3.1 Induction of mutants
The mutants of P. commune NRC 2016 were developed by using various mutagenesis strategies and selected initially based on growing on specific lipid media. Several potent mutants thus selected were evaluated for their lipid production ability. Table (3) indicated the lipid production from the various potent mutants obtained through physical (Ɣ ray) and chemical mutagenesis (NaN3, Et Br, and EMS). It was observed that lipid productivity of 0.81, 1.47, 0.41, 0.93, and 0.90 g/l were obtained from wild-type, Ɣ ray, NaN3, Et Br, and EMS, respectively. The stability of three generations for lipid production from P. commune NRC 2016 mutants indicated that were given stable characters in the case of Ɣ ray mutants, Et Br mutants, and EMS mutants while the NaN3 mutants resulted in an unstable charter.
3.2 Comparative analysis of P. commune NRC 2016 wild type and its mutants using ISSR analysis
In this study, ISSR molecular marker technique was used to differentiate between the wild type of P. commune NRC 2016and its mutants. A total of 5 primers were used and 53 and 39 bands were recorded from P. commune NRC 2016 shown in Figure (1). The most polymorphic primers were ISSR-1 which produced 6 bands, followed by primer ISSR-4, primer ISSR-5, primer ISSR-6, and primer ISSR-10 which produced 7, 14, 12, and 14 bands, respectively (Table 4). Some primers produced several bands, while others produced a few bands. ISSR-primers produced polymorphism ranging from 16.667 to 100%. Primer ISSR-10 produced the highest percentage of polymorphism (100%) and did not produce any monomorphic band, while primer ISSR-4 produced the lowest percentage of polymorphism (0) with 7 monomorphic bands. The mean band frequency of each primer ranged from 0.458 to 1.
3.3 Optimization of the culture conditions for the lipid production from P. commune NRC 2016 wild-type and its mutants
To reduce the cost of lipid production by P. commune NRC 2016 wild type and mutants, the agriculture waste of bagasse with B. cereus 3SME which produced fermentable sugars was used. The RSM had employed for a statistical optimization of lipid production from P. commune NRC 2016 wild type and its mutants. A total sum of 47 runs with different combinations of five parameters was carried out and the results were shown in Table (5). The lipid content by wild type ranged from 0.005 to 2.01 g/l. The lipid content from the mutant by Ɣ ray ranged from 0.005 to 2.55 g/l. The lipid content from mutant by Et Br ranged from 0.005 to 1.705 g/l. The lipid content from mutant by EMS ranged from 0.005 to 2.27 g/l.
The interactive effects of the five parameters for P. commune NRC 2016 wild type and its mutants were deduced by analysis of variance (ANOVA) of the results, regression coefficient, F values, P values of variables as shown in Tables (6-9).
The wild-type ANOVA data was shown in Tables (6). The model was a significant as F-value was 5.91 and A, C, E, C2, and A2E were all significant model terms. The model R2 was 0.721, adequate precision of 9.538, stander deviation was 0.367, and the mean was 0.426.
The final equation for lipid production by wild type P. commune NRC 2016:
Lipid g/l from P. commune NRC 2016 wild-type = 17.823 - 1.461*A - 6.502*B + 0.003*C + 2.104*E + 1.4621*A * pH - 0.1 * A*C -0.933 *A*E - 0.059*B*C - 0.156*A2 + 0.615*B2 + 0.006*C2 + 0.013*A*B*C+ 0.091 A2*E+ 0.13711*A*B2
The mutant by Ɣ ray ANOVA was shown in Tables (7). The model was significant as F-value was 13.89 and A, AB, AC, BC, A2, ABC, ABD, ACD, A2B, and A2 C were all significant model terms. The model R2 was 0.927, the adequate precision of 15.761, the stander deviation was 0.266, and the mean was 0.404.
The final equation for lipid production by Ɣ ray mutant
Lipid (g/l) from P. commune NRC 2016 Ɣ ray = 34.175 - 8.544*A - 5.639* B - 0.087*C - 4.082* D - 1.549*E +1.169*A*B - 0.010*A*C + 0.671*A*E - 0.053*B*C + 0.207*B*D + 0.08*C*D + 0.048*C*E + 0.527*D*E + 0.895*A2 + 0.406*B2 + 0.021*A*B*C - 0.052*A*B*D - 0.009*A*C*D- 0.016*C*D*E - 0.063*A2*B - 0.013*A2*C - 0.083* A* B2
The mutant by Et Br ANOVA was shown in Tables (8). The model was significant as F-value was 31.31 and B, C, AB, AC, BC, B2, C2, ABC, A2 C, and AB2 were all significant model terms. The model R2 was 0.948, the adequate precision of 20.25, the stander deviation was 0.135, and the mean was 0.27.
The final equation for lipid production by Et Br mutant
Lipid g/l from P. commune NRC 2016 Et Br = -25.141 + 12.896*A + 4.677*B +0.075*C + 1.90675*E - 1.8301*A*B - 0.195*A*C- 0.045*B*C - 0.286*B*E + 0.0597*C*E - 0.614*A2 - 0.151*B2 + 0.007*C2 + 0.012*A*B* C + 0.009*B*C*E + 0.046*A2*B + 0.009*A2*C + 0.065*A* B2
The mutant by EMS ANOVA was shown in Table (9). The model was significant as F-value was 6.99 and C, AB, AC, AD, AE, C2, ABC, ABE, ACD, ACE, and BDE were all significant model terms. The model R2 was 0.884, the adjusted of 0.757, the adequate precision of 10.199, the stander deviation was 0.272, and the mean was 0.343.
The final equation in terms of actual factors for mutant by EMS was as follows:
. Lipid g/l from P. commune NRC 2016 EMS = -25.623 + 7.843*A + 7.854* B–0.153*C - 0.758*D - 1.176*E - 1.622*A*B - 0.102*A*C - 0.143*A*D - 0.044*A*E - 0.061*B*C + 0.378*B*D - 0.144*B*E - 0.0506*C*D+ 0.067*C*E + 0.7*D*E -0.410*B2 + 0.009 *C2 + 0.013234*A*B*C - 0.0382*A*B*D +0.073516*A*B*E + 0.011641*A*C*D -0.013*A*C*D -0.108*B*D* E + 0.077*A*B2
The three-dimensional (3D) response surface plots generated by Design-Expert software as shown in Figure (3) represented the relationships between different factors and their effects on lipid production by P. commune NRC 2016 wild-type and its mutants
The production of lipid from P. commune NRC 2016 wild type and its mutants had been numerically optimized and applied practically to validate the Box-Behnken model. The results in Figure (4) revealed the validation of the model at different conditions for predicted maximizing lipid production compared with actual value, chosen value using 1 g/l carbon and nitrogen source, and synthesis medium. For the wild type, the lipid-increasing rate was 59.70%. The predicted lipid value was 1.46, the maximum lipid 2.01 g/l produced at which incubation time was 7 days, pH 6, at a temperature of 25°C, xylose 3 g/l, and peptone 3 g/l compared to results from the synthetic media before using surface response optimization 0.965 g/l. Because the main purpose of this study was to reduce cost so the run was chosen at 1.895 g/l. since it was very close to the optimum, while 1 g xylose and 1 g peptone were used. For the Ɣ ray mutant lipid, the increasing rate was 42.75%. The predicted lipid was 2.34, the maximum lipid 2.55 g/l was produced at which the incubation time was 7 days, pH 6, at a temperature of 25°C, xylose 3 g/l, and peptone 3 g/l compared to results on synthetic media before using surface response optimization 1.46 g/l. Because the main purpose of this study was to reduce cost so the chosen 2.17 g/l. For the Et Br mutant lipid, the increasing rate was 36.73%. The predicted maximizing lipid was1.47, the maximum lipid 1.705 g/l produced at which incubation time was 5 days, pH 7, at a temperature of 20°C, xylose 2 g/l, and peptone 2 g/l compared to results on synthetic media before using surface response optimization 0.705 g/l. Because the main purpose of this study was to reduce cost so the run was chosen at 1.465 g/l. For the EMS mutant lipid, the increasing rate was 60.35%. The predicted maximizing lipid was 1.62, the maximum lipid 2.27 g/l produced at which incubation time was 7 days, pH 8, at a temperature of 25°C, xylose 1 g/l, and peptone 3 g/l compared to results on synthetic media before using surface response optimization 0.90 g/l. Because the main purpose of this study was to reduce cost so the run was chosen at 1.48 g/l.
3.4 Gas chromatography for P. commune NRC 2016 wild type and its mutants
The composition of fatty acids methyl esters and the profiles were cited in Table (10) for P. commune NRC 2016 wild type and its mutants. The main fatty acids composition for biodiesel produced P. commune NRC 2016 wild type was mainly C16-C18. That includes palmitic acid (C16:0), palmitoleic acid (C16:1) stearic acid (C18:0) oleic acid (C18:1), linolelaidic acid (C18:2), linoleic acid (C18:2), cis-11,14-eicosadienoic acid (C20:2) and tricosanoic acid (C23:0).
3.5 Physical properties of the biodiesels
The blending biodiesel (B5) from P. commune NRC 2016 and F. oxysporum NRC 2017 wild types and mutants. Table (11) indicated their physical properties are accepted with ASTM D975 (standard biodiesel for B5).