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Iranica Journal of Energy and Environment 6(4): 334-339, 2015Iranica Journal of Energy & EnvironmentJournal Homepage: www.ijee.netIJEE an official peer review journal of Babol Noshirvani University of Technology, ISSN:2079-2115Cattle urine increases lipid content in Chlorella pyrenoidosa: A low cost medium forbioenergy applicationN. Sharma, M. P. Rai*Amity Institute of Biotechnology (J-3 block), Amity University Uttar Pradesh, Sector-125, Noida, IndiaPAPER INFOPaper history:Received 30 July 2015Accepted in revised form 6 September 2015Keywords:Chlorella pyrenoidosaCattle wasteLipid contentFatty acid methyl estersA B S T R A C TMedia requirement for microalgae cultivation adds most to the cost of biodiesel production atcommercial scale. The present work aims to study the growth of green algae Chlorella pyrenoidosaunder fogg s medium and modified fogg s medium by replacing KNO 3 with urea at differentconcentrations 0.15, 0.20 and 0.25% (w/v). To reduce the cost of urea, cow urine (CU) was utilizedto grow the Chlorella sp. in different volume fractions as 5, 7.5, 10, and 12.5 % (v/v). Biomassproduction in 7.5% cow urine was achieved 1.93 g l-1 that is almost double in comparison to normalFogg’s medium (0.82 g l-1). Cellular biochemical components such as lipid, protein and carbohydrateare determined quantitatively. The lipid content is found to be 32.7 % in 10 % of cow urine and 22.7% in 7.5 % cow urine that is much higher than the Chlorella sp. grown under Fogg s media (7%).The protein content is enhanced to 50.17% and carbohydrate reduced by half in comparison tonormal fogg’s medium cells. The extracted lipid is converted into fatty acid methyl esters (FAME)and characterized by GC-MS. The FAME produced from cow urine grown cells showed suitablecomposition to prove its application as biofuel.doi: esel from microalgae is a new promising industryfor sustainable energy source because of their highphotosynthetic efficiency and ability to grow in extremeenvironmental conditions [1]. The ability of algae tosurvive under different environmental conditions, to alarge extent is due to changing pattern of cellular lipidsas well as by modifying lipid pathways [2]. Researchersare focused in developing new and cheap technologiesto produce biodiesel from micro algae not only due tohigh lipid productivity but also lack of competition withfood, water and land [3]. By adopting integrativeapproach, a simple solution can be designed for largescale production of microalgal biodiesel.Algae has shown huge growth potential undernutrient deficient conditions, which makes it the rolemodel for studying its molecular pathways forenhancing desired lipids. Recently conductedexperimentation reveals that various physiochemicalstress conditions can induce varying expression patterns* Corresponding author: Monika Prakash RaiE-mail: mprai@amity.edu; monika1778@gmail.com, Phone: 91-120 4392721,mobile: 91-9313887818for lipids [4]. Literature shows improvement inmicroalgae biomass and lipid production by modifyingvarious nutrient conditions such as nitrogen [5, 6],carbon source2 [7]; salinity and iron content of themedium also affect algae growth [8]. Extensive work isdone on production of biodiesel from algae but the fuelis still costlier than normal diesel with the price of US 1.25/lb and US 0.43/lb, respectively, which obstructslarge-scale applications of algae biofuel [9]. Mediacomposition requires the expensive operation cost andtherefore limits its availability at larger scale. There is aneed to use cost effective media for growth of algae forbioenergy application.The present study focused on the cultivation ofChlorella pyrenoidosa using an alternative low costnitrogen source that is a waste and which can beadvantageous for commercial production of biodiesel.Cattle urine is a rich source of nitrogen and othermicroelements; total N ranging from 6.8 to 21.6 g l 1, ofwhich an average of 69% is present in the form of urea[8]. Among the organic nitrogen sources, urea is 2010,Please cite this article as: N. Sharma, M. P. Rai, 2015. Cattle urine increases lipid content in Chlorella pyrenoidosa: A low cost medium forbioenergy application, Iranica Journal of Energy and Environment 6 (4): 334-339.

Iranica Journal of Energy and Environment 6(3): 334-339, 2015the best nitrogen source for culturing Chlorella sp. [10,11]. We have chosen Chlorella pyrenoidosa for ourstudy, as the species has potential to grow under varietyof environmental conditions with high lipidaccumulation [12, 13]. A comparative analysis is donefor growth and biochemical composition of theChlorella sp. under cow urine (CU) medium and normalFogg s medium for its biofuel application.for 24 h at room temperature to dissolve the lipidsproperly. The mixture was centrifuged at 3000 rpm for10 min. Supernatant was separated 2 ml of chloroformwas again added to the pellets and shaken properly. Itwas again centrifuged at 3000rpm for 5 mins andsupernatant was separated. After adding 2 ml of 1% KClto the supernatant separate layers will be formed. Lowerlayer will be pipette out and weighed.Comparison of media costIn our present study, cow urine is used for cultivation ofChlorella pyrenoidosa. The cost of 10 % CU medium isestimated approximately 0.025 l-1, which is almost halfof the price of Fogg’s medium that costs approximately 0.04 l-1.ProteinThe crude protein was determined by Lowry method bytaking 0.5 ml of algal culture [15]. The absorbance ofthe sample was checked and the concentration wasdetermined using standard curve.CarbohydrateThe content of carbohydrate is estimated by themodified method of 3, 5- Dinitrosalicylic acidcolorimetry using 100 mg of dry algal powder [16]. Thecarbohydrate content was estimated using DNS reagentand optical density of the sample was determinedagainst the blank at 540 nm in a UV-visiblespectrophotometer.Lipid Content (%) wt. of lipid (g) 100/ wt. of culture(g)Total Protein Content wt. of protein (from BSA curve)X 100/ dry cell mass (g)Carbohydrate Content (%) wt. of carbohydrate (fromGlucose standard curve) X 100/ dry cell mass (g)MATERIALS AND METHODSMicroalgae and culture conditionsFresh water green alga Chlorella pyrenoidosa wasgrown in 1000 ml Erlenmeyer flasks containing 500 mlFogg’s medium which contained (g l-1) KNO3, 0.2;MgSO4.7H2O , 0.2; CaCl2.2H2O, 0.1; K2HPO4, 0.2;Na2EDTA, 0.0745 and 1.0 ml of microelement solutionconsisting of H3BO3 2.86; MnCl2.4H2O O0.0494;FeSO4.7H2O0.0557;CuSO4.5H2O 0.079 pH adjusted to 7.2. The algaegrowth was also conducted in modified Fogg’s mediumby replacing nitrate with urea. The effect of urea on cellgrowth was investigated by varying the concentrationbetween 0-0.25% (w/v). Chlorella sp was grown in cowurine medium (CU) at different concentrations ranging5, 7.5, 10, 12.5 and 15 v/v % without adding anynutrient. The media were sterilized prior to inoculatingwith exponential phase fresh cells. The cultures weregrown in laboratory conditions for 12 days under 24 hfluorescent illuminations (40 watt, white light) at 28 C.Conversion of algal lipid to FAMEThe lipid extract was esterified under acidic conditionusing standard method [12]. The dried oil (300 mg) wasrecovered with chloroform and 6 ml of NaOH (0.5 moll-1) in methanol was added. The mixture was then heatedunder reflux for 15 mins. After that, 18 ml oftransesterification reagent (prepared from 2g ammoniumchloride, 60 ml of methanol and 3 ml of sulphuric acid)was added and heated under reflux for another 15 minsand was subsequently transferred to the separatingfunnel. Separation of biodiesel was done using hexaneand distilled water. A clear yellowish layer is recoveredin the organic layer containing the FAMEs (biodiesel).After 2-3 times washing of biodiesel with water, organiclayer was collected and dried in rotary evaporator. Themethyl esters were then solubilised in hexane for gaschromatography- mass spectroscopy (GC-MS) analysis.Cell growth analysisThe growth of algae and biomass concentration wasmonitored by optical density measurement at 660 nmusing UV/visible spectrophotometer (Shimadzu UV1650). Cells were concentrated by centrifugation(Eppendorf R 5810), washed with de-ionized water anddried (60 C) to determine dry weight (expressed as g l 1).Characterization of FAME by GC-MSFatty acid composition was determined using GC-MS.The amount of sample injected was 1µl. Fatty acidmethyl esters (FAMEs) were identified by comparisonof the retention times with those of the standard(Supelco TM 37 component FAME mix, SigmaAldrich Co.)Quantification of biochemical compositionLipidThe lipid was extracted through Bligh and Dyer method[14]. A mixture of 2ml methanol and 1 ml chloroformwas made and added to 1 g algal biomass. It was kept335

Iranica Journal of Energy and Environment 6(3): 334-339, 2015RESULT AND DISCUSSION2.5Biomass and lipid accumulation of Chlorella sp.under different growth mediumThe growth curve of Chlorella pyrenoidosa was plottedunder different media formulations i.e. Fogg s mediaand cow urine supplemented medium (CU). Theautotrophic cells showed higher cell growth and latestationary phase in cow urine as compared to Fogg smedia. The growth of Chlorella sp. under 10 % cowurine medium reached 1.811g l-1 while it is only 0.768 gl-1 under Fogg s medium on 10th day after inoculation.The growth of Chlorella sp is found faster in cow urinemedium as it reached its maxima within 10 days ofinoculation. As shown in Figure 1, lag period of thecells grown under CU medium is shorter compared tothe cells grown under normal Fogg’s medium that alsocontribute to higher cell growth.Biomass (g/l)205%DCW (g/L)0.504681012.50%The highest growth was obtained in 7.5 and 10%concentration of cow urine medium among differentconcentration applied. The lipid production wascompared in cells kept in these two concentrations ofcow urine and found tremendous increase than normalfogg s medium cells. The lipid production was recorded32.7% in 10% CU medium, 22.7% in 7.5% CU mediumand 7% in normal fogg’s medium cells (Figure 3).Biomass production is slightly higher and lipid is lowerunder 7.5% CU in comparison to cells grown under10% CU medium. Increased lipid content is an attractivefinding regarding bio-diesel application; henceconcentration of cow urine was further optimized to getmaximum lipid content in cells.This is the first study on use of cow urine for thegrowth and lipid production of Chlorella pyrenoidosa.The cost of cultivation medium is very importantcomponent in capital cost for biofuel production; usingcow urine as growth of algae will definitely decrease thetotal production cost.Control210%Figure 2. Biomass production of microalga in differentconcentrations of cow urinecowurine07.50%Concentration of CU medium2110.52.51.51.512DaysFigure 1. Growth of Chlorella sp. in Fogg s medium and 10%cow urine mediumThe growth of Chlorella sp was optimized by varyingthe concentration of cow urine (5, 7.5, 10 and 12.50%).The dry cell weight (DCW) of algae was obtained 0.34g l-1 in 5%, 1.93 g l-1 in 7.5%, 1.80 g l-1 in 10% and0.60 g l-1 in 12.50 % of cow urine supplementedmedium (Figure 2). The cell growth in Fogg’s mediumwas recorded as 0.82 g l-1 within the same time interval.Cow urine is not a toxic waste material; possess 95% ofwater, 2.5% nitrogen sources, and the remaining 2.5%mixture of glucose, minerals, salts, hormones andenzymes [17]. Antimicrobial and germicidal propertiesof cow urine (gomutra) are due to the presence of urea(strong effect), creatinine, swarn kshar (aurumhydroxide), carbolic acid, other phenols, calcium andmanganese [18]. The nitrogen content of 7.5 and 10%CU medium are found quite suitable for growth ofChlorella pyrenoidosa.45%Lipid Content (%)40%35%30%25%20%15%10%5%0%7% concentration10% on lipidFigure 3. Effect ofControlcow urine (CU)content of Chlorella pyrenoidosa CU medium (%)336

Iranica Journal of Energy and Environment 6(3): 334-339, 2015To verify the component present in the CU mediumresponsible for increased growth and lipid accumulationin Chlorella sp., the cells were grown under pure urea atdifferent concentrations (Figure 4). In this study, thegrowth of cell is directly proportional to theconcentration of urea applied. The highest biomassproduction (0.9 g l-1) was obtained at the concentrationof 0.25 g l-1. The biomass production was only 0.145 g l1under urea starvation, and was increased to 0.57 g l-1and 0.713 g l-1 at 0.15 and 0.20% (w/v) of urea,respectively. This finding may be attributed to thegrowth of the Chlorella pyrenoidosa under urea andalso the cell growth may be supported by ammonia ifproduced as a hydrolysed product of urea and also foundin cattle urine.Lipid content was also affected by ureasupplemented medium at different concentrations.Figure 4 shows lipid production of 0.022, 0.12, 0.09 and0.04 g l-1 in 0, 0.15, 0.20, 0.25 % (w/v) of urearespectively. The maximum lipid content (21%) wasestimated at 0.15 % urea (w/v) and decreases withincrease in urea concentration from 0.20 to 0.25 % (dataare not shown). Literature shows, urea is a low costnitrogen source to support growth and lipidaccumulation in various Chlorella species [11, 19, 20].1.2presence of cow urine the excess carbon fromphotosynthesis is channelled into storage molecule(TAGs), and carbohydrate content may be reduced [21].An increase in protein may be induced due to highnitrogen supplementation in CU medium [22].70.00%Fogg s MediumAmount (%)60.00%0.00%Biochemical ComponentsFigure 5. Biochemical components of C. pyrenoidosa inFogg’s medium and cow urine (CU medium).In the present work, cells grown in CU medium doesnot show decrease in protein while there was decrease incarbohydrate and increase in lipid, so the growth has notbeen disturbed by increase in lipid. Similar resultobtained in C. vulgaris when grown in presence ofacetate and glycerol [23].0.8Biomass/Lipid 0.6productio0.4n(g/l)FAME analysisFatty acid profile of the algae grown in Fogg’s mediumand CU medium was determined and the results arepresented in Table 1 (a & b). The fatty acid compositionwas calculated based on specific fatty acid percentageover the total fatty acid of lipids of each sample. Thefatty-acid produced from cells grown under fogg’smedium showing linolenic acid (18:3; 37.55%) as thehighest content followed by palmitic (16:0;14.3%), oleic(18:1; 12.3%) and linoleic acid (18:2; 8.7%) (Table 1a).The FAME profile of cells grown under CU medium isquite different showing highest content of palmitic(16:0; 21.56%) and oleic (18:1; 21.33%) followed bylinoleic (18:2; 13.69%) and linolenic acid (18:3;10.39%). Two fatty acids palmitoleic (16:2; 5.58%) andstearic (18:0; 3.26%) are also present in CU cells thoseare not present in fogg’s cells (Table 1, b). In presenceof CU medium, the total content of saturated fatty acidis found to be 25 % , monounsaturated fatty acids is 24% and polyunsaturated fatty acids is 31% while in cellsThe proportion of saturated, monounsaturated andpolyunsaturated fatty acids are better in CU grown cells,which meets the requirements of the European Standard0.200.1530.00%10.00%Lipid Content040.00%20.00%Cell Biomass1CU medium50.00%0.20.25Urea Concentration (%W/V)Figure 4. Effect of urea concentration on biomass and lipidproduction in C. pyrenoidosaDetermination of biochemical componentsThe cellular components may vary with the growthmedia and culture conditions. The cell grown in cowurine supplemented medium showed high protein incomparison to cells grown in normal fogg’s medium(Figure 5). Protein content was estimated 50.176% inCU medium whereas it was approximately 37% in caseof fogg s medium. The lipid accumulation wasenhanced to 22.7% which is much greater than the lipidextracted from cells grown in control medium (7%),while the carbohydrate was reduced from 40 to 23% inpresence of CU medium. Results showed that in337

Iranica Journal of Energy and Environment 6(3): 334-339, 2015PeakRetentionNum-timeCompound(b) C. pyrenoidosa grown under CU mediumAreaEN 14214 for biodiesel production [25]. In our studycow urine; a rich source of nitrogen is used to cultivatealgae and the effect is found to be very positive inrespect to both biomass and lipid .42hexadecen-1-ol (C20H40O)1217.33Hexadecanoic acid methyl ester21.56CONCLUSION2.94The proportion of saturated, monounsaturated andpolyunsaturated fatty acids are better in CU grown cells,which meets the requirements of the European StandardEN 14214 for biodiesel production. In our study cowurine; a rich source of nitrogen is used to cultivate algaeand the effect is found to be very positive in respect toboth biomass and lipid accumulation.(C17H34O2)1317.577- hexadecenoic acid methyl ester(C17H32O2)1518.879,12- hexadecadienoic acid methyl1719.829,12,15-octadecatrienoic acid1921.91Octadecanoic acid methyl ester2022.269-octadecenoic acid methyl5.58ester (C17H30O2)10.39methyl ester his work is financially supported by a project grantfrom Department of Science and Technology, Govt. ofIndia, New-Delhi (Ref. No. DST/TSG/AF/2009/101).Authors are thankful to Advanced InstrumentationResearch Facility, Jawaharlal Nehru University, NewDelhi, India for providing the facility of Gaschromatography-Mass enoic acid methylester acid methyl ester (C21H32O2)TABLE 1. Fatty acid profile(a) C. pyrenoidosa grown under Fogg’s -tetramethyl-2-hexadecen-REFERENCES1.741-ol (C20H40O)1.914.481-octadecyl isocyanate(C19H37NO)1.741217.66Hexadecanoic acid methyl ester14.311317.947- hexadecenoic acid methyl trienoic .579-octadecenoic acid methyl2123.449,12-octadecadienoic acid methyl2425.235,8,11,14,17-Eicosapentaenoic acidBastianoni, S., F. Coppola, E. Tiezzi, A. Colacevich, F. Borghiniand S. Focardi, 2008. Biofuel potential production from theOrbetello lagoon macroalgae: A comparison with sunflowerfeedstock. Biomass and Bioenergy, 32(7): 619-628.2. Becker, E.W., Microalgae: biotechnology and microbiology.Vol. 10. 1994: Cambridge University Press.3. Bhadauria, H., 2002. Cow urine-a magical therapy. Int J CowSci, 1: 32-36.4. Bligh, E.G. and W.J. Dyer, 1959. A rapid method of total lipidextraction and purification. Canadian journal of biochemistryand physiology, 37(8): 911-917.5. Bristow, A.W., D.C. Whitehead and J.E. Cockburn, 1992.Nitrogenous constituents in the urine of cattle, sheep and goats.Journal of the Science of Food and Agriculture, 59(3): 387-394.6. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnologyadvances, 25(3): 294-306.7. Fan, J., Y. Cui, M. Wan, W. Wang and Y. Li, 2014. Lipidaccumulation and biosynthesis genes response of the oleaginousChlorella pyrenoidosa under three nutrition stressors.Biotechnol. Biofuels, 7: 17.8. Guschina, I.A. and J.L. Harwood, 2006. Lipids and lipidmetabolism in eukaryotic algae. Progress in lipid research, 45(2):160-186.9. He, P., B. Mao, C. Shen, L. Shao, D. Lee and J. Chang, 2013.Cultivation of Chlorella vulgaris on wastewater containing highlevels of ammonia for biodiesel production. Bioresourcetechnology, 129: 177-181.10. Hu, H. and K. Gao, 2006. Response of growth and fatty acidcompositions of Nannochloropsis sp. to environmental l ester (C19H32O2)3.73acid methyl ester (C23H34O2)12.35ester(C19H36O2)8.73ester (C19H34O2)1.91methyl ester (C21H32O2)338

Iranica Journal of Energy and Environment 6(3): 334-339, 201511.12.13.14.15.16.17.18.under elevated CO2 concentration. B

N. Sharma, M. P. Rai* Amity Institute of Biotechnology (J-3 block), Amity University Uttar Pradesh, Sector-125, Noida, India A B S T R A C T P A P E R I N F O Media requirement for microalgae cultivation adds most to the cost of biodiesel production at Paper history: Received 30 July 2015 Accepted in revised form 6 September 2015 Keywords: - Chlorella pyrenoidosa Cattle waste Lipid content .

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