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Photoautotrophic cultures of the host and transformed cells of Marchantia polymorpha under controlled incident light intensity
JOURNAL OF BIOSCLENCE Vol. 88, No. 5, 582-585.
AND
BIOENGINEERING
1999
Photoautotrophic Cultures of the Host and Transformed Cells of Marchantia polymorpha under Controlled Incident Light Intensity JUN-ICHI HATA,’ MASAHITO TAYA,‘* KATSUJI TANI, AND MASAO NASU2 Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka S60-8531’ and Department of Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 5654871 ,2 Japan Received13 May 1999/Accepted24 August 1999 Photoautotrophic cultures of the host and transformed cells of the liverwort, Marchantiapolymorpha, were examined. In cultures in flat glassflasks under various light intensities, it was found that the growth rates of both the cells increased with increase in light intensity in the range of 0 to 25 W/m2, but further increase in light intensity caused photoinhibition of the growth of the cells. Cultures of both the types of cells under lightcontrolled conditions using an externally illuminated bioreactor were carried out taking into consideration the inhibition of cell growth by excessivelight and the light intensity distributions in the cell suspensions. In these cultures, 2.1 (transformed cells) and 3.3 (host cells)kg dry cell weight per m3 were harvested at culture times of 9.0 and 10 d, respectively. These values were larger than those obtained in cultures of the respective cells at a lixed incident light intensity of 25 W/m2. [Key words: photoautotrophic culture, Marchantia polymorpha, transformed cells, light intensity control] One of the important functions of higher terrestrial plants is photosynthesis, by which the cells make use of CO2 and light as carbon and energy sources, respectively. From a biotechnological viewpoint, in vitro cultures of photoautotrophic cells of higher terrestrial plants have been investigated for the effective utilization of their photosynthetic ability. In recent years, photoautotrophic cells of liverworts like Marchantiapaleacea and Marchantia polymorpha have been identified as promising culture materials capable of propagating actively while retaining abundant chlorophyll in their cells in the presence of light (1, 2). Cell cultures of liverworts have attracted increasing interest as biological resources since the cells accumulate physiologically active compounds including unsaturated fatty acids, steroids and antibiotics (3, 4). As reported by Nasu et al. (5), moreover, photoautotrophic cells of M. polymorpha are suitable as host cells into which foreign genes can be introduced and expressed by means of recombinant DNA technology. In our previous work (l), we demonstrated that light was an important factor for the photoautotrophic growth of M. paleacea cells. Furthermore, a strategy for the control of incident light was proposed to diminish the effect of photoinhibition of cell growth (6). In the present study, photoautotrophic cultures were conducted using the host and transformed cells of M. polymorpha and the extents of photoinhibition of the growth of these cells were compared. It was verified that the intensity of light for the photoautotrophic cells could be effectively controlled using an externally illuminated bioreactor. Photoautotrophic liverwort cells, M. polymorpha HYA-2F (7), were used as the host cells. A binary vector plasmid pBI 121 harboring neomycin phosphotransferase (NPTII) and ,&glucuronidase (GUS) genes was introduced into the host cells using the Agrobacterium-mediated transformation method as described previously (5). A * Correspondingauthor.
transformed cell line (referred to as M. polymorpha S210) was chosen based on a relatively high level expression of the introduced genes in the cells. The host cells were cultivated in MSK-12 medium (7), and the transformed cells were grown in the same medium but with added geneticin (10mg/dm3). The pH of the medium was adjusted to 6.5 prior to autoclaving at 121°C for 20 min. In experiments to examine the effect of light intensity on the growth rates, M. polymorpha HYA-2F and S-210 cells were cultivated at 25°C in flat flasks (1611-120A, Sibata Scientific Technology Ltd., Tokyo) containing 27 cm3 of the medium. During the cultures, C02-enriched air (1 vol % CO3 was supplied at a flow rate of 10 cm3/ min, and the flasks were shaken at 30 rpm under illumination from a bank of tubular white fluorescent lamps (FL 2OSS.W, Matsushita Electric Industrial Co., Osaka). Light intensity was regulated by changing the distance between the flasks and lamps, or the number of lamps. Cultures in a bioreactor were conducted in a glass vessel (11 cm in diameter, working volume: 1.Odm3) under external light irradiation. The specifications of the bioreactor were described in our previous work (6). Air containing 1~01% CO2 was introduced into the glass vessel at a flow rate of 150 cm3/min, and the vessel was illuminated by six circular white fluorescent lamps (FCL 4OW/38, Matsushita Electric Industrial Co.). The temperature was kept constant at 25°C and the pH of the medium was regulated at 7.0t0.5 by the addition of 1 kmol/m3 HN03 during the cultures. The intensity of light entering the vessel was regulated on-line by a process controller (Type DPC-2, Able Co., Tokyo) which was connected to a PC-9821 computer (NEC Co., Tokyo) for data processing and calculation of the incident light intensity. The dry cell weight (DCW) and chlorophyll content of the cells were determined as described elsewhere (1). GUS activity was assayed at 37°C by the fluorometric 582
VOL.
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method using 4-methylumbebelliferyl-b-D-glucuronide as the substrate (8, 9). One unit of the enzyme activity represented 1 pmol of 4-methylumbelliferone liberated from the substrate in 1 min. The light intensity and effective absorption coefficient of light by the cell suspensions were determined as shown previously (1). The procedure used for controlling the light intensity in the photoautotrophic cultures of M. polymorpha HYA3F and S-210 cells is illustrated in Fig. 1. By measuring the electrical conductivity of culture medium, the cell concentration was determined employing Eq. 1 as shown previously (10). The light intensity distribution in the reactor was calculated from Eqs. 2 and 3 from the values of incident light intensity and cell concentration. The specific growth rate at a radial position in the reactor was estimated from the relationship represented by Eq. 4, and then the average specific growth rate in the reactor was obtained by integrating Eq. 4 as expressed by Eq. 5. Thus, when the values of the cell concentration were provided, the value of the incident light intensity maximizing the average specific growth rate could be
r--l
Estimation of cell concentration by the measuring
electrical conductivity of the culture medium
t-7 ,
II
1
X=~(AK)+X,
I -
I
(1)
4 Calculation of light intensity distribution in a bioreador
I
at a given cell concentration
583
determined through repeated computation of the average specific growth rate in terms of feasible values of the incident light intensity in the reactor system. In order to evaluate the distribution of the light intensity, light absorption by the cell suspensions of M. polymorpha HYA-2F and S-210 was investigated. Attenuation of light intensity at a given cell concentration was expressed by the following equation based on the Lambert-Beer law. In -$- =-aL (6) ( 1 The effective absorption coefficient of light, (Y, was experimentally determined for various cell concentrations. As shown in Fig. 2, the effective absorption coefficient of the transformed cells was lower than that of the host cells for a given cell concentration, suggesting that light attenuation was significantly greater in cell suspensions of the host cells as compared with that in cell suspensions of the transformed cells. The difference in n values could be explained by the respective average aggregate sizes of these cells (average diameters of the host and transformed cells: 0.12 and O.l6mm, respectively). In the range of examined cell concentrations (X=0-3 kg/m3), linear relationships were observed between the values of a and X for both the host and transformed cells. By fitting the data to Eq. 2, the empirical values of b and c were determined as follows: b=79.0 m2/kg and c= 8.32 m-l for S-210 cells, and b= 135 m2/kg and c=8.91 m-l for HYA-2F cells (Table 1). The specific growth rates of M. polymorpha HYA-2F and S-210 in flat flasks were investigated at various light 400
a=b Xtc
(21
Z(r)=y[exp{-a(R-r)]+ex&a(R+r)I]
(3) ,
I I
Calculation of specific growth rate at a radial position
X[Wm31
and the average specific growth rate in a bioreactor
(4)
-1
0
1
Determination of incident light intensity
at
which the
FIG. 1. Procedure for the control of incident light intensity in photoautotrophic cultures of M. polymotpha S-210 and HYA-2F in a bioreactor.
50 100 I[W/m2]
150
FIG. 2. Determination of parameters for calculation of incident light intensity in a light-controlled culture. (a) Plots of the effective absorption coefficient of light in cell suspensions of M. polymorpha S-210 and HYA-2F against the cell concentrations. The lines were fitted by Eq. 2. @) Plots of specific growth rates of M. polymorpha S210 and HYA-2F against light intensity. The lines were fitted by Eq. 4. Symbols: 0 and -, transformed cells (S-210); 0 and ----, host cells (HYA-2F).
584
HATA ET AL.
TABLE
J. BIOSCI. BIOENG.,
1. Parameters determined for calculation of incident light intensity
Transformed cells (S-2101 In Eq. 1 a=14.9 kg-DCW/(m2.S) In Eq. 2 b=79.0 m2/kg c=8.32 m- I In Eq. 4 1,=83.0 W/m* KS= 12.1 W/m* m=O d&’ ,umax=l.Od--’
Host cells (HYA-2F) a=12.9 kg-DCW/(m*.S) b= 135 m2/kg c=8.91 m--l I, = 209 W/m2 Ks=3.51 W/m* m=O d-’ pmax=0.82 d-’
intensities, to correlate the specific growth rates with the light intensity expressed by Eq. 4 in Fig. 1. As shown in Fig. 2, the specific growth rates of the host cells were higher than those of the transformed cells in the range of light intensities examined in the present study. The relatively lower growth rate of the transformed cells may be attributable to the physiological load of carrying the foreign genes within them. The specific growth rates of both the cells increased with increase in light intensity in the range of 0 to 25 W/m2. However, with further increase in light intensity, the growth rates decreased because of photoinhibition. The degree of photoinhibition of the growth of the transformed cells was greater than that of the host cells. The phenomenon of photoinhibition under higher light intensities was considered to be attributable to reactive oxygen species produced during the course of the photosynthetic reactions in the cells (11). Hirayama et a[. (12) detected hydroxyl radical species in vivo in photoautotrophic cells of Chlorella sp., and demonstrated that increase in light intensity caused an increase in hydroxyl radical production and deterioration in the efficiency of photosynthesis. However, further examination from a biochemical viewpoint is needed to better understand the difference in the degree of photoinhibition in cultures of the host and transformed cells of M. polymorpha. As indicated by the lines in Fig. 2, the experimental data on S-210 and HYA-2F cells were fitted in Eq. 4 using the non-linear least squares method. As a result, the values of the parameters in Eq. 4 were determined to be as follows: 1,=83.0 W/m2, Ks=12.7W/m2, m=Od-’ and pmax=l.Od-l for the transformed cells, and I,=209 W/m2, j&=3.51 W/m2, m= 0 d-l and I-(,,,~=0.82 d-l for the host cells (Table 1). Photoautotrophic cultures of M. polymorpha S-210 and HYA-2F were carried out in a bioreactor under external irradiation with control of the incident light intensity, taking into consideration the light intensity distribution in the cell suspensions and the dependence of the growth rates of the host cells on light intensity. The calculation for control of the incident light intensity was performed employing the procedure shown in Fig. 1 and the values of the parameters shown in Table 1. In these cultures, the values of X0 and R were 0.15 kgDCW/m3 and 5.5 x 10e2 m, respectively. Figure 3 shows the time-courses of changes in cell concentration in photoautotrophic cultures of the transformed (S-210) and host (HYA-2F) cells, where incident light intensity was controlled. The X values of the transformed and host cells reached 2.1 and 3.3 kg-DCW/m3 at culture times of t=9.0 and lOd, respectively. As reference cultures, the transformed and host cells were cultivated while keeping
t [d I FIG. 3. Photoautotrophic cultures of M. polymorpha S-210 and HYA-2F in a bioreactor with and without control of incident light intensity. Symbols: 0 and -, transformed cells (S-210) in lightcontrolled culture; 0 and ----, transformed cells (S-210) in constantlight culture; 0 and - - - -, host cells (HYA-2F) in light-controlled culture; + and --m-, host cells (HYA-2F) in constant-light culture.
the light intensity constant at I,=25 W/m2. In these cultures, the X values of the transformed and host cells were 1.4 and 1.1 kg-DCW/m3 at t= 12 and 9.5 d, respectively, as shown in Fig. 3. Exponential growth of the cells was observed during the early stage (t=l-7 d) in these four cultures. In constant-light cultures, the observed specific growth rates, rl(&s, for S-210 and HYA-2F cells were 0.24 and 0.35d-‘, respectively. On the other hand, the &bs values of S-210 and HYA-2F cells in cultures carried out under control of incident light were 0.38 and 0.50d-l, respectively. It was recognized that the &,bsvalues of both the cells were enhanced in the cultures carried out under control of incident light intensity, compared with those in cultures not carried out under control of incident light intensity. Table 2 summarizes the results obtained from cultures of the transformed and host cells with and without light intensity control in a bioreactor. Under the conditions of controlled and constant light intensity, the chlorophyll contents in the transformed cells were 23-24 g/kg-DCW, and these values were relatively high in comparison with those in the host cells. Concerning GUS activity, no significant difference was observed between cultures of the transformed cells with and without light intensity control. The values of the cell yields based on the irradiated light energy, YxlE, were calculated from the amounts of cells grown and the total light energy illumination throughout the cultures. It was found that the values were in the range of YX/~=l.l-1.4~ 10e9 kg-DCW/J, irrespective of the type of cells and the manner of light illumination. In conclusion, photoautotrophic cultures of the host and transformed cells of liverwort (M. polymorpha HYA-2F and S-210) were conducted using the externally illuminated bioreactor where the incident light intensity was controlled to obtain effective growth of the cells. The amounts of the cells obtained in the light-controlled cultures of the host and transformed cells were larger
VOL.
88, 1999 TABLE
2.
NOTES Summary of photoautotrophic
585
cultures of M. polymorpha HYA-2F and S-210 in a bioreactor with and without control of incident light intensity
Transformed cells (S-210) Host cells (HYA-2F) Controlled light Constant light Controlled light Constant light (17-68 W/m2) (25 W/m2) (25-178 W/m2) (25 W/mZ) 9.0 12 10 9.5 Culture time (d) 1.4 Cell concentration (kg-DCW/m3) 2.1 3.3 1.1 16 17 Chlorophyll content (g/kg-DCW) 23 24 GUS activity (103 U/kg-DCW) 0.72 0.71 N.D. N.D. Irradiated light energy, ET (106 J) 1.4 0.92 2.9 0.74 1.3 1.1 1.3 Cell yield based on irradiated light energy, Yx, (lo+ kg-DCW/J) 1.4 Initial values for transformed cells: X0=0.15 kg-DCW/m3, chlorophyll content=24 g/kg-DCW and GUS activity=0.69 X lo3 U/kg-DCW. Initial values for host cells: X,=0.15 kg-DCW/m3 and chlorophyll content=21 g/kg-DCW. N.D.: Not determined. ET =A S(r,,(t)dt; Y,,, = (X-X0) 0
V/ET
than those in the cultures carried out under constant light intensity. It was verified that culture under lightcontrolled conditions could be effectively applied to the photoautotrophic growth of both the host and transformed cells. NOMENCLATURE
: irradiated surface area of photobioreactor, m2 : empirical constant in Eq. 1, kg-DCW/(m2. S) b : empirical constant in Eq. 2, m2/kg-DCW : empirical constant in Eq. 2, m-l * irradiated light energy, J k I I light intensity, W/m2 IO : incident light intensity, W/m2 IIll : parameter in Eq. 4, W/m2 KS : saturation constant in Eq. 4, W/m2 L : light path length, m : parameter in Eq. 4, d-l R” : radius of the bioreactor, m t : culture time, d v : working volume of photobioreactor, m3 X : cell concentration, kg-DCW/m3 x0 : initial cell concentration, kg-DCW/m3 Y X,~: cell yield based on irradiated light energy, kgDCW/J (Y : effective absorption coefficient of light in cell suspension, m-l AK : change in electrical conductivity of medium, S/m I-I . specific growth rate, d-l ,umaxI maximum specific growth rate in Eq. 4, d-l /*&s : observed specific growth rate, d-l A
a
REFERENCES 1. Taya, M., Miya-oka, M., Toyo-oka, Y., Kino-oka, M., Tone, S., and Ono, K.: Growth characteristics of liverwort cells, Marchantia paleacea var. dipteru, in a photoautotrophic suspension culture. J. Ferment. Bioeng., 80, 580-585 (1995). 2. Katoh, K.: Kinetics of photoautotrophic growth of Marchantia polymorpha cells in suspension culture. Physiol. Plant, 59, 242-248 (1983). 3. Hartmann, E., Beutelmann, P., Vandekerkhove, O., Eoler, R., and Kahn, G.: Moss cell cultures as sources of arachidonic and eicosapentaenoic acids. FEBS Lett., 198, 51-55 (1986). 4. Ono, K., Toyota, M., and Asakawa, Y.: Constituents from cell suspension cultures of selected liverworts. Phytochemistry, 31, 1249-1250 (1992). 5. Nasu, M., Tani, K., Hattori, C., Honda, M., Skimaoka, T., Yamaguchi, N., and Katoh, K.: Efficient transformation of Marchantia polymorpha that is haploid and has very small genome DNA. J. Ferment. Bioeng., 84, 519-523 (1997). 6. Hata, J., Toyo-oka, Y., Taya, M., and Tone, S.: A strategy for control of light intensity in suspension culture of photoautotrophic liverwort cells, Marchantia pa[eacea var. diptera. J. Chem. Eng. Japan, 30, 315-320 (1997). 7. Nasu, M., Hattori, C., Katoh, K., Miyamoto, K., and Kondo, M.: Carbon dioxide fixation by cultured cells of Marchantia polymorpha. CELSS J., 5, 51-56 (1992). 8. Uchiyama, H.: Manual of gene manipulation for plants, 1st ed., p. 65-67. Kodansha, Tokyo (1990). (in Japanese) 9. Kosugi, S.: An improved assay for B-glucuronidase (GUS) reporter gene expression in plant cells. Plant Cell Technol., 4, 209-212 (1992). (in Japanese) 10. Taya, M., Hegglin, M., Prenosil, J. E., and Bourne, J. R.: On-line monitoring of cell growth in plant tissue cultures by conductometry. Enzyme Microb. Technol., 11, 170-176 (1989). 11. Fischer, U. and Alfermann, A. W.: Cultivation of photoautotrophic plant cell suspensions in the bioreactor: influence of culture conditions. J. Biotechnol., 41, 19-28 (1995). 12. Hirayama, S., Ueda, R., and Sugata, K.: Effect of hydroxyl radical on intact microbial photosynthesis. Energy Convers. Mgmt., 36, 685-688 (1995).
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Photoautotrophic Cultures of the Host and Transformed Cells of Marchantia polymorpha under Controlled Incident Light Intensity JUN-ICHI HATA,’ MASAHITO TAYA,‘* KATSUJI TANI, AND MASAO NASU2 Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka S60-8531’ and Department of Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 5654871 ,2 Japan Received13 May 1999/Accepted24 August 1999 Photoautotrophic cultures of the host and transformed cells of the liverwort, Marchantiapolymorpha, were examined. In cultures in flat glassflasks under various light intensities, it was found that the growth rates of both the cells increased with increase in light intensity in the range of 0 to 25 W/m2, but further increase in light intensity caused photoinhibition of the growth of the cells. Cultures of both the types of cells under lightcontrolled conditions using an externally illuminated bioreactor were carried out taking into consideration the inhibition of cell growth by excessivelight and the light intensity distributions in the cell suspensions. In these cultures, 2.1 (transformed cells) and 3.3 (host cells)kg dry cell weight per m3 were harvested at culture times of 9.0 and 10 d, respectively. These values were larger than those obtained in cultures of the respective cells at a lixed incident light intensity of 25 W/m2. [Key words: photoautotrophic culture, Marchantia polymorpha, transformed cells, light intensity control] One of the important functions of higher terrestrial plants is photosynthesis, by which the cells make use of CO2 and light as carbon and energy sources, respectively. From a biotechnological viewpoint, in vitro cultures of photoautotrophic cells of higher terrestrial plants have been investigated for the effective utilization of their photosynthetic ability. In recent years, photoautotrophic cells of liverworts like Marchantiapaleacea and Marchantia polymorpha have been identified as promising culture materials capable of propagating actively while retaining abundant chlorophyll in their cells in the presence of light (1, 2). Cell cultures of liverworts have attracted increasing interest as biological resources since the cells accumulate physiologically active compounds including unsaturated fatty acids, steroids and antibiotics (3, 4). As reported by Nasu et al. (5), moreover, photoautotrophic cells of M. polymorpha are suitable as host cells into which foreign genes can be introduced and expressed by means of recombinant DNA technology. In our previous work (l), we demonstrated that light was an important factor for the photoautotrophic growth of M. paleacea cells. Furthermore, a strategy for the control of incident light was proposed to diminish the effect of photoinhibition of cell growth (6). In the present study, photoautotrophic cultures were conducted using the host and transformed cells of M. polymorpha and the extents of photoinhibition of the growth of these cells were compared. It was verified that the intensity of light for the photoautotrophic cells could be effectively controlled using an externally illuminated bioreactor. Photoautotrophic liverwort cells, M. polymorpha HYA-2F (7), were used as the host cells. A binary vector plasmid pBI 121 harboring neomycin phosphotransferase (NPTII) and ,&glucuronidase (GUS) genes was introduced into the host cells using the Agrobacterium-mediated transformation method as described previously (5). A * Correspondingauthor.
transformed cell line (referred to as M. polymorpha S210) was chosen based on a relatively high level expression of the introduced genes in the cells. The host cells were cultivated in MSK-12 medium (7), and the transformed cells were grown in the same medium but with added geneticin (10mg/dm3). The pH of the medium was adjusted to 6.5 prior to autoclaving at 121°C for 20 min. In experiments to examine the effect of light intensity on the growth rates, M. polymorpha HYA-2F and S-210 cells were cultivated at 25°C in flat flasks (1611-120A, Sibata Scientific Technology Ltd., Tokyo) containing 27 cm3 of the medium. During the cultures, C02-enriched air (1 vol % CO3 was supplied at a flow rate of 10 cm3/ min, and the flasks were shaken at 30 rpm under illumination from a bank of tubular white fluorescent lamps (FL 2OSS.W, Matsushita Electric Industrial Co., Osaka). Light intensity was regulated by changing the distance between the flasks and lamps, or the number of lamps. Cultures in a bioreactor were conducted in a glass vessel (11 cm in diameter, working volume: 1.Odm3) under external light irradiation. The specifications of the bioreactor were described in our previous work (6). Air containing 1~01% CO2 was introduced into the glass vessel at a flow rate of 150 cm3/min, and the vessel was illuminated by six circular white fluorescent lamps (FCL 4OW/38, Matsushita Electric Industrial Co.). The temperature was kept constant at 25°C and the pH of the medium was regulated at 7.0t0.5 by the addition of 1 kmol/m3 HN03 during the cultures. The intensity of light entering the vessel was regulated on-line by a process controller (Type DPC-2, Able Co., Tokyo) which was connected to a PC-9821 computer (NEC Co., Tokyo) for data processing and calculation of the incident light intensity. The dry cell weight (DCW) and chlorophyll content of the cells were determined as described elsewhere (1). GUS activity was assayed at 37°C by the fluorometric 582
VOL.
NOTES
88, 1999
method using 4-methylumbebelliferyl-b-D-glucuronide as the substrate (8, 9). One unit of the enzyme activity represented 1 pmol of 4-methylumbelliferone liberated from the substrate in 1 min. The light intensity and effective absorption coefficient of light by the cell suspensions were determined as shown previously (1). The procedure used for controlling the light intensity in the photoautotrophic cultures of M. polymorpha HYA3F and S-210 cells is illustrated in Fig. 1. By measuring the electrical conductivity of culture medium, the cell concentration was determined employing Eq. 1 as shown previously (10). The light intensity distribution in the reactor was calculated from Eqs. 2 and 3 from the values of incident light intensity and cell concentration. The specific growth rate at a radial position in the reactor was estimated from the relationship represented by Eq. 4, and then the average specific growth rate in the reactor was obtained by integrating Eq. 4 as expressed by Eq. 5. Thus, when the values of the cell concentration were provided, the value of the incident light intensity maximizing the average specific growth rate could be
r--l
Estimation of cell concentration by the measuring
electrical conductivity of the culture medium
t-7 ,
II
1
X=~(AK)+X,
I -
I
(1)
4 Calculation of light intensity distribution in a bioreador
I
at a given cell concentration
583
determined through repeated computation of the average specific growth rate in terms of feasible values of the incident light intensity in the reactor system. In order to evaluate the distribution of the light intensity, light absorption by the cell suspensions of M. polymorpha HYA-2F and S-210 was investigated. Attenuation of light intensity at a given cell concentration was expressed by the following equation based on the Lambert-Beer law. In -$- =-aL (6) ( 1 The effective absorption coefficient of light, (Y, was experimentally determined for various cell concentrations. As shown in Fig. 2, the effective absorption coefficient of the transformed cells was lower than that of the host cells for a given cell concentration, suggesting that light attenuation was significantly greater in cell suspensions of the host cells as compared with that in cell suspensions of the transformed cells. The difference in n values could be explained by the respective average aggregate sizes of these cells (average diameters of the host and transformed cells: 0.12 and O.l6mm, respectively). In the range of examined cell concentrations (X=0-3 kg/m3), linear relationships were observed between the values of a and X for both the host and transformed cells. By fitting the data to Eq. 2, the empirical values of b and c were determined as follows: b=79.0 m2/kg and c= 8.32 m-l for S-210 cells, and b= 135 m2/kg and c=8.91 m-l for HYA-2F cells (Table 1). The specific growth rates of M. polymorpha HYA-2F and S-210 in flat flasks were investigated at various light 400
a=b Xtc
(21
Z(r)=y[exp{-a(R-r)]+ex&a(R+r)I]
(3) ,
I I
Calculation of specific growth rate at a radial position
X[Wm31
and the average specific growth rate in a bioreactor
(4)
-1
0
1
Determination of incident light intensity
at
which the
FIG. 1. Procedure for the control of incident light intensity in photoautotrophic cultures of M. polymotpha S-210 and HYA-2F in a bioreactor.
50 100 I[W/m2]
150
FIG. 2. Determination of parameters for calculation of incident light intensity in a light-controlled culture. (a) Plots of the effective absorption coefficient of light in cell suspensions of M. polymorpha S-210 and HYA-2F against the cell concentrations. The lines were fitted by Eq. 2. @) Plots of specific growth rates of M. polymorpha S210 and HYA-2F against light intensity. The lines were fitted by Eq. 4. Symbols: 0 and -, transformed cells (S-210); 0 and ----, host cells (HYA-2F).
584
HATA ET AL.
TABLE
J. BIOSCI. BIOENG.,
1. Parameters determined for calculation of incident light intensity
Transformed cells (S-2101 In Eq. 1 a=14.9 kg-DCW/(m2.S) In Eq. 2 b=79.0 m2/kg c=8.32 m- I In Eq. 4 1,=83.0 W/m* KS= 12.1 W/m* m=O d&’ ,umax=l.Od--’
Host cells (HYA-2F) a=12.9 kg-DCW/(m*.S) b= 135 m2/kg c=8.91 m--l I, = 209 W/m2 Ks=3.51 W/m* m=O d-’ pmax=0.82 d-’
intensities, to correlate the specific growth rates with the light intensity expressed by Eq. 4 in Fig. 1. As shown in Fig. 2, the specific growth rates of the host cells were higher than those of the transformed cells in the range of light intensities examined in the present study. The relatively lower growth rate of the transformed cells may be attributable to the physiological load of carrying the foreign genes within them. The specific growth rates of both the cells increased with increase in light intensity in the range of 0 to 25 W/m2. However, with further increase in light intensity, the growth rates decreased because of photoinhibition. The degree of photoinhibition of the growth of the transformed cells was greater than that of the host cells. The phenomenon of photoinhibition under higher light intensities was considered to be attributable to reactive oxygen species produced during the course of the photosynthetic reactions in the cells (11). Hirayama et a[. (12) detected hydroxyl radical species in vivo in photoautotrophic cells of Chlorella sp., and demonstrated that increase in light intensity caused an increase in hydroxyl radical production and deterioration in the efficiency of photosynthesis. However, further examination from a biochemical viewpoint is needed to better understand the difference in the degree of photoinhibition in cultures of the host and transformed cells of M. polymorpha. As indicated by the lines in Fig. 2, the experimental data on S-210 and HYA-2F cells were fitted in Eq. 4 using the non-linear least squares method. As a result, the values of the parameters in Eq. 4 were determined to be as follows: 1,=83.0 W/m2, Ks=12.7W/m2, m=Od-’ and pmax=l.Od-l for the transformed cells, and I,=209 W/m2, j&=3.51 W/m2, m= 0 d-l and I-(,,,~=0.82 d-l for the host cells (Table 1). Photoautotrophic cultures of M. polymorpha S-210 and HYA-2F were carried out in a bioreactor under external irradiation with control of the incident light intensity, taking into consideration the light intensity distribution in the cell suspensions and the dependence of the growth rates of the host cells on light intensity. The calculation for control of the incident light intensity was performed employing the procedure shown in Fig. 1 and the values of the parameters shown in Table 1. In these cultures, the values of X0 and R were 0.15 kgDCW/m3 and 5.5 x 10e2 m, respectively. Figure 3 shows the time-courses of changes in cell concentration in photoautotrophic cultures of the transformed (S-210) and host (HYA-2F) cells, where incident light intensity was controlled. The X values of the transformed and host cells reached 2.1 and 3.3 kg-DCW/m3 at culture times of t=9.0 and lOd, respectively. As reference cultures, the transformed and host cells were cultivated while keeping
t [d I FIG. 3. Photoautotrophic cultures of M. polymorpha S-210 and HYA-2F in a bioreactor with and without control of incident light intensity. Symbols: 0 and -, transformed cells (S-210) in lightcontrolled culture; 0 and ----, transformed cells (S-210) in constantlight culture; 0 and - - - -, host cells (HYA-2F) in light-controlled culture; + and --m-, host cells (HYA-2F) in constant-light culture.
the light intensity constant at I,=25 W/m2. In these cultures, the X values of the transformed and host cells were 1.4 and 1.1 kg-DCW/m3 at t= 12 and 9.5 d, respectively, as shown in Fig. 3. Exponential growth of the cells was observed during the early stage (t=l-7 d) in these four cultures. In constant-light cultures, the observed specific growth rates, rl(&s, for S-210 and HYA-2F cells were 0.24 and 0.35d-‘, respectively. On the other hand, the &bs values of S-210 and HYA-2F cells in cultures carried out under control of incident light were 0.38 and 0.50d-l, respectively. It was recognized that the &,bsvalues of both the cells were enhanced in the cultures carried out under control of incident light intensity, compared with those in cultures not carried out under control of incident light intensity. Table 2 summarizes the results obtained from cultures of the transformed and host cells with and without light intensity control in a bioreactor. Under the conditions of controlled and constant light intensity, the chlorophyll contents in the transformed cells were 23-24 g/kg-DCW, and these values were relatively high in comparison with those in the host cells. Concerning GUS activity, no significant difference was observed between cultures of the transformed cells with and without light intensity control. The values of the cell yields based on the irradiated light energy, YxlE, were calculated from the amounts of cells grown and the total light energy illumination throughout the cultures. It was found that the values were in the range of YX/~=l.l-1.4~ 10e9 kg-DCW/J, irrespective of the type of cells and the manner of light illumination. In conclusion, photoautotrophic cultures of the host and transformed cells of liverwort (M. polymorpha HYA-2F and S-210) were conducted using the externally illuminated bioreactor where the incident light intensity was controlled to obtain effective growth of the cells. The amounts of the cells obtained in the light-controlled cultures of the host and transformed cells were larger
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88, 1999 TABLE
2.
NOTES Summary of photoautotrophic
585
cultures of M. polymorpha HYA-2F and S-210 in a bioreactor with and without control of incident light intensity
Transformed cells (S-210) Host cells (HYA-2F) Controlled light Constant light Controlled light Constant light (17-68 W/m2) (25 W/m2) (25-178 W/m2) (25 W/mZ) 9.0 12 10 9.5 Culture time (d) 1.4 Cell concentration (kg-DCW/m3) 2.1 3.3 1.1 16 17 Chlorophyll content (g/kg-DCW) 23 24 GUS activity (103 U/kg-DCW) 0.72 0.71 N.D. N.D. Irradiated light energy, ET (106 J) 1.4 0.92 2.9 0.74 1.3 1.1 1.3 Cell yield based on irradiated light energy, Yx, (lo+ kg-DCW/J) 1.4 Initial values for transformed cells: X0=0.15 kg-DCW/m3, chlorophyll content=24 g/kg-DCW and GUS activity=0.69 X lo3 U/kg-DCW. Initial values for host cells: X,=0.15 kg-DCW/m3 and chlorophyll content=21 g/kg-DCW. N.D.: Not determined. ET =A S(r,,(t)dt; Y,,, = (X-X0) 0
V/ET
than those in the cultures carried out under constant light intensity. It was verified that culture under lightcontrolled conditions could be effectively applied to the photoautotrophic growth of both the host and transformed cells. NOMENCLATURE
: irradiated surface area of photobioreactor, m2 : empirical constant in Eq. 1, kg-DCW/(m2. S) b : empirical constant in Eq. 2, m2/kg-DCW : empirical constant in Eq. 2, m-l * irradiated light energy, J k I I light intensity, W/m2 IO : incident light intensity, W/m2 IIll : parameter in Eq. 4, W/m2 KS : saturation constant in Eq. 4, W/m2 L : light path length, m : parameter in Eq. 4, d-l R” : radius of the bioreactor, m t : culture time, d v : working volume of photobioreactor, m3 X : cell concentration, kg-DCW/m3 x0 : initial cell concentration, kg-DCW/m3 Y X,~: cell yield based on irradiated light energy, kgDCW/J (Y : effective absorption coefficient of light in cell suspension, m-l AK : change in electrical conductivity of medium, S/m I-I . specific growth rate, d-l ,umaxI maximum specific growth rate in Eq. 4, d-l /*&s : observed specific growth rate, d-l A
a
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