A standard vegetative inoculum of Aspergillus niger has been developed for the submerged citric acid fermentation of sugar beet molasses. Increasing the ferrocyanide content and the pH of the seed mash, within limits, decreased the rate of development of the mold pellets whereas increasing the spore inoculum increased the rate of development. Using this "standard" inoculum, an average yield of 8.9% anhydrous citric acid (68.5% conversion of available sugar) was obtained in 116 hr. The standard deviation between runs was 4.5% of the mean citric acid yield. An abrupt breakdown of the fermentation process decreased the yield by 50% and increased the standard deviation to 28.5% of the mean yield. The breakdown was overcome by changing the sporulation medium from a chemically-defined synthetic type to a nutritionally-richer type. Results indicate that serial transfer on a synthetic medium weakened the culture and made the fermentation unstable.

SummaryDissolved oxygen measlirements were made in pilot (20 and 250 1,) arid production scale (15,000 1.) novobiociri fermentations. Bulk mixing was found to be incomplete in pilot tanks with turbine impellers of D / T = 0.40 (where D is impeller diameter, and T is tank diameter) but appeared homogeneous with impellers of D / T = 0.69. I n the former case, the respiration rate was presumably limited by insufficient oxygen supply in areas of poor bulk mixing, whereas, in the latter case, the major resistance was between the bulk of the liquid and the cell (intrachrmp resistance). Higher agitator speeds decreased the gas-liquid resistance proportionally more than they reduced the liquid-cell resistance. I n production fermentors, dissolved oxygen measurements indicated that bulk mixing was complete with each of three impeller sizes tested (D/T = 0.28, 0.33, and 0.43), but that the respiration rate was again limited, mainly by a resistance between the bulk of the liquid arid the cell.

SummaryStudies wert: made of oxygen transfer and power in 20-1. novobiocin fermentations with five sizes (23-60% of the tank diameter) of geometrically similar flatblade turbine impellers. The influence of changes in apparent viscosity on oxygen transfer and power was found to be related t o impeller diameter. At constant agitator speed the oxygen availability rate (OAR) decreased with increase in apparent viscosity, but the magnitude of the reduction was less with a small impeller than with a large one. At constant power input the small-diameter impeller (23% of tank diameter) gave a n OAR about 8 times that of the large one (60% of tank diameter) when tested in viscous beer, whereas with sulfite or yeast all impeller diameters gave the same OAR. Impeller tip velocity correlated with OAR independently of diameter for the five turbine sizes examined. The power required for optimal novobiocin yields increased with increase in turbine diameter, but the yields were independent of diameter at equal OAR.

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SummaryThe performance of a multiple-rod mixing impeller was compared to that of conventional turbine impellers in viscous novobiocin beers. The advantages of the multiplerod impeller were found to be: ( I ) the power requirement was independent of changes in apparent viscosity of the fermentation beer; and ( 2 ) it gave the same novobiocin yield and oxygen-availability rate at about one-half of the power required by turbines.During the past few years, we have reported on the problem of oxygen transfer using the novobiocin fermentation as an example of a viscous non-Newtonian system.1-4 The general picture which emerged for turbine impellers under fully baffled conditions is this : In pilot-scale equipment, at least up to 250-1. batches, the oxygen requirement of the culture may be limited primarily by two main factors. The relative importance of these factors varies with the geometry of the system. First, with turbines of about 40% or less of the tank diameter, the oxygen requirement may be limited by poor bulk mixing. This situation is indicated by dissolved oxygen measurements that are low in one part of a fermentor and high in another part of a fermentor (see also ref. 5). Or, second, with largediameter turbines up to 69% of the tank diameter, where bulk mixing is apparently homogeneous as indicated by dissolved oxygen measurements, the respiration rate may be limited primarily by a resistance between the bulk of the liquid and the site of reaction in the cell. This type of behavior is indicated by uniform dissolved oxygen measurements throughout the fermentor, but with an abnormally high "apparent" critical dissolved-oxygen concentrat-ion, for example, values up to 50 or 60% of saturation. This is also the type of behavior we have observed in a 15,000-1. production fermentor.109

SUMMARY:The occurrence has been studied of mutants which did not produce extracellular cellulose in cultures of two strains of Acetobacter acetigenum (NCIB 8132, NCIB5346) and one strain of A. zylinum var. africanum (NCIB7029). With about 150 glucose yeast-extract (GYE) medium static cultures of organism NCIB 8132, prepared over a period of about one year, cellulose pellicles were produced in each case. In contrast, the loss of cellulose-forming ability and the appearance of diffuse general growth were noted with 6 out of 26 tests made with static cultures in an ethanol yeast-extract (EYE) medium, 3 out of 905 isolates selected from platings of GYE shaken cultures, and 1 out of 3 tests made with EYE shaken cultures. By a 'mass-plating' technique it was found that mutants which did not produce cellulose pellicles (' celluloseless mutants ') were present in cellulose-producing cultures. The two other cultures (NCIB 5846, NCIB 7029) underwent mutation spontaneously in GYE static cultures on several occasions.The present work was undertaken to obtain mutants of Acetobacter ucetigenum and A. xylinum var. africanum which did not form extracellular cellulose as did the parent organisms; these mutants will be referred to as ' celluloseless '. Schramm & Hestrin (1954) reported the occurrence of celluloseless mutants of A. xylinum; the mutants appeared when the wild-type organism was grown by serial transfer in 'swirl' cultures on a medium containing glucose, yeast-extract, peptone and salts, Creedy, Jowett & Walker (1954) isolated a celluloseless mutant of A. acetigenum when the parent organism was cultivated in the presence of sodium arsenite (0.0005 M). Further, Wright & Walker (1955) found that isolates incapable of forming cellulose were present when A. acetigenum was grown under agitated and aerated conditions. The present paper deals primarily with the occurrence of celluloseless mutants when the wild-type of A. acetigenum was grown under various cultural conditions. METHODSOrganisms. Two strains of Acetobacter acetigenum (NCIB 8132, NCIB 5346) and one of A. xylinum var. africanum (NCIB 7029) were obtained from the National Collection of Industrial Bacteria, (NCIB), Teddington, Middlesex. All cultures were replated and re-isolated twice and examined microscopically for purity before use.Media. Glucose yeast-extract (GYE) medium (pH 7.0) contained 3 yo (w/v) glucose and 0.5 yo (w/v) Difco yeast extract; for solid media 2 yo (w/v) of agar was added. Ethanol yeast-extract (EYE) medium contained 2 % (v/v) ethanol and 0.5 yo yeast extract, the sterile ethanol being added aseptically after sterilization of the basal medium. Tests were also made with ethylene glycol or n-propanol (both at 2yo, v/v) substituted for ethanol.

Factors affecting the production of citric acid in the submerged fermentation of ferrocyanide-treated beet molasses by Aspergillzts ~ziger were studied in 2 . 5 and 36 liter fermcnters. The small fermenters were used to determine the effects of changes in sterilization technique, phosphate supplement, fcrrocyanide treatment, inoculum level, initial pI-I, fermentation temperature, and aeration rate. The relation between ferrocyanide concentration and inoculum level \\,as also studied. Four different samples of molasses were fermented successfully. An average yield of 8.2% citric acid (64% conversion) was obtained fro111 51 small-scale fermentations. Comparable yields were obtained in the large ferlnenters under comparable conditions. Most of the information obtained with the small fermenters mas applicable to the larger-scale fermenters, but in the latter the fermentation was significantly more stable. Aeration was the main problem in the scale-up and aeration rates approximately double those calculated on a fermenter cross-sectional area basis were required for comparable results in the large fermenters.

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