Original Article. Areeg Abd Elhafiz 1, Afnan Abd Elhafiz 1, Suruchika S. Gaur 2, Noor Hamdany 1, Maryam Osman 1, T. V.

Transcription

1 Originl Artile Reent Reserh in Siene n Tehnology 2015, 7: oi: /rrst.2015.v Chlorell vulgris n Chlorell pyrenoios live ells pper to e promising sustinle iofertilizer to grow rie, lettue, uumer n eggplnt in the UAE soils Areeg A Elhfiz 1, Afnn A Elhfiz 1, Suruhik S. Gur 2, Noor Hmny 1, Mrym Osmn 1, T. V. Rjy Lkshmi 1 * 1 Stuents, College of Biotehnology, University of Moern Sienes, Dui, UAE, 2 Lortory Tehniin, College of Biotehnology, University of Moern Sienes, Dui, UAE Reeive: Aepte: Pulishe: *Aress for orresponene: T. V. Rjy Lkshmi, College of Biotehnology, University of Moern Sienes, Dui, UAE. E-mil: v.lkshmi@ums.e ABSTRACT The ulture onitions of Chlorell vulgris n Chlorell pyrenoios uner lortory n fiel onitions hve een stnrize. C. pyrenoios oul survive uner fiel onitions t 40 C in 1 L ultures ottles, wheres C. vulgris oul not survive t this temperture. When the slt tolerne of the two speies ws teste ginst three levels of slt, C. vulgris oul survive in 1.0% NCl with ensity of /ml ells while C. pyrenoios i not survive t this level of slt. The totl hlorophyll ontent (hlorophyll n hlorophyll ) of C. pyrenoios is higher (1780 mg/g fresh weight) thn tht of C. vulgris (12360 mg/g fresh weight) for the sme mss of ells. When hlorophyll rih, slt tolernt C. pyrenoios live ells ( /ml) were use s iofertilizer, rie seelings h enhne root system, uumer sees h quiker germintion, n 1 month ol seelings h green leves with 2 times higher hlorophyll ontent thn the respetive ontrol seelings. Enrihment of slt ffete soils with these green lge C. vulgris n C. pyrenoios my e possile option to grow these rops in the UAE. KEY WORDS: Biofertilizer, Chlorell pyrenoios, Chlorell vulgris, hemoytometer, moleulr mrkers, slt tolerne INTRODUCTION The UAE soils re infertile without humus n the high tempertures uring summer sesons o not promote miroorgnisms tht n enrih the soils. In ition to this onition, soils lso ontin high mount of slt, more thn 8000 prts million (8-16 S/m), whih is thret to griulture (Henzell, 2009). Efforts to grow vegetle rops in these soils re eing explore through ifferent wys, of whih enrihing the soils with iofertilizers is one metho. Supplementing the soils my meet the nutrient nee of the rop n llow the rop with etter yiel. Nitrogen or phosphorous fertilizers lso n e e to soil, however, iofertilizers re preferre to hemil fertilizers euse they o not use verse effets to soil or environment or the rops. Usully, nitrogen fixing teri n growth promoting teri, myorrhize, n free floting ferns re use s iofertilizers for ifferent rops. During the lst ee, hlorophyen memers were lso eing explore s iofertilizer s they re rih in rohyrtes, proteins, lipis, n growth hormones. Among the memers of Chlorophyt, Chlorell strins re onsiere s ompetent nites, whih promote plnt growth when use iniviully s in lettue (Fhee n A-El Ftth, 2008) or long with rhizoteri s in lovers (Rposo n De Moris, 2011). Speies of Chlorell hve vrie tolerne to slt n lso to rnge of temperture from 25 C to 40 C (Snkr n Rmsurmnin, 2012; Swnt et l., 2014). In this stuy, n ttempt is me to ulture Chlorell vulgris n C. pyrenoios uner lortory n fiel onitions with the use of C. pyrenoios s iofertilizer to grow vegetle rops viz., lettue, rie, eggplnt n uumer in slt ffete soils. C. vulgris ws hosen for 14 Reent Reserh in Siene n Tehnology Vol

2 the present stuy euse it ontins high mounts of minerls, protein, vitmins n rotenois, n is lso reporte to survive in ultures with 5% petrol/kerosene n up to 15 ys in 20% petrol/kerosene (Agrwl et l., 2007). C. pyrenoios ws etter thn fytozyme to enhne the yiel of soyen (Duey n Duey, 2010). As it is extremely iffiult to ientify the phytoplnkton through light mirosopi stuy, we use speifi 18S DNA primer to uthentite the genus (Swnt et l., 2014) n ttempte to ientify the strin with mirostellite mrkers (Jo et l., 2014). The present work inlues: Authentition of Chlorell Stnriztion of ulture onitions for the two speies n Use of live ells of C. pyrenoios s iofertilizer, forlettue, rie, eggplnt, n uumer. To the est of our knowlege, this is the first report on the use of Chlorell live ells s iofertilizer to grow these rops in slt ffete, infertile UAE soils. MATERIALS AND METHODS Soure of Chlorell Cells C. vulgris ulture ws otine from Shlimr Biotehnology LLC, lote in Dui, UAE. C. pyrenoios ells were retrieve from the tlets of Sun Chlorell A, USA. One 200 mg tlet ws issolve in 1 L of wter; the ottles were mintine in the lortory t 22 ± 1 C or in the fiel with 14/10 h light/rk onitions. The temperture of the fiel vrie etween 32 C n 40 C uring Ferury to My The ulture ws heke perioilly every 3 ys one for the retrievl of ells. Pre Inoulum n Mintenne of Mother Culture Two speies of Chlorell ells were grown in wter in the lortory n lso in the fiel onitions. The ells were suspene in fresh wter one week. The ells were lso sreene ginst three onentrtions of slt viz., 0.01%, 0.1%, n 1.0%. A smll volume of 20 µl of pre-inoulum ws heke uner light mirosope for the presene of single ells, uto-sporngi, n kinetes efore it ws use s pre-inoulum to inoulte the ifferent ulture mei. Mei Preprtion n Culture of Chlorell Cells in Bol s Bsl n Chu 10 Mei Bol s sl meium (Bishoff n Bol, 1963) n Chu 10 meium (Chu et l. 1942), were the most ommon use nutrient mei for ulture of green lgl ells. Bol s n Chu 10 mei, eh supplemente with 0.0, n/or 0.01%, n/or 0.1%, n/or 1.0% NCl ws use to ulture the two speies of Chlorell ells in the present stuy. The ph of the meium ws juste to 7.0 with 1 N NOH efore the meium ws utolve. Aout 10% of the 1-week ol Chlorell ulture ws use s pre-inoulum. For instne, 10 ml of the ulture ws inoulte with 1 ml of pre-inoulum. The ells were then hrveste y spinning the ulture t rpm for 30 s, the superntnt is isre, n the pellet is wshe twie using the meium, the loose pellet fter the 2 n wsh ws use s pre-inoulum, to ulture the mei. The ulture vils n ottles were mintine with one set in the growth hmer t 22 ± 1 C with light intensity 150 mol/m 2 /S, photoperio 14 h/10 h light/rk; while the other set of ultures were mintine in the fiel onitions t C, with 14 h/10 h light/rk yle. The urtion of light/rk yle in the growth hmer ws so juste to mth with the photoperio of the fiel onitions. The experiment is repete 3 times, ell ounts men n stnr evitions were me for eh replite. Light Mirosopy n Determintion of Cell Density on Hemoytometer For light mirosopy, the ells were mounte on flt glss slies n exmine uner light mirosope (XSZ-106T, NO ). Photogrphs were tken uner 40 n oil immersion 100 ojetive lens. The ell ensity for eh of the mei ws estimte using hemoytometer (Neuuer-improve). The formul use for ounting the ell etermintions ws: Numer of ells in the originl smple = Numer of ells ounte 10 ( Proportion of hmer ounte ) 4 ( Volume of squres ounte) Culture of Chlorell Cells in Soli Mei C. vulgris n C. pyrenoios ells were hrveste from 1 ml ultures y spinning the ultures t rpm for 30 s; 100 µl of the loose pellet ws streke on Bol s sl or Chu 10 meium eh supplemente with 0.1% NCl. The ulture pltes were then mintine in the growth hmer t 14 h/10 h light/rk t 22 ± 1 C temperture. After the evelopment of olonies on the soli mei, to otin pure ultures, the single olonies from eh plte were ulture in 10 ml liqui ultures for week. Reent Reserh in Siene n Tehnology Vol

3 Verifition of Chlorell Cells with 18S rdna Primer n Strin Ientifition with Speifi Moleulr Mrkers DNA extrtion DNA ws isolte from C. vulgris n C. pyrenoios ells using Auvis io DNA isoltion kit (Norgen, Cn) following the mnufturer s instrutions. The isolte DNA smples were eletrophorese on 2% grose gel t 100 V, the gels were then viewe n photogrphe uner UV illumintion. Polymerse Chin Retion (PCR) Amplifition PCR retion ws performe in 20 µl retion mixture ontining 1 µl (15-25 ng) of extrte genomi DNA, 10 µl 2 PCR mster mix from Auvis Bio, 1 µl (10 µm) of eh primer set n 7 µl of sterile istille wter)using therml yler progrmme for 35 yles (Veriti PCR Mhine, Applie Bio systems, USA). A smple without templte DNA ws inlue in the retion s negtive ontrol. The PCR mplifition onitions were 95 C for 5 min, followe y 35 yles t 95 C for 30 s, 55 C for 30 s, n 72 C for 30 s with finl extension t 72 C for 7 min. The PCR prouts were eletrophorese on 1% grose gel t 100 V, the gels were viewe n photogrphe uner UV illumintion (Tle 1). Estimtion of Chlorophyll Content in C. vulgris n C. pyrenoios Aout 500 mg of C. vulgris n of C. pyrenoios ells were hrveste from the respetive ultures, inute in 80% etone for 3 h n/or 24 h in the rk. Totl hlorophyll ontent of the ells ws estimte y mesuring the sorption t 652 nm (Gillmrtin n Bowler, 2002) on spetrophotometer (Digitl UV Plus RS, UV 2502 moel from Lome) implementing the following the formul (Jiwn, 1990). C = Chlorophyll ontent µg/ml C = A 652/36 etone extrts C = (1000 A 652 nm/36) 500 mg/g fresh weight of lef tissue C. pyrenoios s Bio-fertilizer See germintion in Petri ishes Sees of uumer, eggplnt, rie n lettue were inute in eh Petri plte on Whtmn filter pper, n wtere with 2 ml of wter or 2 ml of C. vulgris solution ontining live ells /ml n/or C. pyrenoios of ell ensity /ml twie y. The experiments were one in three replites of out 20 sees per eh replite. The perentof see germintion ws lulte for eh plnt. See germintion in potte soils As the hlorophyll ontent of C. pyrenoios ws higher, only C. pyrenoios ws use s iofertilizer for potte plnts. The sees were sown in potte soil n were wtere with wter or C. pyrenoios solution ( ells). The seelings were grown up to 4 weeks; the hlorophyll ontent ws estimte from the leves of the ontrol n the trete plnts. The protool for hlorophyll estimtion ws similr to tht rrie for Chlorell ells. For eh plnt 500 mg of leves ws weighe n inute in 10 ml of 80% etone in rk onitions for perio of 3 h n 24 h, from whih the totl hlorophyll ontent ws estimte. RESULTS Pre Inoulum n Mintenne of Mother Culture C. vulgris ells oul grow in wter n lso in wter ontining NCl uner the lortory 22 ± 1 onitions (Tle 2). Light mirosopi oservtion revele the ells whih re green, spheril eh with hloroplst, nuleus n olorless ell wll. 1-week ol ultures h single ells n kinetes without nyutosporngi. 1-month ol mother ulture h 63.6% of single ells n 36% of kinetes (Tle 2). Presene of kinetes inites unfvorle onitions; proly the wter ws eplete of nutrients resulting in high per ent (36%) of kinetes. Tle 1: List of PCR primers n their sequenes use in this stuy Primer Sequene from 5 to 3 18S rdna mchl 001 mchl 002 mchl 005 mchl 011 mchl 0012 PCR: Polymerse hin retion F: CTG CGA ATG GCT CAT TAA ATC R: AAG GCC AGG GAC GTA ATC AA F: CCT ATT GCT CTA TGT TAA CAT ATG R: GTT TTG AAT TTT TCC CCA TTG CTG F: ACA GGC CAG TCA ATT TAT TT R: CAC TAC ATC GTC TAT TTG ACA TTG AG F: CAA GCC AAT TTT ATT TAA AAT C R: AGG TTC ACC TCT TCG CCT AA F: CAG TAT AGA GTA CAC GAT TTT CC R: GAG CGT GTA ATT GTT ATA ACT TC F: CGC TAT AGT CAT AGC GTC GTG ACG R: CTT GAA AGC TTC ATG AGG AGT GCC Tle 2: Slinity tolerne of Chlorell vulgris ells in wter fter 1 month of ulture Cell ulture in % of single ells % of kinetes Totl numer of ells/ml Wter Wter+0.01% NCl Wter+0.1% NCl Wter+1.0% NCl Reent Reserh in Siene n Tehnology Vol

4 The perent of kinetes ws reue in the 0.01% n lso in 0.1% of NCl onentrtions initing tht kinetes oul isperse the spores resulting in inrese single ells n erese in per ent of kinetes. However, in 1% NCl, high per ent of kinetes n erese in totl ell numers revel unsuitle ulture onitions. The ells in the fiel onitions i not survive in wter with or without NCl, inferring their sensitivity to high temperture 35 C. Retrievl of C. pyrenoios Cells C. pyrenoios ells oul e retrieve from the tlet in wter uner the fiel onitions in 4 weeks (Figure 1). The retrieve ells were ulture in Bol s sl meium n Chu 10 meium eh ontining ifferent onentrtions of slt. Culture of C. vulgris n C. pyrenoios ells in Bol s Bsl n Chu 10 Mei 1-week ol C. vulgris ells grew etter in Chu 10 meium with the highest ell numer (Figure 2 n ). A few tetrsporngi n few uto-sporngi n 4-ellesporngi were lso oserve in the mei (Figure 1 n ). Sporngi with 4 spores (ells) were more frequent thn the multispore uto-sporngi. Although, C. vulgris ell numers were higher in Bol + 1.0% NCl meium (Figure 2), utosporngi were not etete in ny of the three replites fter 1-week of ulturing. This inites tht the initil sporngi must hve relese the spores n new sporngi re not forme t this slt onentrtion. The ells i not survive when the ultures were in the fiel t C. C. pyrenoios ells grew etter in Bol s sl meium, tolerting slinity up to 0.01% only in the lortory onitions (Figure 2 n ). Uner fiel onitions, the ells survive in oth the mei n lso in wter, only in 1 L ulture ottles without slt. The ells i not survive in smll-sle ultures (10 ml n 100 ml), proly euse the smll volume of the ultures get hete up to 40 C, with the outsie environment whih must e etrimentl to ell survivl (Figure 3, n f). After the C. pyrenoios ells were retrieve from the tlet, they i not retin their olonil form, ut only single ells, ys n tetrs n uto sporngi were oserve in the wter n mei ultures (Figure 1 n e). Culture of Chlorell Cells in Soli Mei C. vulgris olonies evelope in 2 ys in the soli mei uner lortory onitions (Figure 3) while C. pyrenoios olonies evelope in 2-3 weeks (Figure 3e). C. pyrenoios ultures when oserve uner the light mirosope, the ultures of seeme to e mintine solonies on soli ulture mei (Figure 3). The growth of C. vulgris in soli mei is fster thn the growth of C. pyrenoios uner similr ulture onitions. DNA Isoltion n PCR Verifition of Chlorell Genomi DNA of the two speies of Chlorell were isolte n mplifie with the 18S rdna n speifi mirostellite primer (4), of the prout size of 175 ps (Figure 4-). Estimtion of Chlorophyll Content in C. vulgris n C. pyrenoios The estimte Chlorophyll ontent of 500 mg of Chlorell ells inute in 80% etone for 3 h smples ws lesser ompre to 24 h inution smples in oth the speies. The hlorophyll estimtes were me from 24 h inute smples. The pigment onentrtion (Chlorophyll n ) Tle 3: See germintion perent in ontrol n trete plnts of eggplnt, oriner, lettue n uumer (Petri pltes re t 22 C, 14 h/10 light/rk) Plnt Numer of sees inute Wter e Figure 1: Chlorell ells in ulture mei. ( n ) Chlorell vulgris ells in Chu 10 ulture meium, mgnifition 100 µ () single multielle uto-sporngium, mgnifition 10 µ, (-e) Chlorell pyrenoios olonil ells retrieve from the tlet issolve in Bol s sl meium fter 20 ys of ulture in 1 L ulture uner fiel onitions. () 2 Colonies, iniviul ells n e seen in olony 1 (rrow) mgnifition 10 µ. ( n e) Dys () n tetrs (e) mgnifition 10 µ % of see germintion C. vulgris/ Wter C. pyrenoios wter C. vulgris C. pyrenoios Lettue Cuumer Egg plnt Rie C. vulgris: Chlorell vulgris, C. pyrenoios: Chlorell pyrenoios Reent Reserh in Siene n Tehnology Vol

5 Figure 2: (-) Cell ensities (men n stnr evition) of 1 week ol ultures of Chlorell vulgris n Chlorell pyrenoios in Bol s sl n Chu 10 mei ± NCl. (Cultures were t 22 C, 14 h/10 h light/rk) (Figure 6 n ). The germinte seelings of lettue survive for week when they were not wtere with Chlorell solution while the ontrol seelings withere in 3-4 ys (Figures 6 n ). Three to 4 weeks ol seelings of uumer n egg plnts in pots h greener n igger leves (Figure 7- n e); uumer seelings were isese resistnt (Figure 7) when wtere with hlorell solutions. e Figure 3: Liqui n soli ultures of Chlorell vulgris n Chlorell pyrenoios. (, n f) 10 ml n 1 L (f) ultures uner fiel onitions. () Colonies of C. pyrenoios from ulture pltes, mgnifition 100 µ. () C. vulgris olonies in Chu 10 pltes 2 ys fter ulture, t 22 ± 1 C with 14/10 h light/rk. (e) C. pyrenoios olonies in Bol s sl meium 3 weeks fter ulture, t 22 ± 1 C with 14/10 h light/rk of C. vulgris is lesser thn tht of C. pyrenoios for sme mss of ells (500 mg) in ll three replites (Figure 5). See Germintion in Petri Dishes n Pots See germintion in the trete smples is either the sme or higher thn in the ontrolsin ll rops exept in lettue (Tle 3). Trete 2 weeks ol seelings of rie in the petri ishes were helthier with enhne root system f Estimtion of Chlorophyll Content in 1-month Ol Seelings The estimte hlorophyll ontent from 3 h inution smples were very low (875 mg/g vs mg/g fresh weight) heneforth, the experiments were ontinue with 24 h inution smples. The Chlorophyll n ontent ws higher in the trete lef smples thn in the ontrols in ll the rops exept in rie (5), the ifferene is more evient in uumer seelings (Figures 5 n 7). Although the totl hlorophyll ontent of rie seelings ws lesser in the trete rie seelings thn in their ontrols, the enhne root growth reflets the effet of C. pyrenoios s the iofertilizer. Eh smple h 20 sees, the experiment is one in 3 replites n the t for the 3 replites is poole. 18 Reent Reserh in Siene n Tehnology Vol

6 Figure 4: Agrose gels (1%) showing genomi DNA n polymerse hin retion (PCR) prouts. () Genomi DNA of C. vulgris n Chlorell pyrenoios. () PCR mplifition prouts with 18S rdna primer. () Lne 4 n 9 showing PCR prouts with primer set 4 Figure 5: Estimtion of Chlorophyll n () Chlorophyll ontent in Chlorell vulgris n Chlorell pyrenoios. () Chlorophyll ontent in ontrol n trete uumer, eggplnt, oriner n lettuex Figure 6: See germintion in Petri pltes (t 22 ± 1 C) n in pots (35 40 C), oth t 14 h/10 h light/rk () 4 ys ol seelings of lettue t the eginning of wter stress ( n ) 2 weeks ol rie seelings (), trete seelings with enhne root system (). () Wilte (ontrol) n helthy (trete) seelings fter 7 ys of wter stress t 22 ± 1 C DISCUSSION C. vulgris ells grew in oth the mei - Bol s sl meium n Chu 10 meium uner the lortory onitions. Chu 10 meium is reporte to e the most suitle meium for some strins of C. vulgris (Shrm et l., 2011) n the present strin is in orne with the report. e Figure 7: 3 to 4 weeks ol seelings in pots irrigte with iofertilizer n/or wter uner fiel onitions: (t C, n 14 h/10 h light/ rk). ( n ) 3 weeks ol seelings of lettue ontrol () n trete (). () 3 weeks ol uumer seelings, with greener leves in trete (right sie). () 3 week ol uumer seelings, trete re isese resistnt. (e) 4 weeks ol eggplnt seelings with igger leves in trete Although, the tolerne of C. vulgris to 1.0% slt ws etter in Bol s sl meium, no uto-sporngi oul e etete in ny of the three replites, in 1-week ol ultures, initing tht they require longer urtion to reproue or fil to o so uner the slt stress. The mirolgl growth is lso ontrolle y the light n temperture esies the nutrient mei, C. vulgris ells grew etter uner nturl light t C thn t C (Shrm et l., 2012). The present strin of C. vulgris lso is temperture sensitive, s they i not Reent Reserh in Siene n Tehnology Vol

7 survive t 35 C. Nutrient mei, temperture n light intensity re reporte to hve n effet on the phenotype of the mirolge (Soylu n Gonulol, 2012), whih oul e possile reson for the loss of olonil stte of C. pyrenoios, in the present stuy. DNA Isoltion n PCR Verifition of Chlorell PCR mplifition of the genomi DNA of the two speies with 18SrDNA primers onforms tht the ells re Chlorell. However, it nnot e onlue t this stge tht C. pyrenoiosis strin of C. vulgris n not ifferent speies unless the PCR prouts re sequene, whih is not the im of the present work. So lso, the mplifition prout of primer 1 ws lesser thn the expete 154 ps size, hene it nnot e onlue t this stge whether it h less numer of repets (TTA) or it ws nonrnom mplifition prout of PCR. See Germintion in Petri Dishes n Pots The trete seelings of the four rops h positive effet of the iofertilizer whih were with greener leves n were helthy. Similr response of greter numer of leves with igger surfe re ws oserve in soyen seelings irrigte with C. pyrenoios, n our present stuy is similr to this erlier report (Duey n Duey 2010). Although infetion ws not inue in the uumer seelings, the ontrol seelings h infetions while the trete seelings were helthy. Resistne to isese mong rops irrigte with Chlorell ells is not new phenomenon s ws reporte in Grpe vines (Bilev, 2012). Lettue seelings were positively ffetewith inrese fresh n ry weight when wtere with C. vulgris (Fhee n A-El Ftth, 2008). In the present stuy, lettue seelings wtere with C. pyrenoios solution uner lortory onitions oul withstn wter efiit up to 7 ys. Tolerne of wter stresses with Chlorell solution is eing reporte for the 1 st time. Estimtion of Chlorophyll Content in 1-month Ol Seelings The hlorophyll ontent of the trete seelings is higher in ll rops exept in rie seelings; however, the rie seelings h enhne root growth initing iofertilizer effet. Enhne root system with rhizoteri pplition in rie fiels is n estlishe ft, n reently in omintion with mirolge is eing experimente; positive effet on plnt growth n root system in reporte in lover plntlets (Rposo et l., 2005). Although the yiel of the four rops hs not een otine, se on the seeling growth, hlorophyll ontent n rought tolerne, irrigtion of seelings with C. pyrenoios seems to e promising tool to grow these vegetle rops in the UAE soils. Ientifition of the strin of C. vulgris is mntory to reommen it s iofertilizer whih is in the proess. Further pplition of Chlorell speies in hyroponis s nutrient supplement will e interesting to explore. CONCLUSIONS To the est of our knowlege, this is the first report on the use of live Chlorell ells s iofertilizer to grow rops in the UAE soils. The stuy opens the possiility of utilizing C. pyrenoios live ells s potentil soure of iofertilizer without using environmentl pollution. Further stuies on evlution of yiel n tste of these vegetle rops re impertive to onlue its use n to reommen to fruit rops. ACKNOWLEDGMENT The uthors thnk UMS for proviing the filities n supporting this work. They re lso thnkful to Dr. Algppn Kumrn for helping the sttistil nlysis. REFERENCES Agrwl SC, Mnish. Growth, survivl n reproution in Chlorell vulgris n C. vriegt with respet to ulture ge n uner ifferent hemil ftors. Foli Miroiol (Prh) 2007;52: Bilev T. Influene of green Alge Chlorell vulgris on infeste Xiphinem inex grpe seelings. J Erth Si Clim Chnge 2013;4:2. Bishoff HW, Bol HC. Physiologil stuies IV. Some Soil Alge from Enhnte Rok n Relte Algl Speies. Vol Texs: University of Texs Pulitions; p Chu SP. The influene of the minerl omposition of the meium on the growth of plnktoni lge. Prt I. Methos n ulture mei. J. Eol 1942;30: Duey A, Duey DK Evlution of ost effetive orgni fertilizer p Aville from: Orgprints.org. [Lst esse August, 2015]. Fhee FA, A-El Ftth Z. Effet of Chlorell vulgris s iofertilizer on growth prmeters n metoli spets oflettue plnt. J Agri So Si 2008;4: Gillmrtin P, Bowler C. Chlorophyll etermintion s mrker for hloroplsts. Moleulr Plnt Biology: A Prtil Approh. Vol. 2. Oxfor University Press; p Henzell J. Soil smples inite ln egrtion. In: UAE Ntionl; Aville from: e/news/ue-news/environment/soil-smples-initeln-egrtion. 20 Reent Reserh in Siene n Tehnology Vol

8 Jiwn PP. Instrumenttion for stuying vegettion nopies for remote sening in optil n therml infrre regions. Remote Sens Rev 1990;5: Jo BH, Lee CS, Song HR, Lee HG, Oh HM. Development of novel mirostellite mrkers for strin-speifi ientifition of Chlorell vulgris. J Miroiol Biotehnol 2014;24: Rposo MF, Oliveir SA, Moris RM. Evlution of soil fertiliztion with enpsultes of Chlorell vulgris n C. vulgris/pseuomons puti Consortium for the Growth of Meow Clover Plntlets; Rposo MF, De Moris RM. Chlorell vulgris s Soil menment: Influene of enpsultion n enrihment with rhizoteri. Int J Agri Biol 2011;13: Snkr M, Rmsurmnin V. Biomss proution of ommeril lge Chlorell vulgris on ifferent ulture mei. E J Life Si 2012;1: Swnt SS, Joshi AA, Bhgwt A, Kelkr-Mne V. Tpping the ntioxint potentil of novel isolte- Chlorell emersonii. Worl J Phrm Res 2014;3: Shrm R, Singh GP, Shrm VK. Comprison of ifferent mei formultions on growth, morphology n hlorophyll ontent of green lg, Chlorell vulgris. IIJBS 2011;2:B Shrm R, Singh GP, Shrm VK. Effets of ulture onitions on growth n iohemil profile of Chlorell vulgris. J Plnt Pthol Miro 2012;3:1-6. Soylu EN, Gonulol A. Morphologil n 18S rrna nlysis of oi green lge isolte from lkes of Kizihrmk Delt. Turk J Biol 2012;36: Reent Reserh in Siene n Tehnology Vol