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Subject: search.filters.subject.Clostridium acetobutylicum

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    Methodology of culture maintenance and inoculum development for production of solvents by Clostridium acetobutylicum : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Biotechnology at Massey University
    (Massey University, 1985) Gutierrez, Noemi A
    Various methods of culture maintenance and inoculum develop­ment were evaluated for their effectiveness in conserving and improving the property of 2 strains of Clostridium acetobutyicum, namely NCIB 2951 and NRRL B-594, to produce solvents by fermentation of whey permeate. The majority of the methods were effective in maintaining the viability and solventogenic property of the organism. However, since in some cases the viability was maintained but the solventogenic property was not, it is clear that the latter should be used as the index in determining the storage life and time of reprocessing of the stock culture. The methods of culture maintenance investigated included refrigeration at 4°c in distilled water, in phosphate buffer and in Cooked Meat Medium containing glucose ( CMMG) ; by freezing at -20°c in distilled water and in phosphate buffer; by drying in soil and by lyophilization ( freeze drying); and by periodic transfer in CMMG and in whey permeate containing yeast extract. Maintenance of the stock cultures at -20° C in distilled water was found to be the most efficient for the storage stability of both strains of organism. The viability and the potential to produce high solvent concentrations, primarily butanol were maintained without any significant loss after 9 months and 12 months, for strain NCIB 2951 and strain NRRL B-594, respectively. The criteria important for a commercial fermentation, i.e., sugar utilization, yield and butanol production rate, remained stable during storage by this method. It was observed that periodic transfer was a poor method as the culture lost their solventogenic property despite remaining viable. The other preservation methods were not as satisfactory as freezing in distilled water at -20°c since the fermentation ability degenerated to some extent after 9 months of storage. Therefore, after such a period reprocessing of the stock cultures kept by these methods is necessary to revive the cultures and minimize degeneration. The repeated use of the stock cultures was found to be deleterious and should be avoided. The inoculum development procedure investigated to maximize fermentation efficiency included the conventional heat shocking of the stock culture; variation in the number of culture stages; use of gassing as an index of transfer time; and the use of different levels of inoculum size. The strain differences which exist between NRRL B-594 and NCIB 2951 influenced how the inocula from these strains should be propagated prior to fermentation. Strain NRRL B-594 responded to heat shocking while strain NCIB 2951 did not. Neither ethanol nor butanol treatment of the stock cultures of the latter were advantageous. Using a 3-stage inoculum development procedure, the ferment­ation efficiency of strain NRRL B-594 was improved by employing heat shocking at 80C for 15 min in the revival stage of the stock culture. The germination factors for the spores of NCIB 2951 await identification. However, by using the presence of highly motile cells as an index in transferring from the revival stage, the inoculum develop­ment procedure resulted in a significantly higher butanol concentration value and production rate. Thus, the revival stage was the most critical.
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    Nutrient requirements for acetone-butanol-ethanol production from whey permeate by Clostridium acetobutylicum P262 in a range of bioreactors : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand
    (Massey University, 1991) Kanchanatawee, Sunthorn
    The acetone-butanol-ethanol (ABE) fermentation process, using Clostridium acetobutylicum P262, was studied. Experiments were conducted in a traditional batch fermentation using freely-suspended cells in a defined medium with lactose as carbon source. Solvent production did occur under conditions of nitrogen-, phosphate- or iron-limitation. However, the optimum conditions were observed when all nutrients were present slightly in excess of growth requirements. A greater excess of nutrients caused the fermentation to be acidogenic rather than solventogenic. Sulphuric acid casein whey permeate, without nutrient supplementation, proved to be a poor substrate for growth and solvent production by this organism. However, the addition of yeast extract (5 g/1) led to strong solventogenesis. The deficiency in the whey permeate was shown to be iron rather than assimilable nitrogen, phosphate or vitamins. Experiments were also performed in a defined medium in continuous culture using freely-suspended cells in a CSTR. An inverse relationship was observed between the biomass concentration and the specific butanol productivity. It is suggested that this was due to the cell population not being homogeneous, and that a change in the nutrient balance led to a change in the relative proportions of acidogenic, solventogenic and inert cells (spores). The addition of supplementary yeast extract during continuous solvent production from whey permeate using free cells in a CSTR showed that yeast extract supplementation of less than 1 g/1 favoured acid production rather than solvent production. It is unlikely that high solvent productivities can be attained with this substrate in a single stage continuous culture system with freely-suspended cells. A maximum solvent productivity of 0.05 g/l.h was observed in continuous culture compared to 0.06 g/l.h in batch culture. A continuous fermentation process was investigated using cells immobilized by adsorption onto bonechar and operated in a packed bed reactor. Three nutrients (i.e. nitrogen, phosphate and iron) were selected for this investigation. Solvent production was favoured by high concentrations of these nutrients in the influent medium. It was not possible to restrict the supply of phosphate or iron in the reactor due to leaching of the bonechar. However, conditions where biomass growth was restricted by a restriction in the nitrogen supply were not conducive to solvent production. This reactor was also operated using whey permeate as the substrate. The biomass build-up was controlled by minimizing the supplementary yeast extract concentration of the feed, but this had deleterious effects on solvent production. Hence, when producing solvent from whey permeate in this type of reactor a compromise must be made between strong solventogenesis and reactor longevity.
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    Investigation of lactose utilization genes in Clostridium acetobutylicum : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand
    (Massey University, 1988) Hancock, Kerrie Ruth
    Preparatory to constructing a genomic library of Clostridium acetobutylicum, cell lysis and DNA preparation conditions leading to undegraded DNA were examined. Early to mid-exponential phase cells lysed more readily than cells at late-exponential or stationary phase. Lysis was facilitated by 0.3% w/v glycine in the growth medium. Achromopeptidase was a more effective lytic enzyme than either mutanolysin or lysozyme. All strains of C. acetobutylicum produced high levels of DNase activity, coinciding with the late-exponential and stationary growth phases. Chromosomal DNA isolated from all strains of C. acetobutylicum was rapidly degraded. This degradation was not prevented by the use of various protein inactivating agents. The adverse effect of oxygen and related radicals on the DNA of this strict anaerobe was considered to be responsible. Undegraded DNA was isolated by protecting the cells to be lysed from oxygen. A genomic library of C. acetobutylicum NCIB2951 in the cosmid vector pLAFR1, constituting 3,500 recombinant clones, was prepared. Clones from this library complemented various Escherichia coli auxotrophic mutations, showing that C. acetobutylicum genes are expressible in E. coli. Recombinant clones coding for the β-galactosidase of NCIB2951 were isolated from the genomic library using the chromogenic substrate X-gal. The lacY mutation of HB101 could not be complemented by these clones, suggesting that a classical lac operon system does not exist in C. acetobutylicum. The β-galactosidase (cbg) gene was further subcloned on a 5.2 kb EcoRI fragment, and was expressed when the fragment was cloned in either orientation. Cbg was thus expressed from its own promoter. The cbg gene is inducible by lactose in C. acetobutylicum. When cloned into E. coli, however, this gene was expressed constitutively, the level being unaffected by the presence of the inducer, IPTG or glucose. Six strains of C. acetobutylicum possessed a sequence highly homologous to the cloned β-galactosidase fragment. The β-gatactosidase gene region of NCIB2951 showed only low homology to the DNA from other Gram-positive bacteria (Streptococcus lactis ATCC7962, Streptococcus thermophilus DRI1424, Lactobacillus bulgaricus DRI20056, Lactobacillus helviticus DRI20064), and no detectable homology to DNA from Gram-negative bacteria (E. coli DC272 or Rhizobium loti PN2231). The β-galactosidase activity of the 5.2 kb fragment was inactivated by Tn5 insertion at either of two loci. Locus I (400 bp) was approximately 500 bp from locus II (approximately 3.2 kb). Maxi-cell analysis identified a 100 kDa protein as the β-galactosidase gene product. The 5.2 kb fragment was sequenced and analyzed. Three ORF's were identified. ORF1 (cbgA) coded for the structural β-galactosidase gene. Significant amino acid homology was detected with the amino acid sequences of the lacZ, ebgA (E. coli) and lacZ (Klebsiella pneumoniae). ORF2 (cbgR) coded for a small regulatory protein which shared homology with the amino acid sequence of the "0.3 kb gene" from Bacillus subtilis. ORF3 coded for a truncated protein which shared significant homology with the N-terminal amino acid regions of spo0A and spo0F (B. subtilis), two regulatory proteins of the two-component system. Hence, no lac operon exists in C. acetobutylicum.
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    Intensification of the acetone, butanol, ethanol fermentation using whey permeate and Clostridium acetobutylicum : a preliminary study : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University
    (Massey University, 1987) Ennis, Brett Mills
    The use of whey permeate as the fermentation substrate for the production of acetone:butanol:ethanol (solvents), using C. acetobutylicum P262 was studied. Initial experiments were conducted in a batch mode using sulphuric acid casein whey permeate medium, in an attempt to optimize the culture conditions for maximal extent of lactose utilization and solvents production. A high initial lactose concentration (65-75 g/l) in combination with a culture pH maintained in the region pH 5.4 to 5.6 were the most favourable conditions for solvent production. An inverse relationship between the lactose utilization rate and solvents yield was observed. Solvent productivities were only 60% however, of that achievable with this strain of organism on an industrial scale using a molasses medium, but comparable productivities were obtained using a semi-synthetic medium containing glucose. Hydrolysed-lactose sulphuric acid casein whey permeate medium was investigated as a medium for solvent production. Glucose and galactose were utilized simultaneously, although glucose was used preferentially. Only a small increase in solvents productivity was obtained compared with that obtained using non-hydrolysed permeate. Experiments were performed in continuous culture using cheese whey permeate medium and alginate-immobilized cells. Significantly greater solvent productivities were obtained, compared with those achieved using free cells in batch culture. Fermentations were operated for over 650 hours with no detectable loss in fermentation performance. The extent of lactose utilization was low, however (less than 40%), and attempts to increase this by the use of pH regulation or a two-stage process were unsuccessful. This fermentation process was described as a biomass volume process (volumetric fraction of alginate beads in the reactor), where the lactose utilization and hence the solvents production, was defined by an inhibitory concentration of butanol, approximately 5 g/l. An alternative continuous fermentation process using free cells and cheese whey permeate medium was investigated. External cell recycle using cross-flow microfiltration (CFM) membrane plant to continuously separate cells from the fermentation culture and recycle them back to the fermenter was utilized. Biomass was continuously removed from the fermenter in order to achieve a stable biomass concentration. Stable solvents production was not achieved under the range of culture conditions investigated; culture degeneration was attributed to the complex interactive morphological cyclic behaviour of the organism. A tubular CFM unit which could be periodically backflushed to maintain the filtrate flux, was found to be the most suitable of those tested. The integration of in-situ or in-line solvents recovery with batch culture using free cells, and continuous fermentation using cells immobilized by adsorption to bonechar, was investigated in order to remove toxic solvents and so increase the extent of lactose utilization and solvents productivity. A novel process using gas-stripping with an inert gas, and solvents recovery from the vapour phase by condensation using a cold trap, was described. An increase in lactose utilization and solvents productivity was achieved in both fermentation modes compared with control fermentations. The use of adsorbent resins and a molecular sieve for integrated fermentation solvents recovery was also demonstrated. However, the adsorption of medium components may mitigate against the usefulness of such a process option. The batch refermentation of batch fermentation effluent treated by gas-stripping to remove solvents was investigated. However, solvent production was favoured only when lactose and nutrients were supplemented to concentrations similar to those present originally. Conversely, fermentation medium treated by gas-stripping to remove solvents could be readily refermented to produce solvents when an existing cell population was used, suggesting that this option of an integrated continuous fermentation-product recovery process may be promising for whey permeate solvent production.
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    Role of motility and chemotaxis in solvent production by Clostridium acetobutylicum : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University
    (Massey University, 1989) Gutierrez, Noemi A.
    The motility of Clostridium acetobutylicum P262 and its relationship to solventogenesis were investigated. Motility was monitored in a typical batch fermentation process using sulphuric acid casein whey permeate as substrate. The motile behaviour of C. acetobutylicum was characterized by "runs" wherein the cells were observed to swim in a long, smooth line, then the cells "tumbled" by thrashing around for a few seconds before running again. The "runs" were particularly associated with the early phase of sugar utilization and acid production, while "tumbles" were associated with the onset of solventogenesis. During solvent production, the cells tumbled more frequently and the runs progressively became shorter and slower. The proportion of cells in the culture which exhibited motility increased to almost 100% up to 13h after inoculation, but decreased considerably after this time. Assays for positive chemotaxis (chemoattraction) and negative chemotaxis (chemorepulsion) were performed to identify the chemoeffectors of C. acetobutylicum. Motile cells of C. acetobutylicum were observed to migrate towards glucose, galactose, and lactose. These sugars were identified as attractants. Acetate and butyrate elicited a dual response. Cells were repelled from the dissociated form and attracted towards the undissociated form above a minimum threshold concentration. Chemoattraction to butyric acid was observed at a threshold concentration of 9 x 10-2 M which is similar to the concentration of undissociated butyric acid inside the cell (1.3 x 10-2 M) at which solventogenesis is reported to be initiated, suggesting that the intracellular butyric acid concentration is the likely switch for solventogenesis to commence. The solvents acetone, butanol and ethanol were identified as repellents. The behavioural response of C. acetobutylicum towards the sugars, acids and solvents demonstrates that the motility observed during fermentation is a chemotactic response. Chemotaxis appears to provide survival advantage to C. acetobutylicum. A non-motile mutant was isolated by mutagenesis using ethyl methane sulfonate. This mutant was morphologically indistinguishable from the motile parent strain, such that it possesses flagella in typical number and shape as those of the parent, and is capable of producing clostridial forms and endospores. This type of mutant is a paralyzed mutant and the mutation may be a defect in any of the genes that code for flagellar rotation. The non-motile mutant was capable of solvent production suggesting that motility is not a regulatory mechanism for the switch to solvent production, but merely a behavioural chemotactic response. However, the maximum butanol concentration achieved, the initial rate of butanol production, the yield, and the sugar utilization observed with the mutant were poorer than those of the parent strain. These confirm the positive relationship between motile, chemotactic cultures and solvent production. The low butanol production by the non-motile mutant suggests that the mutant has a lower butanol tolerance than does the parent. Inhibition studies have shown that both growth and solvent production of C. acetobutylicum are subject to end-product toxicity. Growth inhibition studies confirmed that the non-motile mutant was less tolerant to solvents than was the parent. A deficiency in membrane-bound ATPase activity was observed with the non-motile mutant but not in the parent strain. This deficiency in ATPase activity, lack of motility, and lower butanol tolerance may explain the low butanol production by the mutant. On a percentage basis, greater inhibition of solvent production was observed in the parent than in the mutant suggesting that butanol toxicity during the solvent production phase is more profound in the presence of another target site (i.e. ATPase) in addition to the cell membrane. It was further suggested that during growth, butanol inhibition due to membrane disruption was more important than inhibition of ATPase. Thus, chemotaxis prevents C. acetobutylicum from being confined in a toxic situation. Motile cells are more solventogenic because they can chemotactically respond to changes in their environment, and are less susceptible to product inhibition.