World Journal of Microbiology & Biotechnology 10, 112-113
Short C o m m u n i c a t i o n
Improved culture media for growth of
Bradyrhizobium japonicum J.G.C. Pradella,* M.S. Oliveira, M. Zuccolo, A.C.R. Severo and A. Bonomi A freshly-prepared yeast extract at 30 or 50 g/1 improved the growth of Bradyrhizobiumjaponicum SEMIA 587 in a 5-1 stirred fermenter. Monosodium glutamate or a commercial yeast extract at 2.0 g/1 almost doubled cell mass productivity and cell viability when added at the end of the first exponential growth phase.
Key words: Bradyrhizobium japonicum, culture media, growth kinetics.
There are few publications describing the growth kinetics of Bradyrhizobium. Lopreto et al. (1972) showed that the concentration of yeast extract in the culture medium strongly influenced the generation time of B. japonicum. Mazza et aI. (1976) identified successive exponential growth phases with decreasing specific growth rates for B. japonicum growing in a glycercol/yeast extract culture medium. Utilizing this same culture medium, Bonomi (1986) observed two successive exponential growth phases with decreasing maximum specific growth rates, followed by a very long linear growth phase. In this communication, we describe the growth kinetics characteristics of B. japonicum SEMIA 587 (an industrial strain for soybean inoculant production in Brazil) related to the concentrations of freshly-prepared and commercial yeast extracts and monosodium glutamate added to the culture medium during the fermentation run.
Materials and Methods Culture Conditions Bradyrhizobium japonicum SEMIA 587 was grown in a 5.0-1 working volume fermenter (30°C, 5.0 1 air/min, 300 rev/min) with a 10% (v/v) inoculum. The culture medium contained (g/l): glycerol, 10.0; KzHPO4, 0.5; MgSO 4. 7HzO, 0.2; NaC1, 0.1; KNO 3, 0.8; (NH4)zHPO4, 0.3; and 0.1 ml 10% (w/v) MnSO4/1 and 0.1 ml 10% (w/v) FeCI3 (Mazza et al. 1976). The initial culture pH was 6.8. The culture medium was supplemented with a yeast extract that was freshly prepared by autoclaving 150 g of dried distillery yeast The authors are with the Divis&o Biotecnologia, Instituto de Pesquisas Paulo, S/A.-IPT-Cldade Universit~.ria 01064-970, S&o Paulo, SP, Brazil; fax: author.
de Quirnica, Agrupamento de Tecnol6gicas do Estado de S~o s/n., Caixa Postal 7141, CEP 0055-11-869-3353. *Corresponding
(~ 1994 Rapid Communications of Oxford Ltd
112
World Journal of Microbiology & Bio~echnology, Vol 10, 1994
in 350 ml of distilled water. After autoclaving, the volume was made up to 11 with water and the suspension centrifuged (2900 x g, 20 min). The supematant was used to prepare media with 10, 30 or 50 g freshly-prepared yeast extract/l. In some experiments carried out with 50 g fleshly-prepared yeast extract/l, monosodium glutamate (food grade; Ajinomoto do Brasil) or yeast extract (Difco) was also added after 28 h of fermentation at 2.0 g/I.
Analytical Methods Dry weight determinations were performed by filtration of culture samples (20 mI) through a 0.22-#m pore membrane. In cell viability measurements, samples were serially diluted with sterile saline solution and dropped onto plates with yeast extract/mannitol/agar medium (Miles & Misra 1938). The concentration of glycerol was measured in the supematant produced by centrifugation of the yeast suspension, according to the volumetric method of the American Oil Chemists' Society (1982).
Results and Discussion The preparation of an efficient agricultural inoculant requires a fermentation broth with a large viable cell concentration. A very high correlation (r = 0.954) was observed between cell density concentration (X) and viable cell count concentration (N) [N = (--0.395 + 4.938 X) x 109]. Addition of freshly-prepared yeast extract at 30 and 50 g/1 to the B. japonicum growth medium gave similar results whereas 10 g/1 gave a considerably lower cell growth rate [Figure l(a)]. The B. japonicum had a clear exponential growth phase up to 24 to 30 h, with a /~maxbetween 0.04 and 0.06 h-1. At the end of the exponential growth phase, 80% of the initial glycerol was still present in the medium, and thus, to have attained a cell density of 1.0 to 1.5 g/l, the freshly-prepared yeast extract must have been used as a source of carbon.
Growth kinetics of B. japonicum
6.0
(a)
2~C
i
hOo,s0.6"
YE~
30A"
o
0,2 / 6,0 4,0
~
(b)
2.0
i 0,8 1,0 0,6 0~4 0~2
I
I0
I
l
20 30
I
I
40 50
I
I
I
I
60 70 80 90 Time
100 (h)
Figure 1. Cell growth in (a) medium with 10 (A) 30 (G) or 50 g ( 0 ) freshly prepared yeast extract/I and (b) medium with 50 g fresh yeast extract/I supplemented with 2.0 g commercial yeast extract (YE)/I (A) or 2.0 g monosodium glutamate (Glu)/I ( 0 ) at 28 h.
After 30 h of fermentation, the cells started a second exponential growth phase lasting up to 45 to 49 h. The values of/Jmax and Yx
Short C o m m u n i c a t i o n
Improved culture media for growth of
Bradyrhizobium japonicum J.G.C. Pradella,* M.S. Oliveira, M. Zuccolo, A.C.R. Severo and A. Bonomi A freshly-prepared yeast extract at 30 or 50 g/1 improved the growth of Bradyrhizobiumjaponicum SEMIA 587 in a 5-1 stirred fermenter. Monosodium glutamate or a commercial yeast extract at 2.0 g/1 almost doubled cell mass productivity and cell viability when added at the end of the first exponential growth phase.
Key words: Bradyrhizobium japonicum, culture media, growth kinetics.
There are few publications describing the growth kinetics of Bradyrhizobium. Lopreto et al. (1972) showed that the concentration of yeast extract in the culture medium strongly influenced the generation time of B. japonicum. Mazza et aI. (1976) identified successive exponential growth phases with decreasing specific growth rates for B. japonicum growing in a glycercol/yeast extract culture medium. Utilizing this same culture medium, Bonomi (1986) observed two successive exponential growth phases with decreasing maximum specific growth rates, followed by a very long linear growth phase. In this communication, we describe the growth kinetics characteristics of B. japonicum SEMIA 587 (an industrial strain for soybean inoculant production in Brazil) related to the concentrations of freshly-prepared and commercial yeast extracts and monosodium glutamate added to the culture medium during the fermentation run.
Materials and Methods Culture Conditions Bradyrhizobium japonicum SEMIA 587 was grown in a 5.0-1 working volume fermenter (30°C, 5.0 1 air/min, 300 rev/min) with a 10% (v/v) inoculum. The culture medium contained (g/l): glycerol, 10.0; KzHPO4, 0.5; MgSO 4. 7HzO, 0.2; NaC1, 0.1; KNO 3, 0.8; (NH4)zHPO4, 0.3; and 0.1 ml 10% (w/v) MnSO4/1 and 0.1 ml 10% (w/v) FeCI3 (Mazza et al. 1976). The initial culture pH was 6.8. The culture medium was supplemented with a yeast extract that was freshly prepared by autoclaving 150 g of dried distillery yeast The authors are with the Divis&o Biotecnologia, Instituto de Pesquisas Paulo, S/A.-IPT-Cldade Universit~.ria 01064-970, S&o Paulo, SP, Brazil; fax: author.
de Quirnica, Agrupamento de Tecnol6gicas do Estado de S~o s/n., Caixa Postal 7141, CEP 0055-11-869-3353. *Corresponding
(~ 1994 Rapid Communications of Oxford Ltd
112
World Journal of Microbiology & Bio~echnology, Vol 10, 1994
in 350 ml of distilled water. After autoclaving, the volume was made up to 11 with water and the suspension centrifuged (2900 x g, 20 min). The supematant was used to prepare media with 10, 30 or 50 g freshly-prepared yeast extract/l. In some experiments carried out with 50 g fleshly-prepared yeast extract/l, monosodium glutamate (food grade; Ajinomoto do Brasil) or yeast extract (Difco) was also added after 28 h of fermentation at 2.0 g/I.
Analytical Methods Dry weight determinations were performed by filtration of culture samples (20 mI) through a 0.22-#m pore membrane. In cell viability measurements, samples were serially diluted with sterile saline solution and dropped onto plates with yeast extract/mannitol/agar medium (Miles & Misra 1938). The concentration of glycerol was measured in the supematant produced by centrifugation of the yeast suspension, according to the volumetric method of the American Oil Chemists' Society (1982).
Results and Discussion The preparation of an efficient agricultural inoculant requires a fermentation broth with a large viable cell concentration. A very high correlation (r = 0.954) was observed between cell density concentration (X) and viable cell count concentration (N) [N = (--0.395 + 4.938 X) x 109]. Addition of freshly-prepared yeast extract at 30 and 50 g/1 to the B. japonicum growth medium gave similar results whereas 10 g/1 gave a considerably lower cell growth rate [Figure l(a)]. The B. japonicum had a clear exponential growth phase up to 24 to 30 h, with a /~maxbetween 0.04 and 0.06 h-1. At the end of the exponential growth phase, 80% of the initial glycerol was still present in the medium, and thus, to have attained a cell density of 1.0 to 1.5 g/l, the freshly-prepared yeast extract must have been used as a source of carbon.
Growth kinetics of B. japonicum
6.0
(a)
2~C
i
hOo,s0.6"
YE~
30A"
o
0,2 / 6,0 4,0
~
(b)
2.0
i 0,8 1,0 0,6 0~4 0~2
I
I0
I
l
20 30
I
I
40 50
I
I
I
I
60 70 80 90 Time
100 (h)
Figure 1. Cell growth in (a) medium with 10 (A) 30 (G) or 50 g ( 0 ) freshly prepared yeast extract/I and (b) medium with 50 g fresh yeast extract/I supplemented with 2.0 g commercial yeast extract (YE)/I (A) or 2.0 g monosodium glutamate (Glu)/I ( 0 ) at 28 h.
After 30 h of fermentation, the cells started a second exponential growth phase lasting up to 45 to 49 h. The values of/Jmax and Yx
