Biotechnol. Prog. 1995, 11, 589-591
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A Modified Growth Medium for Bacillus thuringiensis Wei-Ming Liu and m e s h K. Bajpai' Department of Chemical Engineering, University of Missouri, Columbia, Missouri 65211
A published medium for the cultivation of Bacillus thuringiensis was modified for use in high-density fed-batch fermentations. This medium was diagnosed to have a complex nutrient limitation in batch and continuous cultivations. By selective additions of different medium constituents to a chemostat culture, it was established that the limitations were alleviated only by the addition of yeast extract or corn steep liquor; neither the addition of any salt nor that of individual amino acids influenced the culture behavior. In shake flask cultures, a reduction in glucose concentration and addition of corn steep liquor made the medium carbon-limited; enrichment with corn steep liquor alone showed dual limitations. These modified media resulted in higher cell densities and showed an increase in the amounts as well as the potency of insecticidal protoxins.
Introduction Goldberg et al. (1980) reported a continuous culture technique to determine the limiting nutrient of a medium. In this technique, a nutrient is identified to be limiting if an addition of the nutrient to a continuous culture results in an increase in cell concentration and a decrease in glucose concentration. We modified this technique by using the concentration of dissolved oxygen to monitor the response of the culture to additions of nutrients. Dissolved oxygen can be measured on-line with a relatively rapid response compared to the measurements of cell and substrate concentrations. It was used in this work to identify the nature of nutrient limitations in the cultivation of an industrially important sporeformer, Bacillus thuringiensis (Bt). Bt has been cultivated for many years to produce insecticidal protoxins which are formed in sporulating cells. These protoxins, commonly known as crystals, are proteinacious in nature and express toxicity to larvae of Lepidoptera or Diptera species after ingestion by the larvae (Bulla et al., 1980). Arcas et al. (1984)have proposed a medium (hereafter designated as Arcas' medium) for the cultivations of Bt. This medium produces more spores and insecticidal protoxins than any other published medium and contains (in g/L) glucose (lo),yeast extract (41, ( N H d 2 S 0 4 (l),KH2PO4 (31, K2Hp04(31, MgSO4*7Hz0(4), CaClz-2HzO (0.041), MnS040H20(0.030). The objective of this study was to improve upon this medium; the modified media were shown superior in terms of productions of cells mass and protoxins, and potency of the protoxins. Materials and Methods Microorganism. Bacillus thuringiensis subspecies Kurstaki HD-1 (serotype 3a,3b) was obtained from the Bacillus Genetic Stock Center, Ohio State University (stock no. 4D6). Maintenance of the strain has been reported elsewhere (Liu et al., 1994). Batch and Continuous Cultures. These experiments were conducted in a l-L (working volume) Virtis Omni-Culture fermentor operated at 900 rpm agitator speed, 2 L airlmin aeration rate, and 30 "C. Arcas' medium was used in the cultures. The dissolved oxygen level in the culture broth was monitored with a DO controller (NBS Model D081) with a galvanic-type DO
probe (NBS part no. M1016-5002) having a 2-mil Teflon membrane. Glucose and MgS04*7H20were autoclaved separately from the other medium components and were added to the medium after cooling. The fermentor was inoculated with 50 mL of broth from a 10-h-old shake flask culture with Arcas' medium. The pH of the culture broth was automatically controlled above 6.5 by addition of a 2 N KOH solution. During continuous operation, the dilution rate was set at -0.3 h-l, at which all the cells remained vegetative. Shake Flask Cultures. These experiments were performed with 50-mL medium in 250-mL baffled Erlenmeyer flasks. The compositions of the media in the flasks are given in Table 1. The pH of the media in the flasks was adjusted to 7.5 before autoclaving with a KOH solution and was not controlled during the shake flask cultivations. Each flask was inoculated with 5 mL of broth from a 10-h-old shake flask culture with Arcas' medium. The flasks were incubated in a rotary shaker (NBS Model G-25) operated at 30 "C and 200 rpm. All the experiments were conducted repeatedly and showed excellent reproducibility. Analyses of Samples. The methods for the analyses of the samples for cell and glucose concentrations, total crystal protein, and potency of the crystals have been reported elsewhere (Liu et al., 1994).
Results and Discussion Batch Culture. The concentration profiles of cell mass and glucose in a batch culture with Arcas' medium are shown in Figure 1. The results were reproducible with repeated experiments. The cell growth stopped and sporulation started while glucose was still abundant at about the seventh hour after inoculation. This implied that the medium was not carbon-limited. Evidently, some other nutrient was responsible for the cessation of the growth as well as the induction of the sporulation of the cells. The nutrient limitation was explored by a modified continuous culture technique of Goldberg et al. (1980). Continuous Culture. On the basis of calculations of the maximum possible yields from the different nutrients in Arcas' medium, N, Fe, Ca, and probably Mn were suspected to be limiting. Solutions of these nutrients were prepared at concentrations such that 5 mL of
8756-7938/95/3011-0589$09.00/0 0 1995 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1995, Vol. 11, No. 5
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Table 1. Effect of Medium Compositions on Shake Flask
100
Culturesa
flask glucose, g/L yeast extract, g/L corn steep liquor, g/L maximum cell concentration.. B(dry weight)/L crystal protein, @mL of broth uotencv, kIU/mL of broth HpecifiE potency, IU/pg of crystal protein
control 10 4
Y E.:2.Og
1
2
4.21
10 4 4 5.98
8 4 4 5.80
492 145 295
1258 824 814 559 647 678
The mineral concentrations in each flask are the same as those of Arcas' medium.
&
+--
1- Vegetative cells->+12
Free spores-
-sporangia-
-+
i Ink-.
10
;4
--2
0
10
15
20
'0 25
0)
: lj
5
Time, hr
Figure 1. Growth pattern of Bacillus thuringzensis in a minifermentor with 1 L of Arcas' medium.
solution injected into the fermentor delivered the same amount of the particular nutrient as in l-L Arcas' medium. However, additions of these nutrients, individually and in combination with each other, did not show any response in dissolved oxygen concentration nor was there any increase in the concentration of cells, indicating that none of these were limiting in the medium. Since it has been reported that Bt requires organic nitrogen (in the form of amino acids) for growth (Nickerson and Bulla, 19741, the attention was focused on yeast extract which is frequently incorporated in culture media to provide organic nitrogen and growth factors. When a 5-mL solution containing 2 g of yeast extract was injected into the chemostat, the dissolved oxygen concentration in the broth responded immediately (Figure 2a). ARer a sharp increase, it decreased slowly to a level significantly below the original steady state value and then gradually increased to the original steady state. At the nadir of the dissolved oxygen concentration, the cell concentration in the broth increased from a steady-state level of 2.6 to 4.0 g (dry weight)/L. Repeated tests with additions of yeast extract in a chemostat consistently showed the same transient variations in the dissolved oxygen concentration and increase in cell mass, implying that yeast extract was truly the supplier of the limiting nutrient(s) in Arcas' medium. In a separate test, addition of yeast extract to a cell-free broth did not cause any variation in the dissolved oxygen concentration, indicating that the observed variations in the dissolved oxygen concentration after addition of yeast extract were indeed manifestations of changes in cellular metabolism. Similar tests with aspartic acid, glutamic acid, and leucine (the amino acids predominant in yeast extract (Bridson and Brecker, 197011, and with salts of Zn, Cu, and Co did not show any limitations by these major amino acids and minerals of yeast extract. Hence, yeast extract as a whole was considered limiting. Bt subspecies kurstaki does not need vitamins for growth (Nickersonand Bulla, 1974). Corn steep liquor, also a good source of organic nitrogen and growth factors, was used as a partial substitute for
20 0'
C:4.0 9 D W l L
--Time E'
loo[
+
w
(b)
Figure 2. Transient response of Bacillus thuringiensis in a chemostat to (a) an addition of yeast extract and (b) successive additions of corn steep liquor. Quantities at the moments indicated by arrows are C: cell concentration, G glucose concentration, Y.E.: amount of yeast extract injected, and CSL: amount of corn steep liquor injected.
yeast extract in the following injection tests. These tests showed the same response in dissolved oxygen and cell concentrations as yeast extract (Figure 2b), suggesting that corn steep liquor could also alleviate the nutrient limitation. The yeast extract (4 g/L) in Arcas' medium was retained in these and all further experiments. Successive injections of corn steep liquor were performed to determine the extent of need for corn steep liquor in this medium. Four doses of 2 g of corn steep liquor were injected successively to a chemostat (Figure 2b). Both the dissolved oxygen and glucose concentrations in the broth kept decreasing, while cell mass increased with each successive addition of corn steep liquor. Further additions of corn steep liquor were not attempted due to dissolved oxygen limitations. By taking the washing-out effect into consideration, a rough estimation of the final corn steep liquor concentration after the fourth injection was about 4 g/L. Shake Flask Cultures. Other nutrient limitations have also been examined in shake flask cultures. Table 1 shows the medium compositions used in the flasks. Arcas' medium was used as the control (10 g/L glucose and 4 g/L yeast extract). Based upon the chemostat studies, 4 g/L corn steep liquor was added to flasks 1and 2. Glucose concentration in flask 1was 10 g/L (same as in Arcas' medium). It was reduced to 8 g/Lin flask 2, reflecting the last measured residual glucose concentration (1g/L) in Figure 2b plus 1 g additional reduction. Figure 3 shows the cell and glucose concentration profiles in the flasks. When the culture reached the maximum cell concentration, glucose was consumed totally only in flask 2 while plenty of glucose remained in the control flask. In effect, the complex limitation present in the control flask was shifbd to carbon limitation in flask 2 by adding 4 g/L corn steep liquor and reducing glucose concentration to 8 g/L. The medium in flask 1appeared t o have dual limitations. Other results of the shake flask cultures are also summarized in Table 1. It is obvious that the maximum cell concentrations correspond to the variations in the medium compositions, with corn steep liquor being the major factor when flasks 1 and 2 are compared to the control flask, and glucose the minor one when flask 1is compared to flask 2. It is understood that not all protein in the crystals show insecticidal activity (Scherrer et al., 1973). The specific potency, obtained by dividing the crystal potency by the amount of crystal protein in Table 1, can serve as an index of the ('quality"of the crystals. Good-quality crystals were obtained in flasks 1and 2 and poor-quality crystals in the control flask. The medium
Biotechnol. Prog., 1995, Vol. 11, No. 5
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Literature Cited 5.0
Flask #2
Control flask
=
1.0
3
0.0
