OPTIMIZATION OF GROWTH CONDITIONS OF DIFFERENT ALGAL STRAINS AND DETERMINATION OF THEIR LIPID CONTENTS ABSTRACT

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1 Munir et l., The Journl of Animl & Plnt Sienes, 25(2): 2015, Pge: J Anim. Plnt Si. 25(2):2015 ISSN: OPTIMIZATION OF GROWTH CONDITIONS OF DIFFERENT ALGAL STRAINS AND DETERMINATION OF THEIR LIPID CONTENTS N. Munir, A. Imtiz, N. Shrif nd S. Nz Deprtment of Biotehnology nd Miroiology, Lhore College for Women University, Lhore Corresponding uthor s emil: neelm.munir@yhoo.om ABSTRACT Aim of the present study ws olletion nd identifition of ntive lgl speies, optimiztion of their growth onditions nd omprison of their oil ontent. From the olleted 20 smples 3 different lgl speies were identified. The effet of growth medi, temperture, ph level, light intensity nd ertion on growth of Spirogyr sp. nd Oedogonium sp. nd Chlorell sp. ws reorded. It ws oserved tht oth Oedogonium sp. nd Chlorell sp. grow well in Blue green medium while Spirogyr sp. showed etter growth in terms of fresh weight in Bold Bsl medium. Algl growth optimum temperture ws etween o C. It ws lso oserved tht neither very low nor very high ph is suitle for lgl growth nd t ph 7.5 lge were le to grow t mximum with fresh weights of 4.89±0.091 g nd 4.79±0.021 g for Spirogyr sp. nd Oedogonium sp. respetively. Artifiil fluoresent light resulted in n inresed growth of lge s ompred to sunlight or when lge pled ner window nd exposed to indiret sunlight. The oil ontent of the three lgl speies is in order Chlorell sp > Oedogonium sp. Spirogyr sp. Hene, Chlorell sp. ws found to hve the highest lipid ontent with 15.46±0.240% yield for lipid ontent. Key words: Alge; Biofuel; Chlorell.; Oedogonium.; Optimiztion; Spirogyr. INTRODUCTION Alge re group of orgnisms tht hve een generlly desried s photoutotrophi, simple mirosopi or mrosopi, uniellulr to multiellulr plnts nd re ompetent onverters of sun energy to useful iohemil produts like oil (Shenk et l., 2008). Alge re minly wter dwelling orgnisms lking omplex morphologil orgniztion (Brsnti nd Gultieri, 2006). Alge either mro or miro, hve gret potentil for iofuel prodution. Mirolge re vried group of single-elled orgnisms; lso they re ple to offer rnge of solutions for our energy demnds through numer of wys (Bl, 2006). Alge effiiently use CO 2 from tmosphere nd they re responsile for more thn 50% of the totl glol ron fixtion (Feng, 2011). Alge re onsidered s third genertion iofuel. They n inrese iomss very quikly nd few of lgl speies n doule their iomss in s few s 5 hours while severl speies re reveling two doulings eh dy (Huer, 2009). Altogether, lge possess the pity to yield energy-rih oils nd numer of mirolge were found to nturlly gther high oil ontent in their totl dry weight (Rodolfi et l., 2008). Alge re esy to grow nd ultivte nywhere with less energy requirements nd using very few of the nutrients. The idel growth onditions for mirolgl ultures re strin speifi nd the iomss produtivity depends upon mny ftors. These inlude ioti ftors like temperture, minerls, ron dioxide, ph, wter qulity, light yle nd intensity. The ioti ftors inlude ell frgility nd ell density. Mehnil ftors inlude ontinuous mixing, gs ule size nd distriution nd mss trnsfer, ll these re of prtiulr onern in photo-ioretors (Shenk et l., 2008). Light nd temperture re the two most importnt ftors tht ffet lge iomss produtivity. The energy for growing lge is provided y light vi photosynthesis. Suffiient light energy must e effetively utilized to hieve higher iomss produtivity. Temperture influenes the rtes of ll hemil retions relted to lgl growth nd its metolism (Sndnes et l., 2005). Chnge in temperture ffets the iohemil omposition of the ells speifilly lipids nd proteins. Thus light nd temperture hve signifint effet in the metolism, enzyme tivities nd ell omposition of lge. Alge ultivtion lso depends on ph levels nd optimum ph influenes the ron vilility, metolism nd iohemil omposition of ells (Rihmond, 2000). For effiient use of lge s soure of iodiesel it is very importnt to fous on the ntive lgl speies nd to selet tht lgl speies whih not only hs high growth rte ut hs greter lipid ontent. Identifition of lol lgl speies, optimiztion of onditions for ntive lgl speies nd omprison of their oil ontents is the prt of this study. Hene, present study is signifint step forwrd in utiliztion of lge s soure of renewle energy. MATERIALS AND METHODS Smpling: During the present study, totl 30 smples were olleted from 10 different lotions of Lhore 546

2 Munir et l., J. Anim. Plnt Si. 25(2):2015 inluding ponds nd dmp soil ples. The smpling time ws from the month of Septemer, 2011 to Mrh, Smples were olleted in glss jrs, Plsti gs nd ottles nd then trnsferred to vrious nutrient medi stndrdized during the present work using flsks, jrs nd plsti ottles. Identifition: Speies were identified sed on the morphologil hrteristis. These were studied y prepring slides nd oserving under y 10X nd 40 X powers of Mirosope Irrmeo GmH Gemny model IM Cultivtion: Blue-green (BG) medium nd Bold sl (BB) medium (Bold 1949) were tested for the lgl growth nd support. Two growth medium re mostly dpted to freshwter lge. Growth experiments were performed in onil flsks of 250 ml. Eh flsk ws filled with 100 ml of medi nd initilly 2 g of lgl smple ws dded to eh flsk. Every experiment ws performed in replites nd oth the medi were ompred for eh ulture onditions. Optimiztion of different ulture onditions: The effet of different prmeters on the growth rte of lge ws ssessed y following ompletely rndomized experimentl design. In these experiments only one ftor ws vrile while ll other onditions were kept onstnt. The ulture onditions whih were ontrolled for the lgl growth were nutrients, light, temperture, ph nd onstnt mixing or ertion. Culture medium/nutrients: For monitoring the est growth of lge oth the medi (BB nd BG) were used for ulturing lgl ells nd the est growth rte ws estimted fter required period of time hs een pssed. The first lultion ws mde on 7 th dy nd the rte ws estimted on the sis of the rte of inrese in fresh weight of iomss. The ell mss ws seprted through filtrtion nd then weighted fter lotting the exess wter. Temperture: The tempertures were mintined t 16, 20, 24, 28 nd 32 o C. Temperture vritions were monitored regulrly using thermometer. Chnges were reorded nd the lgl growth rtes were found y mesuring their fresh weights. ph: Both the ulture medi were djusted with vrious ph rnges i.e., 6.5, 7, 7.5, 8, 8.5, nd 9. Alge smples were grown within these ph rnges nd the effet ws oserved nd mesured y lulting the fresh weight of smples fter the speified time period hs een elpsed. Light: Algl ultures were pled out door in open sunlight, inside the l ner window nd under rtifiil light provided y fluoresent tues. The effet of light on the growth of lge ws lulted y mesuring the lgl iomss. Aertion/mixing: Different mens of mixing were used for this purpose. For mixing oritl shker OS 5 si yellow line ws used nd the flsks were kept over the shker t 300 rpm. Aertion ws supplied through erting pumps. These ultures were pled indoor where onstnt sunlight ws given. The growth results were then ompred nd ultures with mximum growth were ontinued Oil Extrtion: The lgl ultures grown on optimized onditions were further tested for their oil ontents. Filmentous lge were dried y spreding over lotting pper nd pling the mteril under running fn until ll of the wter ontent ws removed. In se of Chlorell sp. uniellulr lge the ell iomss ws olleted together nd dried in oven t 65 o C for 3 hours. All types of lge were ground to fine powder with motor nd pestle. To extrt oil from the lgl smples Bligh nd Dyer method (1959) ws used with slight modifitions. Five grms of dried smple ws homogenized in 15 ml of hloroform-methnol (1:2, v/v). After this the mixture ws entrifuged for 10 min t 3000 rpm nd then filtered. The residul iomss ws re homogenized with 5 ml of hloroform nd then entrifuged gin (10 min, 3000 rpm). This mixture ws filtered nd olleted together with the previous filtrte nd entrifuged. The lower phse ws olleted nd kept for drying to evporte ll the hloroform nd the remining ontent ws mesured y using the following formul Lipid ontent (%) = lipid extrted (g)/originl smple (g) 100 RESULTS AND DISCUSSION Inresed oil pries, depletion of fossil fuels nd high energy demnd hve mde the reserhers to look for n lterntive soure. Algl fuel might e n lterntive to fossil fuel euse these feedstoks re muh more relile nd using them s iofuel prodution n derese the dependene on fossil fuels (Bnsl nd Shrm, 2005; Kulkrni nd Dli, 2006). Mny lge iofuel studies hve shown tht the quntity nd qulity of lipids within the lgl ell iomss n vry s result of hnges in growth onditions (temperture, ph nd light intensity) or nutrient medi hrteristis (onentrtion of nitrogen, iron nd phosphtes) (Illmn et l., 2000; Liu et l., 2008). Ojetive of this study ws to ollet nd optimize the growth onditions nd determine the oil ontents of different lgl strins. During the present work 30 smples were olleted from 10 different lotions of Lhore. These smples were identified sed on morphologil hrters s given y (Shrm, 1986) nd vouher speimen Spirogyr sp., Oedogonium. sp. nd Chlorell sp. ws preserved s BT-Od-1, BT-Sp-1 nd BT-Ch-1 in Algl Biotehnology L, Deprtment of 547

3 Munir et l., J. Anim. Plnt Si. 25(2):2015 Biotehnology, Lhore ollege for women University. Lhore. The growth of lgl smples ws oserved y lulting the inrese in fresh weight of lge. Spirogyr sp. grown in oth medium hs shown vried results. It is evident from tle 1 tht medium hs signifint effet on fresh weights of lgl iomss. Initil weight of lge tken for eh experiment ws 0.5 g whih lmost eme douled (1.02±0.029 g) fter one week when lge were grown in BB medium. It ws lso oserved tht fter 1 week fresh weight of lgl iomss in BG medium ws greter (1.54±0.042 g) s ompred to BB medium (Tle 1 ). Generlly the sme trend ws oserved with the inrese in time intervl t week 2, 3 nd week 4. Similrly Oedogonium sp. ws ultured in BB nd BG medium. The results hve depited tht oth the medium hve signifint effet on lgl growth nd inresed their ell iomss y 1.28±0.022 g fter 1 week. Oedogonium sp. grown in BB medium ws hving more inresed growth rte s ompred to BG medium i.e. 1.28±0.022 g nd 1.03±0.032 g respetively. At week 4 the iomss eme muh denser with 4.75±0.020 g for BB medium while for BG medium 3.03±0.020 g (Tle 1). In se of uniellulr lge i.e. Chlorell. sp. growth ws mesured in terms of sorne t 600 nm using spetrophotometer Chlorell sp. ells were suspended in BB nd BG medium nd their sorne ws mesured dily in order to find the etter results for oth medium. Figure 4 shows the vlues for sorne t 600 nm nd it is evident from the results tht Chlorell sp. ells grow etter in BG medium s ompred to BB medium. These results show similrity with those reveled y Held (2011 ). Cultures of Chlorell sp. nd Spirogyr sp. were grown in BG medium nd light sttering nd fluoresene lirtion urves were plotted for growth rte. Results indited tht Chlorell sp. ells did not show inrese in growth urve while Spirogyr sp. were le to grow nd inrese in iomss (Groelr, 2000; Henderson et l., Frooq et l (2013) lso reported tht syntheti medium gives good results for lgl growth. Effet of temperture on lgl growth: Alge were grown in vrile temperture rnges. These were 16, 20, 24, 28 nd 32 o C. All other ftors were mde onstnt nd the ultures were oserved with vried temperture effet. The effet of temperture ws found to e signifint on lgl growth nd oth the filmentous forms of lge hve shown ovious effet of temperture on their growth rte in terms of inrese in fresh weight. Minimum temperture ws set t 16 o C nd the growth rte ws oserved to e not very high i.e. 0.96±0.012 g for Spirogyr sp. nd 0.99±0.040 g for Oedogonium sp. t week 1. The growth ontinued nd fter three weeks it ws mesured 2.89±0.36 g nd 3.05±0.133 g for Spirogyr sp. nd Oedogonium sp. respetively. Although growth ws oserved to inrese y inresing the temperture from 16 o C to 20 o C thus the fresh weight of Spirogyr sp. t week 4 eme 3.93±0.240 g t 20 o C while t 24 o C it ws lulted s 4.79±0.097 g (Tle 2). Similrly Oedogonium sp. ws found with inresed growth t 20 o C (4.59±0.021 g). Oedogonium sp. ws oserved to est grow t 24 o C nd the fresh weight ws mesured s 4.96±0.089 g t week 4. At 28 o C the effet ws lso signifint nd oth the lge were grown with inresed growth 4.66±0.226 g nd 4.91±0.052 g for Spirogyr sp. nd Oedogonium sp. respetively. Aove this rnge i.e. t 32 o C the lge gin showed derese in growth nd it ws found to e 2.93±0.087 g for Spirogyr sp. nd 3.59±0.069 g for Oedogonium sp. (Tle 2). Similr results were reveled for effet temperture hnges on lgl ells in work on N. oult nd Chlorell sp. ells. The growth ppered to e ffeted t temperture ove 30 o C. At temperture of 35 o C the mirolge did in ft exhiit 17% deline in its growth nd further inrese in temperture (38 o C) led to deth of lgl ells (Co verti et l., 2009). In nother study sientists hve found tht the optiml temperture for growing for most speies of lge is etween 20 o C to 30 o C (Konopk nd Brok, 1978) Effet of vrious ph levels on lgl growth: Alge when grown in different ph rnges show signifint effet of it. Tle 4 gives the results for effet of ph on lgl growth nd it is evident tht growth ws influened y djusting the ph t different rnges. The experiments were onduted with different ph rnges inluding 6.5, 7.0, 7.5, 8.0, 8.5, 9.0. It ws oserved tht t minimum ph level tested during the present work (6.5), Spirogyr sp. nd Oedogonium sp. were oserved with low growth in terms of fresh weight with 3.23±0.022 g nd 3.16±0.021 g fresh weight respetively fter week 4. They were grown est nd to the mximum t ph 7.5 nd 8.0. Spirogyr sp. ws hving lmost the sme vlues for fresh weight i.e. 4.89±0.091 g nd 4.81±0.052 g t ph 7.5 nd 8.0 respetively (Tle 3 ). The sme effet ws oserved for Oedogonium sp. when their fresh weights were mesured nd they were found to e 4.79±0.021 g nd 4.70±0.021 g t ph 7.5 nd 8.0 respetively. At ph 9 growth ws deresed nd it ws mesured s 2.88±0.065 g for Spirogyr sp. nd 3.52±0.015 g for Oedogonium sp. (Tle 3). The findings of our work re in greement with those puliized y Zhu (2010) whose results depited tht lge grew est in medi djusted with n initil ph of 7.10 nd thus lge hve shown fstest growth when the ph ws ner 8. He lso onluded tht in growth medium with ph of 5.0 nd 9.0 lge did not show pprent growth, thus the desired ph for growing lge is 7-8. Similr results were found in nother study where mirolge ws le to grow t ph 7.5 nd ove 8 the ells showed deline in growth rte (Gri et l., 2000). 548

4 Munir et l., J. Anim. Plnt Si. 25(2):2015 It is lso reported tht ph is the third most importnt ftor fter temperture nd light, for growing lge (Bjhiy et l., 2010). Effet of light on lgl growth: Algl ultures were pled under different light onditions (diret sunlight, indiret sunlight light, fluoresent light nd under drk). No growth ws reorded for ny lgl speies under omplete drk onditions so it ws onluded tht light is n essentil ftor for lgl growth. Algl ultures kept in the room ner window showed growth to some extent with men vlue of 3.13±0.017 g for Spirogyr sp. s well s Oedogonium sp. But this ws less s ompred to fresh weights of lge when pled under diret sunlight. When lgl ultures were exposed to diret sunlight 4.30±0.044 fresh weight ws reorded oth for Spirogyr sp. nd 4.32±0.040 g for Oedogonium sp. Most suitle light ondition for fresh nd helthy growth of lgl iomss found during the present study ws floresent light (Tle 4 ). At ll the time intervls ultures pled under floresent light hd higher fresh weights. Hene highest iomss reorded during present work for Oedogonium sp. ws 3.98±0.020 g t week 4. And for Spirogyr sp. it ws 4.52±0.018 g. Thus it is evident from results tht light hs signifint effet on growth of lge (Tle 4). The growth of lge delines due to dmge in light pigments t high light intensity (Jnssen et l., 1999). Other importnt findings hve grphed out the importne of light intensity nd its effet on lgl growth. Their results hve onluded tht very high nd intense light n inhiit the growth of lge, lso the sun light sometimes n hrm the lgl ells ( Converti et l., Thus it is depited tht light exposure, intensity nd penetrtion re importnt ftor for lgl ultivtion (Shrm et l., 2008). Another work done y Hu et l., (2008) hve onluded tht lge when grown t vrious light intensities hve shown remrkle hnges in their growth. They pled lgl ultures in diret sunlight nd under fluoresent (rtifiil) light. They hve oserved tht lthough lge were le to grow nd undne in their iomss under sunlight ut fter few dys lge strted to deteriorte euse too muh of sunlight ws not fvorle for long survivl of lge. Although when oserved under rtifiil light, it ws found tht lgl iomss exhiited etter growth. Thus these results re in ordne with our proposed effet of light (Newsted, 2004). Effet of ertion on lgl growth: Uniellulr form of lge ws provided with onstnt ertion through pumps. This is n importnt ftor euse in the sene of ny ertion or mixing mens, the ells eme ded nd no more growth n e reorded. A omprison ws mde to see the effet of ertion y supplying on set of Chlorell sp. ultures with ertion pumps nd the other set ws left stti (Figure 5). Asorne ws mesured t 600 nm nd the inrese in ell numer ws oserved in terms of inresed sorne rte. At dy 1 the sorne ws mesured 0.31±0.003 with erting pump while 0.27±0.145 ws found when Chlorell sp. were grown without ertion. At dy 5 the growth inresed nd ell numer were found to e more y lulting the sorne s 0.70± Results were found to e signifint nd it ws oserved tht Chlorell sp. ells n only grow when they re provided with onstnt ertion. Severl reserhers hve reported the sme effet of ertion nd they sid tht it is highly reommended to provide lgl ulture with some mixing or ertion mehnism (Gri et l., 2005; Chen et l. 2006). Ip et l., (2004) used severl mens of ertion in their experiments nd pddle wheels were used in photoioretors nd thus lgl ulture were ontinuously mixed when wheel were rotted inside the ultures. Oil Extrtion: Bligh nd Dyer (1959) extrtion method ws performed to extrt the lipids from lge. All the three types of lge ( Spirogyr, Oedogonium nd Chlorell.) were extrted y using this method. The results for oil extrtion using Bligh nd Dyer method re shown in Figure 5. It is evident from the results tht Chlorell sp. sp. hs the highest lipid ontent followed Oedogonium sp. nd Spirogyr sp. Similr results were found y Hossin et l., (2008) nd showed tht iodiesel prodution ws found mximum in Oedogonium nd minimum in Spirogyr Bligh nd Dyer method ws studied y Woertz (2007) nd his results were in greement with those proposed y our findings nd the totl lipid ontent ws found to e 14% for Oedogonium while Chlorell ws hving higher lipid ontent nd ws given s 24%. In reent study oil extrtion hs een mde from Pkistni lgl speies nd reported highest lipid prodution in oedogonium. (Khol nd Ghzl, 2012). 549

5 Munir et l., J. Anim. Plnt Si. 25(2):2015 Fig. 1() Alge smple from Jillni Prk ()Alge smples from Jillni Prk Fig 2. Algl ultures pled ner window for light exposure ) Spirogyr sp. ) Oedogonium sp. )Chlorell sp. Fig.3 () Spirogyr sp., () Oedogonium sp., () Chlorell sp. 550

6 Munir et l., J. Anim. Plnt Si. 25(2):2015 Figure 4. Effet of Medium nd ertion on growth of Chlorell sp. Figure 5: Perentge yield otined from lgl isoltes y Bligh nd Dyer method Tle 1. Effet of medium on growth of Spirogyr sp. nd Oedogonim sp. Fresh weight (g) Growth medium Spirogyr sp. Oedogonim sp. Week1 Week2 Week3 Week4 Week1 Week2 Week3 Week4 Distilled Wter 0.5± ± ± ± ± ± ± ± BB medium 1.02± ± ± ± ± ± ± ± 0.02 BG medium 1.54± ± ± ± ± ± ± ± 0.02 Effet of medium (with 1 nd 8 df) * * * * * * * * Initil weight of eh smple ws 0.5 g Dt were reorded for 10 replites for eh experiment * Signifint t 5% proility level ording to on-wy ANOVA with df mentioned. Tle 2. Effet of temperture on growth of Spirogyr sp. nd Oedogonium sp. Fresh weight (g) Tempertur Spirogyr sp. Oedogonium sp. e Week1 Week2 Week3 Week4 Week1 Week2 Week3 Week4 16 o C 0.96± ± ± ± ± ± ± ± o C 1.20± o C 1.73± o C 1.51± o C 0.92±0.062 Effet of temp (with 4 nd 10 df) 2.00± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.069 * * * * * * * * 551

7 Munir et l., J. Anim. Plnt Si. 25(2):2015 Tle 3: Effet of ph on growth of Spirogyr sp. nd Oedogonium sp. Fresh weight (g) Spirogyr sp. Oedogonim sp. Week1 Week2 Week3 Week4 Week1 Week2 Week3 Week ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.015 Effet of ph (with 5 nd 12 df) * * * * * * * * Tle 4: Effet of light on growth of Spirogyr sp. nd Oedogonium sp Fresh weight (g) Spirogyr sp. Oedogonim sp. Week1 Week2 Week3 Week4 Week1 Week2 Week3 Week4 Diret 1.14± ± ± ± ± ± ± ±0.040 sunlight Indiret 0.95± ± ± ± ± ± ± ±0.017 sunlight Fluoresent 1.29± ± ± ± ± ± ± ±0.020 light Effet of light (with 2 nd 6 df) * * * * * * * * Dt were reorded for 10 replites for eh experiment * Signifint t 5% proility level ording to on-wy ANOVA with df mentioned. Tle 5. Effet of ertion on growth of Chlorell sp. Aertion onditions Asorne y Chlorell sp. sp. t 600 nm Dy 1 Dy 2 Dy 3 Dy 4 Dy 5 Aertion pump 0.31± ± ± ± ±0.009 Without pump 0.27± ± ± ± ±0.006 Effet of light (with 1 nd 4df) NS * * * * Dt were reorded for 10 replites for eh experiment * Signifint t 5% proility level ording to on-wy ANOVA with df mentioned. Conlusion: From the present work it n e suggested tht Spirogyr sp. nd Oedogonium sp. nd Chlorell sp. re ommon speies of Lhore. Eh of the lgl speies requires different set of growth onditions if ultured in the lortory. The omprison of oil ontents indite tht Chlorell sp. hs higher oil ontent s ompred to other lgl speies tested during the present work. REFERENCES Bjhiy, A.K., S.K. Mndotr, M.R. Suseel, K. Toppo, nd S. Rnde (2010). Algl Biodiesel: the next genertion iofuel for Indi. Asin. J. Exp. Biol. Si. 1(4): Bl, B.K. (2006). Studies on iodiesel from trnsformtion of vegetle oils for diesel engines. Energy Edu Si. Tehnol. 15: Bnsl, B.K., nd M.P. Shrm (2005). Prospets of iodiesel prodution from vegetle oils in Indi. Renew. Sustin. Energy. Rev. 9: Brsnti, L., nd P. Gultieri ( 2006). Alge iotehnology. In: Alge: Antomy, Biohemistry, nd Biotehnology CRC Press Tylor nd Frnis Group, Bo Rton. pp: Bligh, E nd W. Dyer (1959). A rpid method for the totl lipid extrtion nd purifition. Cn. J. Biohem. Physiol

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