research article growth, fatty acid, and lipid composition...

Research ArticleGrowth Fatty Acid and Lipid Composition ofMarine Microalgae Skeletonema costatum Available inBangladesh Coast Consideration as Biodiesel Feedstock

Tania Sharmin1 Chowdhury Md Monirul Hasan1 Sheikh Aftabuddin2

Md Atiar Rahman1 and Mala Khan3

1Department of Biochemistry amp Molecular Biology University of Chittagong Chittagong 4331 Bangladesh2Institute of Marine Sciences and Fisheries University of Chittagong Chittagong 4331 Bangladesh3Bangladesh Council of Scientific and Industrial Research (BCSIR) Dr Qudrat-i-Khuda Road Dhanmondi Dhaka 1205 Bangladesh

Correspondence should be addressed to Chowdhury Md Monirul Hasan monirkyushugmailcom

Received 20 October 2015 Revised 30 January 2016 Accepted 2 February 2016

Academic Editor Garth L Fletcher

Copyright copy 2016 Tania Sharmin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Among the various potential sources of renewable energy biofuels are of most interest Marine microalgae are the most promisingoil sources for making biofuels which can grow very rapidly and convert solar energy to chemical energy via CO

2fixation The

fatty acid profile of almost all the microalgal oil is suitable for the synthesis of biofuel In this research fatty acid and lipid contentsof Bangladeshi strains of marine microalgae Skeletonema costatumwere performed For this the crude oil was extracted by Soxhletextractionmethod using threemost common solvent systems pure hexane andmixture of CHCl

3MeOH (2 1) and hexane EtOH

(3 1) one by one Highest oil recovery (1537) came from CHCl3MeOH (2 1) solvent system from dry biomass whereas the

lowest (249) came from n-hexane fromwet biomassThe qualitative analysis of the extracted oil by GCMS analysis revealed thatit contained significant amount of myristic acid (C140) palmitic acid (C160) stearic acid (C180) and palmitoleic acid (C161)It also indicated presence of hexadecatrienoic acid benzenedicarboxylic acid oleic acid arachidonic acid eicosapentaenoic acid(EPA) 9-Octadecenoic acid methyl ester (C

19H36O2) and so forth The obtained fatty acid profile indicates high potentiality of

S costatum species to be used as promising biofuel feedstock a little improvisation and substantially it can replace diesel in nearfuture

1 Introduction

Energy demand worldwide is increasing continuously at arapid rate with the increase of urbanization in developingcountries Majority of the worldrsquos energy needs are suppliedthrough petrochemical sources coal and natural gases withthe exception of hydroelectricity and nuclear energy andthese sources are finite and at current usage rates will beconsumed shortly [1] Heavy dependence on petroleum-based fuels is not sustainable due to increasing fuel costsdiminishing crude oil reserves and the environmental impactof fossil fuel usage [2ndash4] Crude oil reserves are beingdepleted at a rate of approximately 85ndash90million barrels of oilper day and it is possible that the reserve will be completely

depleted within the next 50 years [5] Therefore alternativesources of energy will need to be researched and developedwithin the next 50 years Continued researchwill facilitate thedevelopment and implementations of renewable fuels to helplessen the worldrsquos dependence on fossil fuels

Biodiesel is a biofuel consisting of monoalkyl esters thatare derived from organic oils plant or animal through theprocess of trans-esterification [6] It is also biodegradableand nontoxic and has low emission profile as compared topetroleum diesel [7] Biodiesel can be burned in existingdiesel engines with no modifications and can be blended inany proportion with petroleum diesel This allows for its usein the existing fuel distribution infrastructure and giving itvalue as a fuel extender Shay [8] reported that algae are one

Hindawi Publishing CorporationJournal of Marine BiologyVolume 2016 Article ID 6832847 8 pageshttpdxdoiorg10115520166832847

2 Journal of Marine Biology

of the best sources of biodiesel In fact algae are the highestyielding feedstock for biodiesel It can produce 250 timesmore than the amount of oil per acre as soybeans and 7 to 31time greater oil than palm oil [9] In fact biodiesel from algaemay be the only way to produce enough automobile fuels toreplace current gasoline usage The best algae for biodieselwould be microalgae [10] Microalgae have much more oilthan macroalgae and it is much faster and easier to grow andharvest [8]

Microalgae can also provide several different types ofrenewable biofuels These include methane produced byanaerobic digestion of the algal biomass [11] biodiesel derivedfrom microalgal oil [12ndash17] and photobiologically producedbiohydrogen [18ndash22] The idea of using microalgae as asource of fuel is not new [2 16 23] but it is now beingtaken seriously because of the escalating price of petroleumand more significantly the emerging concern about globalwarming that is associated with burning fossil fuels [14]

Depending on species microalgae produce many differ-ent kinds of lipids hydrocarbons and other complex oils [1224] Not all algal oils are satisfactory formaking biodiesel butsuitable oils occur commonly Another important fact is thatusing microalgae to produce biodiesel will not compromisewith the production of food fodder and other productsderived from crops The objective of this investigation was toidentify locally available microalgal strains with sustainablelipid content for biofuel production and evaluate the biofuelproductivity of that strain For this purpose the present workwas carried out with marine microalgae S costatum that aredominant in the coastal area especially in Coxrsquos Bazar coastline of Bangladesh shown in Figure 1

2 Materials and Methods

The present investigation was carried out in the Centre forResearch Excellence (CRE) Department of Biochemistry andMolecular Biology University of Chittagong ChittagongBangladesh

21 Sample Collection S costatum sample water was col-lected from Bakkhali River of Coxrsquos Bazar (Figure 1) Bangla-desh in January 2014 Microscopic isolation and identifica-tion were done at Institute of Marine Sciences and FisheriesUniversity of Chittagong

22 Culture of Skeletonema costatum For isolation standardf2 medium [25] nutrients were added with the samplewater and left under light until diatom bloom Bloomed Scostatum was then isolated and purified by serial dilutionand micropipette method [26 27] In micropipette methodsingle cells or filaments were picked up under a dissectingmicroscope from the enriched sample water by using 5mLsyringe The individual cell was transferred to fresh sterilef2 medium containing 100mL flasks and kept under lightat 25∘C for 1 week For serial dilution 5 sterilized test tubesfilledwith 9mL sterilized seawater enrichedwith f2mediumwere marked as 10minus1 up to 10minus5 serially and 1mL of samplecontaining bloomed diatom was transferred to 10minus1 test tubethen 1mL from 10minus1 to 10minus2 and so on The clones were

0 50

(km)

Figure 1 Location of the S costatum sample collection area in thesoutheastern region Bangladesh

kept in 24 hours photoperiod to develop for 10 to 15 daysas stock culture For large scale S costatum production 200liters FRP tanks were used according to the procedure ofAftab Uddin and Zafar [28] Cell counts were observed by acompoundmicroscope and concurrently salinity (25ndash28 ppt)temperature (25ndash27∘C) and pH (76ndash82) were maintainedduring the entire culture period and sufficient aeration wasprovided

23 Biomass Processing At first the biomass was dewateredby filtration with a Whatman filter paper Then the wet algalbiomass was collected from the filter paper with a spatula andtaken in Petri dishes The biomass was then dried at 80∘C for2 hours and kept in the desiccator for whole night so thatthere is no water left in the sample The dry mass was thengrounded properly andweighed immediatelyThedrying stepwas skipped in case of wet extraction process

24 Soxhlet Extraction of Fatty Acid and Lipids Extensivestudy and research were conducted earlier to determine thebest extraction method over wet and dry algae biomass Inthis experiment the effect of three different solvents that isn-hexane [29] the mixture of chloroform and methanol at2 1 vv ratio [30] and n-hexane and ethanol at 3 1 vv ratio[31] had been studied overwet and dry biomass of S costatumby using Soxhlet apparatus [32 33] For this 5 g of dry weightand 10 g of wet fresh algae paste were taken into the extractionchamber separately Both ends of the chamber were enclosedwith cotton balls to withstand any solid algae dischargeThe bulb was filled with 180mL of one of the three organicsolvents and heated at 80∘C The solvent gradually startedto vaporize and after condensation it fell into the extractionchamber containing the algae and release lipids into thechamber The solvent-lipid mixture was then reached to acritical height within the chamber and the siphoning process

Journal of Marine Biology 3

was initialized that is the mixture was drawn back to thebulbThe system recirculates the solvent by constantly boilingand condensing it At the end of this extraction processa mixture of solvent and oil was left in the bulb and thealgae biomass was left in the chamber The processing beforeevaporation was performed as described by Kumar et al [34]The crude extract was taken into a separating funnel andwashed with 1 aqueous sodium chloride solution (50mL)twice The aqueous layer was removed and the solvent layerwas passed through a layer of anhydrous sodium sulfate bytaking it in a glass funnel blocked with cotton plug

25 Evaporation The solvent-oil mixture was placed into apreweighed flask for evaporation In the laboratory setup thesolvent and oil mixture was exposed to a vacuum and thenheated at 60∘C by using a vacuum pump Rotary Evaporator(RE 200 Bibby Sterling UK) in order to remove all thesolvent After evaporation the flask was weighed again andcompared with the first weight In this way the weight of theoil content was calculated The oil was recovered by usingdichloromethane and was collected into a small vial Thepercentage of oil extraction was calculated by the followingformula

of total oil recovered

=

Weight of crude lipid extractedWeight of dry algae biomass

times 100

(1)

26 Quantitative Analysis of Algal Oil

261 TLC Analysis The components of extracted lipids wereseparated by modified thin-layer chromatography (TLC)method [35]This was done by TLC silica gel plates (50mm times100mm in size) and was diffused by two mixed solvents [36]The first eluent was the mixture of ethyl acetate isopropanolchloroform methanol and KCl (025 solution) in a ratio of25 25 25 10 9 (vvvvv) running to a height of 10 cm fromthe origin and the second eluent was the mixture of hexanediethyl ether and acetic acid in a ratio of 80 20 2 (vvv)to a height of 18 cm from the origin After dried the platewas kept in a TLC jar with iodine powder The individuallipid bands became detectable as yellow bands within 5minIn iodine vapor exposure four individual bands were foundfor CHCl

3MeOH (2 1) extract six bands for hexane EtOH

(3 1) extract and a single band for pure hexane werefound For the recovery of individual lipid component thecorresponding silica gel was removed from the plates andwashed it with chloroformmethanol (2 1 vv) [36]The lipidcomponent separated by TLC as described above was thenkept for the fatty acid composition analysis by GCMS

262 Sample Preparation for GCMS The fatty oils wereesterified with methanol prior to Gas Chromatography-MassSpectroscopy (GCMS) analysis to make the fatty oils morevolatile and to avoid the acidic attack to the stationaryphasecolumn For this 10mg (2-3 drops) of fatty oil wastaken in a screw capped glass tube and 1mL of Borontrifluoride methanol complex was added The mixture was

then heated at 100∘C for 1 hr using water bath and cooled at25∘C Then 1mL of deionized water and 2mL hexane wereadded and vortexed for 1min Then the mixture was cen-trifuged at low rpm for 2min and upper layer was collecteddiluted with hexane and filtered for GCMS analysis

263 GCMS Analysis Thin-layer chromatographic (TLC)separation was analyzed by GC-MS with electron impactionization (EI) method on a gas chromatograph (GC-17AShimadzuCorporationKyoto Japan) coupled to amass spec-trometer (GC-MS QP 5050A Shimadzu Corporation KyotoJapan) A fused silica capillary column (30m times 25mm025m film thickness) is coated with DB-1 (JampW) The inlettemperature was set at 260∘C and the oven temperature wasprogrammed as 70∘C (0min) 10∘C 150∘C (5min) 12∘C200∘C (15min) 12∘C 220∘C (5min) The column flow ratewas 06mLmin He gas at a constant pressure of 90KPaThe aux (GC to MS interface) temperature was set to 280∘CThe MS was set in scan mode with a scanning range of 40ndash350 amu the ionization mode was EI (electron ionization)type The mass range was set in the range of 50ndash550mz Theprepared sample was then run for GCMS analysis One120583Lsample was injected in spilt fewer modes Vial containinglipid was used for GCMS analysis to make lipid profile Thislipid profile indicates the fatty acids which are important toevaluate the biomass for biofuel production

27 Statistical Analysis All the data were expressed as meanplusmn SD and they were analyzed by using Microsoft Excel 2007The significance of differencewas calculated by Studentrsquos 119905 testand the values 119875 lt 005 were considered to be significant

3 Results

The result of total oil recovery by three different solventsis shown in Table 1 It showed that every solvent systemgives maximum oil recovery for dry sample and relativelylow recovery for wet sampleThe total oil recoveries obtainedfrom dry and wet biomass by using pure hexane were 1124and 249 by CHCl

3MeOHwere 1537 and 670 and by

hexane EtOHwere 1167 and 532 respectivelyThe resultshowed that in both wet and dry conditions CHCl

3MeOH

(2 1) solvent system gives the highest yieldThe comparison of oil content and oil contents from

dry and wet sample of different solvent extracts is shownin Figure 2 It is clearly evident that both oil content and oil content of dry wet from CHCl

3MeOH extract are

significantly (119875 lt 005) different from those of two othersolvent systems Similarly their contents in wet sample arealso higher in CHCl

3MeOH although the values were not

statistically significant (Figure 2)The fatty acid methyl ester (FAME) composition analysis

of the extracted oil was very important for the evaluation of abiofuel feedstock For this purpose the extracted algae oil wasanalyzed by GCMS to obtain the fatty acid profile of S costa-tum The result showed the fatty acid and lipid profile frompure n-hexane extract presented in Table 2 CHCl

3MeOH

extract in Table 3 and hexane ethanol extract in Table 4


Table 1 Percentage of total oil recovered by the three different solvents

Solvent Physical condition Sample wt (g) Oil content (g) of oil content

n-Hexane Dry 500 plusmn 000 0563 plusmn 0052 1124 plusmn 120Wet 1000 plusmn 000 0247 plusmn 0115 249 plusmn 129

CHCl3MeOH (2 1) Dry 500 plusmn 000 077 plusmn 0054 1537 plusmn 123

Wet 1000 plusmn 000 067 plusmn 0094 670 plusmn 106

n-Hexane EtOH (3 1) Dry 500 plusmn 000 058 plusmn 0089 1167 plusmn 198Wet 1000 plusmn 000 053 plusmn 0151 533 plusmn 168

a

a

b

b

dd

c

c0

4

8

12

16

20

Dry Wet Dry Wet Dry Wetn-Hexane

Oil content

Relat

ive c

onte

nt o

f oil

Oil content ()

a

aaa

n-Hex EtOHCHCl3 MeOH

Figure 2 Comparative oil contents in dry and wet sample of Scostatumdue to solvent variationData are shown asmeanplusmn SD (a bc and d) with different superscript letters over the bars for both dryand wet samples are significantly different from each other (paired119905-test 119875 lt 005)

Most importantly the fatty acids suitable for biodiesel pro-duction such as myristic acid (C140) palmitic acid (C160)palmitoleic acid (C161) and stearic acid (C180) were themost frequent entities encountered in S costatum profile

Table 2 showed that myristic acid (C140) and palmiticacid (C160) were the dominants in pure hexane extract about3593 and 2721 respectively The fatty acid profile ofCHCl

3MeOH extract (presented in Tables 3 and 4) showed

that the main constituents of this extract are myristic acid(C140) palmitic acid (C160) and stearic acid (C180) up to3639 25 and 2772 respectively Palmitoleic acid oleicacid arachidonic acid pentadecanoic acid eicosapentaenoicacid and other fatty acids were also present in low content

4 Discussion

Bioenergy is one of the most important sources that areconcerning the scientists and industrial sector In view of everincreasing global demand for energy there has been substan-tial interest in developing renewable biologically producedfuelMarine algae are one such emerging resource consideredas an alternative for biofuel production Oil extracted fromalgal biomass can be turned into biodegradable and carbonneutral biodiesel through transesterification reaction [2 37]

To be an ideal source of sustainable biodiesel selectedmicroalgal species should contain sufficient lipid with suit-able fatty acids for good biodiesel properties Several workswere carried out to extract algal oil for biofuel productioncommonly using different organic solvents and the lipidcontents were often reported as the weight ratio of the crudeextract and the dry biomass In this study the algal strainswere cultured and their oil extractionwas done by using threedifferent solvent systems pure hexane [29] CHCl

3MeOH

[35] and hexane EtOH [31] to compare their oil recoveryThe result showed that every solvent system recovered theirhighest yield from dry biomass and in both dry and wetconditions CHCl

3MeOH (2 1) and n-hexane EtOH (3 1)

were comparatively more effective than pure n-hexane recov-ering about 15367 and 11673 oil respectively Howeverthe oil content in the dry sample through CHCl

3 CH3OH

is significantly (119875 lt 005) different from that of two othersolvent systems This may be because CHCl

3MeOH (2 1)

and n-hexane EtOH (3 1) both are the mixture of polar(MeOH EtOH) and nonpolar (CHCl

3 n-hexane) solvents

thus both neutral and polar lipids could be extracted by thesetwo solvent systems On the other hand the nonpolar solventn-hexane could preferably dissolve only nonpolar lipids in themicroalgae However it could not dissolve all the oil contentfrom the cells alone

A systematic analysis of the fatty acid methyl ester(FAME) composition and comparative fuel properties is veryimportant for species selection for biodiesel production Ithas been suggested that the higher the degree of unsaturationof the FAMEs of a biodiesel the higher the tendency of thebiodiesel to oxidize There are however other parameterswhich also define the oxidation stability of the fuel forexample natural antioxidant and free fatty acid content [38ndash40]Themost common fatty acids ofmicroalgae are palmitic-(hexadecanoic-C160) stearic (octadecanoic-C180) oleic(octadecenoic-C181) linoleic-(octadecadienoic-C182) andlinolenic-(octadecatrienoic-C183) acids [41]Most algae haveonly small amounts of eicosapentaenoic acid (EPA) (C205)and docosahexaenoic acid (DHA) (C226) however in somespecies of particular genera these PUFAs can accumulate inappreciable quantities depending on cultivation conditions[42] Consistently the oil extract of S costatum showedthe prevalence of methyl palmitate methyl stearate andmethyl myristate when methyl myristate methyl palmitateand methyl 6912-hexadecatrienoate were 3593 2721and 1236 in the n-hexane fraction respectively WhileCHCl

3MeOH fraction showed methyl palmitate (2241)


Table 2 Fatty acid profile of pure n-hexane extract

S number Retention time Area Compound name1 16699 3593 Methyl myristate2 18045 873 trans-2-(3-Methoxyphenyl)-3-phenyl oxirane3 19225 1236 Methyl 6912-hexadecatrienoate4 19418 794 Methyl palmitoleate5 19860 2721 Methyl palmitate6 21964 192 5H-Benzofuro[32-e]imidazo[12-a]pyrazin-4-one7 22585 112 (trans)-2-(31015840-Chlorophenyl)-1-nitroethylene8 24903 338 Methyl stearate

Table 3 Fatty acid profile of lipid from CHCl3MeOH extract

S number Retention time Area Compound name1 9523 1153 12-Benzenedicarboxylic acid dimethyl ester2 10731 372 Phenol 24-bis(11-dimethylethyl)-3 10869 219 Phenol 26-bis(11-dimethylethyl)-4-methyl-4 15461 438 Methyl myristate5 16841 246 Pentadecanoic acid methyl ester6 16855 347 22-Dimethyl-67-dimethoxychromanone

7 16862 234 26-Dimethoxy-3-(31015840-methyl-21015840-butenyl)-14-benzoquinone

8 17113 146 1-(4-Fluorophenyl)-2-[(4-hydroxy-6-methylpyrimidin-2-yl)thio]ethan-1-one


10 17510 204 2-Cyclohexen-1-one 44-dimethyl-11 17655 996 Methyl 6912-hexadecatrienoate12 17762 139 (E)-24-Phytadiene m-Menth-1(7)-ene (R)-(minus)-13 17772 691 Methyl palmitoleate14 17810 835 912-Octadecadien-1-ol (ZZ)-15 17983 2241 Methyl palmitate

16 19201 324 Methyl(5R)-4-hydroxy-56-(isopropylidenedioxy)-2-methylenehexanoate

17 19294 139 Methyl 3-methylquinoxaline-2-carboxylate18 19318 1499 Methyl N-[(1-naphthyl)methyl]carbamate19 19698 230 Methyl oleate20 19860 2474 Methyl stearate21 20967 168 Ethoxylate ester of methyl oleate22 21005 348 Methyl arachidonate

23 21084 772 cis-58111417-Eicosapentaenoic acid methyl ester (EPAmethyl ester)

24 22464 340 3-cis-Methoxy-5-cis-methyl-1R-cyclohexanol25 28253 240 Propane 11-dibromo-2-chloro-

methyl N-[(1-naphthyl)methyl]carbamate (1499) and 12-Benzenedicarboxylic acid as prevalent fatty acids Howeverin hexane EtOH fraction methyl stearate (2064) is onlythe fatty acid present in considerable quantity

The abovementioned FAs of marine microalgae S costa-tum found from the lab-scale examination are dominantFAs which are known to be most common fatty acidswith good biofuel properties [39] Other FAs detected were

hexadecatrienoic acid benzenedicarboxylic acid oleic acidarachidonic acid and eicosapentaenoic acid (EPA) Indeedmost of the microalgae investigated have similar fatty acidprofile but the percentage content of fatty acid for eachmicroalga is very differentThis mainly depends on the strainused and culture conditions [43 44] All of the microalgaehave the same fatty acid profile in chain C16 and C18Palmitic fatty acid (C160) is a predominant fatty acid in


Table 4 Fatty acid profile of lipid band no 1 of Hexane EtOH extract

SN Retention time Area Compound name1 10728 340 Phenol 24-bis(11-dimethylethyl)-2 10862 320 Butylated Hydroxytoluene3 10866 229 Phenol 26-bis(11-dimethylethyl)-4-methyl-4 11007 154 Methyl laurate5 15199 143 Cyclohexanecarboxylic acid 22-dimethylpropyl ester6 15461 408 Methyl myristate7 16858 162 10-Ethyl-33699-pentamethyl-210-diazabicyclo[40]-1-decene8 17979 177 Methyl palmitate9 19856 2064 Methyl stearate10 20967 144 Methoxylate ester of methyl oleate11 17082 114 2-Pentadecanone 61014-trimethyl-12 17800 171 Methyl palmitoleate13 18773 114 (E)1-Allyl-2-methylcyclohexanol14 19232 107 Sulfurous acid di(2-ethylhexyl) ester15 19297 600 14-Dihydropyrano[34-b]indol-3-on16 19459 114 Methyl 4-oxododecanoate17 19698 240 Methyl oleate18 21043 121 Butyl-2-ethylhexyl phthalate

most microalgae research Some critical fuel parameterslike oxidation stability cetane number iodine value andviscosity were correlated with the methyl ester compositionand structural configuration From the current literature itwas found that the FAs indicated by the fatty acid profile of Scostatum are of better fuel properties and thus it is likelihoodto be a viable fuel source for internal combustion engines

The present study offers a scientific basis of the use of thismicroalga as a feedstock for biofuel Still it is in a preliminarystage that requires further study on other parameters It alsoindicates that it may be possible in the future to improve theproperties of biodiesel bymeans of genetic engineering of theparent oils which could eventually lead to a fuel enrichedwith a combination of improved fuel properties

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

Chowdhury Md Monirul Hasan and Tania Sharmin havedesigned the study and performed the experimental works inlaboratory Sheikh Aftabuddin has assisted in the collectionand identification of microalgae strains and prepared theirgrowth environment Mala Khan has provided her labora-tory facilities in analyzing the compounds Chowdhury MdMonirul Hasan Sheikh Aftabuddin Md Atiar Rahman andTania Sharmin drafted the paper Md Atiar Rahman hasendeavored in data analysis and interpretation of the resultsAll authors read and approved the final version of the paper

Acknowledgments

The authors thank the authority of BCSIR Dhaka Chairmanof the Department of Biochemistry and Molecular BiologyandDirector of the Institute ofMarine Sciences and FisheriesUniversity of Chittagong for providing laboratory facilitiesand their kind support in augmentation of the research

References

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[2] M Y Chisti ldquoAn unusual hydrocarbonrdquo Journal of RamsaySociety vol 27-28 pp 24ndash26 1981

[3] A Demirbas and M F Demirbas ldquoImportance of algae oil asa source of biodieselrdquo Energy Conversion and Management vol52 no 1 pp 163ndash170 2011

[4] P T Pienkos and A Darzins ldquoThe promise and challenges ofmicroalgal-derived biofuelsrdquo Biofuels Bioproducts and Biorefin-ing vol 3 no 4 pp 431ndash440 2009

[5] A Z Abdullah N Razali H Mootabadi and B SalamatinialdquoCritical technical areas for future improvement in biodieseltechnologiesrdquo Environmental Research Letters vol 2 no 3Article ID 034001 6 pages 2007

[6] A Demirbas ldquoImportance of biodiesel as transportation fuelrdquoEnergy Policy vol 35 no 9 pp 4661ndash4670 2007

[7] M J Ramos C M Fernandez A Casas L Rodrıguez and APerez ldquoInfluence of fatty acid composition of raw materials onbiodiesel propertiesrdquo Bioresource Technology vol 100 no 1 pp261ndash268 2009


[8] E G Shay ldquoDiesel fuel from vegetable oils status and opportu-nitiesrdquo Biomass and Bioenergy vol 4 no 4 pp 227ndash242 1993

[9] A B M S Hossain A Salleh A N Boyce P Chowdhury andM Naqiuddin ldquoBiodiesel fuel production from algae as renew-able energyrdquo American Journal of Biochemistry and Biotechnol-ogy vol 4 no 3 pp 250ndash254 2008

[10] A K Bajhaiya S K Mandotra M R Suseela K Toppo and SRanade ldquoAlgal biodiesel the next generation biofuel for IndiardquoAsian Journal of Experimental Biology vol 1 no 4 pp 728ndash7392010

[11] P Spolaore C Joannis-Cassan E Duran and A IsambertldquoCommercial applications of microalgaerdquo Journal of Bioscienceand Bioengineering vol 101 no 2 pp 87ndash96 2006

[12] A Banerjee R Sharma Y Chisti and U C Banerjee ldquoBotry-ococcus braunii a renewable source of hydrocarbons and otherchemicalsrdquo Critical Reviews in Biotechnology vol 22 no 3 pp245ndash279 2002

[13] TGDunahay E E Jarvis S SDais andPG Roessler ldquoManip-ulation of microalgal lipid production using genetic engi-neeringrdquo Applied Biochemistry and Biotechnology vol 57-58pp 223ndash231 1996

[14] M Gavrilescu and Y Chisti ldquoBiotechnologymdasha sustainablealternative for chemical industryrdquo Biotechnology Advances vol23 no 7-8 pp 471ndash499 2005

[15] P G Roessler L M Brown T G Dunahay et al ldquoGenetic engi-neering approaches for enhanced production of biodiesel fuelfrommicroalgaerdquo in Enzymatic Conversion of Biomass for FuelsProduction vol 566 of ACS Symposium Series pp 255ndash270American Chemical Society 1994

[16] S Sawayama S Inoue Y Dote and S-Y Yokoyama ldquoCO2fixa-

tion and oil production through microalgardquo Energy Conversionand Management vol 36 no 6-9 pp 729ndash731 1995

[17] J Sheehan T Dunahay J Benemann and P Roessler ldquoAlook back at the US Department of Energyrsquos Aquatic SpeciesProgram-biodiesel from algaerdquo Report NRELTP-580-24190National Renewable Energy Laboratory Golden Colo USA1998

[18] I Akkerman M Janssen J Rocha and R H Wijffels ldquoPhoto-biological hydrogen production photochemical efficiency andbioreactor designrdquo International Journal of Hydrogen Energyvol 27 no 11-12 pp 1195ndash1208 2002

[19] A S Fedorov S Kosourov M L Ghirardi and M SeibertldquoContinuous hydrogen photoproduction by Chlamydomonasreinhardtii using a novel two-stage sulfate-limited chemostatsystemrdquo Applied Biochemistry and Biotechnology vol 121 no 1ndash3 pp 403ndash412 2005

[20] M L Ghirardi L Zhang J W Lee et al ldquoMicroalgae a greensource of renewable H

2rdquo Trends in Biotechnology vol 18 no 12

pp 506ndash511 2000[21] I K Kapdan and F Kargi ldquoBio-hydrogen production from

waste materialsrdquo Enzyme and Microbial Technology vol 38 no5 pp 569ndash582 2006

[22] A Melis ldquoGreen alga hydrogen production progress chal-lenges and prospectsrdquo International Journal of Hydrogen Energyvol 27 no 11-12 pp 1217ndash1228 2002

[23] N Nagle and P Lemke ldquoProduction of methyl-ester fuel frommicroalgaerdquoApplied Biochemistry and Biotechnology vol 24-25pp 355ndash361 1990

[24] P Metzger and C Largeau ldquoBotryococcus braunii a rich sourcefor hydrocarbons and related ether lipidsrdquoAppliedMicrobiologyand Biotechnology vol 66 no 5 pp 486ndash496 2005

[25] R R L Guillard ldquoCulture of phytoplankton for feeding marineinvertebratesrdquo in Culture of Marine Invertebrate Animals pp29ndash60 Plenum Press New York NY USA 1975

[26] E J Allen and E W Nelson ldquoThe artificial culture of marineplankton organismsrdquo Journal of the Marine Biological Associa-tion of the UK vol 8 no 5 pp 421ndash474 1910

[27] M R Droop ldquoA note on the isolation of small marine algae andflagellates for pure culturesrdquo Journal of the Marine BiologicalAssociation of the United Kingdom vol 33 no 2 pp 511ndash5141954

[28] S Aftab Uddin and M Zafar ldquoLive feed production in differentnutritive condition as diet for Penaeus monodon in ShrimpHatchery Bangladeshrdquo International Journal of Agriculture andBiology vol 8 no 4 pp 493ndash495 2006

[29] X Miao and Q Wu ldquoBiodiesel production from heterotrophicmicroalgal oilrdquo Bioresource Technology vol 97 no 6 pp 841ndash846 2006

[30] J Folch M Lees and G H Sloane Stanley ldquoA simple methodfor the isolation and purification of total lipides from animaltissuesrdquo Journal of Biology and Chemistry vol 226 no 1 pp497ndash509 1956

[31] M Chen T Liu X Chen et al ldquoSubcritical co-solvents extrac-tion of lipid fromwet microalgae pastes ofNannochloropsis sprdquoEuropean Journal of Lipid Science and Technology vol 114 no 2pp 205ndash212 2012

[32] R G Ackman ldquoExtraction and analysis of omega-3 fatty acidsprocedures and pitfallsrdquo in Omega-3 Fatty Acids Metabolismand Biological Effects C A Drevon I Baksaas and H EKrokan Eds pp 11ndash20 Birkhauser Basel Switzerland 1993

[33] H Gunnlaugsdottir and R G Ackmanj ldquoThree extractionmethods for determination of lipids in fish meal evaluation ofa hexaneisopropanol method as an alternative to chloroform-based methodsrdquo Journal of the Science of Food and Agriculturevol 61 no 2 pp 235ndash240 1993

[34] P Kumar M R Suseela and K Toppo ldquoPhysico-chemicalcharacterization of algal oil a potential biofuelrdquoAsian Journal ofExperimental Biological Sciences vol 2 no 3 pp 493ndash497 2011

[35] A Vieler C Wilhelm R Goss R Suszlig and J Schiller ldquoThelipid composition of the unicellular green alga Chlamydomonasreinhardtii and the diatom Cyclotella meneghiniana investigatedby MALDI-TOFMS and TLCrdquo Chemistry and Physics of Lipidsvol 150 no 2 pp 143ndash155 2007

[36] E Ryckebosch KMuylaert and I Foubert ldquoOptimization of ananalytical procedure for extraction of lipids from microalgaerdquoJournal of the American Oil Chemistsrsquo Society vol 89 no 2 pp189ndash198 2012

[37] J Jones S Manning M Montoya K Keller and M PoenieldquoExtraction of algal lipids and their analysis by HPLC and massspectrometryrdquo Journal of theAmericanOil Chemistsrsquo Society vol89 no 8 pp 1371ndash1381 2012

[38] L C Meher D V Sagar and S N Naik ldquoTechnical aspects ofbiodiesel production by transesterificationmdasha reviewrdquo Renew-able and Sustainable Energy Reviews vol 10 no 3 pp 248ndash2682006

[39] S K Hoekman A Broch C Robbins E Ceniceros and MNatarajan ldquoReview of biodiesel composition properties andspecificationsrdquo Renewable and Sustainable Energy Reviews vol16 no 1 pp 143ndash169 2012

[40] G Knothe ldquoDependence of biodiesel fuel properties on thestructure of fatty acid alkyl estersrdquo Fuel Processing Technologyvol 86 no 10 pp 1059ndash1070 2005


[41] M Lapuerta J Rodrıguez-Fernandez and E Font de MoraldquoCorrelation for the estimation of the cetane number ofbiodiesel fuels and implications on the iodine numberrdquo EnergyPolicy vol 37 no 11 pp 4337ndash4344 2009

[42] R Huerlimann R de Nys and K Heimann ldquoGrowth lipidcontent productivity and fatty acid composition of tropicalmicroalgae for scale-up productionrdquoBiotechnology andBioengi-neering vol 107 no 2 pp 245ndash257 2010

[43] I Tzovenis N De Pauw and P Sorgeloss ldquoOptimation of T-ISO biomass production rich in essential fatty acids II Effectof different light regimes on growth and biomass productionrdquoAquaculture vol 216 no 1ndash4 pp 223ndash242 2003

[44] S M Renaud L-V Thinh G Lambrinidis and D L ParryldquoEffect of temperature on growth chemical composition andfatty acid composition of tropical Australian microalgae grownin batch culturesrdquo Aquaculture vol 211 no 1ndash4 pp 195ndash2142002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


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BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


of the best sources of biodiesel In fact algae are the highestyielding feedstock for biodiesel It can produce 250 timesmore than the amount of oil per acre as soybeans and 7 to 31time greater oil than palm oil [9] In fact biodiesel from algaemay be the only way to produce enough automobile fuels toreplace current gasoline usage The best algae for biodieselwould be microalgae [10] Microalgae have much more oilthan macroalgae and it is much faster and easier to grow andharvest [8]

Microalgae can also provide several different types ofrenewable biofuels These include methane produced byanaerobic digestion of the algal biomass [11] biodiesel derivedfrom microalgal oil [12ndash17] and photobiologically producedbiohydrogen [18ndash22] The idea of using microalgae as asource of fuel is not new [2 16 23] but it is now beingtaken seriously because of the escalating price of petroleumand more significantly the emerging concern about globalwarming that is associated with burning fossil fuels [14]

Depending on species microalgae produce many differ-ent kinds of lipids hydrocarbons and other complex oils [1224] Not all algal oils are satisfactory formaking biodiesel butsuitable oils occur commonly Another important fact is thatusing microalgae to produce biodiesel will not compromisewith the production of food fodder and other productsderived from crops The objective of this investigation was toidentify locally available microalgal strains with sustainablelipid content for biofuel production and evaluate the biofuelproductivity of that strain For this purpose the present workwas carried out with marine microalgae S costatum that aredominant in the coastal area especially in Coxrsquos Bazar coastline of Bangladesh shown in Figure 1

2 Materials and Methods

The present investigation was carried out in the Centre forResearch Excellence (CRE) Department of Biochemistry andMolecular Biology University of Chittagong ChittagongBangladesh

21 Sample Collection S costatum sample water was col-lected from Bakkhali River of Coxrsquos Bazar (Figure 1) Bangla-desh in January 2014 Microscopic isolation and identifica-tion were done at Institute of Marine Sciences and FisheriesUniversity of Chittagong

22 Culture of Skeletonema costatum For isolation standardf2 medium [25] nutrients were added with the samplewater and left under light until diatom bloom Bloomed Scostatum was then isolated and purified by serial dilutionand micropipette method [26 27] In micropipette methodsingle cells or filaments were picked up under a dissectingmicroscope from the enriched sample water by using 5mLsyringe The individual cell was transferred to fresh sterilef2 medium containing 100mL flasks and kept under lightat 25∘C for 1 week For serial dilution 5 sterilized test tubesfilledwith 9mL sterilized seawater enrichedwith f2mediumwere marked as 10minus1 up to 10minus5 serially and 1mL of samplecontaining bloomed diatom was transferred to 10minus1 test tubethen 1mL from 10minus1 to 10minus2 and so on The clones were

0 50

(km)

Figure 1 Location of the S costatum sample collection area in thesoutheastern region Bangladesh

kept in 24 hours photoperiod to develop for 10 to 15 daysas stock culture For large scale S costatum production 200liters FRP tanks were used according to the procedure ofAftab Uddin and Zafar [28] Cell counts were observed by acompoundmicroscope and concurrently salinity (25ndash28 ppt)temperature (25ndash27∘C) and pH (76ndash82) were maintainedduring the entire culture period and sufficient aeration wasprovided

23 Biomass Processing At first the biomass was dewateredby filtration with a Whatman filter paper Then the wet algalbiomass was collected from the filter paper with a spatula andtaken in Petri dishes The biomass was then dried at 80∘C for2 hours and kept in the desiccator for whole night so thatthere is no water left in the sample The dry mass was thengrounded properly andweighed immediatelyThedrying stepwas skipped in case of wet extraction process

24 Soxhlet Extraction of Fatty Acid and Lipids Extensivestudy and research were conducted earlier to determine thebest extraction method over wet and dry algae biomass Inthis experiment the effect of three different solvents that isn-hexane [29] the mixture of chloroform and methanol at2 1 vv ratio [30] and n-hexane and ethanol at 3 1 vv ratio[31] had been studied overwet and dry biomass of S costatumby using Soxhlet apparatus [32 33] For this 5 g of dry weightand 10 g of wet fresh algae paste were taken into the extractionchamber separately Both ends of the chamber were enclosedwith cotton balls to withstand any solid algae dischargeThe bulb was filled with 180mL of one of the three organicsolvents and heated at 80∘C The solvent gradually startedto vaporize and after condensation it fell into the extractionchamber containing the algae and release lipids into thechamber The solvent-lipid mixture was then reached to acritical height within the chamber and the siphoning process





=


times 100

(1)









3 Results




3MeOH



3MeOH extract are





3MeOH









a

a

b

b

dd

c

c0

4

8

12

16

20


Oil content

Relat

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onte

nt o

f oil

Oil content ()

a

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4 Discussion



3MeOH




3 CH3OH


3MeOH (2 1)































Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





=


times 100

(1)









3 Results




3MeOH



3MeOH extract are





3MeOH









a

a

b

b

dd

c

c0

4

8

12

16

20


Oil content

Relat

ive c

onte

nt o

f oil

Oil content ()

a

aaa







4 Discussion



3MeOH




3 CH3OH


3MeOH (2 1)































Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








a

a

b

b

dd

c

c0

4

8

12

16

20


Oil content

Relat

ive c

onte

nt o

f oil

Oil content ()

a

aaa







4 Discussion



3MeOH




3 CH3OH


3MeOH (2 1)































Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article growth, fatty acid, and lipid composition...

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