CURRENT MICROBIOLOGY,Vol. 11 (1984), pp. 175-178
Current Microbiology 9 Springer-Verlag 1984
Effect of Oxygen on Growth, Sporulation, and Mosquito Larval Toxin Formation by Bacillus sphaericus 1593 A l l a n A. Y o u s t e n , 1 D a v i d A. Wallis, 2 a n d S a m u e l Singer 3 ~Microbiology Section, Biology Department, and 2Department of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia; and 3Biology Department, Western Illinois University, Macomb, Illinois, USA
Abstract. T h e effect of o x y g e n o n g r o w t h , s p o r u l a t i o n , a n d m o s q u i t o larval t o x i n s y n t h e s i s b y
Bacillus sphaericus 1593 g r o w n in a small f e r m e n t o r was i n v e s t i g a t e d . W i t h air as the s o u r c e of o x y g e n , a b o u t o n e - h a l f of the cells s p o r u l a t e d a n d 1022 units of toxicity/rag of cell dry w e i g h t w e r e f o r m e d . A shift to 100% o x y g e n in the gas s t r e a m m a i n t a i n e d a higher level of d i s s o l v e d o x y g e n in the m e d i u m , b u t this p r o d u c e d a late b l o c k in s p o r u l a t i o n ; h o w e v e r , t o x i n s y n t h e s i s was n o r m a l . T h e m e c h a n i s m of o x y g e n i n h i b i t i o n of s p o r u l a t i o n b y B. sphaericus is u n k n o w n , b u t the s a m e effect was o b s e r v e d in B. subtilis 168. S t o p p i n g of the air flow at 8 h, after f o r e s p o r e s w e r e c o m p l e t e d in a b o u t o n e - h a l f the cells, i n h i b i t e d the c o m p l e t i o n of s p o r u l a t i o n , b u t did n o t d e c r e a s e t o x i n p r o d u c t i o n .
S e v e r a l s t r a i n s of Bacillus sphaericus that h a v e b e e n isolated f r o m m o s q u i t o larvae or f r o m larval h a b i t a t s h a v e b e e n s h o w n to p r o d u c e a s u b s t a n c e that is toxic u p o n i n g e s t i o n by m o s q u i t o larvae [9, 10, 11]. T h e toxic s u b s t a n c e is p r o d u c e d at the o n s e t of s p o r u l a t i o n [7] a n d is p r e s e n t in the walls of s p o r u l a t i n g cells [6] a n d in the p a r a s p o r a l i n c l u s i o n s that are a s s o c i a t e d with the spores in the highly toxic strains [1, 2, 8, 12]. B e c a u s e of the toxicity of t h e s e b a c t e r i a , t h e y are b e i n g c o n s i d e r e d for use as m o s q u i t o larvicides. T o x i c cells of B. sphaericus that h a v e b e e n u s e d for l a b o r a t o r y a n d field studies h a v e b e e n p r o d u c e d in small f e r m e n t o r s with a v a r i e t y of m e d i a a n d g r o w t h c o n d i t i o n s [11]. T h e r e has b e e n little s y s t e m a t i c study of the effect of g r o w t h c o n d i t i o n s o n the toxicity of the r e s u l t i n g cell m a s s . A p r e l i m i n a r y s t u d y d e m o n s t r a t e d that t o x i n s y n t h e s i s (or stability) was limited at i n c u b a t i o n t e m p e r a t u r e s a b o v e 35~ a n d that c o n t r o l of the p H n e a r n e u t r a l i t y e n h a n c e d t o x i n a c c u m u l a t i o n b y the cells [13]. Bacillus sphaericus is a n a e r o b i c b a c t e r i u m , a n d we r e p o r t here the effect of varied levels of o x y g e n o n s p o r u l a t i o n a n d o n the p r o d u c tion of the m o s q u i t o - l a r v a l toxin. Materials and M e t h o d s Bacteria and growth conditions. Bacillus sphaericus 1593 and B. sabtilis 168 were maintained on slants of NYSM agar (nutrient agar supplemented with 0.05% yeast extract, 5 • 10 5M MnC12, 7 x 10-4 M CaCI2, 1 • 10 3M MgC12). Fermentor studies were
performed in 1 liter of NYSM broth in a New Brunswick F-2000 Multigen fermentor operated at 30~ 300 rpm, and 0.8 liter/rain aeration with either air or 100% oxygen. The level of dissolved oxygen and the pH in the fermentor were measured continuously. The fermentor was inoculated with 30 ml of 6-h cells grown with shaking (220 rpm) at 30~ in NY broth (nutrient broth supplemented with 0.05% yeast extract). Growth was determined by measurement of the A660. Spore counts were made by heating 1.5 ml of a cell suspension at 80~ for 12 rain. The heated sample was sonicated for 30 s with the small probe of a Fisher model 300 dismembrator to unclump spores. Diluted samples were then plated on NY agar. Bioassay. Bacterial cells were recovered from broth by centrifugation, washed twice with sterile distilled water, and resuspended in sterile distilled water for bioassay. Dilutions of the bacteria were made in dechlorinated, sterile tap water, and 5 ml of the diluted suspensions were placed in plastic cups with 45 ml ot" sterile tap water, 1 drop of 10% wt/vol debinered brewers yeast, and 10 second-instar larvae of Culex quinquefaciatus. Three cups at each dilution and ten cups of untreated control larvae were held at 25~ for 48 h when mortality was determined. Data were corrected for control mortality with Abbotts' formula, and LCs0 values were determined by probit analysis. Toxicity units were determined by comparing the LCs0 of a fermentor sample with the LCs0 produced by a standard B. sphaericus 1593 powder designated RB-80, which was assigned a value of 1000 toxic U/rag. The RB-80 powder was obtained from H. de Barjac (Institut Pasteur, Paris). Chemical analyses. Protein was determined by the method of Lowry et al. [5]. Amino acid analyses were carried out on a Beckman amino acid analyzer, model 121 (Beckman Instruments, Inc., Palo Alto, CA) following sample hydrolysis in 6 N HC1 for 24 h. Oxidation of various substrates by bacterial cells suspended in 0.01 M 3(N-morpholino)propanesulfonic acid
Address reprint requests to: A. A. Yousten, Biology Department, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
176
CURRENT MICROBIOLOGY,Vol. 11 (1984)
I-
Table 1. Changes in the amino acid composition of NYSM broth after 11 h of growth by Bacillus sphaericus 159M
co
,~f
Z
~
!
/i
90 D 80 70
/
o--o
D
60 50 40 30
/ c~
0
20
D I0
O. 0
n-|--I
2
4
;--~-I 6
8
llh
Amino acid
Total
Solubleb
Total
Solubleb
Aspartic acid Alanine Arginine Glutamic acid Glycine Half cystine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Tyrosine Valine
3453 2938 1168 8943 2393 ND c 999 2376 4462 3477 1224 1967 6688 892 3193
367 1769 570 1247 522 ND 988 750 297 1400 522 1364 537 590 1177
1132 648 103 4441 822 ND 208 679 1524 1990 860 1566 1259 600 681
55 ND ND 38 ND ND ND 41 960 674 33 1207 ND 564 54
>-
I00
I
Oh
--n I0
I
I
1
12
14
16
I 24
0
Amino acid concentrations are expressed as nmol/ml. b Following precipitation of protein with 20% sulfosalicylic acid. c Not detected.
HOURS
Fig. 1. Growth of Bacillus sphaericus 1593 in a fermentor supplied with air or with 100% oxygen. Growth in the presence of air (ll) and 100% oxygen (O). Dissolved oxygen levels in fermentors supplied with air (D) and with 100% oxygen (9
buffer (pH 7.0) was carried out with a YSI model 53 oxygen monitor (Yellow Springs Instrument Co., Yellow Springs, OH). Organic acids in culture supernatants were determined by highperformance liquid chromatography in a Bio-Rad HFX-87 column (Bio-Rad Laboratories, Richmond, CA) held at 38~ Samples were eluted with 0.013 N H2804 in 5% acetonitrile, and detection was done at 214 nm. Dipicolinic acid was determined by the method of Janssen et al. [4]. Bacterial cell dry weights were determined in triplicate after drying of samples of the cells suspended in distilled water at 110~ for 24 h.
Results G r o w t h with air. W i t h air as the o x y g e n s o u r c e a n d w i t h o u t i m p o s i n g p H c o n t r o l on the g r o w i n g b a c t e ria, t h e p a t t e r n o f g r o w t h s h o w n in Fig. 1 w a s obtained. Exponential growth lasted approximately 4 h a n d w a s a c c o m p a n i e d b y an i n c r e a s e in p H to 8.9 b y 24 h. T h e p r o t e i n c o n t e n t o f the m e d i u m d e c l i n e d f r o m 2.7 mg/ml at the t i m e o f i n o c u l a t i o n to 1.7 mg/ml b y 10 h. It r e m a i n e d at 1.7 mg/ml f r o m 10 to 24 h. L a t e in the e x p o n e n t i a l p h a s e o f g r o w t h , the r a t e o f o x y g e n u t i l i z a t i o n b y t h e cells w a s so r a p i d r e l a t i v e to the r a t e o f o x y g e n t r a n s f e r into the liquid
that t h e r e w a s no l o n g e r a d e t e c t a b l e level o f d i s s o l v e d o x y g e n in the b r o t h . This c o n d i t i o n prevailed until 11 h, w h e n t h e d i s s o l v e d o x y g e n l e v e l rapidly increased. Under these growth conditions, a b o u t 50% o f the cells d e v e l o p e d into h e a t - r e s i s t a n t s p o r e s (5.8 • 10S/ml), a n d t h e cell m a s s c o n t a i n e d 1022 t o x i n U / m g o f cell d r y w e i g h t at 24 h. T h e i n c r e a s e in d i s s o l v e d o x y g e n m i g h t h a v e b e e n c a u s e d b y the e x h a u s t i o n o f o x i d i z a b l e subs t r a t e , b y l o s s o f o x i d a t i v e c a p a b i l i t y b y the s p o r u l a t i n g cells, o r b y b o t h . A l t h o u g h p r o t e i n r e m a i n e d in t h e m e d i u m at t h e t i m e t h e level o f dissolved oxygen increased, amino acid analysis of the s p e n t b r o t h at 1 l h r e v e a l e d t h a t s e v e r a l s o l u b l e a m i n o a c i d s (not p r e c i p i t a b l e with 20% sulfos a l i c y l i c acid) h a d b e e n c o m p l e t e l y d e p l e t e d ( T a b l e 1). In p a r t i c u l a r , large a m o u n t s o f g l u t a m i c acid a n d p r o l i n e w e r e utilized d u r i n g g r o w t h o f the b a c t e r i a . T h e s e a m i n o a c i d s m a y s e r v e as p r i m a r y c a r b o n and e n e r g y s o u r c e s for t h e s e b a c t e r i a , w h i c h do not use c a r b o h y d r a t e s . T h e o x i d a t i v e a b i l i t y o f B. sphaericus 1593 g r o w n in N Y S M b r o t h w a s t e s t e d with an o x y g e n e l e c t r o d e , a n d it w a s f o u n d that the Qo: o f 5-h cells w a s h i g h e r t h a n that o f 10-h cells. For e x a m p l e , with g l u t a m i c a c i d as s u b s t r a t e , t h e Q < o f 5-h cells w a s 144, w h e r e a s that o f 10-h cells was 19. W i t h the s u p e r n a t a n t f r o m a c e n t r i f u g e d , a u t o c l a v e d f i s h m e a l s u s p e n s i o n , the Qo= o f 5-h cells was 239, w h e r e a s that o f 10-h cells w a s 68.
A. A. Yousten et al.: Effect of Oxygen on Bacillus sphaericus Mosquito Pathogen
Growth with 100% oxygen. To determine whether limited oxygen availability was influencing sporulation and toxicity, 100% oxygen was used for aeration at the same flow rate and agitation rate as had been used with air. The pattern of growth and the rise in pH to 8.9 obtained under these conditions were similar to those obtained with air (Fig. 1). The dissolved oxygen level declined during exponential growth, but did not decrease below 60% saturation, and it then increased after 6 h as the cells entered the stationary phase. It should be noted t h a t the dissolved oxygen level at 60% saturation with 100% oxygen was approximately three times the level at 100% saturation when air was used for aeration. Unexpectedly, the formation of mature, heatresistant spores was inhibited by about 99% (to 5.0 x 106/ml) by the presence of a high level of dissolved oxygen. H o w e v e r , the toxicity of the cells was 1324 toxin U/rag, a level similar to that of air-grown cells. Phase contrast microscopic observation of the 100% oxygen-grown cells revealed that the cells had initiated the sporulation sequence and had formed swollen cells containing nonrefractile forespores. Cells grown for 24 h with air or with 100% oxygen were analyzed for their content of dipicolinic acid (DPA). It was found that 2.7% of the dry weight of the air-grown cells was DPA, whereas only 0.1% of the dry weight of the oxygengrown cells was DPA. No DPA was detected in the culture supernatant of the oxygen-grown cells. Thus, the low DPA content appeared to be due to inhibition of synthesis rather than to failure of the spore to retain the DPA. When B. subtilis 168 was grown with the same high level of dissolved oxygen, its sporulation was also blocked. Growth with air supplied for 8 h. Since an increase in dissolved oxygen inhibited the completion of sporulation and neither enhanced nor inhibited toxicity, limitation of oxygen rather than supplementation was investigated. B. sphaericus 1593 was again grown with air flowing at 0.8 liter/rain; however, after the first 8 h of growth (early stationary phase), the air flow was stopped and the bacteria were grown for an additional 16 h with agitation (300 rpm) alone. The level of dissolved oxygen remained close to 0% for this final 16-h period. The pH rose from 6.6 at the time of inoculation to 7.9 at 8 h, fell to 7.3 at 10 h, and then to 7.0 at 24 h. This was in contrast to the usual continuous rise to pH 8.9 at 24 h. Analysis of the culture supernatant showed no accumulation of acetic acid, lactic acid, pyruvic acid, or of related acidic metabolic by-products,
177
which might have accounted for the drop in pH. If the fermentor was sparged with nitrogen for 15 min after the pH had fallen, the pH quickly rose again during the sparging. The final cell population (1.3 x 109/ml) was similar to that achieved with air flow for the entire 24-h incubation period. At 8 h, when the air flow was stopped, about 50% of the cells had terminal swelling indicative of the onset of sporulation, and there were 1.5 x 104 heat-resistant spores/ml. This increased to only 8.4 x 106/ml at 24 h. At 8 h, however, the toxicity was already 1458 U/rag, and this remained essentially unchanged up to 24 h, when it was 1418 U/rag.
Discussion The rate and extent of growth of B. sphaericus 1593 were about the same whether air or 100% oxygen (both at 0.8 liter/min) was used as the oxygen source. When grown with air, the level of dissolved oxygen rapidly declined and remained at a low level until late in the stationary phase of growth (I 1 h). It was shown that certain amino acids were largely depleted from the medium by 11 h and that the oxidative ability of the stationary-phase cells was less than that of younger cells. Thus, both depletion of oxidizable substrate and a lowered oxidative ability probably contributed to the decrease in oxygen uptake and the subsequent increase in dissolved oxygen after 11 h. About one-half of the bacteria were able to produce heat-resistant spores under these conditions. When 100% oxygen was supplied to the fermentot, a high level of dissolved oxygen was maintained in the broth throughout growth and sporulation. H o w e v e r , the amount of mosquito larval toxin formed was about the same as in air-grown cells. The development of heat-resistant spores was inhibited at some point later than stage III (engulfment of the lbrespore). This inhibition is compatible with the normal development of toxicity observed, since it has been shown that the parasporal inclusions of these bacteria form during forespore engulfment [1, 2, 12]. Although the level of DPA in the inhibited cells was low and DPA was not excreted into the medium, the site of oxygen inhibition may not have been in the biosynthetic pathway for this compound. As in the case of pleiotropic mutations in sporulation [3], a block at one point in the developmental sequence could prevent the occurrence of all later events. The actual site and mechanism of this inhibition are unknown. The inhibition of sporulation by the presence of a high level of
178 dissolved oxygen is not a phenomenon peculiar to B. sphaericus, since B. subtilis 168 was similarly affected. When the air flow was stopped early in the stationary phase of growth (8 h), most of the bacteria had already initiated sporulation and formed the forespore. However, only a few cells went on to develop heat-resistant spores. The drop in pH following the cessation of air flow was not caused by the production of organic acid. It might have been due to CO2 accumulation in the broth if the absence of sparged air reduced the rate of CO2 transport away from the metabolizing cells. This possibility is supported by the increase in pH during sparging with nitrogen. The final toxin level was about the same as when air was supplied for the entire 24 h. In this experiment, air flow was stopped after 8 h to assure that the cells had completed the exponential phase of growth. It is possible that air flow could have been stopped even earlier, though at some point this would limit the amount of cell mass produced and might inhibit the onset of sporulation. It appears from these experiments that adequate oxygen supply is required for the completion of the developmental events of sporulation, but that an excessively high level is inhibitory. With respect to the large-scale production of B. sphaericus for use as a mosquito larvicide, there appears to be no advantage in increasing aeration or even in continuing the expense of aeration after forespore development has been completed, unless heat-resistant spores are desired. Under the growth conditions reported here, toxicity developed early in the sporulation sequence and increased little or not at all as incubation was continued, regardless of oxygen supply. ACKNOWLEDGMENT We thank Ms. Susan Fretz, Mr. Buddy Bolton, Ms. Susan Honeycutt, Mr. Scott Jelley, and Ms. Sandra Holloway for
CURRENT MICROBIOLOOY, Vol. 11 (1984)
technical assistance. This investigation received support from the Vector Biology and Control Component of the UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases.
Literature Cited 1. Davidson, E., Myers, P. 1981. Parasporal inclusions in Bacillus sphaericus. FEMS Microbiology Letters 10:261-265. 2. de Barjac, H., Charles, J-F. 1983. Une nouvelle toxine active sur les moustiques presente dans des inclusions crystalline produites par Bacillus sphaericus. Comptes Rendus Hebdomadaires des Seances de l'Academie des Sciences 296:905-910. 3. Hoch, J . A . 1976. Genetics of bacterial sporulation. Advances in Genetics 18:69-98, 4. Janssen, F., Lund, A., Anderson, L. 1958. Colorimetric assay for dipicolinic acid in bacterial spores. Science 127:26-27. 5. Lowry, O. H., Rosebrough, N., Farr, A., Randall, R. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193:265-275. 6. Myers, P., Yousten, A. 1980. Localization of a mosquitolarval toxin of Bacillus sphaericus 1593. Applied and Environmental Microbiology 39:1205-1211. 7. Myers, P., Yousten, A., Davidson, E. 1979. Comparative studies of the mosquito-larval toxin of Bacillus sphaericus SSII-1 and 1593. Canadian Journal of Microbiology 25:1227-1231. 89 Payne, J., Davidson, E. 1984. Insecticidal activity of the crystalline parasporal inclusions and other components of the Bacillus sphaericus 1593 spore complex. Journal of Invertebrate Pathology 43:383-388. 9. Singer, S. 1980. Bacillus sphaericus for the control of mosquitoes. Biotechnology and Bioengineering 22:1335-1355. 10. Yousten, A. 1984. Bacteriophage typing of mosquito pathogenic strains of Bacillus sphaericus. Journal of Invertebrate Pathology 43:124-125. 11. Yousten, A. 1984. Bacillus sphaericus: microbiological factors related to its potential as a mosquito larvicide. In: Mizrahi, A., Van Wezel, A. (eds.), Advances in Biotechnological Processes, vol. 4. New York: Alan R. Liss (in press)9 12. Yousten, A., Davidson, E. 1982. Ultrastructural analysis of spores and parasporal crystals formed by Bacillus sphaericus 2297. Applied and Environmental Microbiology 44:144%1455. 13. Yousten, A., Madhekar, N., Wallis, D. 1984. Fermentation conditions affecting growth, sporulation, and mosquito larval toxin formation by Bacillus sphaericus. Developments in Industrial Microbiology 25:757-762.