Culture of Spirogyra africana from farm ponds for long- term ...

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426 Biotechnol. Agron. Soc. Environ. 2013 17(3), 423-430 Gallego I., Casas J.J., Fuentes-Rodriguez F. et al.and number of rhizoids per filament. When ANOVAsshowed significant differences between groups (culturemedia, chlorination conditions), Tukey’s HSD posthoctests were used. All analyses were performed withSTATISTICA 7.1 software (Statsoft Inc., 2005).a0.040.02b3. RESULTS3.1. Effect of culture media on S. africana growthrateThe three culture media showed substantial differencesin nutrient composition (Table 1). HSCHU#10 andsD11 contained N:P molar ratios that varied from aP-impoverished FPW medium to a N-depleted ratioin sD11, whereas HSCHU#10 showed an N:P rationearest to the optimal Redfield ratio for phytoplankton,namely 16:1.At the end of the experiment, S. africana onlyshowed a positive growth rate in HSCHU#10(Figure 1). All replicates in sD11 and FPW showednegative growth rates. The two-factor ANOVA testshowed significant differences in S. africana growthrate between culture media (F = 429.784; P < 0.001),between chlorination treatments (F = 152.025;P < 0.001) and in the interaction of both factors(F = 53.315; P < 0.001).Table 1. Nutrient concentration (mg.l -1 ), N:P molar ratioand pH of the three culture media tested to determineits effect on growth, filament survival and rhizoiddevelopment of Spirogyra africana — Concentration deséléments nutritifs (mg.l -1 ), rapport molaire N:P et ph destrois milieux de culture testés pour déterminer son effet surla croissance, la survie des filaments et le développementdes rhizomes de Spirogyra africana.ParameterMediumHSCHU#10 sD11 FPWCa 2+ 4.89 16.98 221.52Mg 2+ 1.23 9.86 130.07Fe 3+ 0.14 1.00 n.m.K + 0.56 41.54 30.02Na + 4.52 13.84 330.55Cl - 0.09 7.13 472.75NO 3- 7.56 26.26 1.27PO 43-1.36 81.78 0.001HCO 3-+ CO 32-5.66 21.43 48.3N:P molar 8.5:1 1:2 1088:1pH 7.3 7.1 8.4n.m. = not measured value — valeur non mesurée.Growth rate (d -1 )0.00-0.02-0.04-0.06-0.08HSCHU#10sD11FPWHSCHU#10sD11FPWFigure 1. Growth rate (expressed as d -1 ) of Spirogyraafricana in three culture media tested, in non-chlorinated(a) and chlorinated samples (b) — Taux de croissance (parjour) de Spirogyra africana dans trois milieux de culturetestés, dans des échantillons non chlorés (a) et chlorés (b).Bars denote standard error — Les barres indiquent l’erreurstandard; *** P < 0.001: two-way ANOVA — ANOVA à deuxfacteurs.3.2. Effect of culture media on live filamentsThe type of culture media also affected the percentageof live filaments (ANOVA F = 150.093; P < 0.001),with HSCHU#10 proving to be the highest and FPWthe lowest (Figure 2). Furthermore, the effect of culturemedia and time was significant (ANOVA F = 53.452;P < 0.001), since HSCHU#10 showed asymptoticvalues around 90% of the initial value after 10 weeksand both sD11 and FPW showed percentages close to40% at the end of the experiment. Chlorination hadno effect on the percentage of live filaments (ANOVAF = 2.665; P > 0.05) but the interaction of time andchlorination showed a positive effect (ANOVAF = 19.019; P < 0.001).3.3. Effect of culture media on rhizoid productionRhizoid genesis was observed from the first week afterinoculation (Figure 3). The percentage of rhizoids perlive filament was affected by the type of culture mediaused (ANOVA F = 4.044; P < 0.05). Tukey’s HSDpost hoc test showed that sD11 was the medium with
Culture of Spirogyra africana 427a100HSCHU#10 sD11 FPWb100Life filaments (%)80604020Life filaments (%)8060402000 1 2 3 4 5 6 7 8 9 10Weeks after the inoculation00 1 2 3 4 5 6 7 8 9 10Weeks after the inoculationFigure 2. Percentage of live filaments of Spirogyra africana in HSCHU#10, sD11 and FPW culture media, in non-chlorinated(a) and chlorinated samples(b) during 10 weeks — Pourcentage de filaments vivants de Spirogyra africana dans les milieux deculture HSCHU#10, sD11 et FPW, dans des échantillons non chlorés (a) et chlorés (b) durant 10 semaines.Mean values of the four replicates are shown — Les valeurs moyennes des quatre répétitions sont indiquées; Bars indicate standarderror — Les barres indiquent l’erreur standard.HSCHU#10 sD11 FPWa1b1Rhizoids per filament0.80.60.40.2Rhizoids per filament0.80.60.40.200 1 2 3 4 5 6 7 8 9 10Weeks after the inoculation00 1 2 3 4 5 6 7 8 9 10Weeks after the inoculationFigure 3. Evolution of rhizoid production (number of rhizoids per filament) in HSCHU#10, sD11, FPW, in non-chlorinated(a) and chlorinated samples (b) during 10 weeks — Évolution de la production de rhizoïdes (nombre de rhizoïdes par filament)dans HSCHU#10, sD11, FPW, dans des échantillons non chlorés (a) et chlorés (b) pendant 10 semaines.Mean values of the four replicates are shown — Les valeurs moyennes des quatre répétitions sont indiquées; Bars indicate standarderror — Les barres indiquent l’erreur standard.the lowest number of rhizoids per filament (P < 0.05),whereas both HSCHU#10 and FPW showed nosignificant differences (P > 0.05). Chlorinationincreased rhizoid production (ANOVA F = 23.956;P < 0.001). However, the interaction of medium andchlorination was not statistically significant (F = 1.731;P > 0.05).4. DISCUSSION4.1. Suitability of the culture mediaOur results show that HSCHU#10 is the mostsuitable of the three assayed media for S. africanaculture, since both growth rate and percentage of live
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