. niger.