The Potential of a Brown Microalga Cultivated in High Salt ...downloads.hindawi.com/journals/bmri/2017/4018562.pdf · 2 BioMedResearchInternational dependentontheirphysiologicalstate.Likewise,theirsec-ondarymetabolismcaneasilybetriggeredbymostformsof

Research ArticleThe Potential of a Brown Microalga Cultivated in High SaltMedium for the Production of High-Value Compounds

Saoussan Boukhris1 Khaled Athmouni2 IbtissemHamza-Mnif1 Rayda Siala-Elleuch1

Habib Ayadi2 Moncef Nasri1 and Alya Sellami-Kamoun1

1Laboratory of Enzyme Engineering and Microbiology University of Sfax National Engineering School of SfaxBP 1173 3000 Sfax Tunisia2Laboratory of Biodiversity and Aquatic Ecosystems Ecology and Planktonology Department of Life Sciences Sfax UniversitySoukra Km 35 BP 1171 3000 Sfax Tunisia

Correspondence should be addressed to Alya Sellami-Kamoun kamoun alyatunettn

Received 9 February 2017 Accepted 27 April 2017 Published 22 May 2017

Academic Editor Marıa M Yust

Copyright copy 2017 Saoussan Boukhris et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Amphora sp was isolated from the Sfax Solar Saltern and cultivated under hypersaline conditions It contains moderate rates ofproteins lipids sugars and minerals and a prominent content of bioactive compounds polyphenols chlorophyll a carotenoidsand fatty acids The analysis of fatty acids with GCMS showed that the C16 series accounted for about 75 of Amphora sp lipidsSaturated fatty acids whose palmitic acid was the most important (2741) represented 4131 Amphora sp was found to be rich inmonounsaturated fatty acids with dominance of palmitoleic acid It also contains a significant percentage of polyunsaturated fattyacids with a high amount of eicosapentaenoic acid (236) Among the various solvents used ethanol at 80 extracted the highestamounts of phenols and flavonoids that were 3827 mg gallic acid equivalent and 1769 mg catechin equivalent gminus1 of dried extractrespectively Using various in vitro assays including DPPH and ABTS radicals methods reducing power assay and 120573-carotenebleaching assay the 80 ethanolic extract showed high antioxidant activity A strong antibacterial activity was checked againstGram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and Gram-negative bacteria (Klebsiella pneumoniae andSalmonella enterica)These results are in favor of Amphora sp valorization in aquaculture and food and pharmaceutical industries

1 Introduction

Microalgae are considered as an important source of bioac-tive compounds with a wide range of applications andcommercial use They produce proteins lipids vitaminspigments and other molecules exploited for health forfood and feed additives and for energy production Dueto their wide diversity and high storage capacity for lipidsparticularly eicosapentaenoic acid (EPA) and amino acidsmicroalgae have pharmaceutical and cosmetic applications [12] Furthermore the protective effects of natural antioxidantsagainst chemically induced oxidative damage have been paidattention nowadays especially when the generation of freeradicals is involved Benthic diatoms have become goodsources of natural antioxidants as revealed by a number

of studies [3ndash6] Moreover microalgae represent a largeand underexplored resource of antimicrobial compounds[7 8] Potentially microalgal extracts are already beingtested as additives for food and feed formulations in anattempt to replace the currently used synthetic antimicrobialcompounds including subtherapeutical doses of antibioticsemployed as a prophylactic measure in animal breeding[9] Microalgae are ubiquitously distributed throughout thebiosphere where they have been adapted to survival undera large spectrum of environmental stresses such as heatcold anaerobiosis salinity photooxidation osmotic pressureand exposure to ultraviolet radiation [10] Hence they maygrow essentially under most of the environmental conditionsavailable ranging from freshwater to extreme salinity Theypossess the extra advantage of substantialmetabolic plasticity

HindawiBioMed Research InternationalVolume 2017 Article ID 4018562 10 pageshttpsdoiorg10115520174018562

2 BioMed Research International

dependent on their physiological state Likewise their sec-ondary metabolism can easily be triggered by most forms ofexternally applied stress [7]

Among the large variety of microalgae CyanophyceaeChlorophyceae Bacillariophyceae (diatoms) and Chryso-phyceae are the most studied for biodiesel production [11]It is estimated that diatoms produce about 25 of the globalprimary biomass [12] In the Sfax Solar Saltern Bacillario-phyceae dominatedwithDinophyceae in the least salty pondsbut they are rarely abundant in hypersaline environments[13]

Unlike the universalmacromolecules of primarymetabo-lism (eg monosaccharides polysaccharides amino acidsproteins and lipids) which are commonly present in allorganisms secondarymetabolites have a farmore limited dis-tribution in nature They are not necessarily produced underall conditions and can only be found in specific organismsor a group of organisms The organism can produce thesecompounds either to protect itself within its own livingecosystem or to play a basic role in its everyday existenceThis reveals bioactive molecules in unrelated biologicalsystems [14] The medicinal and pharmacological actions areoften dependent on the presence of bioactive compoundscalled secondary metabolites [15] The growth of algae isrelatively rapid and it is possible to control the productionof their bioactive compounds such as polyphenols andpigments by modifying culture conditions [10]

In the present workAmphora sp (Bacillariophyceae) wascultivated in a hypersaline medium and the physicochemicalproperties fatty acids profile bioactive compounds andsome biological activities were determined

2 Materials and Methods

21 Microalga Amphora sp is a Bacillariophyceae that wasisolated via micromanipulation and serial dilution from awater sample collected in the pond C4-1 of the Sfax SolarSaltern with average salinity of 107 psu (practical salinityunit) The Sfax Solar Saltern is located in the central easterncost of Tunisia at about 34∘391015840N and 10∘421015840E The ponds areshallow (20ndash70 cm depth) and have various salinities rangingfrom 40 to 400 psu [16] They are connected by pipes andchannels along a 12 km section of sea cost

22 Culture Conditions The microalga was cultivated inbatch in autoclaved artificial seawater which was enrichedwith F2 nutrient medium sodium silicate (Na2SiO3) andtrace metals solution [17 18] Cultures were conducted for 15days at the salinity of 100 psu 25∘C light dark (L D) cycleof (16 h 8 h) and cool white fluorescent light intensity of60 120583moles photonsmminus2 sminus1 The biomass was separated fromthe culture media by centrifugation (4500timesg 10min) andthe pellet was washed with distilled water and centrifugedagain at 4500timesg for 10min (the washing was repeated twotimes) The pellet was freeze-dried and stored at minus70∘C23 Determination of Dry Matter Ash Proteins Lipidsand Total Sugars The dry matter and ash content weredetermined according to the AOAC standard methods [19]

Total nitrogen contents of Amphora sp crude and undi-gested protein substrates were determined using the Kjeldahlmethod according to the AOAC method number 98413 [19]with equipment of BUCHI Digestion Unit K-424 (Switzer-land) Crude proteins were estimated by multiplying the totalnitrogen content by the factor of 625

Lipids content was determined gravimetrically after theSoxhlet extraction of dried samples with hexane for 2 hoursusing Nahita Model 655 (Navarra Spain)

As regards sugars they were estimated by phenol-sulfuricacid method [20] using glucose as a standard

24 Determination of Pigments Content Chlorophylls andCarotenoids Pigments were determined according to themethod described by Lichtenthaler [21] Two milliliters ofculture was centrifuged at 5500timesg for 5min and the super-natant was discarded and the pellet was mixed with 999methanol and incubated in the dark for 24 hours at 45∘CAfter incubation pigments content was determined measur-ing absorbances at 470 6524 and 6652 nm which werecorrected for turbidity by subtracting absorbance at 750 nm

25 Determination of Mineral Content The analysis ofsodium potassium calcium magnesium iron copper andzinc contents inAmphora sp was carried out using the induc-tively coupled plasma optical emission spectrophotometer(ICP-OES)Model 4300DV PerkinElmer (Shelton CTUSA)according to the method of AOAC 1999 [22] Measurementswere performed in triplicate and the results were the averageof three values

26 Determination of Fatty Acids Profile by GC-MS Fattyacids methyl esters (FAME) were prepared by basic trans-esterification protocol The extracted lipids were collectedin 10mL heptane and introduced into a 50mL flask Avolume of 10mL of KOH solution (11 g Lminus1) in methanolwas added with a few antibumping beads The mixture wasboiled under reflux condenser for 10min Then 5mL ofboron trifluoride (BF3) methanol complex (150 g Lminus1) wasintroduced through the condenser by a graduated syringeand themixture was boiled for 2minThemixture was cooledunder room temperature and 15 to 20mL of saturated sodiumsulfate solutionwas added and shakenwell Furthermore thissolution was added until the liquid level reached the neckof the flask After the phase separation the upper layer (n-heptane) was collected by a Pasteur pipette and evaporatedunder nitrogen flow The FAME were redissolved in 500120583Lhexane for GC-MS analysis

The analysis of FAME was performed with a 6890GC5973 MSD GCMS system from Agilent Technologiesequipped with an HP-INNO Wax Polyethylene Glycol cap-illary column (30m length 250 120583m diameter 025 120583m filmthickness) Helium was used as a gas carrier with a flowrate through the column of 1mLmin Column temperaturewas held initially for 1min at 200∘C and increased to 250∘Cat 10∘Cminminus1 and was then held at 250∘C for 15min Theinjector was kept at 260∘C using a splitless mode and thena sample of 1 120583L was injected The ion source temperature

BioMed Research International 3

was set at 230∘C The mass spectra were obtained in fullscan mode MS and scan range was (119898119911) 50ndash600 atomicmass units (AMU) under electron impact (EI) ionization(70 eV) Data were exploited using the National Instituteof Standards and Technology (NIST) Mass Spectral SearchProgram (version 20g)

27 Determination of the Total Phenolic Content The totalphenols content in microalga was determined by theFolinndashCiocalteu method [23] Briefly 02mL of extract wasmixed with 1mL of FolinndashCiocalteu reagent (diluted 1 10vv) followed by the addition of 08mL of sodium carbonate(75 wv) After incubation in the dark the absorbancewas measured at 760 nm The total phenolic content of algalextract was expressed as mg of gallic acid equivalent per gof dry extract (mgGAE gminus1 extract) using a calibration curvewith gallic acid All samples were analyzed in triplicate

28 Determination of the Total Flavonoid Content The totalflavonoid content was determined according to the modifiedmethod of Zhishen et al [24] Briefly 04mL of the extractwasmixedwith 120120583L of 5 sodiumnitrite and 120120583L of 10aluminum chloride followed by the addition of 08mL of 1Msodium hydroxide After the incubation of reaction mixtureat room temperature for 6min absorbance was measuredat 510 nm The total flavonoids content in the extract wasexpressed in terms of catechin equivalent (mg gminus1 of dryextract) All samples were analyzed in triplicate

29 Extraction of Antioxidants The extracts from the bio-mass samples were obtained by two solid-liquid extractionprocedures inspired from themethod of Goiris et al [25] In afirst step both apolar and polar compoundswere extracted by80 ethanol For this 2 g of freeze-dried biomass was groundusing a pestle and mortar and extracted under agitationin the dark with 20mL ethanolwater (41 vv) mixtureat 50∘C for 1 hour After centrifugation (4500timesg 10min)the pellet was resuspended in 2mL of the ethanolwatermixture and extracted for a second time with macerationThe two extracts were pooled and stored at minus20∘C prior toanalysis The second procedure aimed to separate polar fromapolar compounds after sequential extraction in solventswithincreasing polarity hexane ethyl acetate and water Freeze-dried biomass (2 g) was ground in a mortar and extractedwith 20mL of hexane for 1 hour at 50∘C After centrifugationthe pellet was resuspended in hexane and extracted for asecond time and both extracts were combined The biomasspellet was subsequently extracted twice with 2 times 20mL ethylacetate using the sameprocedure andfinallywith 2times 20mLofwater at 50∘C for 1 hour All extracts were concentrated underreduced pressure by a rotary evaporator to a dry condensedresidue The dried samples were weighed and then stored atminus20∘C prior to analysis

210 Antioxidant Property Four in vitro assays were used toevaluate the antioxidant property of the 80 ethanolic extractof Amphora sp

2101 22-Diphenylpicrylhydrazyl (DPPH) Free Radical Scav-enging Assay Free radicals scavenging activity was assessedaccording to Blois [26] with some modifications 1mL ofthe extract at different concentrations (006ndash1mgmLminus1) wasmixed with 1mL of 01mM DPPH in ethanol and 450 120583Lof 50mM Tris-HCl buffer (pH 74) The solution was incu-bated at 37∘C for 30min and the reduction of DPPH freeradicals was evaluated by reading the absorbance at 517 nmThe DPPH scavenging activity is given as percentage andcalculated according to the following equation

DPPH scavenging = [(119860control minus 119860 test)119860control ] times 100 (1)

where 119860control is the absorbance of the reaction medium and119860 test is the absorbance of the reaction test containing theextractThe antioxidant activity of each extract was expressedas IC50 defined as the concentration of extract required tocause a 50 decrease in initial DPPH concentration Buty-lated hydroxytoluene (BHT)was used as positive control andeach sample was analyzed three times

2102 ABTS Radical Scavenging Activity The Trolox equiva-lent antioxidant capacity (TEAC) assay measuring the reduc-tion of the 22-azinobis-(3-ethylbenzothiazoline-6-sulfonicacid) ABTS radical cation by antioxidants was derived fromthe method of Katalinic et al [27] with minor modificationsBriefly ABTS radical cation (ABTSsdot+)was produced by react-ing ABTS stock solution with 245mM potassium persulfateallowing themixture to stand in the dark at room temperaturefor 12ndash16 hours before use ABTSsdot+ solution was dilutedwith saline phosphate buffer (PBS pH 74) to absorbance of070 (plusmn002) at 734 nm After the addition of 2mL of dilutedABTSsdot+ solution to 100120583L of each extract or Trolox standardthe reaction mixture was incubated for 30min in a glassspectrophotometer cell at 30∘C The decrease in absorbancewas recorded at 734 nm Measurements were performed intriplicate Radical scavenging activitywas calculated using theformula below

Inhibition = [(119860control minus 119860 test)119860control ] times 100 (2)

where 119860control is the absorbance of the reaction medium and119860 test is the absorbance of the reaction medium with theextract Each sample was analyzed three times

2103 Ferric Reducing Antioxidant Power (FRAP) Thereducing power of the extract was determined according tothe method described by Yildirim et al [28] Briefly 05mLof 80 ethanolic extract solution at various concentrations(01ndash05mgmLminus1) was mixed with 125mL of 02M phos-phate buffer (pH 66) and 125mL of 1 (wv) potassiumferricyanideK3[Fe(CN)6]Themixturewas incubated at 50∘Cfor 20min and then 125mL of 10 (wv) trichloroaceticacid was added to the mixture which was then centrifugedat 3500timesg for 10min The supernatant (125mL) was mixedwith 125mL distilled water and 025mL of 01 FeCl3 (wv)The absorbance was measured at 700 nm Ascorbic acid wasused as a reference The increase of the absorbance in the


reaction indicates the increase of the iron reduction Thevalues presented are the mean of triplicate analysis

2104 120573-Carotene-Linoleic Acid Assay The ability of theextract to prevent the bleaching of 120573-carotene was assessedas described by Koleva et al [29] A stock solution of 120573-carotenelinoleic acid mixture was prepared by dissolving05mg of 120573-carotene in 1mL of chloroform 25 120583L of linoleicacid and 200120583L of Tween 80The chloroformwas completelyevaporated under vacuum in a rotary evaporator at 40∘C andthen 100mL of distilled water was added and the resultingmixture was vigorously stirred The emulsion obtained wasfreshly prepared before each experiment Aliquots (25mL) ofthe 120573-carotenelinoleic acid emulsion were transferred to testtubes containing 01mL of extract at different concentrations(01ndash05mgmLminus1) Following incubation for 2 h at 50∘C theabsorbance of each test was measured at 470 nm Vitamin Cwas used as a positive standardThe control tube containednosample Antioxidant activity in 120573-carotene bleaching modelexpressed as percentage was calculated with the followingequation

120573-carotene bleaching inhibition ()= [1 minus 1198600 minus 119860 11990511986010158400 minus 1198601015840119905 ] times 100

(3)

where 1198600 and 11986010158400 are the absorbance of the sample and thecontrol respectively measured at time zero and 119860 119905 and 1198601015840119905are the absorbance of the sample and the control respectivelymeasured after 2 hours Tests were carried out in triplicate

211 Antibacterial Assays The operative pathogens wereGram-positive (Bacillus cereus (ATCC11778) Bacillussubtilis (JN934392) Micrococcus luteus (ATCC4698) andStaphylococcus aureus (ATCC6538)) and Gram-negative(Klebsiella pneumoniae (ATCC13883) Salmonella enterica(ATCC43972)) bacteria The antimicrobial activity of 80ethanolic extract was evaluated using agar well diffusionmethodThewells were then filled with 60120583L of the extract at20mgmL in 5 DMSO and 5 DMSO was used as negativecontrol Besides plates were incubated for 24 hours at 37 plusmn1∘C for bacterial strains Then the diameters of inhibitionzones (IZD) were measured

212 Statistical Analysis The data for biological and bio-chemical parameters are expressed as mean plusmn SD Tests werecarried out in triplicate The IC50 values were calculated byProbit Analysis with a reliability interval of 95

3 Results and Discussion

31 Physicochemical Characterization of Amphora sp Somephysicochemical characteristics ofAmphora sp are presentedin Table 1 The obtained results have shown that the biomassof Amphora sp contains moderate amounts of lipids pro-teins carbohydrates ashes and minerals and an importantpercentage of chlorophyll a and carotenoids The dry mattercontent of 7 is close to that found for other strains 8

Table 1 Physicochemical characteristics of Amphora sp

Component Amphora spDry matter ( FW) 7 plusmn 045Proteins ( DW) 2762 plusmn 03Lipids ( DW) 1114 plusmn 019Total sugars ( DW) 1260 plusmn 076Ashes ( DW) 3778 plusmn 043Chlorophyll a ( DW) 4945 plusmn 02Chlorophyll b ( DW) 0666 plusmn 005Carotenoids ( DW) 1083 plusmn 005Sodium (gKgminus1 DW) 1125 plusmn 02Potassium (gKgminus1 DW) 0485 plusmn 005Calcium (gKgminus1 DW) 0584 plusmn 005Magnesium (gKgminus1 DW) 0747 plusmn 01Iron (gKgminus1 DW) 0016 plusmn 0002Copper (g Kgminus1 DW) 0008 plusmn 0001Zinc (g Kgminus1 DW) 0008 plusmn 0001Data are expressed as mean plusmn standard deviation of triplicates FW freshweight DW dry weight

for Amphora sp CTM 20023 [30] and 59 for Amphoracoffeaformis [31] However the lipids content of Amphora spof 1114 DW was relatively lower than the values publishedfor other strains of Amphora [32] and it was higher than thatof Amphora coffeaformis (69 DW) [31] A study on eightmarine species of diatoms revealed various lipid contentsranging from 24 to 213 [33]

The proteins level for Amphora sp (2762) was lowerthan that found for other strains of Amphora 54 for CTM20023 [30] and 30ndash40 for Amphora sp [32] Indeed theproteins level was in agreement with the published valuesrange of 12ndash42 for some microalgae [34] and higher thanthe contents found for Amphora coffeaformis [31]

The total sugars content of Amphora sp was 1260 DWwhich is consistent with that of some microalgae (5ndash23DW) [34] Nevertheless Amphora coffeaformis and somediatoms contain a high amount of sugars in the range of135ndash164 [31]

With respect to the ash content of Amphora sp (3778DW) it is in line with that found for another Amphorastrain (30DW) cultivated at salinity ranging between 15 and35 psu [32] Ash content exceeds 50 (558 to 679) of thedry weight for some diatoms [31] Amphora sp has moderateamounts of sodium potassium calcium and magnesium

Moreover Amphora sp was found to be rich in chloro-phyll mainly chlorophyll a and carotenoids In fact itschlorophyll content reached almost 5 of the dry matterwhich is in agreement with other research works [30]Furthermore the carotenoids content was prominent (1083DW) Bacillariophyceae exhibited high levels of carotenoidsnamely 120573-carotene and xanthophylls thus indicating a pow-erful antioxidant activity [35]

32 Fatty Acids Composition of Amphora sp In salinityof 100 psu on F2 medium the fatty acids profile ofAmphora sp was composed of saturated monounsaturated


Table 2 Fatty acids composition of Amphora sp ( of total fattyacids)

Fatty acids Amphora spC140 3623 plusmn 03C150 3418 plusmn 03C160 27427 plusmn 05C170 1664 plusmn 04C180 1974 plusmn 03C200 0734 plusmn 02C240 2468 plusmn 02sum SFA 41308 plusmn 08C141 3386 plusmn 03C161 45089 plusmn 08C171 0521 plusmn 01C181 3658 plusmn 03sumMUFA 52654 plusmn 12C162 1603 plusmn 02C163 0924 plusmn 03C182 0432 plusmn 01C204 0712 plusmn 02C205 2367 plusmn 03sumPUFA 6038 plusmn 05sum SFA total saturated fatty acids sumMUFA total monounsaturated fattyacidssumPUFA total polyunsaturated fatty acids

and polyunsaturated fatty acids (SFA MUFA and PUFA)respectively (Table 2)The C16 fatty acids series (C160 C161C162 and C163) represented more than 75 Moreoverpalmitic acid (C160) and palmitoleic acid (C161) accountedtogether formore than 7251 of fatty acids SFAwere presentat a high level 4131 with 2741 of palmitic acid HenceAmphora sp could be a suitable producer of SFA which areeasily convertible to biodiesel [36] Amphora sp is rich inMUFA with the dominance of palmitoleic acid (4509) andcontains an important percentage of PUFA (603) Highlevels of palmitoleic acid and other bioactive fatty acids werealso detected in the fusiform morphotype of the Bacillario-phyceae Phaeodactylum tricornutum [37] EicosapentaenoicacidC205 (EPA)was produced in a noticeable level (2367)Indeed it is known that EPA is an important PUFA for healthprotection from many pathologies including cardiovasculardiseases [38] and cancer [39]

33 Phytochemical Composition The total phenols andflavonoids contents of Amphora sp extracts are summarizedin Table 3 The 80 ethanolic extract of this Bacillario-phyceae showed the highest phenols and flavonoids contents3827 plusmn 221mgGAE gminus1 extract and 1769 plusmn 070mgCE gminus1extract respectively compared to the extracts obtained byethyl acetate hexane and water The phenolic content ofethanol extracts of some Moroccan marine microalgae wasfound to range from 82 to 32mgGAE gminus1 extract [40] Theauthors noted that the highest phenolic content was found inthe extract of Nannochloropsis gaditana with 32mgGAE gminus1extract The extracts of Dunaliella sp Phaeodactylum tricor-nutum andNavicula sp also contained high phenolic contentof more than 15mgGAE gminus1 extract [40]

The flavonoid content in the 80 ethanolic extract ofAmphora sp was 322mgCE gminus1 DW which is sixfold higherthan that found for Amphora CTM 20023 [30]

The high levels of polyphenols and flavonoids in theAmphora sp 80 ethanolic extract may be due to the cultureconditions under the high salinity of 100 psu and theextraction conditions

34 Antioxidant Property of Amphora sp Due to its highestcontent of phenols and flavonoids the 80 ethanolic extractwas used to check antioxidant activity through four in vitroassays the DPPH and ABTS radical scavenging capacitiesthe ferric reducing antioxidant power and the 120573-carotenebleaching inhibition tests using different concentrationsof the extract The inhibition percentages of the radicalsDPPH and ABTS by the extract and standards (Trolox andBHT) were found to be concentration-dependent (0065 to1mgmLminus1 for DPPH test and 05 to 4mgmLminus1 for ABTS test)as shown in Figure 1Thededuced values of IC50 are presentedin Table 4 A lower value of IC50 indicates a high antioxidantactivity Amphora sp 80 ethanolic extract exhibited highantiradical power with an IC50 value of 023 plusmn 007mgmLminus1for DPPH scavenging activity and an IC50 value of 261 plusmn064mgmLminus1 for ABTS radical scavenging capacity Someauthors have found a strong correlation between the phenoliccontents and the antioxidant activity [41]

The FRAP assay is based on the ability of phenols toreduce yellow ferric tripyridyl triazine complex (Fe(III)-TPTZ) to blue ferrous complex (Fe(II)-TPTZ) by the actionof electron-donating antioxidants [42] Figure 2(a) shows asignificant reducing power compared to the vitamin C usedas a referenceThe reducing power was also dependent on theextract concentration

The effect of antioxidants on DPPH free radical scaveng-ing was considered to be emanating from their hydrogen-donating ability In this investigation the 80 ethanol extractwas proven to exhibit a strong effect of DPPH free radicalscavenging In fact it reached 7434 of inhibition at 1mg ofextract which is considered higher than themethanol extractof someBacillariophyceae tested at 2mg of the extract (227316 and 766 for Amphora coffeaeformis Navicula spand Achnanthes longipes resp) [31] The IC50 value for ABTSscavenging capacity of Amphora sp extract was 26mgmLminus1Many research studies [43] have mentioned IC50 ABTSscavenging capacities for twobrown algae of 529mgmLminus1 forSargassum binderi and 152mgmLminus1 for Turbinaria conoides

The 120573-carotene bleaching inhibition and the reducingpower tests (absorbance at 700 nm) were measured at con-centrations of extract between 01 and 05mgmLminus1 showingits dependency on concentration (Figure 2(b))The IC50 valueof 120573-carotene bleaching test (Table 4) was strong (021 plusmn005mgmLminus1) with about twice the IC50 value of vitaminC used as the standard The absorbance at 700 nm reached avalue of 078 at a concentration of 05mgmLminus1 of the extractwhich is considered high indicating a good antioxidantactivity Therefore Amphora sp extract was confirmed tohave a strong antioxidant capacity The multiple regressionanalysis has proven that both carotenoid and phenolic


Table 3 Extraction yields and polyphenols and flavonoids contents in extracts from Amphora sp with different solvents

Solvent Yield ( DW) Polyphenols (mgGAE gminus1 extract) Flavonoids (mgCE gminus1 extract)80 ethanol 1820 plusmn 021 3827 plusmn 221 1769 plusmn 070Hexane 1495 plusmn 025 1147 plusmn 060 476 plusmn 015Ethyl acetate 278 plusmn 012 1404 plusmn 031 525 plusmn 030Water 2361 plusmn 030 1334 plusmn 024 427 plusmn 020Data are expressed as mean plusmn standard deviation of triplicates GAE gallic acid equivalent CE catechin equivalent

00 05 10 15 20 25 30 35 40 450

20

40

60

80

100

in

hibi

tion

80 ethanolic extractTrolox

Sample concentration (mg mLminus1)

(a)

00 02 04 06 08 10

20

40

60

80

100

in

hibi

tion

80 ethanolic extractBHT


(b)

Figure 1 Anti-free radical effects ofAmphora sp 80 ethanolic extract for ABTS (a) andDPPH (b) compared to respective standards (Troloxand BHT)

Table 4 IC50 values for antioxidant capacities of Amphora spextract compared to standards

Activity Extract (mgmLminus1) StandardDPPH radical scavenging activity 023 plusmn 007 011 plusmn 003ABTS radical scavenging activity 261 plusmn 064 054 plusmn 008120573-Carotene bleaching assay 021 plusmn 005 009 plusmn 001Data are expressed as mean plusmn standard deviation of triplicates

contents significantly contribute to the explanation of thevariation in FRAP and TEAC activity of the extracts Theregression coefficients indicate that phenols and carotenoidsare of similar importance in explaining variation in theantioxidant activity [25]

35 Antibacterial Assay In the present study the antibac-terial activity of Amphora sp 80 ethanolic extract wasscreened against six bacteria and their potency was qual-itatively and quantitatively analyzed by the diameters ofthe inhibition zones (IZD) as summarized in Table 5 The80 ethanolic extract of Amphora sp at a concentrationof 20mgmLminus1 was active against Gram-positive bacteriaMicrococcus luteus and Staphylococcus aureus and inactive

against both strains of Bacillus (cereus and subtilis) Thisextract was active against Gram-negative bacteria testedKlebsiella pneumonia and Salmonella enterica The extractexhibited strong activity against Micrococcus luteus Staphy-lococcus aureus and Klebsiella pneumoniae The IZD forKlebsiella pneumoniae and Staphylococcus aureus of 16mmand 20mm respectively were stronger than those found forthe ethanolic extract of a strain of Amphora sp cultured instandardWalnersquos medium (10mm and 105mm) respectively[44] This difference may be due to the culture conditionsAccording to some authors the antimicrobial activity of algaedepends on the species and the extraction efficiency of theactive compounds as well as survival conditions [45]

The extract used in this study was found to be richin a variety of compounds lipids and fatty acids car-bohydrates polysaccharides polyphenols flavonoids pig-ments and carotenoids which can be indicative for havingantibacterial activity as mentioned for several microalgae[46] The antimicrobial activity of microalgae has beenattributed to compounds belonging to several chemicalclasses including indoles terpenes acetogenins phenolsfatty acids and volatile halogenated hydrocarbons [47 48]Themechanisms of action of some antimicrobial agents weredescribed carotenoids digest the cell wall [49] flavonoids


Table 5 Antibacterial activity of Amphora sp 80 ethanolic extract

Strains Gram +minus IZD (mm)80 ethanol extract of Amphora sp

Bacillus cereus (ATCC11778) + 0 plusmn 0Bacillus subtilis (JN934392) + 0 plusmn 0Micrococcus luteus (ATCC4698) + 16 plusmn 05Staphylococcus aureus (ATCC6538) + 20 plusmn 2Klebsiella pneumoniae (ATCC13883) minus 16 plusmn 05Salmonella enterica (ATCC43972) minus 12 plusmn 1The data are expressed as mean plusmn SD (119899 = 3) IZD inhibition zone diameter

01 02 03 04 0500

02

04

06

08

10

Redu

cing

pow

er (a

bsor

banc

e at 7

00 n

m)

80 ethanolic extractVit C


(a)

0

20

40

60

80

100

Inhi

bitio

n (

)

01 02 03 04 05



(b)

Figure 2 Ferric reducing antioxidant power (FRAP) (a) and beta-carotene bleaching test (b) for 80 ethanolic extract of Amphora spcompared to vitamin C (Vit C) as a standard

increase permeability of the inner bacteria [50] polyphe-nols participate in enzyme inhibition substrate deprivationcomplexing with cell wall and membrane disruption [8]polysaccharides inhibit hyaluronidase [8] while fatty acidsand lipids cause the disruption of the cellular membrane [51]The antimicrobial activity of supercritical extracts obtainedfrom the microalga Chaetoceros muelleri was related to itslipids composition [52] Fatty acids were reported to bebioactive compounds having antibacterial activity in somediatoms Phaeodactylum tricornutum [51 53] and Skeletonemacostatum [54] and other microalgae Haematococcus pluvialis[55] and Dunaliella salina [56]

Microalgae extracts should be tested as additives forfood and feed formulations in an attempt to replace thecurrently used antimicrobial compounds of synthetic originsincluding subtherapeutical doses of antibiotics employed as aprophylactic measure in animal breeding [9]

4 Conclusion

Amphora sp a brown microalga cultivated in high salinitymedium of 100 psu was confirmed to contain a moderatepercentage of proteins lipids sugars and minerals andimportant levels of polyphenols flavonoids chlorophyllcarotenoids and bioactive fatty acids as EPA Besides thismicroalga has important antioxidant and antibacterial activ-ities Prominent contents of bioactive compounds in thismicroalga are in favor of its possible valorization in food addi-tives and pharmaceutical or cosmetic products Moreover itsrichness in saturated fatty acids allows its eventual use forbiodiesel production

Conflicts of Interest

The authors declare no conflicts of interest regarding thepublication of this paper


Authorsrsquo Contributions

Saoussan Boukhris and Khaled Athmouni contributedequally to this work

Acknowledgments

Thisworkwas supported by theMinistry ofHigher Educationand Scientific Research Tunisia The authors would like tothank Professor Noureddine Drira from the Sfax Faculty ofSciences for the availability of the culture chamber and thelaminar flow hood to the maintenance of microalga culturesThey would also like to thank Professor Neji Garsallallah andDr Sabrine Sellimi for their help

References

[1] G N Gupta S K Tiwari K Lawrence and R S LawrenceldquoEffect of silicon on growth and biodiesel production in freshwater diatomsrdquo Plant Archives vol 11 no 2 pp 673ndash676 2011

[2] J M Graham L E Graham S B Zulkifly B F Pfleger S WHoover and J Yoshitani ldquoFreshwater diatoms as a source oflipids for biofuelsrdquo Journal of Industrial Microbiology amp Bio-technology vol 39 no 3 pp 419ndash428 2012

[3] A Affan R Karawita Y-J Jeon B-Y Kim and J-B LeeldquoGrowth characteristics bio-chemical composition and antiox-idant activities of benthic diatom grammatophora marina fromjeju coast koreardquo Algae vol 21 no 1 pp 141ndash148 2006

[4] R Karawita M Senevirathne Y Athukorala et al ldquoProtectiveeffect of enzymatic extracts from microalgae against DNAdamage induced by H2O2rdquo Marine Biotechnology vol 9 no 4pp 479ndash490 2007

[5] S-H Lee R Karawita A Affan J-B Lee B-J Lee and Y-JJeon ldquoPotential antioxidant activites of enzymatic digests frombenthic diatoms Achnanthes longipes Amphora coffeaeformisand Navicula sp (Bacillariophyceae)rdquo Journal of Food Scienceand Nutrition vol 13 no 3 pp 166ndash175 2008

[6] C Bonnineau I G Sague G Urrea and H Guasch ldquoLighthistory modulates antioxidant and photosynthetic responsesof biofilms to both natural (light) and chemical (herbicides)stressorsrdquo Ecotoxicology vol 21 no 4 pp 1208ndash1224 2012

[7] A C Guedes H M Amaro and F X Malcata ldquoMicroalgae assources of high added-value compounds-a brief reviewof recentworkrdquo Biotechnology Progress vol 27 no 3 pp 597ndash613 2011

[8] H M Amaro A C Guedes and F X Malcata ldquoAntimicrobialactivities of microalgae an invited reviewrdquo Science againstMicrobial Pathogens Communicating Current Research AndTechnological Advances vol 3 2011

[9] J Tramper C Battershill W Brandenburg et al ldquoWhat to do inmarine biotechnologyrdquo Biomolecular Engineering vol 20 no4ndash6 pp 467ndash471 2003

[10] E Christaki E Bonos I Giannenas and P Florou-PanerialdquoFunctional properties of carotenoids originating from algaerdquoJournal of the Science of Food and Agriculture vol 93 no 1 pp5ndash11 2013

[11] A Aullon Alcaine Biodiesel frommicroalgae [Doctoral disserta-tion] Universitat Politecnica de Catalunya Escola UniversitariadrsquoEnginyeria Tecnica Industrial drsquoIgualada (ET Industrial espe-cialitat en Quımica Industrial) 2010

[12] S Scala and C Bowler ldquoMolecular insights into the novelaspects of diatom biologyrdquo Cellular and Molecular Life Sciencesvol 58 no 11 pp 1666ndash1673 2001

[13] S Masmoudi E Tastard W Guermazi A Caruso A Morant-Manceau and H Ayadi ldquoSalinity gradient and nutrients asmajor structuring factors of the phytoplankton communities insalt marshesrdquo Aquatic Ecology 2015

[14] R P F F da Silva T A P Rocha-Santos and A C DuarteldquoSupercritical fluid extraction of bioactive compoundsrdquoTrACmdashTrends in Analytical Chemistry vol 76 pp 40ndash51 2016

[15] K Bone and S Mills Principles and Practice of PhytotherapyModern Herbal Medicine Elsevier Limitd 2013

[16] H Ayadi O Abid J Elloumi A Bouaın and T Sime-NgandoldquoStructure of the phytoplankton communities in two lagoonsof different salinity in the Sfax saltern (Tunisia)rdquo Journal ofPlankton Research vol 26 no 6 pp 669ndash679 2004

[17] R R Guillard and J H Ryther ldquoStudies of marine planktonicdiatoms I Cyclotella nana Hustedt and Detonula confervacea(CLEVE) Granrdquo Canadian Journal of Microbiology vol 8 pp229ndash239 1962

[18] R R Guillard ldquoCulture of phytoplankton for feeding marineinvertebratesrdquo in Culture of Marine Invertebrate Animals ani-mals and of Culture marine invertebrate animals Eds pp 29ndash60 Springer 1975

[19] W Horwitz ldquoOfficial methods of analysis of AOAC Interna-tionalrdquo in Proceeding of the AOAC International GaithersburgMd Gaithersburg Md 2000

[20] M DuBois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[21] H K Lichtenthaler ldquoChlorophylls and carotenoids Pigments ofphotosynthetic biomembranesrdquo inMethods in Enzymology vol148 pp 350ndash382 1987

[22] Association of Official AnalyticalOfficial methods of analysis ofAOAC International edited by P Cunniff Washington DC

[23] C-C Chang M-H Yang H-M Wen and J-C ChernldquoEstimation of total flavonoid content in propolis by twocomplementary colorimetric methodsrdquo Journal of Food andDrug Analysis vol 10 no 3 pp 178ndash182 2002

[24] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[25] K Goiris KMuylaert I Fraeye I Foubert J De Brabanter andL De Cooman ldquoAntioxidant potential of microalgae in relationto their phenolic and carotenoid contentrdquo Journal of AppliedPhycology vol 24 no 6 pp 1477ndash1486 2012

[26] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199-1200 1958

[27] V Katalinic D Modun I Music and M Boban ldquoGenderdifferences in antioxidant capacity of rat tissues determined by2 2rsquo-azinobis (3-ethylbenzothiazoline 6-sulfonate ABTS) andferric reducing antioxidant power (FRAP) assaysrdquo ComparativeBiochemistry and Physiology Part C Toxicology Pharmacologyvol 140 no 1 pp 47ndash52 2005

[28] A Yıldırım A Mavi and A A Kara ldquoDetermination ofanti-oxidant and antimicrobial activities of Rumex crispus L


extractsrdquo Journal of Agricultural and Food Chemistry vol 49no 8 pp 4083ndash4089 2001

[29] I I Koleva TA vanBeek J PH LinssenA deGroot andLNEvstatieva ldquoScreening of plant extracts for antioxidant activitya comparative study on three testing methodsrdquo PhytochemicalAnalysis vol 13 no 1 pp 8ndash17 2002

[30] H Chtourou I Dahmen A Jebali et al ldquoCharacterization ofAmphora sp a newly isolated diatom wild strain potentiallyusable for biodiesel productionrdquo Bioprocess and BiosystemsEngineering vol 38 no 7 pp 1381ndash1392 2015

[31] S-H Lee R Karawita A Affan et al ldquoPotential of benthicdiatoms Achnanthes longipes Amphora coffeaeformis and Nav-icula sp (Bacillariophyceae) as Antioxidant SourcesrdquoAlgae vol24 no 1 pp 47ndash55 2009

[32] H Khatoon S Banerjee F M Yusoff and M Shariff ldquoEvalua-tion of indigenous marine periphytic Amphora Navicula andCymbella grown on substrate as feed supplement in Penaeusmonodon postlarval hatchery systemrdquo Aquaculture Nutritionvol 15 no 2 pp 186ndash193 2009

[33] L Ying M Kang-Sen and S Shi-Chun ldquoTotal lipid and fattyacid composition of eight strains of marine diatomsrdquo ChineseJournal of Oceanology and Limnology vol 18 no 4 pp 345ndash3492000

[34] M R Brown ldquoThe amino-acid and sugar composition of 16species of microalgae used in mariculturerdquo Journal of Experi-mental Marine Biology and Ecology vol 145 no 1 pp 79ndash991991

[35] P Kuczynska M Jemiola-Rzeminska and K Strzalka ldquoPhoto-synthetic pigments in diatomsrdquoMarine Drugs vol 13 no 9 pp5847ndash5881 2015

[36] C F Gao Y Zhai Y Ding and Q Wu ldquoApplication of sweetsorghum for biodiesel production by heterotrophic microalgaChlorella protothecoidesrdquo Applied Energy vol 87 no 3 pp 756ndash761 2010

[37] A P Desbois M Walton and V J Smith ldquoDifferential antibac-terial activities of fusiform and oval morphotypes of Phaeo-dactylum tricornutum (Bacillariophyceae)rdquo Journal of the Ma-rine Biological Association of the United Kingdom vol 90 no 4pp 769ndash774 2010

[38] I Iglesia I Huybrechts M Gonzalez-Gross et al ldquoFolate andvitamin B 12 concentrations are associated with plasma DHAand EPA fatty acids in European adolescents the HealthyLifestyle in Europe by Nutrition in Adolescence (HELENA)studyrdquo British Journal of Nutrition vol 117 no 01 pp 124ndash1332017

[39] W Zheng X Wang W Cao et al ldquoE-configuration structuresof EPA and DHA derived from Euphausia superba and theirsignificant inhibitive effects on growth of human cancer celllinesrdquo Leukotrienes and Essential Fatty Acids (PLEFA) vol 117pp 47ndash53 2017

[40] A Maadane N Merghoub T Ainane et al ldquoAntioxidantactivity of some Moroccan marine microalgae pufa profilescarotenoids and phenolic contentrdquo Journal of Biotechnology vol215 pp 13ndash19 2015

[41] K Athmouni T Belghith K Bellassouad A E Feki and HAyadi ldquoEffect of extraction solvents on the biomolecules andantioxidant properties of Scorzonera undulata (Asteraceae)application of factorial design optimization phenolic extrac-tionrdquo Acta Scientiarum Polonorum Technologia Alimentariavol 14 no 4 pp 313ndash320 2015

[42] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of lsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[43] W Boonchum Y Peerapornpisal D Kanjanapothi et al ldquoAnti-oxidant activity of some seaweed from the Gulf of ThailandrdquoInternational Journal of Agriculture and Biology vol 13 no 1pp 95ndash99 2011

[44] R M Appavoo and D G Femi ldquoEurihaline microalgaemdashanovel substance against post operative pathogenrdquo InternationalJournal of Development Research vol 5 no 1 pp 3030ndash30332015

[45] R Lavanya and N Veerappan ldquoAntibacterial potential of sixseaweeds collected from Gulf of Mannar of southeast coast ofIndiardquo Advances in Biological Research vol 5 no 1 pp 38ndash442011

[46] J Pradhan S Das and B K Das ldquoAntibacterial activity offreshwater microalgae a reviewrdquo African Journal of Pharmacyand Pharmacology vol 8 no 32 pp 809ndash818 2014

[47] A M Mayer and M T Hamann ldquoMarine pharmacology in2001-2002 marine compounds with anthelmintic antibacte-rial anticoagulant antidiabetic antifungal anti-inflammatoryantimalarial antiplatelet antiprotozoal antituberculosis andantiviral activities affecting the cardiovascular immune andnervous systems and other miscellaneous mechanisms ofactionrdquo Comparative Biochemistry and Physiology Part C Toxi-cology Pharmacology vol 140 no 3-4 pp 265ndash286 2005

[48] K H Cardozo T Guaratini M P Barros et al ldquoMetabolitesfrom algae with economical impactrdquo Comparative Biochemistryand Physiology Part C Toxicology Pharmacology vol 146 no 1pp 60ndash78 2007

[49] M Cucco B Guasco GMalacarne and R Ottonelli ldquoEffects of120573-carotene on adult immune condition and antibacterial activ-ity in the eggs of the Grey Partridgerdquo Comparative Biochemistryand Physiology Part AMolecular and Integrative Physiology vol147 no 4 pp 1038ndash1046 2007

[50] O K Mirzoeva R N Grishanin and P C Calder ldquoAntimicro-bial action of propolis and some of its components the effectson growth membrane potential and motility of bacteriardquoMicrobiological Research vol 152 no 3 pp 239ndash246 1997

[51] A P Desbois A Mearns-Spragg and V J Smith ldquoA fatty acidfrom the diatom Phaeodactylum tricornutum is antibacterialagainst diverse bacteria including multi-resistant Staphylococ-cus aureus (MRSA)rdquoMarine Biotechnology vol 11 no 1 pp 45ndash52 2009

[52] J A Mendiola C F Torres A Tore et al ldquoUse of supercrit-ical CO2 to obtain extracts with antimicrobial activity fromChaetoceros muellerimicroalga A correlation with their lipidiccontentrdquo European Food Research and Technology vol 224 no4 pp 505ndash510 2007

[53] V J Smith A PDesbois and E ADyrynda ldquoConventional andunconventional antimicrobials from fish marine invertebratesand micro-algaerdquo Marine Drugs vol 8 no 4 pp 1213ndash12622010

[54] MNaviner J-P Berge P Durand andH Le Bris ldquoAntibacterialactivity of the marine diatom Skeletonema costatum againstaquacultural pathogensrdquo Aquaculture vol 174 no 1-2 pp 15ndash24 1999

[55] S Santoyo I Rodrıguez-Meizoso A Cifuentes et al ldquoGreenprocesses based on the extraction with pressurized fluids to


obtain potent antimicrobials from Haematococcus pluvialismicroalgaerdquo LWTmdashFood Science and Technology vol 42 no 7pp 1213ndash1218 2009

[56] M Herrero E Ibanez A Cifuentes G Reglero and S SantoyoldquoDunaliella salina microalga pressurized liquid extracts aspotential antimicrobialsrdquo Journal of Food Protection vol 69 no10 pp 2471ndash2477 2006

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


dependent on their physiological state Likewise their sec-ondary metabolism can easily be triggered by most forms ofexternally applied stress [7]

Among the large variety of microalgae CyanophyceaeChlorophyceae Bacillariophyceae (diatoms) and Chryso-phyceae are the most studied for biodiesel production [11]It is estimated that diatoms produce about 25 of the globalprimary biomass [12] In the Sfax Solar Saltern Bacillario-phyceae dominatedwithDinophyceae in the least salty pondsbut they are rarely abundant in hypersaline environments[13]

Unlike the universalmacromolecules of primarymetabo-lism (eg monosaccharides polysaccharides amino acidsproteins and lipids) which are commonly present in allorganisms secondarymetabolites have a farmore limited dis-tribution in nature They are not necessarily produced underall conditions and can only be found in specific organismsor a group of organisms The organism can produce thesecompounds either to protect itself within its own livingecosystem or to play a basic role in its everyday existenceThis reveals bioactive molecules in unrelated biologicalsystems [14] The medicinal and pharmacological actions areoften dependent on the presence of bioactive compoundscalled secondary metabolites [15] The growth of algae isrelatively rapid and it is possible to control the productionof their bioactive compounds such as polyphenols andpigments by modifying culture conditions [10]

In the present workAmphora sp (Bacillariophyceae) wascultivated in a hypersaline medium and the physicochemicalproperties fatty acids profile bioactive compounds andsome biological activities were determined

2 Materials and Methods

21 Microalga Amphora sp is a Bacillariophyceae that wasisolated via micromanipulation and serial dilution from awater sample collected in the pond C4-1 of the Sfax SolarSaltern with average salinity of 107 psu (practical salinityunit) The Sfax Solar Saltern is located in the central easterncost of Tunisia at about 34∘391015840N and 10∘421015840E The ponds areshallow (20ndash70 cm depth) and have various salinities rangingfrom 40 to 400 psu [16] They are connected by pipes andchannels along a 12 km section of sea cost

22 Culture Conditions The microalga was cultivated inbatch in autoclaved artificial seawater which was enrichedwith F2 nutrient medium sodium silicate (Na2SiO3) andtrace metals solution [17 18] Cultures were conducted for 15days at the salinity of 100 psu 25∘C light dark (L D) cycleof (16 h 8 h) and cool white fluorescent light intensity of60 120583moles photonsmminus2 sminus1 The biomass was separated fromthe culture media by centrifugation (4500timesg 10min) andthe pellet was washed with distilled water and centrifugedagain at 4500timesg for 10min (the washing was repeated twotimes) The pellet was freeze-dried and stored at minus70∘C23 Determination of Dry Matter Ash Proteins Lipidsand Total Sugars The dry matter and ash content weredetermined according to the AOAC standard methods [19]

Total nitrogen contents of Amphora sp crude and undi-gested protein substrates were determined using the Kjeldahlmethod according to the AOAC method number 98413 [19]with equipment of BUCHI Digestion Unit K-424 (Switzer-land) Crude proteins were estimated by multiplying the totalnitrogen content by the factor of 625

Lipids content was determined gravimetrically after theSoxhlet extraction of dried samples with hexane for 2 hoursusing Nahita Model 655 (Navarra Spain)

As regards sugars they were estimated by phenol-sulfuricacid method [20] using glucose as a standard

24 Determination of Pigments Content Chlorophylls andCarotenoids Pigments were determined according to themethod described by Lichtenthaler [21] Two milliliters ofculture was centrifuged at 5500timesg for 5min and the super-natant was discarded and the pellet was mixed with 999methanol and incubated in the dark for 24 hours at 45∘CAfter incubation pigments content was determined measur-ing absorbances at 470 6524 and 6652 nm which werecorrected for turbidity by subtracting absorbance at 750 nm

25 Determination of Mineral Content The analysis ofsodium potassium calcium magnesium iron copper andzinc contents inAmphora sp was carried out using the induc-tively coupled plasma optical emission spectrophotometer(ICP-OES)Model 4300DV PerkinElmer (Shelton CTUSA)according to the method of AOAC 1999 [22] Measurementswere performed in triplicate and the results were the averageof three values

26 Determination of Fatty Acids Profile by GC-MS Fattyacids methyl esters (FAME) were prepared by basic trans-esterification protocol The extracted lipids were collectedin 10mL heptane and introduced into a 50mL flask Avolume of 10mL of KOH solution (11 g Lminus1) in methanolwas added with a few antibumping beads The mixture wasboiled under reflux condenser for 10min Then 5mL ofboron trifluoride (BF3) methanol complex (150 g Lminus1) wasintroduced through the condenser by a graduated syringeand themixture was boiled for 2minThemixture was cooledunder room temperature and 15 to 20mL of saturated sodiumsulfate solutionwas added and shakenwell Furthermore thissolution was added until the liquid level reached the neckof the flask After the phase separation the upper layer (n-heptane) was collected by a Pasteur pipette and evaporatedunder nitrogen flow The FAME were redissolved in 500120583Lhexane for GC-MS analysis

The analysis of FAME was performed with a 6890GC5973 MSD GCMS system from Agilent Technologiesequipped with an HP-INNO Wax Polyethylene Glycol cap-illary column (30m length 250 120583m diameter 025 120583m filmthickness) Helium was used as a gas carrier with a flowrate through the column of 1mLmin Column temperaturewas held initially for 1min at 200∘C and increased to 250∘Cat 10∘Cminminus1 and was then held at 250∘C for 15min Theinjector was kept at 260∘C using a splitless mode and thena sample of 1 120583L was injected The ion source temperature


















(3)




























00 05 10 15 20 25 30 35 40 450

20

40

60

80

100

in

hibi

tion



(a)

00 02 04 06 08 10

20

40

60

80

100

in

hibi

tion



(b)












01 02 03 04 0500

02

04

06

08

10

Redu

cing

pow

er (a

bsor

banc

e at 7

00 n

m)



(a)

0

20

40

60

80

100

Inhi

bitio

n (

)

01 02 03 04 05



(b)




4 Conclusion







Acknowledgments


References




































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















(3)




























00 05 10 15 20 25 30 35 40 450

20

40

60

80

100

in

hibi

tion



(a)

00 02 04 06 08 10

20

40

60

80

100

in

hibi

tion



(b)












01 02 03 04 0500

02

04

06

08

10

Redu

cing

pow

er (a

bsor

banc

e at 7

00 n

m)



(a)

0

20

40

60

80

100

Inhi

bitio

n (

)

01 02 03 04 05



(b)




4 Conclusion







Acknowledgments


References




































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





(3)




























00 05 10 15 20 25 30 35 40 450

20

40

60

80

100

in

hibi

tion



(a)

00 02 04 06 08 10

20

40

60

80

100

in

hibi

tion



(b)












01 02 03 04 0500

02

04

06

08

10

Redu

cing

pow

er (a

bsor

banc

e at 7

00 n

m)



(a)

0

20

40

60

80

100

Inhi

bitio

n (

)

01 02 03 04 05



(b)




4 Conclusion







Acknowledgments


References




































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















00 05 10 15 20 25 30 35 40 450

20

40

60

80

100

in

hibi

tion



(a)

00 02 04 06 08 10

20

40

60

80

100

in

hibi

tion



(b)












01 02 03 04 0500

02

04

06

08

10

Redu

cing

pow

er (a

bsor

banc

e at 7

00 n

m)



(a)

0

20

40

60

80

100

Inhi

bitio

n (

)

01 02 03 04 05



(b)




4 Conclusion







Acknowledgments


References




































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




00 05 10 15 20 25 30 35 40 450

20

40

60

80

100

in

hibi

tion



(a)

00 02 04 06 08 10

20

40

60

80

100

in

hibi

tion



(b)












01 02 03 04 0500

02

04

06

08

10

Redu

cing

pow

er (a

bsor

banc

e at 7

00 n

m)



(a)

0

20

40

60

80

100

Inhi

bitio

n (

)

01 02 03 04 05



(b)




4 Conclusion







Acknowledgments


References




































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





01 02 03 04 0500

02

04

06

08

10

Redu

cing

pow

er (a

bsor

banc

e at 7

00 n

m)



(a)

0

20

40

60

80

100

Inhi

bitio

n (

)

01 02 03 04 05



(b)




4 Conclusion







Acknowledgments


References




































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgments


References




































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

The Potential of a Brown Microalga Cultivated in High Salt ...downloads.hindawi.com/journals/bmri/2017/4018562.pdf · 2 BioMedResearchInternational dependentontheirphysiologicalstate.Likewise,theirsec-ondarymetabolismcaneasilybetriggeredbymostformsof