Synthesis of cellulose by Acetobacter xylinum. 2. Preparation of freeze-dried cells capable of polymerizing glucose to cellulose

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Abstract
A procedure for the preparation of Acetobacter xylinum in the form of a cellulose-free suspension of washed freeze-dried cells is described in detail. Non-viable freeze-dried cells in optimum reaction conditions polymerized glucose to cellulose at a rate of 0.25 [mu]mole/mg/hour, corresponding to a production of cellulose on the bacterial surface at a rate in the order of 102 monomer monolayers/hour. Synthesis was attended by a rapid decline in the turbidity of the cell suspension and formation of a macroscopic cellulose gel body. The gross shape of the latter was a function of the type and intensity of the agitation during synthesis. The polymerization product has been characterized on the basis of solubility, qualitative chemical properties, infrared absorption, X-ray diffraction, and electron micrography, and identified as high-polymer, native, crystalline (type 1) cellulose. At least 78% of the C of the polmer product was derived from exogenously added glucose. Synthesis was negligible in absence of glucose or O2. Synthesis was immediate in onset, progressive in course, variable linearly in rate with cell concentration and essentially irreversible. The optimum pH was in the range 5-7. The rate was about half-maximum at O2 tension 20% and approached maximum at tension 100%. At low initial glucose concentration, the terminal yield of cellulose was 26% of the total glucose. Whereas the enzyme system for synthesis largely withstood freeze-drying, the latter incapacitated > 90% of the cells for proliferation. Kinetics of synthesis, properties of the cellulose, and the negligible cellulose content of the cells suggest that a constituent of the outer surface of the bacteria is a terminal catalyst of the production of cellulose. Similarities between cells of A. xylinum and green plants and the use of A. xylinum as a model for the study of cell-wall production are evaluated.