Batch cultivation of R. erythropolis (ATCC 25544)
The bacteria were grown on the GYS medium in a 2-liter scale fermentor in batch mode operation under pH and temperature controlled conditions. Under this conditions the cell yield was doubled (9.5 mg/ml vs. 4.8 mg/ml dry cell weight) and the cultivation time was reduced to one third (60 vs. 180 hours) as compared with shaken flasks. These results are in good agreement with the literature [12]. We found that addition of 2 g/l cholesterol to the culture broth [12], prepared as an aqueous emulsion with the aid of Tween 80 at a weight ratio 2:1 results in a high yield of COX production [9], but the preparation procedure of that emulsion had a marked influence in the final enzyme yield, although not on the cell weight, as seen in Table 1. The spray-dry method resulted advantageous because the cholesterol :Tween 80 emulsion formed readily and COX production increased in overall by three times with respect to the preparation of the cholesterol:Tween 80 mixture at the flame. Enzyme production improvement resulted larger as cell-linked (3.8-fold) than as extracellular (2.3-fold). This overall increase of COX production can be due to a better availability of cholesterol to the cell since particle size obtained by spray-dry is smaller.
Table 1  Effect of the cholesterol emuIsification method on the production of COX.
COX activity (U/ml)*
Emulsification cholesterol method   Cell-linked   extracellular   Dry weight (mg/ml)
Spray-dry  230  140  8.75
At the flame  60  60  9.05
Improvement  3.8  2.3  0.97
*Enzymatic activity figures correspond to 70 hours of fermentation. Results from a typical batch fermentation are shown in Figure 1. Three stages can be differentiated during the fermentation process, (i) A first stage (0–16 h), in which 02 consumption increases continuously and HCl is consumed to keep the pH constant to 6.75. Buckland et al. [12] found that pH of the culture rose by 0.5 units in the first part of growth, and then fell. An exponential increase of cell mass is observed and low levels of COX activity appear linked to cells, (ii) The second stage (16–45 h) is characterized by a strong 02 consumption and a consumption of base. In this phase aerobic metabolism drives the cell growth but the growth rate is certainly limited by the O2 availability – note pO2 is nearly zero under continuous stirring and air supply-. This stage is also characterized by a high rate of COX production of both types, cell-linked and extracellular, (iii) The third stage (45 h to the end of the process) a second phase of consumption of acid was recorded whereas pO2 increased again to reach saturating levels. The greatest increase of cell-linked COX production was observed in this stage whilst extracellular COX production stopped.
Figure 1  Characterization of the R. erythropolis fermentation process: biomass and production of cell-linked and extracellular COX. Enzyme activities are given as units/ml of cell culture. The data shown are from a single experiment but are representative of three separate replicates. The profile of fermentation was very similar to that obtained by Buckland et al. [12] but differed in the accumulation of extracellular COX: the strain of Nocardia (NCIB 10554) used by these authors produced very low levels of extracellular enzyme while the strain tested in this work produces high levels. They also tested the effect of dissolved oxygen tension on the production of COX and found that in limiting conditions of oxygen supply the production of cell-linked COX was low. As seen in Figure 1, when oxygen supply is limiting (in the second stage) the rate of cell-linked COX production decreases, however is in these conditions when extracellular COX production takes place. Thus, there seem to be some relation between dissolved oxygen tension and extracellular COX production by the strain used in this work.
The results obtained are coherent with those presented in a previous study in shaken flasks [9], where extracellular COX production is large and arises from the partial solubilization of the cell-linked enzyme [20,21]. After 70 hours of fermentation the total enzyme activity obtained was ca. 360 U/ml, being 230 U/ml cell-linked and 130 U/ml extracellular, thus the cell-linked to extracellular ratio is 1.26. This ratio in shaken flasks ranged from 1.26, using the same amount of Tween 80 as in this work (0.1%), to 1.38, using 1% Tween 80, but in the latter the overall yields of COX production were 7-fold smaller [9]. The overall yield obtained in this work is comparable to that obtained by Buckland et al. [12] and by Minut et al. [17] but larger than that of Cheetham et al. [22]. Watanabe et al [24] compared the cell-linked and extracellular COX production of 31 strains of the genus Rhodococcus and Nocardia and found that among the best extracellular COX producers, the strains Rhodococcus sp. N° 31 and R. equi N° 24, displayed the highest cell-linked to extracellular ratio, 1.32 and 2.68 respectively.