The use of correlation techniques in chromatography has been critically examined both in the laboratory and by computer simulation. The origin of a number of previously reported difficulties is established and some basic guide-

Protocatechuate 3,4-dioxygenase (PCD) from p-hydroxybenzoate-induced cells of Acinetobacter calcoaceticus was purified by heat and protamine sulfate treatment, ammonium sulfate fractionation, DEAE-cellulose, and Sephadex G-200 column chromatography. The enzyme appears to be homogeneous by ultracentrifugation and acrylamide gel electrophoresis. This is the first report of PCD purified from Acinetobacter. For comparison, crystalline Pseudomonas PCD was also obtained. The enzymes from Acinetobacter and Pseudomonas are quite similar in their molecular weight, molecular size, and iron content. The specific enzyme activity of PCD from Acinetobacter is about one-third of that from Pseudomonas, despite their similar iron content. Visible and circular dichroism spectra indicate some conformational differences between these two enzymes. Protocatechualdehyde, a competitive deadend inhibitor, binds Pseudomonas PCD more effectively than Acinetobacter PCD. p-Hydroxymercuribenzoate, specific for free-SH groups, inhibits only Acinetobacter PCD and shows no effect on Pseudomonas PCD. Amino acid analyses reveal very low proline and methionine content with higher lysine, glutamic acid, and isoleucine compositions for Acinetobacter PCD. Other properties, including active center conformation, were studied and discussed.

The isolation of an alkane-oxidizing strain of Pseudomonas oleovorans which maintains its viability at 5 C is described. This strain epoxidates 1-octene at a rate five times that of the parent strain. The most efficient substrates for induction of the epoxidase are C7, C,, and C,, although C5 to C12 also serve as growth substrates and inducers. The greater rate may be attributed to an enhanced general stability of the cells as opposed to a modification of the enzyme system involved.

Four strains of the homofermentative, obligately anaerobic thermophile Clostridium thermoaceticum were compared in pH-controlled batch fermentation for their tolerance to acetic acid, efficiency of converting glucose to acetic acid and cell mass, and growth rate. At pH 6 (and pH 7) and initial acetic acid concentrations of less than 10 g/liter, the four strains had mass doubling times of 5 to 7 h and conversion efficiencies to acetic acid and cell mass of about 90% (70 to 110%) and 10%, respectively. At pH 6 and initial acetic acid concentrations of greater than 10 g/liter, only two of the strains grew, the mass doubling time increased to 18 h, and the conversion efficiencies to acetic acid and cell mass remained unchanged. Both of these strains had been selected for their ability to grow in the presence of acetate at neutral pH. The highest acetic acid concentrations reached were about 15 and 20 g/liter at pH 6 and 7, respectively. C. thermoaceticum is apparently more sensitive to free acetic acid than to either acetate ion or pH. It was also shown that, at pH 6 and 7, the redox potential must be at least as low as −300 and −360 mV, respectively, for growth to occur.

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