Statistical experimental designs; involving (i) a fractional factorial design (FFD) and (ii) a central composite design (CCD) were applied to optimize the culture medium constituents for production of a unique antifreeze protein by the Antartic micro algae Chaetoceros neogracile. The results of the ...
Statistical experimental designs; involving (i) a fractional factorial design (FFD) and (ii) a central composite design (CCD) were applied to optimize the culture medium constituents for production of a unique antifreeze protein by the Antartic micro algae Chaetoceros neogracile. The results of the FFD suggested that NaCl, KCl, $MgCl_2$, and ${Na}_{2}{SiO}_{3}$ were significant variables that highly influenced the growth rate and biomass production. The optimum culture medium for the production of an antifreeze protein from C. neogracile was found to be Kalle's artificial seawater, pH of $7.0{\pm}0.5$, consisting of 28.566 g/l of NaCl, 3.887 g/l of $MgCl_2$, 1.787 g/l of $MgSO_4$, 1.308 g/l of $CaSO_4$, 0.832 g/l of ${K_2}{SO_4}$, 0.124 g/l of $CaCO_3$, 0.103 g/l of KBr, 0.0288 g/l of $SrSO_4$, and 0.0282 g/l of ${H_3}{BO_3}$. The antifreeze activity significantly increased after cells were treated with cold shock (at $-5^{\circ}C$) for 14 h. To the best of our knowledge, this is the first report demonstrating an antifreeze-like protein of C. neogracile.
Statistical experimental designs; involving (i) a fractional factorial design (FFD) and (ii) a central composite design (CCD) were applied to optimize the culture medium constituents for production of a unique antifreeze protein by the Antartic micro algae Chaetoceros neogracile. The results of the FFD suggested that NaCl, KCl, $MgCl_2$, and ${Na}_{2}{SiO}_{3}$ were significant variables that highly influenced the growth rate and biomass production. The optimum culture medium for the production of an antifreeze protein from C. neogracile was found to be Kalle's artificial seawater, pH of $7.0{\pm}0.5$, consisting of 28.566 g/l of NaCl, 3.887 g/l of $MgCl_2$, 1.787 g/l of $MgSO_4$, 1.308 g/l of $CaSO_4$, 0.832 g/l of ${K_2}{SO_4}$, 0.124 g/l of $CaCO_3$, 0.103 g/l of KBr, 0.0288 g/l of $SrSO_4$, and 0.0282 g/l of ${H_3}{BO_3}$. The antifreeze activity significantly increased after cells were treated with cold shock (at $-5^{\circ}C$) for 14 h. To the best of our knowledge, this is the first report demonstrating an antifreeze-like protein of C. neogracile.
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neogracile under cold-shock conditions. To the best of our knowledge, this is the first report describing the statistical optimization of the culture medium for growth of C. neogieacile, with the indication of its possible antifreezelike protein production. Owing to a lack of detailed structural information, further kinetic studies are now in progress for the purification as well as the determination of the consensus sequence of the ice-binding domain of antifreeze protein(s) from C.
제안 방법
E/m2/s using white fluorescent lamps. All experiments were conducted with the seed culture during its exponential growing phase. C.
Based on the results of the FFD, variables of NaCl, KC1, MgCl2, and Na2SiO3 were subsequently employed to determine the independent significance on cell growth. The next experiment was performed to find the factor values more precisely in order to produce a desired response.
The CCD is very useful to acquire data to fit a polynomial. CCD was generated for the four variables (concentrations ofNa2SiO3, NaCl, KC1, and MgCl2) with 25 factorial points, two center points (running in duplicate), and 8 axial points where four factors were set at their center point level (Table 6). The result of central composite design for the four factors is given in Table 7.
). Data from the Coulter counter were collected by AccuComp software and then exported to an Excel spreadsheet to calculate the cell numbers and cell size distribution as well as average cell size [7]. The total cell volume {=cell numbers (cell/ ml)xaverage cell volume (m3/cell)} was used to calculate the specific growth rate.
Taken together, we optimized the culture medium for growth of C. neogracile using the statistical analysis models FFD and CCD, and introduced a possibility for the production of antifreeze-like protein produced from C. neogracile under cold-shock conditions. To the best of our knowledge, this is the first report describing the statistical optimization of the culture medium for growth of C.
independent significance on cell growth. The next experiment was performed to find the factor values more precisely in order to produce a desired response. The CCD is very useful to acquire data to fit a polynomial.
성능/효과
3B). In conclusion, the maximum cell numbers of C. neogracile increased up to 300% after the optimization of the culture medium was compared with the control in the validation experiment (Fig. 3A). These resuIts indicated the importance of the optimization of components in the culture media for growth of C.
In the present study, fractional factorial design and central composite design, known as powerful tools for optimization of medium components, were carried out to maximize the growth rate and biomass production. The results of fractional factorial design showed that NaCl, KC1, MgCl2, and Na2SiO3 were significant factors (see Table 8). In CCD, the central values of the NaCl, KC1, MgCl2, and Na2SiO3 concentrations were 24.
These results indicated that the four variables NaCl, KC1, MgCl2, and Na2SiO3, especially Na2SiO3, may act as a major or limiting component in nutrients of culture medium, and small variations in their components can alter the growth rate and/or production of antifreeze protein from C. neogracile. As expected, this is not surprising because Na2SiO3-9H2O (in group F) is well known as an essential nutrient ft)r composition of the diatom's cell wall, and salts in grgip A (NaCl, KC1, and MgCU) are related to salinity and osmolarity.
neogracile. These results showed the biomass productivity measured as the concentration of chlorophyll a and/or b: 0.78 mg/1 in Lyman, 1.40 mg/1 in Coral, 1.58 mg/1 in Kalle's ASW, 1.30 mg/1 in MBL, 1.18 mg/1 in Millero's ASW. Therefore, for further study, Kalle's ASW was selected as the best culture medium for the growth of C.
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