ReviewArticle - Hindawi Publishing Corporationdownloads.hindawi.com/journals/bmri/2018/1503126.pdf · 12/29/2017 · roles in microalgal metabolism and growth period. Some lipids

Review ArticleDetermination of Microalgal Lipid Content and Fatty Acid forBiofuel Production

Zhipeng Chen LingfengWang Shuang Qiu and Shijian Ge

Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse School of Environmental and Biological EngineeringNanjing University of Science and Technology Xiao Ling Wei 200 Nanjing Jiangsu 210094 China

Correspondence should be addressed to Shuang Qiu qiushuang89njusteducn and Shijian Ge geshijian1221njusteducn

Received 29 December 2017 Revised 12 March 2018 Accepted 4 April 2018 Published 21 May 2018

Academic Editor Xiaoling Miao

Copyright copy 2018 Zhipeng Chen 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

Biofuels produced frommicroalgal biomass have received growingworldwide recognition as promising alternatives to conventionalpetroleum-derived fuels Among the processes involved the downstream refinement process for the extraction of lipids frombiomass greatly influences the sustainability and efficiency of the entire biofuel system This review summarizes and comparesthe current techniques for the extraction and measurement of microalgal lipids including the gravimetric methods using organicsolvents CO

2-based solvents ionic liquids and switchable solvents Nile red lipid visualization method sulfo-phospho-vanillin

method and the thin-layer chromatography method Each method has its own competitive advantages and disadvantages Forexample the organic solvents-based gravimetric method is mostly used and frequently employed as a reference standard to validateother methods but it requires large amounts of samples and is time-consuming and expensive to recover solvents also with lowselectivity towards desired products The pretreatment approaches which aimed to disrupt cells and support subsequent lipidextraction through bead beating microwave ultrasonication chemical methods and enzymatic disruption are also introducedMoreover the principles and procedures for the production and quantification of fatty acids are finally described in detail involvingthe preparation of fatty acid methyl esters and their quantification and composition analysis by gas chromatography

1 Introduction

Nowadays limited stock of petroleum-derived fuel resourcescombined with perpetually increasing demands for energydue to the rapid industrialization and population growthhas troubled many governments and organizations acrossthe world [1] Moreover the combustion of fossil-derivedfuels has led to increasing emission of greenhouse gases suchas carbon dioxide (CO

2) leading to global climate change

and posing threats to the biosphere [2] In order to achievesustainable development the critical issues noted above andthe gradually rising fossil-derived fuel prices have called forthe needs to search for alternative sustainable and renewableenergy sources [3]

Biofuels produced from biomass are promising alterna-tives to fossil-derived fuels due to several distinct advantagesincluding carbon neutrality reduced emissions of gaseouspollutants (eg carbon monoxide CO

2 and sulfur oxides)

continuous availability of biomass feedstocks and their safety

of production by farming [4] According to their physicalcharacteristics biofuels are divided into solid (ie biochar)liquid (ie bioethanol vegetable oil and biodiesel) andgaseous (ie biogas biosyngas and biohydrogen) fuelsBased on the types of used feedstocks biofuels are catego-rized into three generations The first generation feedstocksmainly include food crops such as corn soybean rapeseedsunflower and palm oil The second-generation biofuels arederived from nonedible feedstocks like JatrophaMiscanthusSwitch grass and other organic wastes Nevertheless theexpanding demand for edible feedstocks as food sourcesand their need for large areas of arable land for produc-tion have limited the development of both the first- andsecond-generation biofuels The use of microalgae as a third-generation biofuel feedstock avoids these issues and presentsseveral distinct advantages of not requiring agricultural orarable lands for production high photosynthetic efficienciesand biomass productivities (biomass doubled in less thanone day) and 100 times more lipids per acre of land [5 6]

HindawiBioMed Research InternationalVolume 2018 Article ID 1503126 17 pageshttpsdoiorg10115520181503126

2 BioMed Research International

２

２

２

（2

（2 minus

minus

P

O

O

H

H

H

H

H

C

C

C

O

O

O

C

C CH

O

Phosphate

Fatty acid

Glycerol

Figure 1 Lipid molecules Triacylglycerol (NL) on the left Phospholipid (polar lipid) on the right R1015840 R10158401015840 and R101584010158401015840 in the triacylglycerolmolecule represent fatty acid chains Phospholipid molecule is negatively charged [15]

Moreover the main storage lipids in microalgae are neutrallipids (NLs) or triacylglycerols that can be esterified toFAMEs with the primary profiles of C16 and C18 proven tobe the most suitable for biofuel production [7] Microalgaeexhibit great adaption to various environmental conditionsmaking them easy to cultivate For instance they can growon marginal land in both the open pond and closed systemsusing waste streams like wastewater waste or CO

2-enriched

gas (biogas flue gas) waste organics (ie crude glycerol) andwaste heat to provide nutrients and carbon and temperaturemaintenance (in a cold climate) achieving economicallyfeasible and environmentally sustainable biofuel productionand waste bioremediation [8] Moreover various routes ofmicroalgal metabolisms can be adopted for enhanced growthand lipid production Traditionally phototrophic algae aregrown autotrophically with CO

2as the unique carbon source

and light providing all the energy needed Moreover somemicroalgae species can grow heterotrophically using onlyorganic compounds while others can grow mixotrophicallyusing both organic compounds and CO

2to support growth

[9]Currently the high costs of the important microalgal har-

vesting and lipid extraction processes are the primary obsta-cles impeding on the commercial application of microalgae-derived biofuel production [10ndash12] For example lipid extrac-tion is a high-power-consumption process because lipids arestored in microalgal cells and the cell wall is a thick and rigidlayer composed of complex carbohydrates and glycoproteinswith high mechanical strength and chemical resistanceposing difficulties for lipid extraction [13] Therefore certaincell disruption techniques are generally considered priorto lipid extraction to improve the extraction efficienciesNevertheless the efficiencies of cell disruption and lipidextraction vary with methods selected with different operat-ing conditions (eg temperature atmospheric pressure andhumidity) microalgae species and biomass amount

This review summarizes and compares themethodologiesemployed for the extraction and quantification of microalgallipids The pretreatment methods supporting cell disruptionand subsequent lipid extraction are also included Finally theprinciples and procedures for the production and quantifica-tion of fatty acids in microalgae are discussed in detail

2 Microalgal Lipids

Microalgal lipids can be divided into two groups accordingto their structures nonpolar NLs (acylglycerols sterolsfree fatty acids wax and steryl esters) and polar lipids(phosphoglycerides glycosylglycerides and sphingolipids)Figure 1 shows the structural formula of the polar lipidand NLs [14 15] These lipids play different but importantroles in microalgal metabolism and growth period Somelipids such as phosphoglycerides glycosylglycerides andsterols are imperative structural components of biologicalmembranes while lipids like inositol lipids sphingolipidsand oxidative products of polyunsaturated fatty acids mayact as key intermediates in the cell signaling pathwaysand play a role in sensing changes in the environment[16] The quantities of these microalgal lipids vary withthe type of species growth conditions and ambient envi-ronments It was reported that the lipid contents rangedat 20ndash50 of dry biomass including Chlorella Cryptheco-dinium Cylindrotheca Dunaliella Isochrysis NannochlorisNannochloropsis Neochloris Nitzschia Phaeodactylum Por-phyridium Schizochytrium and Tetraselmis [17]

3 Microalgal Cell Disruption Methods

Specific microalgal cell pretreatment procedures must beconsidered prior to the subsequent lipid extraction due tothe microalgal cell wall structure When the extraction isconducted from the wet biomass the pretreatment step ismandatory to disrupt the microalgal cell walls and allow thelipids to be released into the extracting mixture The com-monly used pretreatment methods are summarized below

31 Bead Beating Bead beating also known as bead mill orball mill disrupts cells by the impact of high-speed spinningof fine beads on the biomass slurry The whole disruptionprocess could be done within minutes and it could beapplied to any kinds of microalgae without preparation [1819] Two common types of bead mills are shaking vesselsand agitated beads [20] Shaking vessels usually consist ofmultiple containers or well-plates on a vibrating platformand the cell disruption is done by shaking the entire vessel

BioMed Research International 3

on a vibrating platform The shaking vessels are usuallyemployed in laboratory as they are only suitable for multiplesamples requiring similar disruption treatment conditionsComparatively the agitated beads type that is made upof a rotating agitator in a fixed vessel filled with beadsand cell culture could achieve better disruption efficienciesHowever the cooling jackets must be equipped to protect theheat-sensitive biomolecules as the rotating agitator generatesheat during disruption process [18 19] The combination ofagitation collision and grinding of the beads could producea higher disruption efficiency [20] To sum up the simplicityof the equipment and the rapidness of the treatment processare the two main advantages of bead beating methods whilethe requirement of an extensive cooling system to protect thetarget products has limited it to scale up [18]

32 Microwave Microwave is an electromagnetic wave withthe frequency ranging between 300MHz and 300GHzwhich is lower than that of infrared and higher than that ofradio waves Microwave-assisted extraction technology hasbeen studied for extracting target compounds in a few fieldsincluding microalgal lipid extraction [50] When microalgalcells are exposed to themicrowavewith the specific frequency(approximately 2450MHz) cell molecules generate a rapidoscillation within the rapidly oscillating electric field result-ing in the heat generation due to the frictional forces from theinter- and intramolecular movements [51] The intracellularheating causes the water to vapor which disrupts the cellsand subsequently opens up the cell membrane This methodexhibits strong advantages of short reaction time low-operating costs and efficient extractionwith all of the speciesbut the requirement of a vast cooling system to protect targetproducts limits its large-scale application [18 19]

33 Ultrasonication Ultrasonication has been a well-knownmethod for the microbial cell disruption due to its shortreaction time with high productivity [52] When the ultra-sound is applied to the liquid cultures small ldquovacant regionsrdquocalled microbubbles are momentarily formed as the liquidmolecules are moved by the acoustic waves Meanwhilethe production of microbubbles causes cavitation which inturn creates pressure on the cells to break up [53] Duringthe treatment the rapid compressiondecompression cyclesof the sonic waves generate transient and stable cavitationThe transient cavitation occurs when oscillations that cavi-tation undergoes are unsteady and implode ultimately Thistype of implosion could produce extremely localized shockwaves and high temperature the conditions of which impartmechanical stress on the cells and crack the cell wall andmembrane [54] On the other hand the cavitation thatoscillates for many cycles is referred to as stable cavitationwhich can produce microscale eddies inducing stress orphysiological changes in microorganisms [55] Ultrasonichorn and bath are the two basic types of sonicators andthey are commonly employed in batch operations but canalso be adapted for continuous operations [56] Horns usepiezoelectric generators which are made of lead zirconatetitanate crystals and vibrate with amplitude of 10ndash15mmAs the energy generated at the horn tip dissipates rapidly

with distance the cavitation must be created with sufficientdisruptive force Transducers placed at the bottom of thesonicator are used in sonicator baths to generate ultrasonicwaves In sonicator baths the number and arrangement oftransducers vary according to the capacity and shape of thesonicator [20] The working conditions of ultrasonicationtreatment are easy to set up and the whole process could bedone in a very short timewhile with high reproducibility [19]However it is difficult to scale up as cavitation the strongeffect of which is able to achieve cell disruption only occursin small regions near ultrasonic probes

34 Chemical Method The rupture of cells occurs whenchemicals are used to increase the permeability of cell up to aparticular value [57] It was reported that through chemicaltreatments with acids (ie HCl and H

2SO4) alkalis (ie

NaOH) and surfactants chemical linkages on the microalgalcell envelope were degraded followed by the lysis of cellwall [58] Comparatively chemical treatment consumes lessenergy because it does not require a large amount of heat orelectricity while showing higher efficiency of cell disruptionHowever the continuous consumption of chemicals chal-lenges the economic sustainability of this method Moreoveracids and alkalis have a high risk of corroding the reactor andattacking microalgal lipids thus ruining the whole process[18]

35 Enzymatic Disruption In addition to the autolysisthe use of foreign lytic enzymes is extensively investigatedbecause enzymes are the commercially available and easilycontrolled biologicalmaterials [59] Specific enzyme is able todegrade certain structural cell components thus improvingthe release of desired intracellular compounds [60] In somecases a mixture of different enzymes is reported to have abetter economic and technical feasibility and the lipid yieldscould be improved when enzymatic hydrolysis is combinedwith acidalkaline pretreatment Compared to the chemicalmethod that possibly destroys every particle existing in thesolution and even induces side-reactions of the target prod-ucts (ie lipids) the reaction condition of enzymatic methodis mild and its selectivity is high with specific chemicallinkages Moreover enzymatic disruption combined withother methods is usually considered for economic processand improved disruption performance [18] However moreresearches need to be conducted to reduce the high cost andrelatively long treatment time which have limited the large-scale application of this method

36 Other Methods Apart from the methods noted abovethere are some other microalgal cell disruption approachesthat have been investigated as well For example when themixture of microalgae and other solvents is sprayed througha narrow tube under high pressure hydraulic shear force isgenerated and high pressure homogenization (HPH) alsoknown as French press makes use of this force to extractinternal substances of microalgae By measuring the increasein the soluble chemical oxygen demand (SCOD) during thecell disruption lots of researchers have evaluated the celldisruption efficiency and found that HPH exhibited high cell


2 CylinderFurnace

ElectricReactor

Gas flow meter

Regulator

PressureSafety valve

PCRM

TRPI

Stirrer

V1

V2

Figure 2 Schematic diagram of thermal pretreatment apparatus[61]

disruption efficiency It is worth mentioning that HPH haslots of advantages such as producing less heat during theextraction process thus requiring less cooling cost and it iseasy to scale up However the pretreatment process relyingon HPH requires a relatively long time and consumes quite afew amount of power Electroporation can achieve permanentcell disruption by applying a much stronger electromagneticfield (EF) to the biomass that can damage the cell envelopesbeyond their healing abilities since the application of an EFof suitable intensity will lead to the formation of pores onthe cell envelopes of the cells and the pores are closed bya healing process when the EF is removed Electroporationis a promising cell disruption method as it requires simpleequipment and operation procedures with high energy effi-ciency [18 20] Thermal treatment could achieve effectiverecovery of hydrocarbons as well and a typical process ofthermal pretreatment is shown in Figure 2 Firstly placethe samples in vessel and replace the air in the vessel bynitrogen gas Then heat vessel to the set temperature andafterwards cool the vessel to the ambient temperature aftermaintaining the samples at the set temperatureThen stir thesamples mechanically Finally open the autoclave and care-fully remove the samples for further analysis [61] Recentlyplenty of work have been done to compare the efficiencyof different cell disruption methods however due to thedifferent species of microalgae used in experiments so asthe different operating temperatures atmospheric pressuresand other influence factors the efficiency of different celldisruption methods is short of comparability

4 Microalgal Lipid Extraction andQuantification Approach

The microalgal lipid extraction refers to the process ofseparating the valuable NLs and fatty acids from the cellu-lar matrix and water As far multiple methods have beenreported for the quantification of microalgal lipids mainlyincluding the conventional gravimetric method using extrac-tion solvents Nile red lipid visualization method SPV andTLC [62ndash64]

static organicsolvent film

bulk organicsolvent

cell membraneand cell wall

cytoplasm

nucleus

1

1

2

2

3

3

4

4

5

5

Figure 3 Schematic diagram of the organic solvent-based microal-gal lipid extraction mechanisms The pathway shown at the top ofthe cell is for nonpolar organic solvent while the pathway shown atthe bottom of the cell is for nonpolarpolar organic solvent mixtureOrange circle lipids white circle nonpolar organic solvent andwhite diamond polar organic solvent [15]

41 Gravimetric Method The gravimetric method is mostwidely used to determine microalgal lipid content It isalso frequently used as a reference standard to validateother methods The gravimetric method consists of the lipidextraction using solvents and lipid quantification achievedby recording the weight of extracted lipids after evaporatingthe extracting solvents The extraction solvents used includethe conventional organic solvents CO

2-based solvents ionic

liquids (ILs) and switchable solvent

411 Organic Solvent Extraction The chemistry conceptof ldquolike dissolving likerdquo is the basic principle underlyingthe organic solvent-based extraction of microalgal lipidsFigure 3 illustrates the principle of the 5-step-microalgaelipid extraction mechanism Typically the organic solventspenetrate through the cell membrane into the cytoplasm(step 1) and interact with the lipid complex (step 2) Duringthis process the nonpolar organic solvent interacts withNLs through van der Waals associations while the polarorganic solvent interacts with the polar lipids by generatinghydrogen bonds that are strong enough to replace the lipid-protein associations that prevent nonpolar organic solventfrom accessing the lipids Subsequently an organic solvent-lipids complex is produced (step 3) followed by the organicsolvent-lipids complex diffusing across the cell membrane(step 4) and the static organic solvent film (step 5) into thebulk organic solvent driven by a concentration gradient

The commonly used organic solvent extraction proce-dures are summarized in Table 1 The nonpolar organicsolvents such as hexane benzene toluene diethyl etherethyl acetate and chloroform are usually combined with thepolar organic solvents to maximize the extraction efficiencyof NLs As such when lipid extraction is achieved withthe use of a nonpolarpolar organic solvent mixture the


Table1Determinationof

microalgallipid

contentu

singconventio

nalorganicsolvents

Lipidextractio

nDeterminationof

lipid

content

Ref

Reagent(s

)Ex

tractio

nprocess

Treatm

entfor

finaldeterm

ination

Expressio

nof

lipid

content

Chloroform

isop

ropano

l(11

vv)

andh

exane

Addsolventm

ixture

tofro

zenpellets

centrifugea

ndtransfe

rsup

ernatantsreextract

pelletswith

hexane

andcentrifugetocollect

supernatants

Dry

thec

ombinedsupernatantinas

peed

vacuum

andrecord

thew

eight

Apercentage

oftotalfresh

weight

(ww

)[21]

Ethylether

Groun

ddrysamples

topo

wdera

ndusethe

Soxh

letextractor

Distill

thes

olventdry

ther

esidueand

record

the

weight

Apercentage

ofdrycellweight(

ww

)[22]

Methano

lchloroform

1NaC

l(22

1vvv)

Extractthe

biom

assw

iththes

olvent

mixture

Evaporatethe

chloroform

layerdrythes

amplea

ndrecord

thew

eight

Thew

eightratioof

extractedlip

idto

lyop

hilized

pellets(

ww

)[23]

Deion

ized

(DI)

waterm

ethano

lchloroform

(12

1vvv)chloroform

Addsolventm

ixture

toharvestedbiom

assa

ndreactfor

24hmixandthen

addsolventto

achievea

finalDIw

aterm

ethano

lchloroform

ratio

of09

11centrifugeremoveandfilter

thelipid-chloroform

layerrepeatthea

bove

steps

forsecon

dextractio

n

Evaporatethe

chloroform

layercoolthetub

eand

record

thew

eight

Apercentage

oftotalbiomass

weight(w

w)

[24]

Chloroform

methano

l(2

1vv)

Mixdryalgalp

owderw

iththes

olvent

mixture

putu

nder

water

bath

with

thea

idof

ultrasou

ndcentrifu

geandrepeatthea

bove

steps

threetim

es

Evaporatethe

organics

olvent

andweight

Apercentage

oftotalbiomass

weight(w

w)

[25]

Chloroform

methano

l(2

1vv)

Extractlipid

from

drybiom

assw

iththes

olvent

mixture

Evaporatethe

solventand

weight

Weightd

ifference

betweenthe

blankflask

andthefl

askcontaining

thee

xtracted

oil

[26]


Table 2 Comparisons between conventional organic solvents extraction and CO2-based solvents extraction approaches

Items Organic solvent scCO2

lCO2

Heavy metal contamination Unavoidable Free of heavy metals Free of heavy metalsInorganic salt content Difficult to avoid Free of inorganic salts Free of inorganic saltsSelectivity Poor selectivity Highly selective Highly selectiveExtracted compounds Polar and nonpolar compounds Nonpolar compounds Nonpolar compoundsSafety Flammable andor toxic Nontoxic and nonflammable Nontoxic and nonflammable

Operation condition Regular temperature and pressure High temperature and pressure Lower temperature and pressurethan scCO

2

Recycling Solvent recovery is expensive CO2could be recycled and reused CO

2could be recycled and reused

Operation cost High power consumption (insolvent recovery) High power consumption Lower than scCO

2

Extraction time Time-consuming Shorter than solvent extraction Shorter than solvent extraction

polar organic solvent is intended to disrupt the neutral-polar lipid complexes while the nonpolar organic solventaims to solubilize the intracellular NLs [65] Moreover thelipid yields vary with the type of used organic solvents andthe ratios of polar solvents to nonpolar solvents Thereforethe final lipid extraction efficiencies using different organicsolvents extractionmethods cannot be impartially comparedIn addition the different experiment steps equipment andexperimental conditions involved in the extraction processalso contribute to various extraction results

The organic solvent-based extraction methods usuallyrequire a relatively large quantity of biomass and have fewenvironmental impacts In addition organic solvents are nothighly selective towards the desired neutral (mono- di-and triacylglycerols) lipids and free fatty acid componentssome of them are not easily removable posing difficultiesto the subsequent process An ideal solvent for the lipidextraction should be free of toxicity easy to remove andmore selective towards target products These characteristicshave been found in CO

2-based solvents ionic liquids and

switchable solvents which will be introduced hereinafter

412 CO2-Based Solvent Extraction The supercritical(scCO

2) and liquid (lCO

2) CO2are able to solubilize many

organic molecules and can be easily recycled at the end ofthe process while leaving no residual solvents making thempromising alternatives to traditional organic solvents

(1) scCO2 Extraction The supercritical fluid extraction (SFE)is a promising green technology that can potentially displacethe use of traditional lipid extraction procedure due toits high selectivity short extraction time and their absentuse of toxic organic solvents [66] As can be seen fromTable 2 scCO

2has been regarded with interest in the field of

SFEs because it offers advantages of negligible environmentalimpact high diffusivity no toxicity no oxidation or thermaldegradation of extracts and easy separation of desired bio-products [67] Moreover scCO

2has high selectivity towards

microalgal NLs (mono- di- and triacylglycerols) and hasbeen used in the lipid extraction of microalgae such as Cylin-drotheca closterium Arthrospira maxima Nannochloropsisoculate Chlorella vulgaris and Spirulina platensis [68ndash71]

For example Halim et al [72] employed scCO2into a wet

Chlorococum sp paste to obtain a yield of 71 wt at atemperature of 333 K and a pressure of 30MPa over an 80minextraction timeMoreover coupling the nonpolar scCO

2with

the polar cosolvents (ie methanol ethanol and toluene)could enhance the affinity towards NLs that form complexeswith polar lipids resulting in a greater biofuel production[73] The general procedure of scCO

2extraction is described

as follows CO2is first condensed to lCO

2and then to the

scCO2 Subsequently the fluid is pumped into the extraction

vessel under the desired and controlled conditions of pressureand temperature After the extraction the extracted lipids areprecipitated and collected into a glass trap cooled in an icebath with the amount assessed by gravimetry It should benoted that the effects of operating conditions (ie extractionvessel size and type pressure and extraction time) involvedin the scCO

2extraction process noted above on the lipid yield

and selectivity should be investigated on a case-by-case basisMoreover the high temperature (ie 100∘C) and pressure(ie 41MPa) requirement are the main concerns that havelimited this approach from industrial-scale application [74]

(2) lCO2 Extraction Comparatively lCO2sharesmany of the

same benefits as scCO2while it requires lower temperature

and pressure than scCO2extraction (Table 2) and therefore

has emerged as a possible substitute Paudel et al [75]recovered about 26wt of the extractable lipids using lCO

2

directly from the wet biomass of Chlorella vulgaris underdifferent pressures (68ndash17MPa) and a constant temperatureof 25∘C An extraction example using lCO

2is described as

follows Firstly lCO2is pressurized to a certain pressure

(ie 68MPa) using the high pressure pump and during thisprocess a coolant (ie 75 ethyleneglycol in distilled water)must be used to keep the pump at minus5∘C to prevent it frombeing heated Secondly the pressurized lCO

2is delivered

through the tube coil to tube vessel aiming to make CO2

coming from the cold pump warm up to the temperatureof the water bath Thirdly the vessel containing dry algaeis heated in water bath at 25∘C for 2 h during which therequired pressure in the system ismaintained by backpressureregulator (BPR) After the extraction the remaining CO

2is

vented into the flask and the remaining extract exiting the


20 40 60 800Pressure (bar)

1

3

5

7

VV

0

EthanolMethanolTetrahydrofuran

Ethyl Acetate

Figure 4 Isothermal volumetric expansion of benign solvents byCO2at 40∘C [77]

BPR is captured separated and dried The extract is thenpreserved in the ice-cold isopropanol

(3) Gas Expanded Liquids Extraction Gas expanded liquids(GXLs) are liquids expanded in volume by the applicationof modest pressures with a compressible gas among whichCO2is one of the most commonly used gases [76]The GXLs

are made up of a mixture of compressed gases and con-ventional solvents Jessop and Subramaniam [77] reportedthat GXL solvents have the combined beneficial properties ofa compressed gas and organic solvent so the properties ofsolvent can be adjusted through variations in the pressureAs can be seen from Figure 4 the gaseous CO

2has a

considerable solubility in many benign organic solvents atadequate pressures (lt8MPa) such as ethanol methanol thatshow a 2- to 3-fold volumetric expansion at relatively mildpressures and moderate temperatures [77] As such variousprinciples and applications of CO

2-expanded liquids (CXLs)

including lipid extraction have been proposed [78 79] Dueto the fact that CXLs can be operated at mild temperaturesand pressures a reduction in process costs and energyconsumption could be realized The mass transfer rates canalso be improved via CXLs by reducing interfacial tensionand viscosity as well as increasing diffusivity [80] Wang et al[78] used the CO

2-expanded ethanol to successfully extract

lipids from Schizochytrium sp with a 357 wt lipid contentof dry biomassTheCO

2-expandedmethanol increased up to

82 of the selectivity of methanol towards the extraction ofbiodiesel-desirable NLs and free fatty acids [75]

413 ILs Extraction ILs are organic salts with the melt-ing point below 100∘C and they typically consist of largeasymmetric organic cations coupled with smaller anions [81]Advantages such as thermal stability synthetic flexibilitynonvolatility nonflammability recyclability and unique sol-vent properties have made ILs promising replacements oftraditional organic solvents in lipid extraction as they can

dissolve highly recalcitrant biopolymers [82] For instanceILs are capable of disrupting cell structure in wet microalgaebiomass under mild conditions This allows either autopar-titioning of the lipids or presumably improving access ofcosolvents to the intracellular lipids thus facilitating theextraction of lipids from microalgae and making it fasterthan organic solvent extraction processes Most solvent-based extraction processes however are incompatible withwet biomass which add significant costs to the overall processsince dewatering and drying processes are thought to beresponsible for up to 70 of the biofuel production cost [83]

A typical lipid extraction procedure with the aid of ILs isdescribed as follows [84] Firstly mix microalgae paste with1 10 mass ratio of dry equivalent microalgae to [C

2mim]

[EtSO4] and incubate the mixture Secondly add water to the

mixture to improve separation after the addition of hexaneand remove the top layer to a new container The proceduresnoted above are repeated three times to achieve a highextraction efficiency After the extraction wash the extractswith NaCl and transfer the target part to a preweighed vesselThe mass of extractable lipids is measured after evaporatingthe solvent For ILs recycling add methanolwater to themixture to precipitate the residual solids and pool hexanewith the previous extraction Subsequently filter the solventand wash with methanolmethanol to collect ILs and thenILs are recovered by evaporation

414 Switchable Solvents Extraction Switchable solventsknown as ldquoreversiblerdquo or ldquosmartrdquo solvents can reversiblychange their properties upon addition or removal of aldquotriggerrdquoThe switchable solvents (subclass of ILs) are dividedinto two categories switchable polarity solvents (SPSs) andswitchable hydrophilicity solvents (SHSs) [85 86] Specifi-cally the polarity of SPSs exhibits variation with the solutionCO2concentration The polarity of the solvents can be

reversed by removing the CO2from the system by heating or

sparging the solution with nonacidic gases SPSs are dividedinto two classes which are either single-component or two-component species In the two-component SPSs a base withan alcohol or with an amine is usually included while single-component SPSs require a primary or secondary amine[85] Unlike SPSs SHSs can change from a hydrophobicsolvent into a hydrophilic one and their potential applica-tions have extended to the extraction of microalgal lipids[87] In the SHSs system the hydrophobic form creates abiphasic mixture with water and the hydrophilic form isthe corresponding bicarbonate salt thus SHSs could alsobe reversibly converted between the above two forms bythe addition or removal of CO

2[88 89] A proposed lipid

extraction procedure using SHSs is illustrated in Figure 5Briefly SHSs are employed to dissolve or extract lipids intheir hydrophobic state the carbonated water is introducedto revert SHSsrsquos hydrophilic form and form a two-phase of thelipid and SHSswater finally the SHSswater mixture wouldbe separated into two components and then be reused byflushing air through [88]

42 Nile Red Lipid Visualization Method Compared withthe mostly used gravimetric methods noted above Nile


Extractwithsolvent

- C2

Add waterC2amp

oil

oil

hydrophilicsolventamp water


oil amphydrophobic

solvent

water

hydrophobicsolvent

Figure 5 The process of SHSs used for soybean oil extraction from soybean flakes without a distillation step The dashed lines indicate therecycling of the solvent and the aqueous phase [88]

red lipid visualization method is more convenient as thenumber of samples and preparation time are greatly reducedThe Nile red (9-diethylamino-5H-benzo[120572]phenoxazine-5-one) is a lipid-soluble probe that fluoresces at the definedwavelengths depending upon the polarity of the surroundingmedium However due to the composition and structure ofthe thick and rigid cell walls in some microalgae speciesNile red is prevented from penetrating the cell wall and cyto-plasmic membrane and therefore lipids cannot provide thedesired fluorescence Thus the dimethyl sulfoxide (DMSO)is introduced to microalgal samples as the stain carrier atan elevated temperature [63] When microalgal lipids aremeasured by Nile red visualization method a standard curvepreparation is included in most cases Table 3 summarizessome specific Nile red lipid procedures for the determinationof microalgal lipids Different solvents are combined withNile red solution to stain microalgal culture samples and thesamples are diluted if necessary The lipid content determi-nation is achieved by comparing the resulting fluorescencevalues to a certain standard curve in which the wavelengthof excitation and emissionmay be different Nevertheless thelipid contents measured by this method are usually interferedby the environmental factors and other components in thecell cytoplasm and the fluorescence intensity varies betweensamples Thus the optimal spectra and reaction conditionsshould be determined for each type of sample prior to thefluorescent measurement [90]

43 SPV Method The colorimetric SPV method is a rapidalternative for lipid measurement because of its fast responseand relative ease in sample handling [91] The SPV reactswith lipids to produce a distinct pink color and the intensity

is quantified using spectrophotometric methods thereforeit is employed for direct quantitative measurement of lipidswithin liquidmicroalgal cultures [40] However the results ofSPV assay can be affected by lots of factors such as the degreeof oil saturation incubation time heating and cooling thusthe SPV assay may give misleading results [27]

The general procedure of SPV method includes the sam-ple addition solvent evaporation sulfuric acid addition sam-ples incubation color developing by adding phosphovanillinreagent absorbance reading and measurement of the lipidcontent based on the standard curve [62] Phosphovanillinreagent is prepared by dissolving vanillin in absolute ethanoland DI water followed by the addition of concentratedH3PO4 To prepare standard lipid stocks canola oil is firstly

added to chloroform and then different amount of standardlipid stocks is added to the tubes After that these tubes aretreated to evaporate the solvent followed by the addition ofwater Subsequently these samples are prepared by followingSPV reaction methods (1) suspend tested samples in waterand place in a glass tube (2) add concentrated sulfuric acidfollowed by heat treatment and ice bath (3) add freshlyprepared phosphovanillin reagent and incubate in incubatorshaker (4) read absorbance at 530 nmanddetermine the lipidcontent by comparing to the standard curve

44 TLC Method TLC is also a promising alternative toconventional lipids measurement approaches as it requiresminimal equipment which is available in most laboratoriesand it can also provide additional information about lipidclasses which is important for biofuel production [92]Among different solvent systems the multi-one-dimensionalTLC (MOD-TLC) separates the lipid classes rapidly and


Table3Nile

redassayprocedures

forthe

determ

inationof

microalgallipids

Reagent(s

)Nile

redlip

idassayprocedure

Fluo

rescence

determ

ination

Ref

Isop

ropylalcoh

ol(IPA

)Nile

redsolutio

nbleach

solutio

nmethano

lcorn

oil(dissolvedin

21

methano

lchloroform)

Suspendlip

idextractsin

chloroform

dilu

teextractswith

methano

ladddilutedsamples

andcorn

oiltothem

icroplate

toachievea

rang

eincubatethep

lateandevaporatethe

solvents

addIPAandcoolthep

lateaddNile

redsolutio

naddbleach

solutio

nto

each

wellincubatethep

late

Determinefl

uorescence

usingap

latereader

with

excitatio

nsetto530n

mandem

issionsetto575n

mwith

a570

nmcut-o

ffread

thep

lateuse

thefi

rstreading

after

the

fluorescencep

eakforq

uantitatio

n

[27]

DMSO

Nile

redsolutio

ntriolein

Placem

icroalgalcellsin

microcentrifugetub

esandpu

tund

ermicrowavetreatmentmixwith

DMSO

put

undersecon

dmicrowavetreatmentu

singthep

reviou

scon

ditio

nsadd

Nile

redsolutio

nandincubatethetub

esin

thed

arkpipetthe

samples

into

96wellm

icroplates

Measure

fluorescenceu

singFluo

rescence

Analyzerw

ithexcitatio

nat535n

mandem

issionat580n

mm

easure

untre

ated

suspensio

nandmedium

containing

Nile

redalon

eas

autoflu

orescenceconvertfl

uorescence

todryweighto

flip

ids

[28]

DMSO

Nile

redsolutio

nvirgin

oliveo

ilStaindilutedmicroalgalculture

samples

with

Nile

redusing

DMSO

ascarriertopup

thev

olum

eand

incubatewith

agitatio

n

Read

thefl

uorescence

usingMicroplateR

eaderw

ithexcitatio

nsetto520n

mandem

issioncaptured

at570n

m

compare

thefl

uorescence

toav

irgin

oliveo

ilsta

ndardcurve

[29

30]

Pure

trioleinchloroformisoprop

anolN

ileredsto

ck(in

100

spectralgradea

cetone)

Standardized

samplefl

uorescenceadd

triolein

tochloroform

anddilutewith

isoprop

anolprepare

workingsta

ndards

bybringing

interm

ediatesto

cksw

ithDIw

ater

NileredassayaddNile

redto

cellsuspensio

nor

toalipid

stand

ard

Measure

fluorescenceu

singspectro

photofl

uorometer

with

excitatio

nsetto475n

mandem

issionsetto580n

mexpress

cellu

larn

eutrallip

idas

triolein

equivalents

[31]


reproducibly The MOD-TLC method can achieve the quan-tification for the majority of microalgal lipids through modi-fications in solvent mixtures and lengths of separation timesand the mass of each resolved lipid band is determined bycomparing band intensities of unknown samples (visualizedby the lipophilic dye primulin followed by an automatedlaser-fluorescence detector scanning) to dilution curves ofauthentic standards Compared to two-dimensional thin-layer chromatography MOD-TLC directly analyzes multiplesamples on a single TLC plate while still providing goodresolution for the quantification of most major classes of lipidspecies [32] SomeTLC running procedures are introduced indetail (Table 4) Usually TLC plates must be activated beforeTLC running and NLs are separated by certain solvents suchas a mixture of chloroform methanol acetic acid water(85 125 125 3 vvvv) The determination of microalgallipid content is finally achieved by comparing the resultingfluorescence values with a standard curve

5 Quantification for Microalgal Fatty Acids

The theoretical biofuel potentials of microalgal biomass areultimately determined by the acyl chains of the lipids andtherefore the lipid contents are quantified as the sum oftheir fatty acid constituents The fatty acids constituents varywith their structural features such as chain length degreeof saturation branching of carbon chain positional isomersconfiguration of double bonds or other chemical groups(ie hydroxy epoxy cyclo and keto) [93] It was reportedthat C16 and C18 are the most abundant microalgal fattyacids including palmitic acid (hexadecanoic C160) stearicacid (octadecanoic C180) oleic acid (octadecenoic C181)linoleic acid (octadecadienoic C182) and linolenic acid(octadecatrienoic C183) The other fatty acids such as C14C20 and C26ndashC32 are relatively in low concentrations [94]

Table 5 summarizes and compares different methods ofthemeasurement and quantification ofmicroalgal fatty acidsThese methods consisted of two steps (1) the preparationof FAMEs (2) quantification and composition analysis ofFAMEs by gas chromatography (GC) In the first step viathe transesterification or in situ transesterification processthe triglycerides contained in algal lipids are reacted withmethanol to produce FAMEs and glycerol Catalysts (acidcatalyst base catalyst or the mixture) and heat (water oroil bath) are usually required during the transesterificationprocess to speed up the reaction The common catalysts forthis transesterification include NaOH [95] HCl [35] H

2SO4

[37] acetyl chloridemethanol (1 10 vv) [42] and a mixtureof methanol H

2SO4 and chloroform (17 03 20 vvv)

[47] In the second step the separation of FAMEs fromthe mixture and their quantification are performed usingGC The procedure noted above is based on the amountof fatty acids after the lipid extraction in algal biomassComparatively the in situ transesterification is a relativelysimpler process and achieves the transesterification to getFAMEs directly from the whole biomass with no requirementof lipid extraction Therefore it is able to obtain all fattyacids in the biomass and accurately represent the reflectionof biofuels potential [49 96] In addition various procedures

for transesterification are followed in terms of microalgalspecies lipid contents and targeted FAMEs fraction Thespecific transesterification procedures performed in literatureare listed in Table 5 In brief the lipid extracts or the algalbiomass are mixed with the catalysts and methanol and arereacted at the conditions of high temperature The producedFAMEs are then recovered in solvents like hexane for furtherpurification and quantification Subsequently the purifiedFAMEs are separated and analyzed by GC equipped withthe flame ionization detector (FID) and specific columnsrunning at various temperatures The identification andquantification standards are required such as the commercial37-component standards pentadecanoic acid and heptade-canoic acid [40 41 47 48 97] Specific FAMEs are quantifiedby comparing their peak areas with those of the standardsIt should be noted that there is still not a routine methodfor the quantification of fatty acids specific to algal biomassissued by Association of Analytical Communities (AOAC)International All the methods noted above vary significantlyincluding the procedures types of used chemicals and theirdoses and analytical apparatus This might result in a lack ofcomparability between FAMEs concentrations obtained fromdifferent methods

6 Conclusion

Microalgae have proven to be one of themost promising feed-stocks for the production of third-generation biofuels that areboth economically feasible and environmentally sustainableRapid accurate sustainable and cost-effective methods forthe lipid extraction and quantification are essential for therational application of microalgae-based biofuel productionGravimetric method is most widely used but requires quite afew amount of samples Nile red lipid visualization methodis rapid as the number of samples and preparation time aregreatly reduced while a correlation between fluorescence andlipid levels must be previously established as the cell stainingvaries among different microalgae species the results of SPVassay can be affected by lots of factors such as the degreeof oil saturation incubation time heating and cooling thusthe SPV assay may give misleading results in addition to thequantitative measurement of microalgal lipids TLC can alsoprovide additional information about lipid classes which isimportant for biofuel productionVarious lipid quantificationmethods could be considered on a case-by-case basis butmore effective and greener techniques (eg CO

2-basedmeth-

ods) for microalgal cell disruption and extraction are stillrequired to maximize lipid yields while avoiding the issues oftoxicity flammability and time consumption for extractionIn addition the identification and quantification of fatty acidsin extracted lipids are also important to evaluate the quality ofmicroalgae-derived biofuel during which transesterificationand in situ transesterification are involved in the preparationof FAMEs for the further the quantification and compositionanalysis of FAMEs by GC It is worth mentioning that in situtransesterification is relatively simpler and more convenientas it does not require lipid extraction thus achieving thetransesterification to get FAMEs directly from the wholebiomass


Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb

yshakinganddegassingaddsolventsto

theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand

pressuredeterminethe

lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse

hexanediethyletheraceticacid

(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020

1hexanediethyletheracetic

acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium

hydroxideaspiratetwo-third

softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg

as(15mLsdotminminus1)

(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax

column(30mtimes320120583

mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing

FAMEsintonewglasstub

e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta

ndardinternalstandard

(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector


Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors would like to graciously acknowledge theNational Natural Science Foundation of China (51708294)the Fundamental Research Funds for the Central Universities(30918011306 and 30918011308) and Research Start-UpGrantof NJUST for the funding and support provided for thisresearch

References

[1] S Shafiee and E Topal ldquoWhen will fossil fuel reserves bediminishedrdquo Energy Policy vol 37 no 1 pp 181ndash189 2009

[2] Y Chisti ldquoBiodiesel from microalgaerdquo Biotechnology Advancesvol 25 no 3 pp 294ndash306 2007

[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] Y Li S Lian D Tong et al ldquoOne-step production of biodieselfrom Nannochloropsis sp on solid base Mg-Zr catalystrdquoApplied Energy vol 88 no 10 pp 3313ndash3317 2011

[5] I Hariskos and C Posten ldquoBiorefinery of microalgae - oppor-tunities and constraints for different production scenariosrdquoBiotechnology Journal vol 9 no 6 pp 739ndash752 2014

[6] M R Tredici ldquoPhotobiology of microalgae mass culturesUnderstanding the tools for the next green revolutionrdquo Biofuelsvol 1 no 1 pp 143ndash162 2010

[7] S Ge P Champagne W C Plaxton G B Leite and F MarazzildquoMicroalgal cultivation with waste streams and metabolic con-straints to triacylglycerides accumulation for biofuel produc-tionrdquoBiofuels Bioproducts andBiorefining vol 11 no 2 pp 325ndash343 2017

[8] S Ge and P Champagne ldquoNutrient removal microalgalbiomass growth harvesting and lipid yield in response tocentrate wastewater loadingsrdquoWater Research vol 88 pp 604ndash612 2016

[9] E Sforza R Cipriani T Morosinotto A Bertucco and G MGiacometti ldquoExcess CO 2 supply inhibits mixotrophic growthof Chlorella protothecoides andNannochloropsis salinardquo Biore-source Technology vol 104 pp 523ndash529 2012

[10] S Ge M Agbakpe Z Wu L Kuang W Zhang and X WangldquoInfluences of surface coating UV irradiation and magneticfield on the algae removal using magnetite nanoparticlesrdquoEnvironmental Science amp Technology vol 49 no 2 pp 1190ndash1196 2015

[11] S Ge M Agbakpe W Zhang and L Kuang ldquoHeteroaggre-gation between PEI-coated magnetic nanoparticles and algaeEffect of particle size on algal harvesting efficiencyrdquoACSAppliedMaterials amp Interfaces vol 7 no 11 pp 6102ndash6108 2015

[12] S Ge P Champagne H Wang P G Jessop and M FCunningham ldquoMicroalgae Recovery from Water for BiofuelProduction Using CO2-Switchable Crystalline NanocelluloserdquoEnvironmental Science amp Technology vol 50 no 14 pp 7896ndash7903 2016

[13] A Steriti R Rossi A Concas and G Cao ldquoA novel cell dis-ruption technique to enhance lipid extraction frommicroalgaerdquoBioresource Technology vol 164 pp 70ndash77 2014

[14] I A Guschina and J L Harwood ldquoComplex lipid biosynthesisand its manipulation in plantsrdquo Improvement of Crop Plants forIndustrial End Uses pp 253ndash279 2007

[15] R Halim M K Danquah and P A Webley ldquoExtractionof oil from microalgae for biodiesel production a reviewrdquoBiotechnology Advances vol 30 no 3 pp 709ndash732 2012

[16] M A Borowitzka and N RMoheimani ldquoAlgae for biofuels andenergyrdquo Algae for Biofuels and Energy pp 1ndash288 2013

[17] T M Mata A A Martins and N S Caetano ldquoMicroalgaefor biodiesel production and other applications a reviewrdquoRenewable amp Sustainable Energy Reviews vol 14 no 1 pp 217ndash232 2010

[18] J Kim G Yoo H Lee et al ldquoMethods of downstreamprocessing for the production of biodiesel from microalgaerdquoBiotechnology Advances vol 31 no 6 pp 862ndash876 2013

[19] R Ranjith Kumar P Hanumantha Rao and M ArumugamldquoLipid ExtractionMethods fromMicroalgae A ComprehensiveReviewrdquo Frontiers in Energy Research vol 2 2015

[20] A K Lee D M Lewis and P J Ashman ldquoDisruption ofmicroalgal cells for the extraction of lipids for biofuels Pro-cesses and specific energy requirementsrdquo Biomass amp Bioenergyvol 46 pp 89ndash101 2012

[21] SOKotchoni EWGachomoK Slobodenko andDH ShainldquoAMP deaminase suppression increases biomass cold toleranceand oil content in green algaerdquo Algal Research vol 16 pp 473ndash480 2016

[22] Y Li M Horsman B Wang N Wu and C Q Lan ldquoEffectsof nitrogen sources on cell growth and lipid accumulation ofgreen alga Neochloris oleoabundansrdquo Applied Microbiology andBiotechnology vol 81 no 4 pp 629ndash636 2008

[23] W Zhang Y Zhao B Cui H Wang and T Liu ldquoEvaluation offilamentous green algae as feedstocks for biofuel productionrdquoBioresource Technology vol 220 pp 407ndash413 2016

[24] A Mehrabadi M M Farid and R Craggs ldquoEffect of CO2addition on biomass energy yield in wastewater treatment highrate algal mesocosmsrdquo Algal Research vol 22 pp 93ndash103 2017

[25] Q Lu W Zhou M Min et al ldquoMitigating ammonia nitrogendeficiency in dairy wastewaters for algae cultivationrdquo Biore-source Technology vol 201 pp 33ndash40 2016

[26] A Silkina M-P Zacharof G Hery T Nouvel and R WLovitt ldquoFormulation and utilisation of spent anaerobic digestatefluids for the growth and product formation of single cellalgal cultures in heterotrophic and autotrophic conditionsrdquoBioresource Technology vol 244 pp 1445ndash1455 2017

[27] B T Higgins A Thornton-Dunwoody J M Labavitch and JS Vandergheynst ldquoMicroplate assay for quantitation of neutrallipids in extracts frommicroalgaerdquoAnalytical Biochemistry vol465 pp 81ndash89 2014

[28] A E M Abdelaziz G B Leite M A Belhaj and P C Hallen-beck ldquoScreening microalgae native to Quebec for wastewatertreatment and biodiesel productionrdquo Bioresource Technologyvol 157 pp 140ndash148 2014

[29] Y-Z Wang P C Hallenbeck G B Leite K Paranjape and D-Q Huo ldquoGrowth and lipid accumulation of indigenous algalstrains under photoautotrophic and mixotrophic modes at lowtemperaturerdquo Algal Research vol 16 pp 195ndash200 2016

[30] G B Leite K Paranjape A E M Abdelaziz and P CHallenbeck ldquoUtilization of biodiesel-derived glycerol or xylosefor increased growth and lipid production by indigenousmicroalgaerdquo Bioresource Technology vol 184 pp 123ndash130 2015


[31] J C Priscu L R Priscu A C Palmisano and C W SullivanldquoEstimation of neutral lipid levels in antarctic sea icemicroalgaeby nile red fluorescencerdquoAntarctic Science vol 2 no 2 pp 149ndash155 1986

[32] T White S Bursten D Federighi R A Lewis and E Nudel-man ldquoHigh-resolution separation and quantification of neutrallipid and phospholipid species in mammalian cells and sera bymulti-one-dimensional thin-layer chromatographyrdquo AnalyticalBiochemistry vol 258 no 1 pp 109ndash117 1998

[33] J Liu J Huang Z Sun Y Zhong Y Jiang and F ChenldquoDifferential lipid and fatty acid profiles of photoautotrophicand heterotrophic Chlorella zofingiensis Assessment of algaloils for biodiesel productionrdquo Bioresource Technology vol 102no 1 pp 106ndash110 2011

[34] C J Hulatt O Berecz E S Egeland RHWijffels andVKironldquoPolar snow algae as a valuable source of lipidsrdquo BioresourceTechnology vol 235 pp 338ndash347 2017

[35] S N Genin J Stewart Aitchison and D Grant Allen ldquoDesignof algal film photobioreactors Material surface energy effectson algal film productivity colonization and lipid contentrdquoBioresource Technology vol 155 pp 136ndash143 2014

[36] I Smid and M Salfinger ldquoMycobacterial identification bycomputer-aided gas-liquid chromatographyrdquo Diagnostic Micro-biology And Infectious Disease vol 19 no 2 pp 81ndash88 1994

[37] H-M PhamH S KwakM-EHong J LeeW S Chang and SJ Sim ldquoDevelopment of an X-Shape airlift photobioreactor forincreasing algal biomass and biodiesel productionrdquo BioresourceTechnology vol 239 pp 211ndash218 2017

[38] A M Santos M Janssen P P Lamers W A C Evers and RH Wijffels ldquoGrowth of oil accumulating microalga Neochlorisoleoabundans under alkaline-saline conditionsrdquo BioresourceTechnology vol 104 pp 593ndash599 2012

[39] D J Pyle R A Garcia and Z Wen ldquoProducing docosahex-aenoic acid (DHA)-rich algae from biodiesel-derived crudeglycerol Effects of impurities on DHA production and algalbiomass compositionrdquo Journal of Agricultural and Food Chem-istry vol 56 no 11 pp 3933ndash3939 2008

[40] S KMishraW I SuhW Farooq et al ldquoRapid quantification ofmicroalgal lipids in aqueous medium by a simple colorimetricmethodrdquo Bioresource Technology vol 155 pp 330ndash333 2014

[41] M Tossavainen A Nykanen K Valkonen A Ojala S Kostiaand M Romantschuk ldquoCulturing of Selenastrum on dilutedcomposting fluids conversion ofwaste to valuable algal biomassin presence of bacteriardquo Bioresource Technology vol 238 pp205ndash213 2017

[42] N D Q Duy D S Francis and P C Southgate ldquoThe nutritionalvalue of live and concentrated micro-algae for early juveniles ofsandfish Holothuria scabrardquo Aquaculture vol 473 pp 97ndash1042017

[43] J A Conlan P L Jones G M Turchini M R Hall and DS Francis ldquoChanges in the nutritional composition of captiveearly-mid stage Panulirus ornatus phyllosoma over ecdysis andlarval developmentrdquo Aquaculture vol 434 pp 159ndash170 2014

[44] N Arora A Patel P A Pruthi and V Pruthi ldquoRecycled de-Oiled algal biomass extract as a feedstock for boosting biodieselproduction from chlorella minutissimardquo Applied Biochemistryand Biotechnology vol 180 no 8 pp 1534ndash1541 2016

[45] N Arora A Patel P A Pruthi and V Pruthi ldquoSynergisticdynamics of nitrogen and phosphorous influences lipid pro-ductivity in Chlorella minutissima for biodiesel productionrdquoBioresource Technology vol 213 pp 79ndash87 2015

[46] P Jain N Arora J Mehtani V Pruthi and C B MajumderldquoPretreated algal bloom as a substantial nutrient source formicroalgae cultivation for biodiesel productionrdquo BioresourceTechnology vol 242 pp 152ndash160 2017

[47] Z Chi D Pyle Z Wen C Frear and S Chen ldquoA laboratorystudy of producing docosahexaenoic acid from biodiesel-wasteglycerol by microalgal fermentationrdquo Process Biochemistry vol42 no 11 pp 1537ndash1545 2007

[48] E Indarti M I A Majid R Hashim and A Chong ldquoDirectFAME synthesis for rapid total lipid analysis from fish oil andcod liver oilrdquo Journal of Food Composition and Analysis vol 18no 2-3 pp 161ndash170 2005

[49] L M L Laurens M Quinn S Van Wychen D W Templetonand E J Wolfrum ldquoAccurate and reliable quantification of totalmicroalgal fuel potential as fatty acid methyl esters by in situtransesterificationrdquoAnalytical and Bioanalytical Chemistry vol403 no 1 pp 167ndash178 2012

[50] S Balasubramanian J D Allen A Kanitkar and D BoldorldquoOil extraction from Scenedesmus obliquus using a continuousmicrowave system - design optimization andquality character-izationrdquo Bioresource Technology vol 102 no 3 pp 3396ndash34032011

[51] F Amarni and H Kadi ldquoKinetics study of microwave-assistedsolvent extraction of oil from olive cake using hexane Compar-ison with the conventional extractionrdquo Innovative Food Scienceand Emerging Technologies vol 11 no 2 pp 322ndash327 2010

[52] A F Ferreira A P S Dias C M Silva and M Costa ldquoEffectof low frequency ultrasound on microalgae solvent extractionAnalysis of products energy consumption and emissionsrdquoAlgalResearch vol 14 pp 9ndash16 2016

[53] K S Suslick and D J Flannigan ldquoInside a collapsing bub-ble Sonoluminescence and the conditions during cavitationrdquoAnnual Review of Physical Chemistry vol 59 pp 659ndash683 2008

[54] F Adam M Abert-Vian G Peltier and F Chemat ldquolsquoSolvent-freersquo ultrasound-assisted extraction of lipids from freshmicroal-gae cells a green clean and scalable processrdquo BioresourceTechnology vol 114 pp 457ndash465 2012

[55] M S Doulah ldquoMechanism of disintegration of biological cellsin ultrasonic cavitationrdquo Biotechnology and Bioengineering vol19 no 5 pp 649ndash660 1977

[56] R Halim A Hosikian S Lim and M K Danquah ldquoChloro-phyll extraction frommicroalgae A review on the process engi-neering aspectsrdquo International Journal of Chemical EngineeringArticle ID 391632 2010

[57] M Vaara ldquoAgents that increase the permeability of the outermembranerdquo Microbiology and Molecular Biology Reviews vol56 no 3 pp 395ndash411 1992

[58] A Sathish and R C Sims ldquoBiodiesel from mixed culture algaevia a wet lipid extraction procedurerdquo Bioresource Technologyvol 118 pp 643ndash647 2012

[59] M Demuez AMahdy E Tomas-Pejo C Gonzalez-Fernandezand M Ballesteros ldquoEnzymatic cell disruption of microalgaebiomass in biorefinery processesrdquo Biotechnology and Bioengi-neering vol 112 no 10 pp 1955ndash1966 2015

[60] A Zuorro G Maffei and R Lavecchia ldquoOptimizationof enzyme-assisted lipid extraction from Nannochloropsismicroalgaerdquo Journal of the Taiwan Institute of Chemical Engi-neers vol 67 pp 106ndash114 2016

[61] K Kita S Okada H Sekino K Imou S Yokoyama and TAmano ldquoThermal pre-treatment of wet microalgae harvest forefficient hydrocarbon recoveryrdquo Applied Energy vol 87 no 7pp 2420ndash2423 2010


[62] Y-S Cheng Y Zheng and J S VanderGheynst ldquoRapid quan-titative analysis of lipids using a colorimetric method in amicroplate formatrdquo Lipids vol 46 no 1 pp 95ndash103 2011

[63] W Chen C Zhang L Song M Sommerfeld and Q Hu ldquoAhigh throughputNile redmethod for quantitativemeasurementof neutral lipids in microalgaerdquo Journal of MicrobiologicalMethods vol 77 no 1 pp 41ndash47 2009

[64] I R Sitepu L Ignatia A K Franz et al ldquoAn improvedhigh-throughput Nile red fluorescence assay for estimatingintracellular lipids in a variety of yeast speciesrdquo Journal ofMicrobiological Methods vol 91 no 2 pp 321ndash328 2012

[65] Y Tang Y Zhang J N Rosenberg N Sharif M J Betenbaughand F Wang ldquoEfficient lipid extraction and quantificationof fatty acids from algal biomass using accelerated solventextraction (ASE)rdquoRSCAdvances vol 6 no 35 pp 29127ndash291342016

[66] L Lardon A Helias B Sialve J P Steyer andO Bernard ldquoLife-cycle assessment of biodiesel production from microalgaerdquoEnvironmental Science amp Technology vol 43 no 17 pp 6475ndash6481 2009

[67] P Mercer and R E Armenta ldquoDevelopments in oil extractionfrom microalgaerdquo European Journal of Lipid Science and Tech-nology vol 113 no 5 pp 539ndash547 2011

[68] R L Mendes A D Reis and A F Palavra ldquoSupercritical CO2

extraction of 120574-linolenic acid and other lipids from Arthrospira(Spirulina)maxima comparison with organic solvent extrac-tionrdquo Food Chemistry vol 99 no 1 pp 57ndash63 2006

[69] G Andrich U Nesti F Venturi A Zinnai and R Fioren-tini ldquoSupercritical fluid extraction of bioactive lipids fromthe microalga Nannochloropsis sprdquo European Journal of LipidScience and Technology vol 107 no 6 pp 381ndash386 2005

[70] C Crampon A Mouahid S-A A Toudji O Lepine and EBadens ldquoInfluence of pretreatment on supercritical CO2 extrac-tion fromNannochloropsis oculatardquoThe Journal of SupercriticalFluids vol 79 pp 337ndash344 2013

[71] A Mouahid C Crampon S-A A Toudji and E BadensldquoSupercritical CO2 extraction of neutral lipids frommicroalgaeExperiments andmodellingrdquoThe Journal of Supercritical Fluidsvol 77 pp 7ndash16 2013

[72] R Halim B Gladman M K Danquah and P A WebleyldquoOil extraction from microalgae for biodiesel productionrdquoBioresource Technology vol 102 no 1 pp 178ndash185 2011

[73] L Soh and J Zimmerman ldquoBiodiesel productionThe potentialof algal lipids extracted with supercritical carbon dioxiderdquoGreen Chemistry vol 13 no 6 pp 1422ndash1429 2011

[74] B-C Liau C-T Shen F-P Liang et al ldquoSupercritical fluidsextraction and anti-solvent purification of carotenoids frommicroalgae and associated bioactivityrdquoThe Journal of Supercrit-ical Fluids vol 55 no 1 pp 169ndash175 2010

[75] A Paudel M J Jessop S H Stubbins P Champagne and PG Jessop ldquoExtraction of lipids from microalgae using CO2-expanded methanol and liquid CO2rdquo Bioresource Technologyvol 184 pp 286ndash290 2015

[76] B Subramaniam RV Chaudhari A S Chaudhari G R Akienand Z Xie ldquoSupercritical fluids and gas-expanded liquids astunable media for multiphase catalytic reactionsrdquo ChemicalEngineering Science vol 115 pp 3ndash18 2014

[77] P G Jessop and B Subramaniam ldquoGas-expanded liquidsrdquoChemical Reviews vol 107 no 6 pp 2666ndash2694 2007

[78] H-C Wang W Klinthong Y-H Yang and C-S Tan ldquoCon-tinuous extraction of lipids from Schizochytrium sp by CO2-expanded ethanolrdquoBioresource Technology vol 189 pp 162ndash1682015

[79] B Subramaniam and G R Akien ldquoSustainable catalytic reac-tion engineering with gas-expanded liquidsrdquo Current Opinionin Chemical Engineering vol 1 no 3 pp 336ndash341 2012

[80] Y-H Yang W Klinthong and C-S Tan ldquoOptimization ofcontinuous lipid extraction from Chlorella vulgaris by CO2-expandedmethanol for biodiesel productionrdquoBioresource Tech-nology vol 198 pp 550ndash556 2015

[81] S Wahidin A Idris and S R M Shaleh ldquoIonic liquidas a promising biobased green solvent in combination withmicrowave irradiation for direct biodiesel productionrdquo Biore-source Technology vol 206 pp 150ndash154 2016

[82] Y Chisti ldquoBiodiesel from microalgae beats bioethanolrdquo Trendsin Biotechnology vol 26 no 3 pp 126ndash131 2008

[83] R E Teixeira ldquoEnergy-efficient extraction of fuel and chemicalfeedstocks from algaerdquo Green Chemistry vol 14 no 2 pp 419ndash427 2012

[84] K Gao V Orr and L Rehmann ldquoButanol fermentation frommicroalgae-derived carbohydrates after ionic liquid extractionrdquoBioresource Technology vol 206 pp 77ndash85 2016

[85] P G Jessop D J Heldebrant X Li C A Eckertt andC L Liotta ldquoGreen chemistry Reversible nonpolar-to-polarsolventrdquo Nature vol 436 no 7054 p 1102 2005

[86] P G Jessop ldquoSwitchable solvents as media for synthesis andseparations An update from the co-creator of GreenCentreCanadardquo Aldrichimica Acta vol 48 no 1 pp 18ndash21 2015

[87] VCAOrr andL Rehmann ldquoIonic liquids for the fractionationof microalgae biomassrdquo Current Opinion in Green and Sustain-able Chemistry vol 2 pp 22ndash27 2016

[88] P G Jessop L Phan A Carrier S Robinson C J Durr and JR Harjani ldquoA solvent having switchable hydrophilicityrdquo GreenChemistry vol 12 no 5 pp 809ndash814 2010

[89] P G Jessop L Kozycz Z G Rahami et al ldquoTertiary aminesolvents having switchable hydrophilicityrdquo Green Chemistryvol 13 no 3 pp 619ndash623 2011

[90] G H Huang G Chen and F Chen ldquoRapid screening methodfor lipid production in alga based on Nile red fluorescencerdquoBiomass amp Bioenergy vol 33 no 10 pp 1386ndash1392 2009

[91] L S Inouye and G R Lotufo ldquoComparison of macro-gravimetric and micro-colorimetric lipid determination meth-odsrdquo Talanta vol 70 no 3 pp 584ndash587 2006

[92] B Fuchs R Suszlig K Teuber M Eibisch and J Schiller ldquoLipidanalysis by thin-layer chromatographymdasha review of the currentstaterdquo Journal of Chromatography A vol 1218 no 19 pp 2754ndash2774 2011

[93] P Pesaresi J S Amthor G Mandolino and etal Improvementof Crop Plants for Industrial End Uses Springer Netherlands2007

[94] H Liu and W Liu ldquoConcentration and distributions of fattyacids in algae submerged plants and terrestrial plants from thenortheastern Tibetan Plateaurdquo Organic Geochemistry vol 113pp 17ndash26 2017

[95] V Kumar M Muthuraj B Palabhanvi A K Ghoshal andD Das ldquoEvaluation and optimization of two stage sequentialin situ transesterification process for fatty acid methyl esterquantification from microalgaerdquo Journal of Renewable Energyvol 68 pp 560ndash569 2014


[96] S Van Wychen K Ramirez and L M Laurens Determinationof Total Lipids as Fatty Acid Methyl Esters (FAME) by In SituTransesterification Laboratory Analytical Procedure (LAP)National Renewable Energy Laboratory (NREL) Golden ColoUSA 2013

[97] G Knothe ldquoImproving biodiesel fuel properties by modifyingfatty ester compositionrdquo Energy amp Environmental Science vol2 no 7 pp 759ndash766 2009

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


２

２

２

（2

（2 minus

minus

P

O

O

H

H

H

H

H

C

C

C

O

O

O

C

C CH

O

Phosphate

Fatty acid

Glycerol

Figure 1 Lipid molecules Triacylglycerol (NL) on the left Phospholipid (polar lipid) on the right R1015840 R10158401015840 and R101584010158401015840 in the triacylglycerolmolecule represent fatty acid chains Phospholipid molecule is negatively charged [15]

Moreover the main storage lipids in microalgae are neutrallipids (NLs) or triacylglycerols that can be esterified toFAMEs with the primary profiles of C16 and C18 proven tobe the most suitable for biofuel production [7] Microalgaeexhibit great adaption to various environmental conditionsmaking them easy to cultivate For instance they can growon marginal land in both the open pond and closed systemsusing waste streams like wastewater waste or CO

2-enriched

gas (biogas flue gas) waste organics (ie crude glycerol) andwaste heat to provide nutrients and carbon and temperaturemaintenance (in a cold climate) achieving economicallyfeasible and environmentally sustainable biofuel productionand waste bioremediation [8] Moreover various routes ofmicroalgal metabolisms can be adopted for enhanced growthand lipid production Traditionally phototrophic algae aregrown autotrophically with CO

2as the unique carbon source

and light providing all the energy needed Moreover somemicroalgae species can grow heterotrophically using onlyorganic compounds while others can grow mixotrophicallyusing both organic compounds and CO

2to support growth

[9]Currently the high costs of the important microalgal har-

vesting and lipid extraction processes are the primary obsta-cles impeding on the commercial application of microalgae-derived biofuel production [10ndash12] For example lipid extrac-tion is a high-power-consumption process because lipids arestored in microalgal cells and the cell wall is a thick and rigidlayer composed of complex carbohydrates and glycoproteinswith high mechanical strength and chemical resistanceposing difficulties for lipid extraction [13] Therefore certaincell disruption techniques are generally considered priorto lipid extraction to improve the extraction efficienciesNevertheless the efficiencies of cell disruption and lipidextraction vary with methods selected with different operat-ing conditions (eg temperature atmospheric pressure andhumidity) microalgae species and biomass amount

This review summarizes and compares themethodologiesemployed for the extraction and quantification of microalgallipids The pretreatment methods supporting cell disruptionand subsequent lipid extraction are also included Finally theprinciples and procedures for the production and quantifica-tion of fatty acids in microalgae are discussed in detail

2 Microalgal Lipids

Microalgal lipids can be divided into two groups accordingto their structures nonpolar NLs (acylglycerols sterolsfree fatty acids wax and steryl esters) and polar lipids(phosphoglycerides glycosylglycerides and sphingolipids)Figure 1 shows the structural formula of the polar lipidand NLs [14 15] These lipids play different but importantroles in microalgal metabolism and growth period Somelipids such as phosphoglycerides glycosylglycerides andsterols are imperative structural components of biologicalmembranes while lipids like inositol lipids sphingolipidsand oxidative products of polyunsaturated fatty acids mayact as key intermediates in the cell signaling pathwaysand play a role in sensing changes in the environment[16] The quantities of these microalgal lipids vary withthe type of species growth conditions and ambient envi-ronments It was reported that the lipid contents rangedat 20ndash50 of dry biomass including Chlorella Cryptheco-dinium Cylindrotheca Dunaliella Isochrysis NannochlorisNannochloropsis Neochloris Nitzschia Phaeodactylum Por-phyridium Schizochytrium and Tetraselmis [17]

3 Microalgal Cell Disruption Methods

Specific microalgal cell pretreatment procedures must beconsidered prior to the subsequent lipid extraction due tothe microalgal cell wall structure When the extraction isconducted from the wet biomass the pretreatment step ismandatory to disrupt the microalgal cell walls and allow thelipids to be released into the extracting mixture The com-monly used pretreatment methods are summarized below

31 Bead Beating Bead beating also known as bead mill orball mill disrupts cells by the impact of high-speed spinningof fine beads on the biomass slurry The whole disruptionprocess could be done within minutes and it could beapplied to any kinds of microalgae without preparation [1819] Two common types of bead mills are shaking vesselsand agitated beads [20] Shaking vessels usually consist ofmultiple containers or well-plates on a vibrating platformand the cell disruption is done by shaking the entire vessel







2SO4) alkalis (ie





2 CylinderFurnace

ElectricReactor

Gas flow meter

Regulator


PCRM

TRPI

Stirrer

V1

V2






bulk organicsolvent


cytoplasm

nucleus

1

1

2

2

3

3

4

4

5

5









microalgallipid

contentu

singconventio

nalorganicsolvents

Lipidextractio

nDeterminationof

lipid

content

Ref

Reagent(s

)Ex

tractio

nprocess

Treatm

entfor

finaldeterm

ination

Expressio

nof

lipid

content

Chloroform

isop

ropano

l(11

vv)

andh

exane

Addsolventm

ixture

tofro

zenpellets

centrifugea

ndtransfe

rsup

ernatantsreextract

pelletswith

hexane


supernatants

Dry

thec


peed

vacuum

andrecord

thew

eight

Apercentage

oftotalfresh

weight

(ww

)[21]

Ethylether

Groun

ddrysamples

topo

wdera

ndusethe

Soxh

letextractor

Distill

thes

olventdry

ther

esidueand

record

the

weight

Apercentage

ofdrycellweight(

ww

)[22]

Methano

lchloroform

1NaC

l(22

1vvv)

Extractthe

biom

assw

iththes

olvent

mixture

Evaporatethe

chloroform

layerdrythes

amplea

ndrecord

thew

eight

Thew

eightratioof

extractedlip

idto

lyop

hilized

pellets(

ww

)[23]

Deion

ized

(DI)

waterm

ethano

lchloroform

(12

1vvv)chloroform

Addsolventm

ixture

toharvestedbiom

assa

ndreactfor

24hmixandthen

addsolventto

achievea

finalDIw

aterm

ethano

lchloroform

ratio

of09


thelipid-chloroform

layerrepeatthea

bove

steps

forsecon

dextractio

n

Evaporatethe

chloroform

layercoolthetub

eand

record

thew

eight

Apercentage

oftotalbiomass

weight(w

w)

[24]

Chloroform

methano

l(2

1vv)

Mixdryalgalp

owderw

iththes

olvent

mixture

putu

nder

water

bath

with

thea

idof

ultrasou

ndcentrifu

geandrepeatthea

bove

steps

threetim

es

Evaporatethe

organics

olvent

andweight

Apercentage

oftotalbiomass

weight(w

w)

[25]

Chloroform

methano

l(2

1vv)

Extractlipid

from

drybiom

assw

iththes

olvent

mixture

Evaporatethe

solventand

weight

Weightd

ifference

betweenthe

blankflask

andthefl

askcontaining

thee

xtracted

oil

[26]




lCO2



2




2







2) and liquid (lCO










2with




2and then to the









2


2is described as



2is delivered



2is




1

3

5

7

VV

0


Ethyl Acetate





2has a











2mim]










Extractwithsolvent

- C2

Add waterC2amp

oil

oil



oil amphydrophobic

solvent

water

hydrophobicsolvent









Table3Nile

redassayprocedures

forthe

determ

inationof

microalgallipids

Reagent(s

)Nile

redlip

idassayprocedure

Fluo

rescence

determ

ination

Ref

Isop

ropylalcoh

ol(IPA

)Nile

redsolutio

nbleach

solutio

nmethano

lcorn

oil(dissolvedin

21

methano

lchloroform)

Suspendlip

idextractsin

chloroform

dilu

teextractswith

methano

ladddilutedsamples

andcorn

oiltothem

icroplate

toachievea

rang

eincubatethep

lateandevaporatethe

solvents

addIPAandcoolthep

lateaddNile

redsolutio

naddbleach

solutio

nto

each

wellincubatethep

late

Determinefl

uorescence

usingap

latereader

with

excitatio

nsetto530n

mandem

issionsetto575n

mwith

a570

nmcut-o

ffread

thep

lateuse

thefi

rstreading

after

the

fluorescencep

eakforq

uantitatio

n

[27]

DMSO

Nile

redsolutio

ntriolein

Placem

icroalgalcellsin

microcentrifugetub

esandpu

tund


DMSO

put

undersecon


singthep

reviou

scon

ditio

nsadd

Nile

redsolutio

nandincubatethetub

esin

thed

arkpipetthe

samples

into

96wellm

icroplates

Measure

fluorescenceu

singFluo

rescence

Analyzerw

ithexcitatio

nat535n

mandem

issionat580n

mm

easure

untre

ated

suspensio

nandmedium

containing

Nile

redalon

eas

autoflu

orescenceconvertfl

uorescence

todryweighto

flip

ids

[28]

DMSO

Nile

redsolutio

nvirgin

oliveo


samples

with

Nile

redusing

DMSO

ascarriertopup

thev

olum

eand

incubatewith

agitatio

n

Read

thefl

uorescence

usingMicroplateR

eaderw

ithexcitatio

nsetto520n

mandem

issioncaptured

at570n

m

compare

thefl

uorescence

toav

irgin

oliveo

ilsta

ndardcurve

[29

30]

Pure


anolN

ileredsto

ck(in

100

spectralgradea

cetone)

Standardized

samplefl

uorescenceadd

triolein

tochloroform

anddilutewith

isoprop

anolprepare

workingsta

ndards

bybringing

interm

ediatesto

cksw

ithDIw

ater

NileredassayaddNile

redto

cellsuspensio

nor

toalipid

stand

ard

Measure

fluorescenceu

singspectro

photofl

uorometer

with

excitatio

nsetto475n

mandem

issionsetto580n

mexpress

cellu

larn

eutrallip

idas

triolein

equivalents

[31]






2SO4





6 Conclusion


2-basedmeth-



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018








2SO4) alkalis (ie





2 CylinderFurnace

ElectricReactor

Gas flow meter

Regulator


PCRM

TRPI

Stirrer

V1

V2






bulk organicsolvent


cytoplasm

nucleus

1

1

2

2

3

3

4

4

5

5









microalgallipid

contentu

singconventio

nalorganicsolvents

Lipidextractio

nDeterminationof

lipid

content

Ref

Reagent(s

)Ex

tractio

nprocess

Treatm

entfor

finaldeterm

ination

Expressio

nof

lipid

content

Chloroform

isop

ropano

l(11

vv)

andh

exane

Addsolventm

ixture

tofro

zenpellets

centrifugea

ndtransfe

rsup

ernatantsreextract

pelletswith

hexane


supernatants

Dry

thec


peed

vacuum

andrecord

thew

eight

Apercentage

oftotalfresh

weight

(ww

)[21]

Ethylether

Groun

ddrysamples

topo

wdera

ndusethe

Soxh

letextractor

Distill

thes

olventdry

ther

esidueand

record

the

weight

Apercentage

ofdrycellweight(

ww

)[22]

Methano

lchloroform

1NaC

l(22

1vvv)

Extractthe

biom

assw

iththes

olvent

mixture

Evaporatethe

chloroform

layerdrythes

amplea

ndrecord

thew

eight

Thew

eightratioof

extractedlip

idto

lyop

hilized

pellets(

ww

)[23]

Deion

ized

(DI)

waterm

ethano

lchloroform

(12

1vvv)chloroform

Addsolventm

ixture

toharvestedbiom

assa

ndreactfor

24hmixandthen

addsolventto

achievea

finalDIw

aterm

ethano

lchloroform

ratio

of09


thelipid-chloroform

layerrepeatthea

bove

steps

forsecon

dextractio

n

Evaporatethe

chloroform

layercoolthetub

eand

record

thew

eight

Apercentage

oftotalbiomass

weight(w

w)

[24]

Chloroform

methano

l(2

1vv)

Mixdryalgalp

owderw

iththes

olvent

mixture

putu

nder

water

bath

with

thea

idof

ultrasou

ndcentrifu

geandrepeatthea

bove

steps

threetim

es

Evaporatethe

organics

olvent

andweight

Apercentage

oftotalbiomass

weight(w

w)

[25]

Chloroform

methano

l(2

1vv)

Extractlipid

from

drybiom

assw

iththes

olvent

mixture

Evaporatethe

solventand

weight

Weightd

ifference

betweenthe

blankflask

andthefl

askcontaining

thee

xtracted

oil

[26]




lCO2



2




2







2) and liquid (lCO










2with




2and then to the









2


2is described as



2is delivered



2is




1

3

5

7

VV

0


Ethyl Acetate





2has a











2mim]










Extractwithsolvent

- C2

Add waterC2amp

oil

oil



oil amphydrophobic

solvent

water

hydrophobicsolvent









Table3Nile

redassayprocedures

forthe

determ

inationof

microalgallipids

Reagent(s

)Nile

redlip

idassayprocedure

Fluo

rescence

determ

ination

Ref

Isop

ropylalcoh

ol(IPA

)Nile

redsolutio

nbleach

solutio

nmethano

lcorn

oil(dissolvedin

21

methano

lchloroform)

Suspendlip

idextractsin

chloroform

dilu

teextractswith

methano

ladddilutedsamples

andcorn

oiltothem

icroplate

toachievea

rang

eincubatethep

lateandevaporatethe

solvents

addIPAandcoolthep

lateaddNile

redsolutio

naddbleach

solutio

nto

each

wellincubatethep

late

Determinefl

uorescence

usingap

latereader

with

excitatio

nsetto530n

mandem

issionsetto575n

mwith

a570

nmcut-o

ffread

thep

lateuse

thefi

rstreading

after

the

fluorescencep

eakforq

uantitatio

n

[27]

DMSO

Nile

redsolutio

ntriolein

Placem

icroalgalcellsin

microcentrifugetub

esandpu

tund


DMSO

put

undersecon


singthep

reviou

scon

ditio

nsadd

Nile

redsolutio

nandincubatethetub

esin

thed

arkpipetthe

samples

into

96wellm

icroplates

Measure

fluorescenceu

singFluo

rescence

Analyzerw

ithexcitatio

nat535n

mandem

issionat580n

mm

easure

untre

ated

suspensio

nandmedium

containing

Nile

redalon

eas

autoflu

orescenceconvertfl

uorescence

todryweighto

flip

ids

[28]

DMSO

Nile

redsolutio

nvirgin

oliveo


samples

with

Nile

redusing

DMSO

ascarriertopup

thev

olum

eand

incubatewith

agitatio

n

Read

thefl

uorescence

usingMicroplateR

eaderw

ithexcitatio

nsetto520n

mandem

issioncaptured

at570n

m

compare

thefl

uorescence

toav

irgin

oliveo

ilsta

ndardcurve

[29

30]

Pure


anolN

ileredsto

ck(in

100

spectralgradea

cetone)

Standardized

samplefl

uorescenceadd

triolein

tochloroform

anddilutewith

isoprop

anolprepare

workingsta

ndards

bybringing

interm

ediatesto

cksw

ithDIw

ater

NileredassayaddNile

redto

cellsuspensio

nor

toalipid

stand

ard

Measure

fluorescenceu

singspectro

photofl

uorometer

with

excitatio

nsetto475n

mandem

issionsetto580n

mexpress

cellu

larn

eutrallip

idas

triolein

equivalents

[31]






2SO4





6 Conclusion


2-basedmeth-



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



2 CylinderFurnace

ElectricReactor

Gas flow meter

Regulator


PCRM

TRPI

Stirrer

V1

V2






bulk organicsolvent


cytoplasm

nucleus

1

1

2

2

3

3

4

4

5

5









microalgallipid

contentu

singconventio

nalorganicsolvents

Lipidextractio

nDeterminationof

lipid

content

Ref

Reagent(s

)Ex

tractio

nprocess

Treatm

entfor

finaldeterm

ination

Expressio

nof

lipid

content

Chloroform

isop

ropano

l(11

vv)

andh

exane

Addsolventm

ixture

tofro

zenpellets

centrifugea

ndtransfe

rsup

ernatantsreextract

pelletswith

hexane


supernatants

Dry

thec


peed

vacuum

andrecord

thew

eight

Apercentage

oftotalfresh

weight

(ww

)[21]

Ethylether

Groun

ddrysamples

topo

wdera

ndusethe

Soxh

letextractor

Distill

thes

olventdry

ther

esidueand

record

the

weight

Apercentage

ofdrycellweight(

ww

)[22]

Methano

lchloroform

1NaC

l(22

1vvv)

Extractthe

biom

assw

iththes

olvent

mixture

Evaporatethe

chloroform

layerdrythes

amplea

ndrecord

thew

eight

Thew

eightratioof

extractedlip

idto

lyop

hilized

pellets(

ww

)[23]

Deion

ized

(DI)

waterm

ethano

lchloroform

(12

1vvv)chloroform

Addsolventm

ixture

toharvestedbiom

assa

ndreactfor

24hmixandthen

addsolventto

achievea

finalDIw

aterm

ethano

lchloroform

ratio

of09


thelipid-chloroform

layerrepeatthea

bove

steps

forsecon

dextractio

n

Evaporatethe

chloroform

layercoolthetub

eand

record

thew

eight

Apercentage

oftotalbiomass

weight(w

w)

[24]

Chloroform

methano

l(2

1vv)

Mixdryalgalp

owderw

iththes

olvent

mixture

putu

nder

water

bath

with

thea

idof

ultrasou

ndcentrifu

geandrepeatthea

bove

steps

threetim

es

Evaporatethe

organics

olvent

andweight

Apercentage

oftotalbiomass

weight(w

w)

[25]

Chloroform

methano

l(2

1vv)

Extractlipid

from

drybiom

assw

iththes

olvent

mixture

Evaporatethe

solventand

weight

Weightd

ifference

betweenthe

blankflask

andthefl

askcontaining

thee

xtracted

oil

[26]




lCO2



2




2







2) and liquid (lCO










2with




2and then to the









2


2is described as



2is delivered



2is




1

3

5

7

VV

0


Ethyl Acetate





2has a











2mim]










Extractwithsolvent

- C2

Add waterC2amp

oil

oil



oil amphydrophobic

solvent

water

hydrophobicsolvent









Table3Nile

redassayprocedures

forthe

determ

inationof

microalgallipids

Reagent(s

)Nile

redlip

idassayprocedure

Fluo

rescence

determ

ination

Ref

Isop

ropylalcoh

ol(IPA

)Nile

redsolutio

nbleach

solutio

nmethano

lcorn

oil(dissolvedin

21

methano

lchloroform)

Suspendlip

idextractsin

chloroform

dilu

teextractswith

methano

ladddilutedsamples

andcorn

oiltothem

icroplate

toachievea

rang

eincubatethep

lateandevaporatethe

solvents

addIPAandcoolthep

lateaddNile

redsolutio

naddbleach

solutio

nto

each

wellincubatethep

late

Determinefl

uorescence

usingap

latereader

with

excitatio

nsetto530n

mandem

issionsetto575n

mwith

a570

nmcut-o

ffread

thep

lateuse

thefi

rstreading

after

the

fluorescencep

eakforq

uantitatio

n

[27]

DMSO

Nile

redsolutio

ntriolein

Placem

icroalgalcellsin

microcentrifugetub

esandpu

tund


DMSO

put

undersecon


singthep

reviou

scon

ditio

nsadd

Nile

redsolutio

nandincubatethetub

esin

thed

arkpipetthe

samples

into

96wellm

icroplates

Measure

fluorescenceu

singFluo

rescence

Analyzerw

ithexcitatio

nat535n

mandem

issionat580n

mm

easure

untre

ated

suspensio

nandmedium

containing

Nile

redalon

eas

autoflu

orescenceconvertfl

uorescence

todryweighto

flip

ids

[28]

DMSO

Nile

redsolutio

nvirgin

oliveo


samples

with

Nile

redusing

DMSO

ascarriertopup

thev

olum

eand

incubatewith

agitatio

n

Read

thefl

uorescence

usingMicroplateR

eaderw

ithexcitatio

nsetto520n

mandem

issioncaptured

at570n

m

compare

thefl

uorescence

toav

irgin

oliveo

ilsta

ndardcurve

[29

30]

Pure


anolN

ileredsto

ck(in

100

spectralgradea

cetone)

Standardized

samplefl

uorescenceadd

triolein

tochloroform

anddilutewith

isoprop

anolprepare

workingsta

ndards

bybringing

interm

ediatesto

cksw

ithDIw

ater

NileredassayaddNile

redto

cellsuspensio

nor

toalipid

stand

ard

Measure

fluorescenceu

singspectro

photofl

uorometer

with

excitatio

nsetto475n

mandem

issionsetto580n

mexpress

cellu

larn

eutrallip

idas

triolein

equivalents

[31]






2SO4





6 Conclusion


2-basedmeth-



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




microalgallipid

contentu

singconventio

nalorganicsolvents

Lipidextractio

nDeterminationof

lipid

content

Ref

Reagent(s

)Ex

tractio

nprocess

Treatm

entfor

finaldeterm

ination

Expressio

nof

lipid

content

Chloroform

isop

ropano

l(11

vv)

andh

exane

Addsolventm

ixture

tofro

zenpellets

centrifugea

ndtransfe

rsup

ernatantsreextract

pelletswith

hexane


supernatants

Dry

thec


peed

vacuum

andrecord

thew

eight

Apercentage

oftotalfresh

weight

(ww

)[21]

Ethylether

Groun

ddrysamples

topo

wdera

ndusethe

Soxh

letextractor

Distill

thes

olventdry

ther

esidueand

record

the

weight

Apercentage

ofdrycellweight(

ww

)[22]

Methano

lchloroform

1NaC

l(22

1vvv)

Extractthe

biom

assw

iththes

olvent

mixture

Evaporatethe

chloroform

layerdrythes

amplea

ndrecord

thew

eight

Thew

eightratioof

extractedlip

idto

lyop

hilized

pellets(

ww

)[23]

Deion

ized

(DI)

waterm

ethano

lchloroform

(12

1vvv)chloroform

Addsolventm

ixture

toharvestedbiom

assa

ndreactfor

24hmixandthen

addsolventto

achievea

finalDIw

aterm

ethano

lchloroform

ratio

of09


thelipid-chloroform

layerrepeatthea

bove

steps

forsecon

dextractio

n

Evaporatethe

chloroform

layercoolthetub

eand

record

thew

eight

Apercentage

oftotalbiomass

weight(w

w)

[24]

Chloroform

methano

l(2

1vv)

Mixdryalgalp

owderw

iththes

olvent

mixture

putu

nder

water

bath

with

thea

idof

ultrasou

ndcentrifu

geandrepeatthea

bove

steps

threetim

es

Evaporatethe

organics

olvent

andweight

Apercentage

oftotalbiomass

weight(w

w)

[25]

Chloroform

methano

l(2

1vv)

Extractlipid

from

drybiom

assw

iththes

olvent

mixture

Evaporatethe

solventand

weight

Weightd

ifference

betweenthe

blankflask

andthefl

askcontaining

thee

xtracted

oil

[26]




lCO2



2




2







2) and liquid (lCO










2with




2and then to the









2


2is described as



2is delivered



2is




1

3

5

7

VV

0


Ethyl Acetate





2has a











2mim]










Extractwithsolvent

- C2

Add waterC2amp

oil

oil



oil amphydrophobic

solvent

water

hydrophobicsolvent









Table3Nile

redassayprocedures

forthe

determ

inationof

microalgallipids

Reagent(s

)Nile

redlip

idassayprocedure

Fluo

rescence

determ

ination

Ref

Isop

ropylalcoh

ol(IPA

)Nile

redsolutio

nbleach

solutio

nmethano

lcorn

oil(dissolvedin

21

methano

lchloroform)

Suspendlip

idextractsin

chloroform

dilu

teextractswith

methano

ladddilutedsamples

andcorn

oiltothem

icroplate

toachievea

rang

eincubatethep

lateandevaporatethe

solvents

addIPAandcoolthep

lateaddNile

redsolutio

naddbleach

solutio

nto

each

wellincubatethep

late

Determinefl

uorescence

usingap

latereader

with

excitatio

nsetto530n

mandem

issionsetto575n

mwith

a570

nmcut-o

ffread

thep

lateuse

thefi

rstreading

after

the

fluorescencep

eakforq

uantitatio

n

[27]

DMSO

Nile

redsolutio

ntriolein

Placem

icroalgalcellsin

microcentrifugetub

esandpu

tund


DMSO

put

undersecon


singthep

reviou

scon

ditio

nsadd

Nile

redsolutio

nandincubatethetub

esin

thed

arkpipetthe

samples

into

96wellm

icroplates

Measure

fluorescenceu

singFluo

rescence

Analyzerw

ithexcitatio

nat535n

mandem

issionat580n

mm

easure

untre

ated

suspensio

nandmedium

containing

Nile

redalon

eas

autoflu

orescenceconvertfl

uorescence

todryweighto

flip

ids

[28]

DMSO

Nile

redsolutio

nvirgin

oliveo


samples

with

Nile

redusing

DMSO

ascarriertopup

thev

olum

eand

incubatewith

agitatio

n

Read

thefl

uorescence

usingMicroplateR

eaderw

ithexcitatio

nsetto520n

mandem

issioncaptured

at570n

m

compare

thefl

uorescence

toav

irgin

oliveo

ilsta

ndardcurve

[29

30]

Pure


anolN

ileredsto

ck(in

100

spectralgradea

cetone)

Standardized

samplefl

uorescenceadd

triolein

tochloroform

anddilutewith

isoprop

anolprepare

workingsta

ndards

bybringing

interm

ediatesto

cksw

ithDIw

ater

NileredassayaddNile

redto

cellsuspensio

nor

toalipid

stand

ard

Measure

fluorescenceu

singspectro

photofl

uorometer

with

excitatio

nsetto475n

mandem

issionsetto580n

mexpress

cellu

larn

eutrallip

idas

triolein

equivalents

[31]






2SO4





6 Conclusion


2-basedmeth-



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





lCO2



2




2







2) and liquid (lCO










2with




2and then to the









2


2is described as



2is delivered



2is




1

3

5

7

VV

0


Ethyl Acetate





2has a











2mim]










Extractwithsolvent

- C2

Add waterC2amp

oil

oil



oil amphydrophobic

solvent

water

hydrophobicsolvent









Table3Nile

redassayprocedures

forthe

determ

inationof

microalgallipids

Reagent(s

)Nile

redlip

idassayprocedure

Fluo

rescence

determ

ination

Ref

Isop

ropylalcoh

ol(IPA

)Nile

redsolutio

nbleach

solutio

nmethano

lcorn

oil(dissolvedin

21

methano

lchloroform)

Suspendlip

idextractsin

chloroform

dilu

teextractswith

methano

ladddilutedsamples

andcorn

oiltothem

icroplate

toachievea

rang

eincubatethep

lateandevaporatethe

solvents

addIPAandcoolthep

lateaddNile

redsolutio

naddbleach

solutio

nto

each

wellincubatethep

late

Determinefl

uorescence

usingap

latereader

with

excitatio

nsetto530n

mandem

issionsetto575n

mwith

a570

nmcut-o

ffread

thep

lateuse

thefi

rstreading

after

the

fluorescencep

eakforq

uantitatio

n

[27]

DMSO

Nile

redsolutio

ntriolein

Placem

icroalgalcellsin

microcentrifugetub

esandpu

tund


DMSO

put

undersecon


singthep

reviou

scon

ditio

nsadd

Nile

redsolutio

nandincubatethetub

esin

thed

arkpipetthe

samples

into

96wellm

icroplates

Measure

fluorescenceu

singFluo

rescence

Analyzerw

ithexcitatio

nat535n

mandem

issionat580n

mm

easure

untre

ated

suspensio

nandmedium

containing

Nile

redalon

eas

autoflu

orescenceconvertfl

uorescence

todryweighto

flip

ids

[28]

DMSO

Nile

redsolutio

nvirgin

oliveo


samples

with

Nile

redusing

DMSO

ascarriertopup

thev

olum

eand

incubatewith

agitatio

n

Read

thefl

uorescence

usingMicroplateR

eaderw

ithexcitatio

nsetto520n

mandem

issioncaptured

at570n

m

compare

thefl

uorescence

toav

irgin

oliveo

ilsta

ndardcurve

[29

30]

Pure


anolN

ileredsto

ck(in

100

spectralgradea

cetone)

Standardized

samplefl

uorescenceadd

triolein

tochloroform

anddilutewith

isoprop

anolprepare

workingsta

ndards

bybringing

interm

ediatesto

cksw

ithDIw

ater

NileredassayaddNile

redto

cellsuspensio

nor

toalipid

stand

ard

Measure

fluorescenceu

singspectro

photofl

uorometer

with

excitatio

nsetto475n

mandem

issionsetto580n

mexpress

cellu

larn

eutrallip

idas

triolein

equivalents

[31]






2SO4





6 Conclusion


2-basedmeth-



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




1

3

5

7

VV

0


Ethyl Acetate





2has a











2mim]










Extractwithsolvent

- C2

Add waterC2amp

oil

oil



oil amphydrophobic

solvent

water

hydrophobicsolvent









Table3Nile

redassayprocedures

forthe

determ

inationof

microalgallipids

Reagent(s

)Nile

redlip

idassayprocedure

Fluo

rescence

determ

ination

Ref

Isop

ropylalcoh

ol(IPA

)Nile

redsolutio

nbleach

solutio

nmethano

lcorn

oil(dissolvedin

21

methano

lchloroform)

Suspendlip

idextractsin

chloroform

dilu

teextractswith

methano

ladddilutedsamples

andcorn

oiltothem

icroplate

toachievea

rang

eincubatethep

lateandevaporatethe

solvents

addIPAandcoolthep

lateaddNile

redsolutio

naddbleach

solutio

nto

each

wellincubatethep

late

Determinefl

uorescence

usingap

latereader

with

excitatio

nsetto530n

mandem

issionsetto575n

mwith

a570

nmcut-o

ffread

thep

lateuse

thefi

rstreading

after

the

fluorescencep

eakforq

uantitatio

n

[27]

DMSO

Nile

redsolutio

ntriolein

Placem

icroalgalcellsin

microcentrifugetub

esandpu

tund


DMSO

put

undersecon


singthep

reviou

scon

ditio

nsadd

Nile

redsolutio

nandincubatethetub

esin

thed

arkpipetthe

samples

into

96wellm

icroplates

Measure

fluorescenceu

singFluo

rescence

Analyzerw

ithexcitatio

nat535n

mandem

issionat580n

mm

easure

untre

ated

suspensio

nandmedium

containing

Nile

redalon

eas

autoflu

orescenceconvertfl

uorescence

todryweighto

flip

ids

[28]

DMSO

Nile

redsolutio

nvirgin

oliveo


samples

with

Nile

redusing

DMSO

ascarriertopup

thev

olum

eand

incubatewith

agitatio

n

Read

thefl

uorescence

usingMicroplateR

eaderw

ithexcitatio

nsetto520n

mandem

issioncaptured

at570n

m

compare

thefl

uorescence

toav

irgin

oliveo

ilsta

ndardcurve

[29

30]

Pure


anolN

ileredsto

ck(in

100

spectralgradea

cetone)

Standardized

samplefl

uorescenceadd

triolein

tochloroform

anddilutewith

isoprop

anolprepare

workingsta

ndards

bybringing

interm

ediatesto

cksw

ithDIw

ater

NileredassayaddNile

redto

cellsuspensio

nor

toalipid

stand

ard

Measure

fluorescenceu

singspectro

photofl

uorometer

with

excitatio

nsetto475n

mandem

issionsetto580n

mexpress

cellu

larn

eutrallip

idas

triolein

equivalents

[31]






2SO4





6 Conclusion


2-basedmeth-



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Extractwithsolvent

- C2

Add waterC2amp

oil

oil



oil amphydrophobic

solvent

water

hydrophobicsolvent









Table3Nile

redassayprocedures

forthe

determ

inationof

microalgallipids

Reagent(s

)Nile

redlip

idassayprocedure

Fluo

rescence

determ

ination

Ref

Isop

ropylalcoh

ol(IPA

)Nile

redsolutio

nbleach

solutio

nmethano

lcorn

oil(dissolvedin

21

methano

lchloroform)

Suspendlip

idextractsin

chloroform

dilu

teextractswith

methano

ladddilutedsamples

andcorn

oiltothem

icroplate

toachievea

rang

eincubatethep

lateandevaporatethe

solvents

addIPAandcoolthep

lateaddNile

redsolutio

naddbleach

solutio

nto

each

wellincubatethep

late

Determinefl

uorescence

usingap

latereader

with

excitatio

nsetto530n

mandem

issionsetto575n

mwith

a570

nmcut-o

ffread

thep

lateuse

thefi

rstreading

after

the

fluorescencep

eakforq

uantitatio

n

[27]

DMSO

Nile

redsolutio

ntriolein

Placem

icroalgalcellsin

microcentrifugetub

esandpu

tund


DMSO

put

undersecon


singthep

reviou

scon

ditio

nsadd

Nile

redsolutio

nandincubatethetub

esin

thed

arkpipetthe

samples

into

96wellm

icroplates

Measure

fluorescenceu

singFluo

rescence

Analyzerw

ithexcitatio

nat535n

mandem

issionat580n

mm

easure

untre

ated

suspensio

nandmedium

containing

Nile

redalon

eas

autoflu

orescenceconvertfl

uorescence

todryweighto

flip

ids

[28]

DMSO

Nile

redsolutio

nvirgin

oliveo


samples

with

Nile

redusing

DMSO

ascarriertopup

thev

olum

eand

incubatewith

agitatio

n

Read

thefl

uorescence

usingMicroplateR

eaderw

ithexcitatio

nsetto520n

mandem

issioncaptured

at570n

m

compare

thefl

uorescence

toav

irgin

oliveo

ilsta

ndardcurve

[29

30]

Pure


anolN

ileredsto

ck(in

100

spectralgradea

cetone)

Standardized

samplefl

uorescenceadd

triolein

tochloroform

anddilutewith

isoprop

anolprepare

workingsta

ndards

bybringing

interm

ediatesto

cksw

ithDIw

ater

NileredassayaddNile

redto

cellsuspensio

nor

toalipid

stand

ard

Measure

fluorescenceu

singspectro

photofl

uorometer

with

excitatio

nsetto475n

mandem

issionsetto580n

mexpress

cellu

larn

eutrallip

idas

triolein

equivalents

[31]






2SO4





6 Conclusion


2-basedmeth-



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table3Nile

redassayprocedures

forthe

determ

inationof

microalgallipids

Reagent(s

)Nile

redlip

idassayprocedure

Fluo

rescence

determ

ination

Ref

Isop

ropylalcoh

ol(IPA

)Nile

redsolutio

nbleach

solutio

nmethano

lcorn

oil(dissolvedin

21

methano

lchloroform)

Suspendlip

idextractsin

chloroform

dilu

teextractswith

methano

ladddilutedsamples

andcorn

oiltothem

icroplate

toachievea

rang

eincubatethep

lateandevaporatethe

solvents

addIPAandcoolthep

lateaddNile

redsolutio

naddbleach

solutio

nto

each

wellincubatethep

late

Determinefl

uorescence

usingap

latereader

with

excitatio

nsetto530n

mandem

issionsetto575n

mwith

a570

nmcut-o

ffread

thep

lateuse

thefi

rstreading

after

the

fluorescencep

eakforq

uantitatio

n

[27]

DMSO

Nile

redsolutio

ntriolein

Placem

icroalgalcellsin

microcentrifugetub

esandpu

tund


DMSO

put

undersecon


singthep

reviou

scon

ditio

nsadd

Nile

redsolutio

nandincubatethetub

esin

thed

arkpipetthe

samples

into

96wellm

icroplates

Measure

fluorescenceu

singFluo

rescence

Analyzerw

ithexcitatio

nat535n

mandem

issionat580n

mm

easure

untre

ated

suspensio

nandmedium

containing

Nile

redalon

eas

autoflu

orescenceconvertfl

uorescence

todryweighto

flip

ids

[28]

DMSO

Nile

redsolutio

nvirgin

oliveo


samples

with

Nile

redusing

DMSO

ascarriertopup

thev

olum

eand

incubatewith

agitatio

n

Read

thefl

uorescence

usingMicroplateR

eaderw

ithexcitatio

nsetto520n

mandem

issioncaptured

at570n

m

compare

thefl

uorescence

toav

irgin

oliveo

ilsta

ndardcurve

[29

30]

Pure


anolN

ileredsto

ck(in

100

spectralgradea

cetone)

Standardized

samplefl

uorescenceadd

triolein

tochloroform

anddilutewith

isoprop

anolprepare

workingsta

ndards

bybringing

interm

ediatesto

cksw

ithDIw

ater

NileredassayaddNile

redto

cellsuspensio

nor

toalipid

stand

ard

Measure

fluorescenceu

singspectro

photofl

uorometer

with

excitatio

nsetto475n

mandem

issionsetto580n

mexpress

cellu

larn

eutrallip

idas

triolein

equivalents

[31]






2SO4





6 Conclusion


2-basedmeth-



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







2SO4





6 Conclusion


2-basedmeth-



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table4Processeso

fTLC

runn

ingforthe

measuremento

fmicroalgallipid

content

PreparationforT

LCanalysis

TLCanalysis

Ref

Preparethe

solventb


theT

LCtank

andequilib

rateTL

Ctank

Addsamples

toplatesdry

thes

amples

andrunthep

lates

inhexane

diethyl

etheraceton

e(60

40

5vvv)dry

plates

andrunin

chloroform

methano

lam

mon

ium

hydroxidewater

(60

354

1vvvv)to

18cm

score

plates

andbreakapartw

ithhand


lineo

na

different

plateo

rspray

onlytheo

utsid

elaneo

fthe

platetobe

cutrotatethelow

erpo

rtionby

180∘

andrunin

chloroform

methano

laceticacidw

ater

(85

125125

3vvvv)

intheo

riginalorientation

drythep

lates

andspraywith

asolutio

nof

prim

ulin

dyevisualizelipid

spotsa

ndscan

thep

latesb

ylaser-excited

fluorescent

detection

quantifyspotsa

ndcompare

tostandard

curves

[32]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyieldNLsdry

NLs

andre-suspend

inchloroform

activate

plates

inan

oven

Subjectthe

NLfractio

nto

TLCforlipid

classseparatio

nandidentifi

catio

nuse


(70

301vv)for

lipid

separatio

nco-chrom

atograph

ywith

pure

stand

ardsstain

band

soflipid

classes

with

27-dichloroflu

oresceinvisu

alizeu

nder

UVlight

[33]

Elutes

ilica

gelw

ithchloroform

elutelip

idsw

ithchloroform

toyield

NLsdry

NLs

andresuspendin

chloroform

activate

plates

inan

oven

Use

8020


acid

forlipid

separatio

nspraythep

late

with

asolutionof

prim

ulin

dyeillum

inatethe

platew

ithUVlight

[2732

33]


Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table5Metho

dsem

ployed

ford

eterminingfatty

acidsa

fterlipid

extractio

ninclu

ding

thetranseste

rificatio

nandin

situtranseste

rificatio

nBo

thmetho

dsconsist

ofthepreparationof

FAMEs

andtheq

uantificatio

nandcompo

sitionanalysisof

FAMEs

byGC

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Transeste

rificatio

n

Redissolve

totallipid

extractsandelu

teneutral

lipidsa

ndpo

larlipidsw

ithdifferent

solvents

after

drying

undern

itrogen

gasderiv

atizetoFA

MEs

andrecoverthe

FAMEs

GCequipp

edwith

FIDandAgilent

CP-W

ax52

CBcolumn

(1)Internalstand

ardtripentadecano

inC

150

triacylglycerolSigm

a-Aldric

h(2)Identificatio

nandqu

antifi

catio

nsta

ndard

Supelco

37-com

ponent

stand

ards

[34]

AddHCl

andmethano

ltolip

idextractsandheat

mixture

with

hexane

andmethyl-tert-b

utylether

washtheu

pper

organicp

hase

with

sodium


softhe

organic

extractsandtransfe

rtoas

amplev

ial

GCequipp

edwith

FIDandas

pecial

perfo

rmance

capillary

column(H

ewlett

Packardmod

elH

P-5MS)

(1)Internalstand

ardhexadecane

(Sigma-Aldric

hH

6703)stand

ard

(2)C

alibratio

nstandardoliveo

il[3536]

AddH2SO4to

lipid

extractsandheatcoo

lthe

sampletoroom

temperature

andmixwith

DI

water

toseparatelip

idextracts

movethe

lower

partliq

uidinto

avial

GCequipp

edwith

FIDandaS

upelc

oNucolTM

column(355

33-03A

film

thickn

ess)usingHelium

asthec

arrie

rgas

(flow

20mLsdotminminus1)

(1)Internalstand

ardpentadecanoica

cid(C

150)

(2)Identificatio

nsta

ndardauthentic

stand

ards

(Sigma-Aldric

hMOU

SA)

[3738]

Addmethano

licHCl

tolip

idextractsandflu

shheadspacew

ithnitro

genandsealtig

htlyandheat

coolvialsa

ndaddaqueou

sK2CO3centrifu

geand

removethe

upperp

hase

anddry

GCequipp

edwith

FIDandaS

GESol

Gel-WaxTM

capillary

columnusingheliu

mas

thec

arrie

rgas

(1)Identificatio

nsta

ndardstandard

fatty

acids

(Nu-Ch

ekPrep

Inc

Elysian

MN)

(2)Q

uantificatio

nsta

ndardheptadecanoica

cid

(C170)

[39]

Addchloroform

containing

heptadecanoica

cid

(C170)methano

landsulfu

ricacid

toeach

tube

andheatcoo

ldow

naddDIcentrifugeand

separatethelow

erchloroform

phasea

ndfilterfor

test

GCequipp

edwith

FIDandHP19091N-213

HP-IN

NOWax

polyethylene

glycolcolumn

(1)Internalstand

ardheptadecanoica

cid(C

170)

(2)Identificatio

nandqu

antifi

catio

nsta

ndard37

compo

nent

FAMEsta

ndardmix(Sup

elco

BellefonteUSA

)

[40]

AddBH

T(butulated

hydroxytoluene1

inmethano

l)to

prevento

xidatio

npriorto

methylatio

n

GCequipp

edwith

FIDandthec

apillary

columnDB-23

(Agilent

Techno

logies)u

sing

heliu

mas

thec

arrie

rgas

(1mLsdotminminus1

splitless)

(1)Q

uantificatio

nsta

ndardC190(non

adecanoic

Acid

72332-1G

-Fanalytic

alsta

ndard

Sigm

a-Aldric

h)

(2)Identificatio

nsta

ndardSupelcoTM

37Com

ponent

FAMEMixSigma

[41]

Addfre

shlyprepared

acetylchlorid

emethano

l(110)m

ethano

lintolip

idextracts

andsealvials

andheatcoo

ldow

nandaddK 2

CO3hexane

centrifugethe

samples

andrecoverthe

hexane

supernatant

GCequipp

edwith

FIDandaB

PX70

capillary

column(120

mtimes025

mm

internal

diam

eter025120583

mfilm

thickn

essSG

EAnalytic

alScienceRing

woo

dVIC

Australia)u

singheliu

mas

carrierg


(1)Internalstand

ardC2

30

(2)Identificatio

nsta

ndardas

erieso

fmixed

and

individu

alstandardsfrom

Sigm

a-Aldric

h[4243]

MixH2SO4m

ethano

lTH

Fandlip

idextracts

reflu

xther

eactionmixture

at90∘Cwith

continuo

usstirr

ingfor3

hneutralizethe

mixture

with

NaH

CO3andextractitw

ithhexane

GCequipp

edwith

FIDandHP-IN

NOWax


mtimes025120583m

film

ofpo

lyethylene

glycol)u

singheliu

mas

carrier

gas

(1)Th

estand

ardFA

MES

C140C

160C

163

C180C1

81C1

82C1

83andC2

00

(2)S

upelco

TM37

Com

ponent

FAMEMix

Sigm

a-Aldric

hTreatextracted

lipidsw

ithmethano

licsulfu

ricacid

andheatrecover

FAMEs

inhexane

centrifugethe

suspensio

nandaspiratehexane

containing


e

GCequipp

edwith

DB-5capillary

column

(30m

m025m

m1120583m)a

ndFIDusing

heliu

mas

carrierg


NISTILSdatabase

[44ndash

46]


Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table5Con

tinued

Metho

dPreparationof

FAMEs

GCop

eration

Standardsu

sed

Ref

Insitutranseste

rificatio

n

Addam

ixture

ofmethano

lsulfu

ricacidand

chloroform

into

driedcellbiom

assa

ndheptadecanoica

cid(C

170)asa

ninternalsta

ndard

andheatcoo

ldow

naddDIw

aterm

ixand

settletransfe

rthe

lower

phasec

ontainingFA

MEs

toac

lean

vialanddrywith

anhydrou

sNa 2SO4

GCequipp

edwith

FIDandSG

ESol

Gel-WaxTM

capillary

column(30mtimes

025

mmtimes025120583m)u

singheliu

mas

the

carrierg

as

(1)Identificatio

nsta

ndardstandard

fatty

acids

(SigmaMO)

(2)Q

uantificatio

nsta


(C170)

[4748]

Therea

remanydifferent

waystoaddac

atalyst

such

asmethano

lichydrogen

chlorid

eNaO

Me

andBF3N

aOMeandtetram

ethylguanidine

and

methano

ltridecanoica

cidmethyleste

r(C

13-FAME)

asan

internalstandard

andheat

GCequipp

edwith

FID(A

gilent

6890NH

P5-MScolumn(A

gilentU

SA)30

m025

mm

IDand025120583m

FT)u

singheliu

mas

the

carrierg


(1)C

8ndashC2

4SIGMAcat

18918

C13ndashC2

1SIGMAcat

1896

[49]

Note(1)Allsamples

prepared

forF

AMEs

containthec

orrespon

ding

internalsta

ndards

thathave

been

listedin

thetableabove(2)F

IDflam

eion

izationdetector




Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





Acknowledgments


References








































































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018











































































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018











































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

ReviewArticle - Hindawi Publishing Corporationdownloads.hindawi.com/journals/bmri/2018/1503126.pdf · 12/29/2017 · roles in microalgal metabolism and growth period. Some lipids