1. Field of the Invention
The present invention relates to a method for synchronizing the growth of clostridia useful in the production of solventogenic cells, enzymes, antibiotics, useful toxic proteins, or refractile endospores. Vegetative cells of bacteria of the genus Clostridium may be massively converted to synchronized solventogenic cells of essentially the same critical length, or the conversion allowed to proceed in a manner such that the production of refractile endospores is selectively induced. More particularly, the bacteria are synchronized in cell number and mass by selective subculturing in a medium containing a slowly metabolizable carbon source to avoid random cell growth. The synchronized cells are elongated to at least three times the length of vegetative cells, at which point they become solventogenic. Synchrony of cell mass and number is stabilized by addition of at least about 0.01M of a divalent cation to the medium. If solventogenesis is to be preserved, growth must be inhibited by chemical or physical means. Where preparation of enzymes, antibiotics or toxic protein producing cells is desired, cell growth may be arrested at selected growth stages beyond the solventogenic stage by inhibition of cell division or DNA replication.
2. Description of the Prior Art
Some anaerobic, thermophilic, endospore-forming bacteria of the genus Clostridium are capable of only limited metabolic production of enzymes, antibiotics, toxic proteins, or for producing solvents by acetone-butanol-ethanol (ABE) fermentation. The special capabilities of this genus are largely attributable to their expanded genetic versatility, where significant production of endospores occurs only under a particular physiological condition. Endospores are characterized by their ability to withstand extreme conditions which would destroy the cells in their vegetative state. The morphological changes exhibited by clostridial cells are related to changes in cellular enzyme activity. Depending upon the culture conditions, these bacteria can enter either an acidogenic phase or a solventogenic phase in the process of growth. Regulation of the overall sporulation process is thus a necessary prerequisite to the commercial production of cellular metabolites.
Some species of bacteria of the genus Clostridium are directly capable of converting low cost biomass wastes, such as xylan, or other pentose polymers, into solvents without prior depolymerization of the substrate. By virtue of the fact that certain clostridia are anaerobic and thermophilic, industrial fermentation processes using this genus may be carried out at relatively high temperatures. As a consequence, recovery of their fermentation products requires less energy because it can be accomplished by vacuum distillation directly from the fermentation vessel. Vacuum recovery also reduces the problems associated with solvent toxicity to the fermenting cells. The clostridia have high metabolic rates thus reducing the required residence time in the bioreactor and the ratio of end products to cells is high, maximizing the total bioreactive output. In addition, the use of a thermophilic system along with a simplified culture medium and defined and massive numbers of inoculated cells, assures that the system is inherently less subject to contamination. Sterilization of the raw materials therefore may be eliminated.
Lignocellulosic biomass material, the cheapest and most abundant feedstock for bacterial fermentation processes, has three major fractions: crystalline cellulose, hemicellulose, and lignin, each of which must be separately processed. Cellulose can be hydrolyzed to glucose with acid or enzyme catalysts. However, acid catalyst continue to degrade the resulting glucose. Furthermore, enzyme processes are not yet well developed and consequently are not cost efficient. Hemicellulose is largely composed of xylan, which is easily hydrolyzed to xylose but difficult to ferment to ethanol with existing fermentation technologies. Lignin is not a sugar polymer and, therefore, cannot be fermented to produce ethanol but can be thermochemically converted for use as a liquid fuel additive.
Use of anaerobic Clostridium acetobutylicum for industrial solvent production began at least as early as the 1920's employing a cane molasses feedstock. However, the best solvent yield obtainable was about 1.8% and even this yield was unreliable and unstable due to the susceptibility of C. acetobutylicum to phage (bacterial virus) infection. The method of the present invention enables control of the rates and yields of product formation and use of cheaper lignocellulose feedstocks. In especially preferred forms, the present invention utilizes thermophilic organisms of the genus Clostridium which, because of their ability to grow at elevated temperatures, make the process more energy efficient. In addition, thermophilic clostridia are not susceptible to phage infections.
One form of synchronous elongation of Clostridium thermosaccharolyticum is described in a chapter by Edward J. Hsu, one of the inventors hereof, in Spore Research, published by Academic Press (London, 1976, pp. 223-242), but that description makes no reference to synchronous growth of the cells in the presence of a divalent cation capable of stabilizing the cells during at least final multiplication thereof.
Ethanol-producing mutants of Clostridium thermosaccharolyticum are described in U.S. Pat. No. 4,652,526 issued to Edward J. Hsu, one of the inventors hereof. This patent also makes no mention of synchronous growth of cells in a growth medium under conditions where a divalent cation is added to stabilize the cells.
Hartmanis, et al. in Applied Microbiology and Biotechnology, Vol. 23 (1986) at pp. 369-371 describe repetitious subculturing of Clostridium acetobutylicum in a growth medium containing quantities of divalent cations. A small amount of calcium was included in the starter culture to prevent degeneration of the cells after only three transfers. The addition of CaCO.sub.3 permitted as much as ten transfers without degeneration. The calcium addition thus eliminated the need for multiple heat shock treatments for the preparation of a starting culture. However, synchronization of growth in the number of cells and their effective mass was not carried out to produce a substantially homogeneous cell population. The authors described a process wherein each subculture was allowed to progress for at least about a 24 hour interval to produce heat resistant spores. It was reported that the minor amount of calcium in the growth medium appeared to render the spores more heat-resistant.
U.S. Pat. No. 4,778,760 to Ishida, et al. indicates that a slight amount of calcium (4 ppm) is useful to stabilize a thermostable .alpha.-amylase-producing thermophilic anaerobic bacteria of the Clostridium class. However, the calcium is not utilized as a component of the growth medium for the bacterial cells.