biochemical modulation of lipid pathway in … › bb95 › 4c1cba22ad25dd34e...of texas at austin,...

Research ArticleBiochemical Modulation of Lipid Pathway in MicroalgaeDunaliella sp for Biodiesel Production

Ahmad Farhad Talebi1 Masoud Tohidfar23 Seyedeh Mahsa Mousavi Derazmahalleh4

Alawi Sulaiman25 Azhari Samsu Baharuddin6 and Meisam Tabatabaei23

1Microbial Biotechnology Department Semnan University Semnan 35131-19111 Iran2Microbial Biotechnology and Biosafety Department Agricultural Biotechnology Research Institute of Iran (ABRII)Karaj 31359-33151 Iran3Biofuel Research Team (BRTeam) Karaj 31438-44693 Iran4Genomics Department Agricultural Biotechnology Research Institute of Iran (ABRII) Karaj 31359-33151 Iran5Tropical Agro-Biomass (TAB) Group Faculty of Plantation and Agrotechnology Universiti Teknologi MARA (UiTM)40450 Shah Alam Malaysia6Faculty of Engineering Universiti Putra Malaysia (UPM) 43400 Serdang Malaysia

Correspondence should be addressed to Azhari Samsu Baharuddin azharisengupmedumy andMeisam Tabatabaei meisam tabyahoocom

Received 31 October 2014 Revised 7 January 2015 Accepted 8 January 2015

Academic Editor Ximing Zhang

Copyright copy 2015 Ahmad Farhad Talebi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Exploitation of renewable sources of energy such as algal biodiesel could turn energy supplies problem around Studies on a locallyisolated strain of Dunaliella sp showed that the mean lipid content in cultures enriched by 200mg Lminus1 myoinositol was raisedby around 33 (15 times higher than the control) Similarly higher lipid productivity values were achieved in cultures treatedby 100 and 200mg Lminus1 myoinositol Fluorometry analyses (microplate fluorescence and flow cytometry) revealed increased oilaccumulation in the Nile red-stained algal samples Moreover it was predicted that biodiesel produced from myoinositol-treatedcells possessed improved oxidative stability cetane number and cloud point values From the genomic point of view real-timeanalyses revealed that myoinositol negatively influenced transcript abundance of AccD gene (one of the key genes involved in lipidproduction pathway) due to feedback inhibition and that its positive effectmust have been exerted through other genesThefindingsof the current research are not to interprete that myoinositol supplementation could answer all the challenges faced in microalgalbiodiesel production but instead to show that ldquothere is a there thererdquo for biochemical modulation strategies which we achievedincreased algal oil quantity and enhanced resultant biodiesel quality

1 Introduction

Commercial and industrial microalgae cultivation is of grow-ing interest for numerous applications including productionof food fertilizer bioplastics and pharmaceuticals as well asalgal fuel [1 2] Among algal species themicroalgaDunaliellaspp are known as the best commercial natural source for theproduction of cis-120573-carotene Besides several biochemicaland genetical engineering investigations have demonstratedthat different species of Dunaliella spp are capable of accu-mulating significant amounts of valuable compound such

as proteins glycerol and also lipids [3] As a result theyare not only a promising feedstock for biofuel productionfor example biodiesel [4] but also could potentially beused for different biotechnological processes such as foreignproteins expression and 120573-carotene production at industriallevel [5] In order to fully and economically exploit theseorganisms however some existing barriers for their large-scale cultivation should be overcome On such basis low costand high quality biomass production is of great significanceTo achieve such goal for biodiesel production for instancethe following strategies could be taken into consideration in

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 597198 12 pageshttpdxdoiorg1011552015597198

2 BioMed Research International

order to boost biomass productivity (BP) and volumetric lipidproductivity (LP) [6]

(A) Introduction of new genes into algal cells by imple-mentation of genetic engineering techniques stimulates somekey metabolic pathways and consequently improves theenergy production phenotypes in green microalgae [7] andultimately produces biofuel at lower cost However despitethe fact that the underlying principles laid out in this strategyare reasonable there has been little achievement made todate For instance the very first genetic engineering attemptsin order to increase microalgal lipid content (LC) by upreg-ulating the first major steps of fatty acid synthesis throughoverexpression of acetyl-CoA carboxylase (ACCase) andmalic enzyme were to some extent effective (12 increase inLC) [8] Blocking-off competing pathways may also enhancethe lipid accumulation in the cells In an attempt Picataggioand coworkers [9] blocked 120573-oxidation in C tropicalis byknocking out the genes encoding acyl-CoA oxidase Despitetheir expectation it was observed that the growth of the cellswas adversely affected Therefore enhanced lipid accumula-tion was rarely reported through overexpression of relevantenzymes andor intermediate products such as FAs becauseof emerging a secondary rate-limiting step Overall given theexisting obstacles facing gene transformation projects suchas biosafety rules correct introduction and maintenance oftransgenes this strategy should not be considered as the firstresort

(B) The second strategy is the optimization of nutrientformulations [10 11] There have been reports that devel-opment of optimum growth condition variables [12 13] hasincreased the lipid pool in some microalgal strains [14 15]Provision of biogenic elements mainly nitrogen is one ofthe main factors affecting algal metabolism The change inthe carbonnitrogen ratio in a media is known to result in achange in the metabolism direction In many algae speciesincreases in this ratio could contribute significantly to theaccumulation of neutral lipids mainly triacylglycerol [16ndash18]On the other hand unfortunately the conditions requiredfor optimal production of algal biomass are different fromthose of lipid production consequently decreasing the costthrough growth condition optimization approaches is notonly time- andmoney-consuming but also inmost cases willnot significantly improve lipid accumulation [16]

(C) The last strategy would be biochemical engineeringapproaches in which a variety of different plant growthregulators (PGR) could be used to support cell growthIt is generally assumed that the genetic background of arespective algal species would determine the compositionof the produced lipids but the lipid amount is mainly aresponse to the growth conditions [19] To channel metabolicflux generated in photobiosynthesis into lipid biosynthesisimplementation of somePGRs vitamins and lipid precursorscould lead to an increase in total catabolism activation andlipid accumulation

Myoinositol as one of the nine stereoisomers of inositolis classified as a member of the vitamin B complex and isrequired for the cell growth as well as other significant bio-logical processes Myoinositol was first used by Jacquiot [20]in order to favor bud formation and retard necrosis in elm

when supplied at 20ndash1000mg Lminus1 however the proliferationof the callus was not improved Letham [21] reported thatmyoinositol acts as second messengers in the primary actionof auxins in plants and its interaction with cytokinin stimu-lates cell division in carrot phloem explants [22] Moreoverinositol is also known as a precursor for phospholipids suchphosphatidylinositol (PI) in the cells In an investigation itwas found out that inositol addition into the growth mediaof yeast led to changes in transcript abundance of over 100genes namely the UASino-containing genes Many of thesegenes encode enzymes involved in lipid metabolism [23 24]For instance in yeast the expression of ACCase gene a keygene in the synthesis of long chain fatty acids is stimulatedby inositol and choline [25]

The present study was set to investigate myoinositol-drivenmodulation of LP and quality (fatty acids (Fas) profile)in cultures of local Dunaliella sp strain isolated from thenorth coast of PersianGulf Nile red staining usingmicroplatefluorescence reading as well as epifluorescent microscopicand flow cytometer analyses was used to monitor the effect ofthemyoinositol supplementation on the algal cellsMoreoverreal-time PCR analysis was performed to look into the impactof myoinositol on one of the key genes involved in lipidsynthesis

2 Methods

21 Strains Cultivation A marine strain (Dunaliella sp) iso-lated from Bandar Lengeh a port city on the northern coastof the Persian Gulf was used in this study Green colonieswere transferred into new flasks containing a media namedLake Media and was kept at 20∘C and a constant (24 0)3klux photon flux of white and red LED lamps [27] Theingredients of Lake Media developed during the course ofthe present study are included Lake salt sediment (60 g Lminus1)NPK fertilizer (2 g Lminus1) and FeSO

4

(005 g Lminus1) The mediarsquospH was set at 75 and the samples were constantly shaking at120 rpm

Beside the strain locally isolated in this study one otherlocal strain D salina (generously provided by Dr ShariatiIsfahanUniversity) and two standard stains CCAP 1918 andUTEX 200 purchased from the Culture Collection of Algaeand Protozoa (Sams Research Scotland) and Universityof Texas at Austin (Austin USA) respectively were alsoincluded in the experiments All the strains were cultivatedin the Lake Media under the above-mentioned conditionsDuring the cultivation period growth kinetic parameterswere recorded for all the strains in triplicateData comparisonwas then carried out using the ANOVA test The calculatedgrowth parameters included BP LC and LP (mg Lminus1 dayminus1)For BP determination algal suspensions were centrifuged(3000 g 10min) and the wet weights were determined gravi-metrically LP was calculated according to the followingequation

LP = BP times LC (1)

Besides to investigate the effect of myoinositol inclusionon lipidmetabolismandbiodiesel properties only the Persian

BioMed Research International 3

Gulf strain was used and cultured in the Lake Media supple-mented with 0 50 100 and 200mg Lminus1 myoinositol

22 Molecular Identification Isolation of total DNA con-tent from the studied strains was carried out by usingthe DNeasy plant minikit (QIAGENE Germany) Species-specific oligonucleotides namely MA1 and MA3 (withoutany restriction site) corresponding to the conserved regionsof 51015840 and 31015840 termini were used to amplify 18S rDNAgene PCRreactions were performed according to themethod describedby Olmos and coworkers [26] The molecular weights ofPCR-amplified productswere calculated and confirmedusinga gel documentation system PCR amplicons were purifiedusing the PCR purification kit (Roche) according to themanufacturerrsquos instructionsThen the purified products weresequenced by Macrogen Company (Korea) Using BLASTsoftware the obtained sequences were compared with thosedeposited in NCBI GenBank as 18S rDNA and ITS regions ofdifferent Dunaliella species

A neighbor-joining tree was constructed using the soft-ware MEGA version 4 Evolutionary distances were com-puted using the maximum composite likelihood modelFor analysis 1000 bootstrap replicates were performed toassess the statistical support for the tree Phylogenetic studiesincluded Chlamydomonas pumilio as the outgroup

23 RNA Extraction Reverse Transcription and Real-TimePCR Analysis To extract RNA from algal cells 50mg wetbiomass (28-day old culture) was harvested by centrifugationat 2000timesg for 10min Separated algae cells inmicrotube weredisrupted by glass rod in liquid nitrogen then 500120583L TRIzolreagent (Invitrogen Carlsbad CA USA) was added andRNA was extracted according to the manufacturerrsquos instruc-tions Concentrations and purity of the total RNA extractedwere measured spectrophotometrically The extracted RNAswere treated with DNase to eliminate genomic DNA contam-ination and continuously reverse transcription (RT) was car-ried out usingQuantiTect Reverse Transcription kit (Qiagen)Real-time PCR was performed using a Bio-Rad iCycler iQreal-time PCR

The first rate-limiting step in the FA biosynthesis pathwaywas regulated by ACCase [29] Since AccD subunit over-expression leads to boosted ACCase activity the effect ofmyoinositol on AccD transcript accumulation was studiedGene-specific primer pairs of AccD and a housekeeping geneused for PCR are listed in Table 5 The 18S rRNA transcriptwas used to normalize the results by eliminating variationsin the quantity and quality of mRNA and cDNA A reactionmixture for each PCR runwas prepared with the SYBRGreensupermix (Bio-Rad) The cycle parameters consisted of onecycle of 100 s at 95∘C and then 35 cycles of 30 s at 95∘Cfollowed by 30 s at 62∘C and 20 s at 72∘C Data were collectedat the end of each extension stepThe relative quantification ofgene expressions among the treatment groups was analyzedby the 2minusΔΔCt method [30] where Ct is the cycle numberat which the fluorescent signal rises statistically above thebackground

24 Fluorescent Measurement of Microalgal Neutral LipidsThe intracellular neutral lipid distribution in microalgal cellswas examined by staining the cells harvested from 500120583Lcell suspension with 300 120583L working solution (1120583M) ofNile red fluorescent dye (Sigma-Aldrich St Louis MO)diluted in Hanks and 20mM Hepes buffer (HHBS) pH 7The stock solution was first prepared by dissolving Nile redin anhydrous DMSO (1mM) The cells were incubated at37∘C for 10min and protected from light To remove theNile red working solution from the cells the cells werewashed and resuspended in HHBS Cells were examined byan epifluorescent microscope Leica DMRXA with a Nikon(DXM 1200) digital camera (Nikon Tokyo Japan) with anexcitation wavelength of 486 nm the emission was measuredat 570 nm following the method of Cooney et al [28]

For fluorescence-based quantification of the accumulatedlipids a high-throughput technique reported by Chen andcoworkers [31] was followed with some modifications Thebase procedure was performed in two models (1) measuringtotal emitted fluorescence by staining same volume of cellsuspension for different treatments (cells at the lag stationaryphase) which would quantify total produced lipid volu-metrically (2) measuring emitted fluorescence by stainingsame cell number (105 cells) for different treatment whichwould quantify stored lipid in cells Both procedures involvedstaining the cell suspensions with Nile red as mentionedabove Fluorescence was measured on a Varian 96-well platespectrofluorometerThe excitation and emission wavelengthsof 522 and 628 nm were selected based on a previouslypublished report [28]

25 Flow Cytometry Study To figure out the duration afterwhich myoinositol supplementation would lead to an im-proving effect on lipid synthesis flow cytometry analysis wasused To investigate that 7- and 35-day cell suspensions atlinear growth phase and lag stationary phase respectivelywere analyzed Cells were stained with working Nile redsolution as explained earlier The optical system used inthe EPICS XL flow cytometer collects yellow light (575band-pass filters) in the FL2 channel corresponding to theNile red fluorescence in a neutral lipid matrix To removenonalgal particles chlorophyll fluorescence characteristicswere considered Approximately 10000 cells were analyzedusing a log amplification of the fluorescence signal Unstainedcells were used as autofluorescence controlThe data usedwasthe arithmetic mean of all cytometric events (10000 cells) in3 repeats and two independent experiments [32]

26 Oil Extraction and Fatty Acid Profile by Gas Chromatog-raphy (GC) Analysis All cultures by three replications wereallowed to grow for 35 days to reach the lag stationary phaseand then cells were harvested for lipid profile analysis By thismeans the effects of growth phase on the total LC and FAprofile were minimized [28] LC reported as percentage ofthe total biomass (dwt) was determined based on the Blighand Dyer method [33] and was obtained in triplicate for thedifferent strains Data comparison was then carried out usingthe ANOVA test


Dunaliella tertiolecta strain CCMP 1320 Dunaliella tertiolecta strain DtsiDunaliella sp ABRIINW-Sh63Dunaliella sp ABRIINW-G4Dunaliella sp ABRIINW-G3Dunaliella sp ABRIINW U11Dunaliella sp ABRIINW M11Dunaliella peircei strain UTEX LB 2192Dunaliella salina strain CCAP 1918

Dunaliella sp ABRIINW M12Dunaliella sp ABRIINW-Ch31

Dunaliella salina strain Persian Gulf Dunaliella parvaDunaliella sp ABRIINW Q11Dunaliella sp ABRIINW-S15

Dunaliella bardawilDunaliella viridis strain CONC002

Chlamydomonas pumilioEttlia carotinosa strain SAG 213-4

100

97

68

100

42

92

0005

Dunaliellaceae

Figure 1 NJ bootstrap consensus tree showing the relationships among Dunaliella salina (Persian Gulf) and other standard and Iranianstrains Bootstrap values were calculated over 1000 replicates Chlamydomonas pumilio and Ettlia carotinosa were considered as outgroups

For GC analysis the direct transesterification methodwas used based on the procedure reported by Lepage andRoy with minor modifications [34] The samples containinghexane and FAME were used for GC analysis The FAsdetermination was carried out on a Varian CP-3800 GC(Varian Inc Palo Alto CA) equipped with a CP-Sil88 fusedsilica column (100m 025mm ID film thickness 025120583m)The oven temperature was programmed as our previousreport [6] Fatty acid peeks were identified by comparison ofthe retention time with FAME standards

27 Biodiesel Properties by Bioprospection Biodiesel qualityparameters that is oxidation stability cold performance(cloud point) and combustion characteristics (cetane num-ber) were estimated based on the fact that they are directlyinfluenced by the molecular structure of FAME like thecarbon chain length and the amount andor position ofdouble bonds [35] Oxidation stability parameters estimatedwere iodine value (IV) APE and BAPE All these parameterswere calculated based on the FAME profile using empiricalequations as detailed in previous report [36]

3 Result and Discussion

31 Identification Molecular identification and discrimina-tion of living eukaryotic organisms based on the comparisonof 18S rDNA gene sequences are a promising method andhave been frequently used for molecular identification of dif-ferent species of Dunaliella [26 37 38] Hereby the chromo-somal DNA of 18S using the forward primerMA1 and reverseprimer MA3 was amplified A single band representing

the amplified DNA product of 17 kb was recorded whichcould be used in the intron sizing method for the identifi-cation of Dunaliella genus [39] The sequence was alignedwith 18 different strains whose 18S sequences were previouslysubmitted at NCBI The 18S sequence of the Dunaliella spPersian Gulf strain was registered in NCBI database with anaccession number of KF477384This sequence exhibited highsimilarities to other members of Dunaliellaceae The highestsimilarity was observed withDunaliella sp ABRIINWQ11 at98 which was isolated from an ancient saline lake locatedin the middle of Iran plateau (Qum Salt Lake) This findingcoupled with themorphological features of the isolated strain(Figure 1) confirmed that the saline water isolate strain is amember of Dunaliella genus The cells lack a cell wall anda well-developed apical papilla and two equal-long (250ndash300 120583m) and smooth flagella also uphold this identification

As for the size number and position of introns of the18S rDNA gene in the Dunaliella sp three types of 18SrDNA structure have been reported by Olmos et al [40] no-intron genes with a size of sim1770 bp one-intron gene witha size of sim2170 bp and finally the genes with two intronsand with a size of sim2570 bp Interestingly using intron-sizingmethod in this genus was applied to an indicator for selectionof hyperproducing species [40] Based on this method thePersian Gulf strain belonged to the first group with no intronin the 18S rDNAgeneThis strain never turned red indicatingthat 120573-carotene was not hyperproduced by this strain Inorder to properly explore the similarity observed among theobtained 18S sequences and moreover to determine theirphylogenetic relationship a phylogenetic tree was recon-structed using the neighbor-joining (NJ) method NJ resultalong with the bootstrap coefficients (replication times1000) is


Table 1 Biomass productivity lipid content and lipid productivity of the microalgae strains

Strains

Parameters

Biomass productivity(BP g Lminus1 dayminus1) Lipid content (LC dwt)lowast

Volumetric lipidproductivity Pb times LC times

1000(LP mg Lminus1 dayminus1)

Dunaliella salina (Shariati) 005A 189 plusmn 11A 1026 plusmn 04A

D salina (UTEX) 015C 24 plusmn 13B 3648 plusmn 06C

D salina (CCAP1918) 014B 251 plusmn 07B 3514 plusmn 02C

Dunaliella sp (Persian Gulf) 015C 22 plusmn 2B 33 plusmn 03B

1 014B 25 plusmn 05C 3615 plusmn 09C

2 014B 27 plusmn 05C 386 plusmn 04D

3 012B 33 plusmn 1D 393 plusmn 06DlowastAll cultures harvested after reaching the stationary phase and LC was determined based on the Bligh and Dyer method [33] Data are expressed as mean plusmnSD (119899 = 3) Means of BP LC and LP are compared using one-way ANOVA and ones with different letter are significantly different (at 119875 lt 005)1 2 and 3 representing 50 100 and 200mg Lminus1 myoinositol implementation in Persian Gulf strain respectively

depicted in Figure 1 In this classification Persian Gulf strainwas under the family of Dunaliellaceae close to Qum strain(Q11) and hypo-120573-carotene producing strains As observedin the figure the Persian Gulf strain is situated close toD parva and D viridis which also confirmed the findingof the intron-sizing method since these two strains havelow 120573-carotene production capacity as well but can grow inhypersaline environments [40]

32 Studying the Algal Species Using Growth ParametersSince the intracellular LC and FAs profile of microalgae areaffected by both culture conditions and growth phase all ofthe studied strains were cultivated under the same conditions(flasks containing Lake Media were kept at 20∘C and a con-stant (24 0) 3klux photon flux of white and red LED lamps)Sole influence of myoinositol addition on lipid metabolismwas also investigated under the same conditions as well Allcultures were harvested after reaching the stationary phaseThe results of LC BP and LP have been presented in Table 1

BP value slightly fluctuated for all the strains between012 and 015 g Lminus1 dayminus1 except in case of D salina (Shariati)where significantly lower value of 005 g Lminus1 dayminus1 wasrecorded Myoinositol addition into the Dunaliella sp (Per-sian Gulf) culture caused a constant but nonmeaningfuldecrease in the BP value proportional to the increasingconcentrations of myoinositol The cells grown in the highestconcentration of myoinositol (200mgLminus1) had 20 less BPin comparison with those grown in myoinositol free culture(012 and 015 g Lminus1 dayminus1 resp)

The lowest amount of LC was obtained for D salina(Shariati) followed byDunaliella sp (Persian Gulf) (189 and22 dwt resp) In contrast the highest LC values were alsoachieved for Dunaliella sp (Persian Gulf) when myoinositolwas added to the culture media More specifically the meanvalue of LC for cultures enriched by 200mg Lminus1 myoinositolwas around 33 This record represents 50 increase inoil accumulation in comparison to the control Similarlythe LP values were classified into four significantly differentgroups The highest LP value belonged to the myoinositol

treatment group These findings revealed the efficiency ofbiochemical engineering strategies This was also reportedin another biochemical modulation study on the algae Csorokiniana using 01 tryptophan supplementation Theauthors recorded 5728 enhancement in LP just 4 days afterthe treatment [41] Their findings as well as those of thepresent study could in a way confirm the promising role ofbiochemical modulation when combined with selection ofproper strains in enriching lipids quantity and consequentlyin biodiesel production

Overall LP as an indicator of the produced oil in termsof both volume and time showed a sharp increase aftermyoinositol addition This parameter has been reported asa suitable variable to evaluate algal species potential forbiodiesel production [42] This would highlight the positiveimpact of myoinositol on increasing algal potential for pro-ducing biodiesel

33 Integrated Growth and Lipid Production Using a NovelAlgal Media Generally a two-stage cultivation is consideredfor algal lipid production growth stage in which high Nconcentration is used to achieve the highest possible BPfollowed by the second stage where N-starvation is imposedto encourage lipid production [8] James and coworkers [43]observed that when nitrogen was deprived for 4 days LCincreased for all the studied strains of C reinhardtii In theirstudy using such 2-stage cultivation strategy the total FAsof the wild-type strains increased 13- and 14-fold Howeverthis would increase the cost and consequently deterioratesthe economic aspect of the algal fuel production scenarioin comparison with single-stage cultivation On the otherhand it has been well documented that algal cultivation ina media with decreased nitrogen content results in a declinein biomass content [44] This is ascribed to the fact thatN-starvation leads to decreased duration of the exponentialgrowth phase [45] Therefore providing high N contentthroughout the cultivation period while encouraging lipidproduction through other alternatives could play a significantrole in achieving an economic algal biodiesel In light of that


Table 2 Real-time PCR analysis of gene expression Values were normalized against 18S rRNA as housekeeping gene and represent therelative mRNA expression (mean standard error) of three replicate cultures

Treatment CTAccD CT 18S ΔCTtreat ΔCTcontrol ΔΔCT 2minusΔΔCt

Control 267 plusmn 046 212 plusmn 11 minus55 mdash mdash mdash50mg 2719 plusmn 033 1938 plusmn 05 minus781 minus231 02100mg 2635 plusmn 041 1887 plusmn 085 mdash minus748 minus198 025200mg 3027 plusmn 062 2243 plusmn 072 mdash minus783 minus233 02

in the present study a newmedia named LakeMedia capableof encouraging high BP was formulized using 2 g Lminus1 of NPKfertilizer as a nitrogen source and a considerable quantity ofnatural lake salt (60 g Lminus1) By considering the different cost ofby-product by Dunaliella sp grown in various batch culturemedia it is obviously clear that implementation of newlydeveloped media in this research Lake Media provides agolden opportunity in large-scale cultivation of this microal-gae and final production economically In another study onthis media implementation of Lake Media declined the costsof carotenoid production to just 00029USD mgminus1 which is200 lower than the standard media (Johnson and OlmosMedia) and it compensates the lower cell density obtained byLake Media (unpublished data)

At the same time andwith simultaneity of BP increase LCwas also successfully encouraged in thismedia bymyoinositolinclusion as lipid precursor As a result the normally usedbiphasic algal cultivation and lipid production were simpli-fied into a single-phased process in which the combination ofthe Lake Media and myoinositol met all the requirements ofthe cells for growth and lipid synthesis simultaneously Thisstrategy could lead to decreasing the final cost of producedbiodiesel

34 Impact of Myoinositol on Acetyl-coA Carboxylase AKey Gene in Lipid Synthesis Quantification of the relative-fold change in mRNA levels of the AccD gene 28 daysafter myoinositol supplementation in the treated sample incomparison with the control group was conducted using thereal-time PCR analysisThe 2minusΔΔCt value decreased from 025for the 100mg Lminus1 myoinositol to 02 for 200mg Lminus1 As pre-sented in Table 2 AccD gene exhibited decreasing responsestomyoinositol supplementation In fact myoinositol resultedin a 75ndash80 decrease in AccD transcript abundance ascompared to the control sample This decrease could beexplained as follows supplementation of myoinositol asa lipid precursor caused an increase in lipid productionand accumulation and this in turn resulted in a feedbackinhibition downregulation of the genes involved in lipidsynthesis including AccD This explanation is in line with thefindings of Al-Feel who investigated the impact of inositolsupplementation on lipid production while monitoring theresponse of another key gene involved in lipid productionpathway acetyl Co-A carboxylase (ACC

1

) [25]They reportedthat ACC1 was repressed due to inositol supplementation butreturned to near basal expression level by steady state

Therefore it could be concluded that inositol and itsstereoisomer myoinositol exert their positive effect on lipid

production through other genes involved in lipid productionpathway and not the AccD and ACC1 genes In case ofthe AccD gene this could also be confirmed by the factthat inositol stimulates transcription of a series genes whichhave a conserved domain in their promoter called UASino(inositol-sensitive upstream activating sequence) elementThis sequence is absent in the AccDrsquos promoter As for theACC1 despite the presence of this element moderate varia-tion in the expression of this gene by inositol supplementationwas reported [24 46]

On the other hand based on the model presented byThomas and Fell concerning regulation of enzymatic path-ways many enzymes are involved in controlling the rate ofa reaction and alteration of one alone may have a smallimpact [47] Therefore one could point out that myoinositolcould have influenced many metabolic processes rather thantargeting a single enzyme in a pathway and as a resultincreased the total lipid accumulation However increasedlipid production and accumulation and consequent feedbackinhibition resulted in the downregulation of the key genesinvolved in lipid production pathway that isAccD andACC1

Such hypothesis could be supported by the findings ofa number of studies revealing that the extent of involvedgenes upregulation was not reflected in the fatty acid (FA)profilecontent [48]

35 Proposed Molecular Mechanisms for Lipid Increase byMyoinositol Supplementation In the present study the totallipid accumulation was sharply increased by 50 in responseto myoinositol implementation which could be attributed tomyoinositol role in simultaneously increasing lipid storageand membrane lipids [49] Previously a survey on Saccha-romyces cerevisiae showed a swift 5-6-fold increase in cellularmembrane phosphatidylinositol (PI) content in responseto inositol inclusion This rise in PI content seems to bepositively correlatedwith FA synthesis [50]More specificallyinositol leads to higher production of negatively chargedPI and consequently lower negative surface charge of themembrane On the other hand the distribution of long-chainacyl-CoA molecules in membrane and activity of ACCaseare both controlled by negative surface charge and thereforemyoinositol supplementation would increase the capacity ofthemembrane for incorporation of long-chain acyl CoAs andconsequently enhance the cellular FFA synthesis [51]

Although mechanisms describing the growth-promotingeffect ofmyoinositol on algal cell have not been described pre-cisely their growth-promoting effects through plant growthregulators (phytohormones) have been previously pointed


Cell wall

PI

PI

PI

Plasma membraneFFAs

FAS

ACCase products

ACCase

Phosphatidylinositol

(1) Stimulating the transcriptionof UASino harboring genes

(2) Auxin related responses

(3) Negatively charged membrane byPI higher FA accumulation capacity

Myoinositol Auxin

MI

MI

NucleusACC

Figure 2 Proposedmechanisms for the lipid-promoting effects of myoinositol (MI) in algal cells (1) through stimulating the transcription ofthe responsive genes (eg ACC) harboring inositol-sensitive upstream activating sequence (UASino) element in their promoters which couldin turn positively impact FA synthesis (FAS) (2) through auxin-related responses and (3) by increasing membrane negative charge regulatedby phosphatidylinositol (PI)

out [52 53] In fact stimulation of signaling pathways byphytohormones plays a vital role in plant responses to envi-ronmental changes Among phytohormones auxin which isinvolved in plant growth and development [54] is regulatedby the concentration of phosphoinositides which are mainlysynthesized from myoinositol [50] In a study Arroussi andcoworkers managed to increase initial lipid content of Dtertiolecta (24) to 38 and 43 after addition of auxinat 05 and 1mgL respectively [55] Therefore one possibleexplanation for the lipid-promoting effect of myoinositol inD salina could be attributed to the action of auxins More-over myoinositol plays a key role in cell membrane chargealteration (by PI) which could consequently increase themembrane capacity for FFAs accumulation Finallymyoinos-itol also stimulates the transcription of the responsive genes(eg ACC) harboring UASino element in their promoterwhich could in turn positively impact FA synthesis (FAS)The proposed mechanisms for the lipid-promoting effects ofmyoinositol are presented in Figure 2

36 FluorescenceMicroscopic Study of Neutral Lipids Nile redhas been widely used to screen wild and mutant variationto explore new candidates for biodiesel production frommicroalgae to dinoflagellate [56 57] In this study theliposoluble fluorescence probe Nile red was used to visualizeneutral lipids in the cells As shown in Figure 3 the lipid

bodies appear as yellow fluorescing circular organelles whilethe red background fluorescence is attributed to chlorophyllautofluorescence The highest LC as determined by Nilered staining was observed in cells treated by 200mg Lminus1myoinositol In these cells small drops of neutral lipidswere seen dispersed throughout the cytoplasm (Figure 3)while cells with no myoinositol addition in the media werecomparatively poor in terms of neutral LC during the lagstationary phase and showed limited number of small lipidbodies in the cells

37 Quantitative Fluorescence Measurement of Neutral Lipids

371 Microplate Fluorometry Result of total lipid measure-ment using microplate fluorometryin the same volume of thecell suspensionwas summarized in Figure 4Thefluorescenceintensity dramatically increased in the samples treated bythe increasing amount of myoinositol More specificallyimplementation of 100mg Lminus1 myoinositol sharply increasedthe recorded fluorescence by 59 and further by 152for 200mg Lminus1 myoinositol in comparison with that of thecontrol All treatments were significantly different (at 119875 lt001)

Effect of cell concentration on the fluorescence of neutrallipids was also taken into consideration by staining the samecell concentration (105 cells mLminus1) for all the investigated


(a) (b)

(c) (d)

Figure 3 Epifluorescent microphotographs (magnification times40) of microalgae stained with fluorochrome Nile red Neutral lipids in cellsare seen as lighter colored drops (a) Bright field image and fluorescence image (b) control (c) 100mg Lminus1 myoinositol supplementation(d) 200mg Lminus1 myoinositol supplementation Microphotographs were taken using a Leica DMRXA compound light microscope with aNikon (DXM 1200) digital camera a band-pass filter with an excitation range of 450ndash490 nm and a long-pass suppression filter with anedge wavelength of 515 nm

samples tomeasure the relative amount of lipid stored in samenumber of cells Similar to the results of total lipid measure-ment in cell suspensionsmyoinositol was proven to stimulatesingle cells to accumulate more lipid in their cytoplasm200mg Lminus1 myoinositol implementation led to 284 raise inthe emitted fluorescence A significant difference between thethree studied levels of myoinositol (50 100 and 200mg Lminus1)could be seen in the same cell number mode (120 increasein fluorescence emission for 100 and 200mg Lminus1) This wasalso confirmed by total extracted lipid data based on which50 100 and 200mg Lminus1 myoinositol supplementation caused13 23 and 50 increase in LC respectively

372 Flow Cytometry Flow cytometry in conjunction withmicroplate fluorometry represents an invaluable tool forscreening and exploiting high lipid-producing microalgaestrains In this study lipid accumulation was studied usingflow cytometry 7 and 35 days after myoinositol supplementa-tion The results obtained revealed that on day 35 the meanfluorescence for the control was 1996 whereas this valuewas at 22565 2636 and 2831 (13 32 and 42 increase incomparison with the control) for the cells treated by 50 100and 200mg Lminus1 myoinositol respectively

Moreover the effect of exposure time to myoinositol andits relation with cell growth phase and lipid accumulationwere also studied by comparing the mean fluorescence ondays 7 and 35 The results clearly confirmed that the positiveeffect of myoinositol supplementation on lipid production isvisible in the stationary phase when algal cells have alreadycompleted their growth or in other words the emittedfluorescence values recorded in the young algal cells (on theday 7) were not significantly different among the treatments(Figure 5)

Overall there was a clear correlation between the fluo-rometry (microplate fluorescence and flow cytometry) resultsand those of LC gravimetrical determination (Figures 4 and5 and Table 1) Therefore fluorometry techniques could besuggested as powerful and high-throughput alternative ana-lytical tools to monitor the effect of chemicals and biologicalmolecules on lipid production and accumulation in algalcells

38 The Role of Myoinositol Treatment on Algal FAs ProfilesFatty acid methyl ester (FAME) profiles for different algalstrains studied in this study are summarized in Table 3 Ithas been frequently reported that 16ndash18 carbon chain FAs are


Table 3 Types of fatty acids produced and properties of algal oil

Strain Fatty acid () SFA MUFA PUFA SFAUSFA16 0 16 1 18 0 18 1 18 2 18 3 20 1

Dunaliella sp(Persian Gulf) 919 plusmn 12 080 plusmn 08 427 plusmn 09 2251 plusmn 07 384 plusmn 04 4431 plusmn 21 142 plusmn 02 1347 2474 4815 016

D salina (Shariati) 1202 plusmn 21 445 plusmn 02 191 plusmn 12 2367 plusmn 16 228 plusmn 11 4036 plusmn 22 140 plusmn 02 1393 2952 4265 016D salina(UTEX200) 1634 plusmn 14 104 plusmn 09 643 plusmn 12 1958 plusmn 11 676 plusmn 12 2771 plusmn 25 228 plusmn 03 2277 2289 3447 028

D salina(CCAP1918) 1587 plusmn 18 ND+ 614 plusmn 13 2139 plusmn 21 1592 plusmn 16 2395 plusmn 19 ND 2201 2139 3987 026

1lowast 705 plusmn 09 625 plusmn 03 155 plusmn 07 2295 plusmn 08 1215 plusmn 03 3766 plusmn 04 112 plusmn 06 860 3032 4981 0102lowast 775 plusmn 06 504 plusmn 08 142 plusmn 05 2527 plusmn 19 1858 plusmn 14 2691 plusmn 19 ND 917 3031 4548 0113lowast 841 plusmn 08 452 plusmn 11 214 plusmn 03 3203 plusmn 22 1358 plusmn 13 2724 plusmn 14 NDminus 1055 3655 4082 012lowast1 2 and 3 representing 50 100 and 200mg Lminus1 myoinositol implementation in Persian Gulf strain respectively+Not detectedminusNon identified Fas which are around 10

05000

10000150002000025000300003500040000

w 50 100 200

Fluo

resc

ence

inte

nsity

TreatmentsCminus C+

Figure 4 Fluorescence emission of Nile red-stained microalgaeThe excitation and emission wavelengths for fluorescence measure-ment were at 522 and 628 nm respectively The cell density of thesuspensions used for analysis was 105 cellmLminus1 Nile red stainingwasconducted based on the procedures described by Cooney et al [28]Data were the mean values of three replicates (vertical dashed linessame volume horizontal dashed lines same cell number) Cminus andC+ represent nonstained and stained cell with no myoinositolinclusion respectively

150170190210230250270290310330

C 50 100 200

Cyto

met

ric F

L2 si

gnal

Myoinositol concentration (mg Lminus1)

Figure 5 Variation of cytometric signal (FL2 yellow fluorescence120582 =575 nm) in cells stainedwithNile red (Dunaliella sp)Horizontaland diagonal bricks represent the sample treated by myoinositol for35 and 7 days respectively

Table 4 Comparison of the estimated properties of algal biodieselfrom cells treated with myoinositol

Strains Biodiesel propertiesCN CP BAPE APE

Dunaliella sp (Persian Gulf) 4375 minus016 9247 11882D salina (Shariati) 4565 133 8301 10896D salina (UTEX200) 5536 360 6217 8851D salina (CCAP1918) 5296 336 6383 101141lowast 4227 minus128 8747 122572lowast 4761 minus091 7239 116233lowast 4716 minus057 6806 11366lowast1 2 and 3 representing 100 and 200mg Lminus1 myoinositol implementation inPersian Gulf strain respectively

dominant in algal cells [6 28 58] The same observation wasmade in this study as well In all the strains investigated theomega-3 fatty acid linolenic acid (18 3) made up the highestportion of the FAME profile Persian Gulf strain grown inthe Lake Media with no myoinositol enrichment was foundto have the highest percentage for this FA (gt44) while thelowest record for 18 3 of around 26 was also observedin the same strain but in presence of myoinositol On thecontrary myoinositol inclusion (200mg Lminus1) led to increasedpercentages of monounsaturated FAs (MUFA) that is palmi-toleic acid (16 1) and oleic acid (18 1) by 46- and 04-folds respectively Moreover in case of linoleic acid (18 2)low concentration of myoinositol (100mg Lminus1) considerablyincreased percentage of this FA while 200mg Lminus1 myoinos-itol implementation led to a significant decrease in 18 2accumulation Overall MUFA were increased continuouslyby myoinositol addition in the media while PUFA were feltdown vice versa

These findings were similar to those of studies in whichN-starvation was applied to enhance lipid accumulationin microalgae Gurr and Harwood [59] reported relativeaccumulations in 18 1 and 18 2 accompanied by a decrease


Table 5 Sequences of primer pairs used in real-time PCR

Primer name Sequence18S rRNA (forward) 51015840-CAGACACGGGGAGGATTGACAGATTGAGAG-31015840

18S rRNA (reverse) 51015840-GCGCGTGCGGCCCAGAACATC-31015840

AccD (forward) 51015840-AAGACGCACAAGAACGAACAG-31015840

AccD (reverse) 51015840-AACTACAGAGCCCATACTTCCC-31015840

in 18 0 acidwhenN-starvationwas usedThey explained thatN-starvation promoted the desaturation pathways beginningwith delta-9 desaturase In a similar study using N-starvationon Botryococcus braunii an increase in the content of SFAs(up to 768) and a substantial decrease in the PUFA content(up to 68) were observed [19] It could be concluded thatmyoinositol might also encourage the desaturation pathways

39 Estimation of Biodiesel Properties The impact of myoin-ositol on key biodiesel quality parameters such as allylicposition equivalents (APE) and bisallylic position equivalents(BAPE) cetane number (CN) and cloud point (CP) wasalso investigated The BAPE and APE values are effectivemeans of predicting oxidative instability of biodiesel Thehighest BAPE and APE values were measured for the controlPersian Gulf (no myoinositol addition) while the lowestvalues were recorded for UTEX200 strain (Table 4) Thiscould be explained by the highest and lowest levels ofunsaturated FAs in particular 18 3 in the control PersianGulf and UTEX200 strain respectively A decreasing trendfor BAPE and APE values and consequently higher oxidativestability were observed when algal cells (Persian Gulf strain)were treated bymyoinositol For instance BAPE decreased by26 at myoinositol concentration of 200mg Lminus1 (Table 4)

CN indicates the combustion quality of diesel fuelsincluding biodiesel In other words the higher this value isthe easier it would be to start a standard diesel engine Anincrease in this parameterwas observedwhere algal cells weretreated by myoinositol Slight improvements in the estimatedCP values were also recorded Overall it was shown thatinclusion of myoinositol led to improved fuel properties

In a different study Ngangkham et al [41] also strived toimprove C sorokiniana oil quality for biodiesel productionbut different treatments from that of this study were appliedIn particular they reported reduced level of PUFA and conse-quent increased oxidative stability of the produced biodieselIn conclusion and based on the findings of Ngangkham et al[41] and those of the present investigation biochemical engi-neering treatments could be regarded as efficient strategiesfor directed improvement of algal oil quality and resultantbiodiesel based on a particular climate condition

4 Conclusion

Thepresent studywas set to evaluate the effects ofmyoinositolon a locally isolated Persian microalgae strainDunaliella spwith a specific focus on LP and biodiesel quality based onFA composition Inclusion ofmyoinositol (200mg Lminus1) in themedia improved the total lipid accumulation (up to 50) and

biodiesel quality parameters that is APE BAPE CN andCP Hypothetically myoinositol treatment led to increasedauxin and PI accumulation in the cells and consequentlymore negatively charged membranesThis in turn resulted inincreased FFA synthesis and lipid accumulation Biochemicalmodulation strategies should still be progressively consideredin hope of finding more efficient and economically feasiblestrategies leading to more viable production systems

Abbreviations

ACCase Acetyl-CoA carboxylaseAPE Allylic position equivalentsBAPE Bisallylic position equivalentsCN Cetane numberCP Cloud pointD DunaliellaFA Fatty acidFAME Fatty acid methyl esterIV Iodine valueLC Lipid contentBP Biomass productivityLP Lipid productivityMSFA Monosaturated FAsMUFA Monounsaturated FAsPGR Plant growth regulatorPI PhosphatidylinositolSFA Total saturated fatty acids

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authorsrsquo appreciation goes to Dr Shariati for providingone of the algal strains used in this study The authorswould also like to thank Biofuel Research Team (BRTeam)and Agricultural Biotechnology Research Institute of Iran(ABRII) for financing this studyThey alsowould like to thankDr S Malekzadeh for his comments on the paper

References

[1] M Y Menetrez ldquoMeeting the US renewable fuel standard acomparison of biofuel pathwaysrdquo Biofuel Research Journal vol1 no 4 pp 110ndash122 2014

[2] F Bux ldquoThe potential of using wastewater for microalgalpropagationrdquo Biofuel Research Journal vol 1 no 4 p 106 2014


[3] E Specht S Miyake-Stoner and S Mayfield ldquoMicro-algaecome of age as a platform for recombinant protein productionrdquoBiotechnology Letters vol 32 no 10 pp 1373ndash1383 2010

[4] K Beetul S Bibi Sadally N Taleb-Hossenkhan R Bhagooliand D Puchooa ldquoAn investigation of biodiesel productionfrom microalgae found in Mauritian watersrdquo Biofuel ResearchJournal vol 1 pp 58ndash64 2014

[5] D R Georgianna M J Hannon M Marcuschi et al ldquoPro-duction of recombinant enzymes in the marine alga Dunaliellatertiolectardquo Algal Research vol 2 no 1 pp 2ndash9 2013

[6] A F Talebi S K Mohtashami M Tabatabaei et al ldquoFatty acidsprofiling a selective criterion for screening microalgae strainsfor biodiesel productionrdquo Algal Research vol 2 no 3 pp 258ndash267 2013

[7] M Tabatabaei M Tohidfar G S Jouzani M Safarnejad andM Pazouki ldquoBiodiesel production from genetically engineeredmicroalgae future of bioenergy in Iranrdquo Renewable amp Sustain-able Energy Reviews vol 15 no 4 pp 1918ndash1927 2011

[8] A F Talebi M Tohidfar A Bagheri S R Lyon K Salehi-Ashtiani and M Tabatabaei ldquoManipulation of carbon fluxinto fatty acid biosynthesis pathway in Dunaliella salina usingAccD and ME genes to enhance lipid content and to improveproduced biodiesel qualityrdquo Biofuel Research Journal vol 1 no3 pp 91ndash97 2014

[9] S Picataggio T Rohrer K Deanda et al ldquoMetabolic engi-neering of Candida tropicalis for the production of long-chaindicarboxylic acidsrdquo BioTechnology vol 10 no 8 pp 894ndash8981992

[10] A F Talebi M Tabatabaei S K Mohtashami M Tohidfarand F Moradi ldquoComparative salt stress study on intracellularion concentration inmarine and salt-adapted freshwater strainsof microalgaerdquo Notulae Scientia Biologicae vol 5 pp 309ndash3152013

[11] G Olivieri A Marzocchella R Andreozzi G Pinto and APollio ldquoBiodiesel production from Stichococcus strains at labo-ratory scalerdquo Journal of Chemical Technology and Biotechnologyvol 86 no 6 pp 776ndash783 2011

[12] S Elumalai and V Prakasam ldquoOptimization of abiotic condi-tions suitable for the production of biodiesel from Chlorellavulgarisrdquo Indian Journal of Science and Technology vol 4 pp91ndash97 2011

[13] D Sasi P Mitra A Vigueras and G A Hill ldquoGrowth kineticsand lipid production using Chlorella vulgaris in a circulatingloop photobioreactorrdquo Journal of Chemical Technology andBiotechnology vol 86 no 6 pp 875ndash880 2011

[14] V H Work R Radakovits R E Jinkerson et al ldquoIncreasedlipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydratesynthesis in complemented strainsrdquo Eukaryotic Cell vol 9 no8 pp 1251ndash1261 2010

[15] N Mallick S Mandal A K Singh M Bishai and A DashldquoGreen microalga Chlorella vulgaris as a potential feedstock forbiodieselrdquo Journal of Chemical Technology and Biotechnologyvol 87 no 1 pp 137ndash145 2012

[16] M Piorreck K-H Baasch and P Pohl ldquoBiomass productiontotal protein chlorophylls lipids and fatty acids of freshwatergreen and blue-green algae under different nitrogen regimesrdquoPhytochemistry vol 23 no 2 pp 207ndash216 1984

[17] W-L Chu S-M Phang and S-H Goh ldquoEnvironmentaleffects on growth and biochemical composition of Nitzschiainconspicua Grunowrdquo Journal of Applied Phycology vol 8 no4-5 pp 389ndash396 1996

[18] G A Thompson Jr ldquoLipids and membrane function ingreen algaerdquo Biochimica et Biophysica Acta Lipids and LipidMetabolism vol 1302 no 1 pp 17ndash45 1996

[19] N O Zhila G S Kalacheva and T G Volova ldquoEffect ofnitrogen limitation on the growth and lipid composition of thegreen alga Botryococcus braunii Kutz IPPAS H-252rdquo RussianJournal of Plant Physiology vol 52 no 3 pp 311ndash319 2005

[20] C Jacquiot ldquoAction of meso-inositol and of adenine on budformation in the cambium tissue ofUlmus campestris cultivatedin vitrordquoComptes Rendus de lrsquoAcademie des Sciences vol 233 pp815ndash817 1951

[21] D S Letham ldquoRegulators of cell division in plant tissuesmdashII Acytokinin in plant extracts isolation and interaction with othergrowth regulatorsrdquo Phytochemistry vol 5 no 3 pp 269ndash2861966

[22] K Watanabe K Tanaka K Asada and Z Kasai ldquoThe growthpromoting effect of phytic acid on callus tissues of rice seedrdquoPlant and Cell Physiology vol 12 no 1 pp 161ndash164 1971

[23] T C Santiago and C BMamoun ldquoGenome expression analysisin yeast reveals novel transcriptional regulation by inositol andcholine and new regulatory functions for Opi1p ino2p andino4prdquoThe Journal of Biological Chemistry vol 278 no 40 pp38723ndash38730 2003

[24] S A Jesch X Zhao M T Wells and S A Henry ldquoGenome-wide analysis reveals inositol not choline as the major effectorof Ino2p-Ino4p and unfolded protein response target geneexpression in yeastrdquo Journal of Biological Chemistry vol 280no 10 pp 9106ndash9118 2005

[25] W Al-Feel J C DeMar and S J Wakil ldquoA Saccharomycescerevisiae mutant strain defective in acetyl-CoA carboxylasearrests at the G2M phase of the cell cyclerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 100 no 6 pp 3095ndash3100 2003

[26] J Olmos J Paniagua and R Contreras ldquoMolecular identifica-tion of Dunaliella sp utilizing the 18S rDNA generdquo Letters inApplied Microbiology vol 30 no 1 pp 80ndash84 2000

[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] M J Cooney D Elsey D Jameson and B Raleigh ldquoFluorescentmeasurement of microalgal neutral lipidsrdquo Journal of Microbio-logical Methods vol 68 no 3 pp 639ndash642 2007

[29] Y Madoka K-I Tomizawa J Mizoi I Nishida Y Naganoand Y Sasaki ldquoChloroplast transformation with modified accDoperon increases acetyl-CoA carboxylase and causes extensionof leaf longevity and increase in seed yield in tobaccordquo Plant andCell Physiology vol 43 no 12 pp 1518ndash1525 2002

[30] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minus998779998779119862119879 methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[31] W Chen C H Zhang L Song M Sommerfeld and H QiangldquoA high throughput Nile red method for quantitative measure-ment of neutral lipids in microalgaerdquo Journal of MicrobiologicalMethods vol 77 no 1 pp 41ndash47 2009

[32] Y-H Lee S-Y Chen R J Wiesner and Y-F Huang ldquoSimpleflow cytometric method used to assess lipid accumulation infat cellsrdquo Journal of Lipid Research vol 45 no 6 pp 1162ndash11672004

[33] E G Bligh and W J Dyer ldquoA rapid method of total lipidextraction and purificationrdquo Canadian Journal of Biochemistryand Physiology vol 37 no 8 pp 911ndash917 1959


[34] G Lepage andC C Roy ldquoDirect transesterification of all classesof lipids in a one-step reactionrdquo The Journal of Lipid Researchvol 27 no 1 pp 114ndash120 1986

[35] A Sarin R Arora N P Singh R Sarin R K Malhotra and KKundu ldquoEffect of blends of Palm-Jatropha-Pongamia biodieselson cloud point and pour pointrdquo Energy vol 34 no 11 pp 2016ndash2021 2009

[36] A F Talebi M Tabatabaei and Y Chisti ldquoBiodieselAnalyzer auser-friendly software for predicting the properties of prospec-tive biodieselrdquo Biofuel Research Journal vol 1 pp 55ndash57 2014

[37] J Olmos-Soto J Paniagua-Michel R Contreras and L Tru-jillo ldquoMolecular identification of 120573-carotene hyper-producingstrains of dunaliella from saline environments using species-specific oligonucleotidesrdquo Biotechnology Letters vol 24 no 5pp 365ndash369 2002

[38] R Raja S Hema D Balasubramanyam and R RengasamyldquoPCR-identification of Dunaliella salina (Volvocales Chloro-phyta) and its growth characteristicsrdquoMicrobiological Researchvol 162 no 2 pp 168ndash176 2007

[39] M A Hejazi A Barzegari N H Gharajeh and M SHejazi ldquoIntroduction of a novel 18S rDNA gene arrangementalong with distinct ITS region in the saline water microalgaDunaliellardquo Saline Systems vol 6 article 4 2010

[40] J Olmos L Ochoa J Paniagua-Michel and R ContrerasldquoDNA fingerprinting differentiation between-carotene hyper-producer strains of Dunaliella from around the worldrdquo SalineSystems vol 5 no 1 article 5 2009

[41] M Ngangkham S K Ratha R Prasanna et al ldquoBiochem-ical modulation of growth lipid quality and productivity inmixotrophic cultures of Chlorella sorokinianardquo SpringerPlusvol 1 no 1 pp 1ndash13 2012

[42] L Rodolfi G C Zittelli N Bassi et al ldquoMicroalgae for oilstrain selection induction of lipid synthesis and outdoor masscultivation in a low-cost photobioreactorrdquo Biotechnology andBioengineering vol 102 no 1 pp 100ndash112 2009

[43] G O James C H Hocart W Hillier et al ldquoFatty acid profilingof Chlamydomonas reinhardtii under nitrogen deprivationrdquoBioresource Technology vol 102 no 3 pp 3343ndash3351 2011

[44] J-Y An S-J Sim J S Lee and B W Kim ldquoHydrocarbonproduction from secondarily treated piggery wastewater by thegreen alga Botryococcus brauniirdquo Journal of Applied Phycologyvol 15 no 2-3 pp 185ndash191 2003

[45] F Brenckmann C Largeau E Casadevall and C BerkaloffldquoInfluence de la nutrition azoteesur la croissance et la pro-duction des hydrocarbures de lrsquoalgue unicellulaire Botryococcusbrauniirdquo Energy from Biomass pp 717ndash721 1985

[46] H J Schuller A Hahn F Troster A Schutz and E SchweizerldquoCoordinate genetic control of yeast fatty acid synthase genesFAS1 and FAS2 by an upstream activation site common to genesinvolved in membrane lipid biosynthesisrdquo EMBO Journal vol11 no 1 pp 107ndash114 1992

[47] S Thomas and D A Fell ldquoThe role of multiple enzyme activa-tion in metabolic flux controlrdquo Advances in Enzyme Regulationvol 38 no 1 pp 65ndash85 1998

[48] A Lei H Chen G Shen Z H Hu L Chen and J WangldquoExpression of fatty acid synthesis genes and fatty acid accu-mulation in Haematococcus pluvialis under different stressorsrdquoBiotechnology for Biofuels vol 5 article 18 2012

[49] M L Gaspar H F Hofbauer S D Kohlwein and S AHenry ldquoCoordination of storage lipid synthesis and membranebiogenesisrdquo Journal of Biological Chemistry vol 286 no 3 pp1696ndash1708 2011

[50] M L Gaspar M A Aregullin S A Jesch and S A HenryldquoInositol induces a profound alteration in the pattern and rateof synthesis and turnover of membrane lipids in Saccharomycescerevisiaerdquo The Journal of Biological Chemistry vol 281 pp22773ndash22785 2006

[51] M Sumper ldquoControl of fatty acid biosynthesis by long chainacyl CoAs and by lipid membranesrdquo European Journal ofBiochemistry vol 49 no 2 pp 469ndash475 1974

[52] J M Stevenson I Y Perera I Heilmann S Persson and WF Boss ldquoInositol signaling and plant growthrdquo Trends in PlantScience vol 5 no 6 pp 252ndash258 2000

[53] Y Luo G Qin J Zhang et al ldquoD-myo-inositol-3-phosphateaffects phosphatidylinositol-mediated endomembrane functionin Arabidopsis and is essential for auxin-regulated embryogen-esisrdquo Plant Cell vol 23 no 4 pp 1352ndash1372 2011

[54] S Zhang and B van Duijn ldquoCellular auxin transport in algaerdquoPlants vol 3 no 1 pp 58ndash69 2014

[55] H El Arroussi R Benhima I Bennis N El Mernissi and IWahby ldquoImprovement of the potential of Dunaliella tertiolectaas a source of biodiesel by auxin treatment coupled to salt stressrdquoRenewable Energy vol 77 pp 15ndash19 2015

[56] C Fuentes-Grunewald E Garces S Rossi and J Camp ldquoUse ofthe dinoflagellateKarlodinium veneficum as a sustainable sourceof biodiesel productionrdquo Journal of Industrial Microbiology ampBiotechnology vol 36 no 9 pp 1215ndash1224 2009

[57] T T Y Doan B Sivaloganathan and J P Obbard ldquoScreeningof marine microalgae for biodiesel feedstockrdquo Biomass andBioenergy vol 35 no 7 pp 2534ndash2544 2011

[58] I Lang L Hodac T Friedl and I Feussner ldquoFatty acid profilesand their distribution patterns in microalgae a comprehensiveanalysis of more than 2000 strains from the SAG culturecollectionrdquo BMC Plant Biology vol 11 article 124 2011

[59] M I Gurr and J L Harwood Lipid Biochemistry An Introduc-tion Chapman amp Hall London UK 4th edition 1991


order to boost biomass productivity (BP) and volumetric lipidproductivity (LP) [6]

(A) Introduction of new genes into algal cells by imple-mentation of genetic engineering techniques stimulates somekey metabolic pathways and consequently improves theenergy production phenotypes in green microalgae [7] andultimately produces biofuel at lower cost However despitethe fact that the underlying principles laid out in this strategyare reasonable there has been little achievement made todate For instance the very first genetic engineering attemptsin order to increase microalgal lipid content (LC) by upreg-ulating the first major steps of fatty acid synthesis throughoverexpression of acetyl-CoA carboxylase (ACCase) andmalic enzyme were to some extent effective (12 increase inLC) [8] Blocking-off competing pathways may also enhancethe lipid accumulation in the cells In an attempt Picataggioand coworkers [9] blocked 120573-oxidation in C tropicalis byknocking out the genes encoding acyl-CoA oxidase Despitetheir expectation it was observed that the growth of the cellswas adversely affected Therefore enhanced lipid accumula-tion was rarely reported through overexpression of relevantenzymes andor intermediate products such as FAs becauseof emerging a secondary rate-limiting step Overall given theexisting obstacles facing gene transformation projects suchas biosafety rules correct introduction and maintenance oftransgenes this strategy should not be considered as the firstresort

(B) The second strategy is the optimization of nutrientformulations [10 11] There have been reports that devel-opment of optimum growth condition variables [12 13] hasincreased the lipid pool in some microalgal strains [14 15]Provision of biogenic elements mainly nitrogen is one ofthe main factors affecting algal metabolism The change inthe carbonnitrogen ratio in a media is known to result in achange in the metabolism direction In many algae speciesincreases in this ratio could contribute significantly to theaccumulation of neutral lipids mainly triacylglycerol [16ndash18]On the other hand unfortunately the conditions requiredfor optimal production of algal biomass are different fromthose of lipid production consequently decreasing the costthrough growth condition optimization approaches is notonly time- andmoney-consuming but also inmost cases willnot significantly improve lipid accumulation [16]

(C) The last strategy would be biochemical engineeringapproaches in which a variety of different plant growthregulators (PGR) could be used to support cell growthIt is generally assumed that the genetic background of arespective algal species would determine the compositionof the produced lipids but the lipid amount is mainly aresponse to the growth conditions [19] To channel metabolicflux generated in photobiosynthesis into lipid biosynthesisimplementation of somePGRs vitamins and lipid precursorscould lead to an increase in total catabolism activation andlipid accumulation

Myoinositol as one of the nine stereoisomers of inositolis classified as a member of the vitamin B complex and isrequired for the cell growth as well as other significant bio-logical processes Myoinositol was first used by Jacquiot [20]in order to favor bud formation and retard necrosis in elm

when supplied at 20ndash1000mg Lminus1 however the proliferationof the callus was not improved Letham [21] reported thatmyoinositol acts as second messengers in the primary actionof auxins in plants and its interaction with cytokinin stimu-lates cell division in carrot phloem explants [22] Moreoverinositol is also known as a precursor for phospholipids suchphosphatidylinositol (PI) in the cells In an investigation itwas found out that inositol addition into the growth mediaof yeast led to changes in transcript abundance of over 100genes namely the UASino-containing genes Many of thesegenes encode enzymes involved in lipid metabolism [23 24]For instance in yeast the expression of ACCase gene a keygene in the synthesis of long chain fatty acids is stimulatedby inositol and choline [25]

The present study was set to investigate myoinositol-drivenmodulation of LP and quality (fatty acids (Fas) profile)in cultures of local Dunaliella sp strain isolated from thenorth coast of PersianGulf Nile red staining usingmicroplatefluorescence reading as well as epifluorescent microscopicand flow cytometer analyses was used to monitor the effect ofthemyoinositol supplementation on the algal cellsMoreoverreal-time PCR analysis was performed to look into the impactof myoinositol on one of the key genes involved in lipidsynthesis

2 Methods

21 Strains Cultivation A marine strain (Dunaliella sp) iso-lated from Bandar Lengeh a port city on the northern coastof the Persian Gulf was used in this study Green colonieswere transferred into new flasks containing a media namedLake Media and was kept at 20∘C and a constant (24 0)3klux photon flux of white and red LED lamps [27] Theingredients of Lake Media developed during the course ofthe present study are included Lake salt sediment (60 g Lminus1)NPK fertilizer (2 g Lminus1) and FeSO

4

(005 g Lminus1) The mediarsquospH was set at 75 and the samples were constantly shaking at120 rpm

Beside the strain locally isolated in this study one otherlocal strain D salina (generously provided by Dr ShariatiIsfahanUniversity) and two standard stains CCAP 1918 andUTEX 200 purchased from the Culture Collection of Algaeand Protozoa (Sams Research Scotland) and Universityof Texas at Austin (Austin USA) respectively were alsoincluded in the experiments All the strains were cultivatedin the Lake Media under the above-mentioned conditionsDuring the cultivation period growth kinetic parameterswere recorded for all the strains in triplicateData comparisonwas then carried out using the ANOVA test The calculatedgrowth parameters included BP LC and LP (mg Lminus1 dayminus1)For BP determination algal suspensions were centrifuged(3000 g 10min) and the wet weights were determined gravi-metrically LP was calculated according to the followingequation

LP = BP times LC (1)

Besides to investigate the effect of myoinositol inclusionon lipidmetabolismandbiodiesel properties only the Persian

















100

97

68

100

42

92

0005

Dunaliellaceae










Strains

Parameters

























1









Cell wall

PI

PI

PI

Plasma membraneFFAs

FAS

ACCase products

ACCase





Myoinositol Auxin

MI

MI

NucleusACC









(a) (b)

(c) (d)














05000

10000150002000025000300003500040000

w 50 100 200

Fluo

resc

ence

inte

nsity

TreatmentsCminus C+


150170190210230250270290310330

C 50 100 200

Cyto

met

ric F

L2 si

gnal


















4 Conclusion



Abbreviations




Acknowledgments


References














































































100

97

68

100

42

92

0005

Dunaliellaceae










Strains

Parameters

























1









Cell wall

PI

PI

PI

Plasma membraneFFAs

FAS

ACCase products

ACCase





Myoinositol Auxin

MI

MI

NucleusACC









(a) (b)

(c) (d)














05000

10000150002000025000300003500040000

w 50 100 200

Fluo

resc

ence

inte

nsity

TreatmentsCminus C+


150170190210230250270290310330

C 50 100 200

Cyto

met

ric F

L2 si

gnal


















4 Conclusion



Abbreviations




Acknowledgments


References
































































Strains

Parameters

























1









Cell wall

PI

PI

PI

Plasma membraneFFAs

FAS

ACCase products

ACCase





Myoinositol Auxin

MI

MI

NucleusACC









(a) (b)

(c) (d)














05000

10000150002000025000300003500040000

w 50 100 200

Fluo

resc

ence

inte

nsity

TreatmentsCminus C+


150170190210230250270290310330

C 50 100 200

Cyto

met

ric F

L2 si

gnal


















4 Conclusion



Abbreviations




Acknowledgments


References





































































1









Cell wall

PI

PI

PI

Plasma membraneFFAs

FAS

ACCase products

ACCase





Myoinositol Auxin

MI

MI

NucleusACC









(a) (b)

(c) (d)














05000

10000150002000025000300003500040000

w 50 100 200

Fluo

resc

ence

inte

nsity

TreatmentsCminus C+


150170190210230250270290310330

C 50 100 200

Cyto

met

ric F

L2 si

gnal


















4 Conclusion



Abbreviations




Acknowledgments


References































































Cell wall

PI

PI

PI

Plasma membraneFFAs

FAS

ACCase products

ACCase





Myoinositol Auxin

MI

MI

NucleusACC









(a) (b)

(c) (d)














05000

10000150002000025000300003500040000

w 50 100 200

Fluo

resc

ence

inte

nsity

TreatmentsCminus C+


150170190210230250270290310330

C 50 100 200

Cyto

met

ric F

L2 si

gnal


















4 Conclusion



Abbreviations




Acknowledgments


References































































(a) (b)

(c) (d)














05000

10000150002000025000300003500040000

w 50 100 200

Fluo

resc

ence

inte

nsity

TreatmentsCminus C+


150170190210230250270290310330

C 50 100 200

Cyto

met

ric F

L2 si

gnal


















4 Conclusion



Abbreviations




Acknowledgments


References





































































05000

10000150002000025000300003500040000

w 50 100 200

Fluo

resc

ence

inte

nsity

TreatmentsCminus C+


150170190210230250270290310330

C 50 100 200

Cyto

met

ric F

L2 si

gnal


















4 Conclusion



Abbreviations




Acknowledgments


References








































































4 Conclusion



Abbreviations




Acknowledgments


References






























































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