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

424 Biotechnol. Agron. Soc. Environ. 2013 17(3), 423-430 Gallego I., Casas J.J., Fuentes-Rodriguez F. et al.case of Andalusian farm ponds, S. africana is one of themost common growing macroalgae, which may formsmonoalgal mats during spring months (I. Gallego,unpubl. data).The genus Spirogyra has recently drawn attentionto researchers due to its various biotechnological andindustrial applications, mostly based on its sorptionproperties. Spirogyra filaments remove heavy metals(Romera et al., 2007; Singh et al., 2007; Kaonga et al.,2008; Pribyl et al., 2008) and other toxic compoundsfrom effluents (Aleissa et al., 2004; Çelekli et al.,2009); as well as they produce allelochemicalsinhibiting microalgal growth (Zakaria, 2002; Trochineet al., 2011), implying important consequences forthe management of aquatic ecosystems. Antiviraland antihelmintic properties have also been reported(Muller-Feuga et al., 2003; Pultz et al., 2004). Morerecently, Spirogyra biomass has been shown to bean efficient energy source for biofuel (Hossain et al.,2008; Eshaq et al., 2010; John et al., 2011).Both stock maintenance and growth of algal speciesare essential for their use in biotechnology. Therefore,the search of the most suitable culture media and easeof laboratory culture are relevant topics. Regardinggenus Spirogyra, culture techniques were firstreported early in the 20 th century (see Kadlubowska,1984 and references herein). Mainly three culturemedia have proved successful for Spirogyra growthunder laboratory conditions in short-term culture:sD11 (Graham et al., 1995), Half-Strength CHU#10(Nalewajko et al., 1989), and Artificial Pond Water(Inoue et al., 2002). However, their suitability for longtermexperiments has not been tested so far, neithercontrasting experiments on different culturing mediahave not been carried out under the same environmentalconditions (e.g. irradiance, temperature).One of the most widely used media for Spirogyracultures nowadays is water from the source ecosystem,e.g. Townsend et al. (2008), sometimes replaced byArtificial Pond Water (Inoue et al., 2002; Ikegaya et al.,2004; Yoshida et al., 2009). sD11 culture medium hasalso proved suitable for Spirogyra culturing (Nalewajkoet al., 1989). It is derived from D11 medium, originallyused for Cladophora Kützing (1843) and slightlymodified for Spirogyra (Graham et al., 1995; O’Nealet al., 1995). Half-Strength CHU#10, henceforthHSCHU#10, has been widely used for a variety ofgreen alga, including Spirogyra spp. (Saygideger,1996).The presence of epibionts in Spirogyra in vitrocultures is one of the major problems in testing filamentsgrowth, despite the continuous mucilage secretion fromSpirogyra cell walls (Simons et al., 1990; Wöber et al.,2007). Among the several techniques used to removeepibionts from macroalgae, chlorination – an oxidantsterilization of filaments with a sodium hypochloritesolution – has been successful for epiphytic algae inseawater (Pang et al., 2007) and is commonly used forobtaining axenic cultures, e.g. García-Jiménez et al.(1999). To our knowledge, no evidence has been foundfor its use in Spirogyra cultures.Rhizoid production has also been included as aparameter for quantifying filament growth and survival,since rhizoids fix the filaments to the substratum andhence facilitate algal survival. Several factors havebeen reported as drivers of rhizoid genesis, such assubstratum properties (Ikegaya et al., 2008) or thelowering of extracellular Ca 2+ concentration (Inoueet al., 2002; Yamada et al., 2003). Release of singlecells resulting from filament-severing (Inoue et al.,2002; Ikegaya et al., 2008) has also been reported as atriggering factor.In the present study, we measured growth rate,percentage of live filaments and rhizoid production ofSpirogyra africana filaments over 10 weeks in threecommonly-used culture media – sD11, HSCHU#10and Filtered Pond Water (FPW) i.e. water from thesource pond where the filaments were gathered – withthe aim of culturing Spirogyra africana for longtermmaintenance, thus facilitating biotechnologicalapplications and stock maintenance. Additionally, wetested the effect of sterilization with NaClO (henceforthchlorination) as a procedure for assuring monoalgalcultures.2. MATERIALS AND METHODS2.1. Sample collection and laboratory analysesFilaments were collected from a small agriculturalpond measuring 20 x 18 x 1.75 m (L x W x D), locatedin Almería, SE Spain. As the filaments were anchoredonto the substratum, a rake was used to collect them.Samples were rinsed in pond water to remove sedimentand immediately taken to the laboratory. For thepreparation and chemical analysis of FPW, 10 l waterwere taken with a centrifugal electric pump from thebottom of the pond, where Spirogyra filaments weregrowing.2.2. Laboratory experimentThe influence of culture media type and chlorinationon Spirogyra africana growth and rhizoid productionwere investigated in a laboratory experiment with a3 x 2 factorial design, with culture media and filamentchlorination as independent factors. Four replicates pertreatment were performed, with a total of 24 cultureflasks.Three different culture media were analyzed: sD11,HSCHU#10 and FPW (pond water filtered through a

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

428 Biotechnol. Agron. Soc. Environ. 2013 17(3), 423-430 Gallego I., Casas J.J., Fuentes-Rodriguez F. et al.filaments were higher in this medium compared to theothers. This can be explained by the difference in thenutrient composition of the three culture media tested,particularly their contrasting N:P molar ratios, sinceboth are essential macronutrients for algal primaryproduction (Sterner et al., 2002; Klausmeier et al.,2004). In the field, a high growth of the Spirogyrafluviatilis Hilse complex was reported after fertilizingwith a molar ratio N:P = 10:1 (Townsend et al., 2008).This approaches the HSCHU#10 of our experiments(N:P = 8.5:1) and both are relatively close to the optimalRedfield ratio of 16:1 proposed for phytoplankton(Klausmeier et al., 2004). However, the N:P supplyratios of FPW and sD11 significantly deviated fromthe ratio both Redfield ratio and the ratio proposed byTownsend et al. (2008), pointing respectively to a caseof P- and N- impoverished media.Spirogyra africana mats grew in the pond withapparently no nutrient limitations during the 10 weeksof our experiment (I. Gallego, unpubl. data) while FPWshowed the poorest result. It is likely that the sedimentwas an important P source for this benthic alga in thenatural system (Townsend et al., 2008). The extremelylow-P FPW probably resulted in a severe nutrientdepletion in our experiment, where the pond sedimentwas not included.Contrary to our expectations, S. africana filamentsdid not grow in sD11 and the percentage of livefilaments was relatively low. The specific sD11 mediumhas been reported as a successful medium to growSpirogyra spp. in vitro cultures (Graham et al., 1995;O’Neal et al., 1995; Berry et al., 2000). However, weobtained poorer results with this medium comparedto HSCHU#10, a medium commonly used on a widerange of algae (Saygideger, 1996). Our experimentalconditions were similar to those used previously withSpirogyra spp. growing in sD11, i.e. temperature,irradiance and a weekly medium replacement.However, our experiment lasted for 10 weeks and halfthe medium was replaced weekly, whilst previous oneslasted only for 2-3 weeks (O’Neal et al., 1995; Berryet al., 2000) and media were entirely replenished every7-10 days (Berry et al., 2000). The effect of N depletionon S. africana growth became evident several weeksafter inoculation in sD11, while filaments in short-termcultures could thrive for a while on cellular N reserves.Our results show that despite the reportedly goodresults of the specific sD11 medium for S. africanacultures, filament growth for long-term cultures is notguaranteed.Indeed, mat cohesiveness was studied by Berryet al. (2000) as an indicator of filament decline thatdepends on irradiance. A higher filament entanglementwas observed in our sD11 replicates than in FPW andHSCHU#10 (data not shown), despite all our replicateswere exposed to the same irradiation. We suggest thatcohesiveness may be also dependent on the mediumnutrient content as well, although further study isrequired in this area.Since there is a lack of studies on irradiance and/or temperature using other culture media apart fromsD11, we can only suggest that the observed matcohesiveness and the low-N composition of sD11 maybe detrimental for S. africana survival.The conditions for avoiding nutrient depletionin our experiment (weekly replacement of half themedium) were clearly insufficient in sD11 and FPW,but seemed to be adequate in HSCHU#10. Indeed, theeffect of mat cohesiveness may interact with nutrientcomposition and nutrient depletion. Thus, long-termcultures can be performed in HSCHU#10 with lesslaboratory manipulation and effort, so we recommendthis medium for long-term experiments or for themaintenance of stock cultures of S. africana.Effectiveness of chlorination on S. africana growth.Chlorination had a positive effect on filaments growth,while the effect of chlorination on the percentage oflive filaments was only significant when interacted withtime. Chlorine is reported as a traditional sterilizingagent for removing epibionts from macroalgae(García-Jiménez et al., 1999; Pang et al., 2007) and freechlorine also inactivates bacteria and cyanoprokariota(Daly et al., 2007; Amiri et al., 2010). The eliminationof these nutrient competitors by chlorination probablyfavored Spirogyra growth. In spite of this apparentlypositive effect, sodium hypochlorite can react withpolysaccharides present in the mucilaginous sheathof the Zygnemataceae (Cheli et al., 1989) and evendamage the cell walls. Thus, the higher mortalityobserved in chlorinated samples at the beginning ofthe experiment could be attributed to this treatment.However, at the end of the experiment, this likelydamage might be counteracted by the elimination ofcompetitors.4.2. Rhizoid genesis in S. africanaOur results on rhizoid production are not as clear asthose on growth and percentage of live filaments.The N:P ratio does not seem to be involved in rhizoidproduction, since both HSCHU#10 and FPW showedsimilar results. A low extracellular Ca 2+ concentrationand release of single cells have also been reported astriggering factors of rhizoid formation (Nalewajkoet al., 1989; Ikegaya et al., 2004). Nonetheless, thelowest concentration of Ca 2+ occurred in HSCHU#10,which showed high rhizoid formation, while singlecells released from filaments were observed in bothsD11 and FPW (I. Gallego, unpubl. data), whichparadoxically showed different results. We suggestthat other unidentified factors, not described in the

Culture of Spirogyra africana 429literature so far, may be involved in the process ofrhizoid genesis. However, chlorination increasedrhizoid genesis, supporting our previous results on thepositive effect of chlorine sterilization on Spirogyrafilaments.5. CONCLUSIONThe search of the most suitable culture media forstock maintenance and optimal growth of S. africanais essential for its subsequent use in biotechnology.For this purpose, we tested the suitability of the threeculture media most commonly used for S. africanagrowth.Half-Strength CHU#10 was the most suitable of themedia assayed for S. africana growth and it seems tobe appropriate for long-term experiments. The widelyused sD11 specifically modified for Spirogyra showedpoorer results.Filament chlorination prior to experimentinoculation showed a positive effect on S. africanagrowth and rhizoid production.We recommend the use of both Half-StrengthCHU#10 and surface decontamination of filamentousalgae with NaClO for Spirogyra cultures. Thus, longertermcultures can be performed with less laboratorymanipulation and effort.AbbreviationsHSCHU#10: Half-Strength CHU#10FPW: Filtered Pond WaterAcknowledgementsThis research has been funded by the Andalusian RegionalGovernment (Junta de Andalucía, Consejería de MedioAmbiente y Consejería de Innovación, Ciencia y Empleo)as part of the project P06-RNM01709. We thank the pondowner for allowing us access and sampling. We sincerelythank Enric Descals for editorial assistance.BibliographyAleissa K.A., Shabana E.S.I. & Al-Masoud F.I.S., 2004.Accumulation of uranium by filamentous green algaeunder natural environmental conditions. J. Radioanal.Nucl. Chem., 260, 683-687.Amiri F., Mesquita M.M.F. & Andrews S.A., 2010.Disinfection effectiveness of organic chloramines,investigating the effect of pH. Water Res., 44, 845-853.APHA, 2005. Standard methods for the examination of thewater and wastewater. 22 th ed. Washington, DC: APHA.Berry H.A. & Lembi C., 2000. Effects of temperatureand irradiance on the seasonal variation of Spirogyra(Chlorophyta) population in a Midwestern lake (USA).J. Phycol., 36, 841-851.Bonachela S. et al., 2013. Pond management and waterquality for drip irrigation in Mediterranean intensivehorticultural systems. Irrig. Sci., 31, 769-780.Casas J.J. et al., 2011. Artificial ponds in a Mediterraneanregion (Andalusia, southern Spain): agricultural andenvironmental issues. Water Environ. J., 25, 308-317.Çelekli A., Yavuzatmaca M. & Bozkurt H., 2009. Kineticand equilibrium studies on the adsorption of reactivered 120 from aqueous solution on Spirogyra majuscula.Chem. Eng. J., 152, 139-145.Cheli F. & De Vecchi L., 1989. An ultrastructural andcytochemical study on Conjugatophycean cell wall.Caryologia, 42, 127-137.Daly R.I., Ho L. & Brookes J.D., 2007. Effect of chlorinationon Microcystis aeruginosa cell integrity and subsequentmicrocystin release and degradation. Environ. Sci.Technol., 41, 4447-4453.Eshaq F.S., Ali M.N. & Mohd M.K., 2010. Spirogyrabiomass a renewable source for biofuel (bioethanol)production. Int. J. Environ. Sci. Technol., 2, 7045-7054.García-Jiménez P., Marian F.D., Rodrigo M. &Robaina R.R., 1999. Sporulation and sterilizationmethod for axenic culture of Gelidium canariensis.J. Biotechnol., 70, 227-229.Graham J.M., Lembi C.A., Adrian H.L. & Spencer D.F.,1995. Physiological responses to temperature andirradiance in Spirogyra (Zygnematales, Charophyceae).J. Phycol., 31, 531-540.Hossain A.B.M.S. et al., 2008. Biodiesel fuel productionfrom algae as renewable energy. Am. J. Biochem.Biotechnol., 4, 250-254.Ikegaya H., Yamada S.Y., Sonobe S. & Shimmen T., 2004.Saponin-induced release of single cells from filamentsand rhizoid differentiation in Spirogyra. J. Plant Res.,117, 443-447.Ikegaya H. et al., 2008. Presence of xyloglucan-likepolysaccharide in Spirogyra and possible involvementin cell-cell attachment. Phycol. Res., 56, 216-222.Inoue N., Yamada S., Nagata Y. & Shimmen T., 2002.Rhizoid differentiation in Spirogyra: position sensing byterminal cells. Plant Cell Physiol., 43, 479-483.Iwata K., Tazawa M. & Itoh T., 2001. Turgor pressureregulation and the orientation of cortical microtubules inSpirogyra cells. Plant Cell Physiol., 42, 594-598.John R.P., Anisha G.S., Nampoothiri K.M. & Pandey A.,2011. Micro and macroalgal biomass: a renewable sourcefor bioethanol. Bioresour. Technol., 102, 186-193.Juan M. et al., 2012. Construction characteristics andmanagement practices of in-farm irrigation pondsin intensive agricultural systems. Agronomic andenvironmental implications. Irrig. Drain., 61(5), 657-665.Kadlubowska J.Z., 1984. Süsswasserflora von Mitteleuropa.Band 16, Chlorophyta VIII. Conjugatophyceae I: