DSpace at VNU: Growth, antioxidant capacity and total carotene of Dunaliella salina DCCBC15 in a low cost enriched natural seawater medium

The efficacy of a new selected low-cost enri-ched natural seawater medium MD4, supplemented with industrial N–P–K fertilizer, was investigated with respect to biomass production, chlorop

S H O R T C O M M U N I C A T I O N

Growth, antioxidant capacity and total carotene of Dunaliella

salina DCCBC15 in a low cost enriched natural seawater medium

Duc Tran•Nguyen Doan•Clifford Louime•

Mario Giordano•Sixto Portilla

Received: 28 February 2013 / Accepted: 21 June 2013

Ó Springer Science+Business Media Dordrecht 2013

Abstract Dunaliella is currently drawing worldwide

attention as an alternative source of nutraceuticals

Com-mercially, b-carotene making up over 10 % of Dunaliella

biomass is generating the most interest These compounds,

because of their non-toxic properties, have found applications

in the food, drug and cosmetic industry The b-carotene

con-tent of Dunaliella cells, however, depends heavily on the

growth conditions and especially on the availability of

nutri-ents, salinity, irradiance and temperature in the growth

med-ium A chemically well defined medium is usually required,

which significantly contributes to the cost of pigment

pro-duction; hence a desire for low cost marine media The present

study aimed at evaluating the suitability of six different media,

especially exploiting local potential resources, for the mass

production of Dunaliella salina DCCBC15 as functional food

and medicine The efficacy of a new selected low-cost

enri-ched natural seawater medium (MD4), supplemented with

industrial N–P–K fertilizer, was investigated with respect to

biomass production, chlorophyll, antioxidant capacity, and total carotene by Dunaliella though culture conditions were not optimized yet This new medium (MD4) appears extre-mely promising, since it affords a higher production of Dunaliella biomass and pigments compared with the control,

a common artificial medium (MD1), while allowing a sub-stantial reduction in the production costs The medium is also recommended for culturing other marine algae

Keywords Dunaliella Carotene  Medium  Cultivation

Introduction

Dunaliella is a cell wall-less green flagellate belonging to the order Volvocales (Chlorophyceae, Chlorophyta) (Oren

2005; Polle et al.2009) There are about 26 saltwater species described for the genus of Dunaliella (Oren 2005; Boro-witzka and Siva 2007; Polle et al.2009) D salina TEO-DORESCO is a model organism and the type species of the genus, whose vegetative cells are under high irradiance, high salinity, or low nitrogen concentrations, are characterized by

an intense orange color due to the intracellular accumulation

of large amounts of b-carotene (Polle et al.2009; Lamers

et al 2010; Fu et al.2013) D salina are among the main sources for natural b-carotene, which is employed in the food, cosmetic and pharmaceutical industries and also used

as colorant, antioxidant and anti-cancer agent (Ben-Amotz

et al.1982,1989; Borowitzka and Borowitzka1988; Sergio and Rubens2010; Fu et al.2013) Dunaliella history, bio-logical characteristics as well as applications were presented

in thorough reviews and publications of authors such as Oren (2005); Polle et al (2009), and others

Various artificial and natural seawater media have been used for Dunaliella cultivation, both in laboratory settings

D Tran ( &)  N Doan

School of Biotechnology, International University, VNU,

Thu Duc Dist., Ho Chi Minh, Vietnam

e-mail: tnducminh@yahoo.com; tnduc@hcmiu.edu.vn

C Louime

FAMU BioEnergy Group, College of Engineering Sciences,

Technology and Agriculture, Florida A&M University,

Tallahassee, FL 32307, USA

M Giordano

Dipartimento di Scienze della Vita e dell’Ambiente (DISVA),

Universita` Politecnica delle Marche, Via Brecce Bianche,

60131 Ancona, Italy

S Portilla

Center for Estuarine, Environmental and Coastal Oceans

Monitoring, Dowling College, 150 Idle Hour Blvd., Oakdale,

NY 11769, USA

DOI 10.1007/s11274-013-1413-2

and in large industrial scales (Ben-Amotz1995; Pisal and

Lele 2005; Garcı´a-Gonza´lez et al 2003; Simental and

Sa´nchez-Saavedra 2003; Fazeli et al 2006; Grobbelaar

1995; Abu-Rezq et al 2010; Ana Prieto et al 2011)

However, in developing countries such as Vietnam, some

of these media are unrealistically expensive and the quest

to exploit local, lower cost medium components may be

crucial for a commercially viable cultivation of Dunaliella

Vietnam has a vast potential for Dunaliella b-carotene

production along its coastal areas This would contribute to

the economic and environmental stability of the country

(and beyond) In the present study, we investigated the

growth of Dunaliella using local seawater enriched with an

industrial fertilizer (N–P–K) commercially available in

Vietnam In addition to N, P and K, this fertilizer contains

micronutrients (Mg, Ca, Zn, Cu, Fe, Mo) present in

artifi-cial medium and essential for Dunaliella growth

(Boro-witzka and Boro(Boro-witzka 1988; Sergio and Rubens 2010)

The growth data showed that our medium allows to attain a

much lower cost per unit of dry mass than more common

artificial media The medium is recommended for the

culturing of other marine algae as well

Materials and methods

Dunaliella salina DCCBC15 growth conditions

Dunaliella salina DCCBC15 was kindly provided by Dr

E.W Polle, Department of Biology, Brooklyn College of

CUNY Brooklyn, NY (USA) The cultures were grown in

batch and maintained in artificial 1.5 M NaCl medium according to Chitlaru and Pick (1989) Briefly, the medium contained 1.5 M NaCl; 0.4 M Tris–HCl, 5 mM KNO3,

5 mM MgSO4, 0.3 mM CaCl2, 0.2 mM KH2PO4, 1.5 lM FeCl3 in 6 lM EDTA, 0.185 mM H3BO3, 7 lM MnCl2, 0.8 lM ZnCl2, 0.2nM CuCl2, 0.2 lM Na2MoO4, 20nM CoCl2, 50 mM NaHCO3

Six different 1.5 M NaCl media were devised for

D salina cultures, including one fully artificial medium (MD1), and five media based on natural seawater, enriched

by the addition of different chemicals (MD2–MD6), the detailed composition of all media is shown in Table1 Components of N–P–K are listed as a note below Table1 MD6 is enriched natural seawater with components according to Garcı´a-Gonza´lez et al (2003) and Ana Prieto

et al (2011) for open pilot culture The N–P–K (code: Dau Trau 501) was bought from Binh Dien fertilizer company Seawater with salinity about 0.5 M collected from Long Hai beach, Vung Tau province, was first filtered to remove sand and other small particles Then all media (added salts

to obtain 1.5 M) were sterile filtered using cellulose acetate filter with pore size 0.45 lm after adding chemicals according to Table1

Experimental design

One liter of algal culture was grown in 2 liter flasks; for each treatment, 3 replicate cultures were used The cultures were maintained at 25°C, and at a photon flux density of

50 lmol m-2 s-1 (PAR), provided by white fluorescence lamps The experiment was run for 24 days

Table 1 Components of the different media tested in this study

Chemicals Medium

Artificial a

(Tris–HCl)

Natural SW added with artificial components as in MD1 (Tris–HCl)

Natural SW added with artificial components as in MD1 (but no Tris–HCl)

Natural SW (no Tris–

HCl) (g/l)

Natural SW (no Tris–

HCl) (g/l)

Natural SW (no Tris– HCl) (g/l)

a 87.75 g/l NaCl; 9.48 g/l Tris–HCl, 0.6675 g/l KNO3, 1.86 g/l MgSO4, 0.06615 g/l CaCl2, 0.022 g/l KH2PO4, 9.75 9 10 -4 g/l FeCl3in 8.76 9 10 -3 g/l EDTA, 6.975 9 10 -4 g/l H3BO3, 9.45 9 10 -5 g/l MnCl2, 8.16 9 10 -6 g/l ZnCl2, 3.834 9 10 -6 g/l CuCl2, 3.6 9 10 -5 g/l

Na2MoO4, 3.567 9 10 -6 g/l CoCl2, 3.15 g/l NaHCO3

b N–P–K (30–15–10): 30 % N, 15 % P2O5, 10 % K2O, 0.05 % Mg, 0.05 % Ca, 0.01 % B, 0.05 % Zn, 0.05 % Cu, 0.05 % Fe, 0.025 % Mn, 0.005 % Mo, a-NAA, b-NOA, GA3

Pigments analysis

Pigment extraction was carried out according to Psal and

Lele (2005) A 5 ml aliquot of Dunaliella salina culture

was centrifuged at 8,000 rpm for 20 min The pellet was

washed with distilled water, suspended in an 80 % acetone

solution, thoroughly vortexed and centrifuged to extract

pigments until the pellets turned clear/white The

absor-bance of the relevant pigments was measured in the extract

using UV–VIS spectrophotometer Chlorophyll and total

carotene were estimated according to Lichtentaler and

Wellburn formulas (1985):

Chla lg=mlð Þ ¼ 11:75 Að 662Þ  2:35 Að 645Þ

Chlb lg=mlð Þ ¼ 18:61 Að 645Þ  3:96 Að 662Þ

Cxþ c lg=mlð Þ ¼ 1000Að 470

2:270 Chla  81:4 ChlbÞ=198 where Chla chlorophyll a, Chlb chlorophyll b, Cx ? c total

carotene

Antioxidant capacity

DPPH solution was prepared by dissolving 0.004 g of

DPPH in 100 ml of methanol Five ml of D salina

cen-trifuged The pellet was extracted with 20 ml of absolute

ethanol and vortexed well The extract was filtered to

remove possible particles would interfere optical density

reading, and dried at a temperature of 55°C The dried

extract was dissolved in 2 ml of absolute ethanol The

absorbance of the extract at 515 nm was determined

spectrophotometrically A blank sample (absolute ethanol)

was also taken as control The antioxidant activity was

calculated based on the inhibition of free radical DPPH in

percent according to the formula I % = (Ablank- Asample)/

Ablank9 100

Growth and biomass determination

Cell counts were performed every 2 days using haemacytometer

For biomass determination, the cultures were filtered through 47 mm glass fiber filters with a 0.7 lm nominal pore size The filter was washed with 5 ml of ammonium formiate (0.5 M), dried at 103°C for 6 h or until weight was stable, for dry weight determination The dry biomass was further burned in a furnace at 550°C to determine the ash weight The biomass was calculated as the difference

of the dry weight and the ash weight

Statistical analysis

Data were analyzed by one-way ANOVA using the SPSS software The level of significance was always set at

P\ 0.05

Results

Growth

Results of the different media with respect to cell density, biomass and chlorophyll a after 24 days of cultivation, which were all in line, are shown in Fig.1 The medium (MD4) enriched with industrial fertilizer N–P–K showed best overall performance The differences of cell density, biomass and chlorophyll a among the different media tes-ted were statistically significant (P = 0.021 \ 0.05) The highest cell numbers, biomass and chlorophyll a in MD5, MD6 were 0.5 9 106/ml, 0.2 g/l and 1 lg/ml respectively, which were three to four times lower than those in MD1, MD4 after 2 weeks (Fig.1a, b)

Antioxidant capacity and total carotene

Similar to growth, antioxidant capacity and carotene of Dunaliella were significantly lower in natural media MD5, MD6 (P = 0.00) These media therefore do not seem to be appropriate for Dunaliella cultivation For other media MD1, MD2, MD3, MD4, though growth of Dunaliella declined after the first initial 2 weeks due to limiting nutrients (Fig 1), antioxidant capacity and carotene con-tinued to accumulate for an additional week due to limiting nutrients (Fig.2b) Among these four media, antioxidant capacity and carotene of Dunaliella were higher in artificial MD1 and natural MD4 after the exponential growth phase

Table 2 Concentration (mg/l) of some components in N–P–K

com-pared with artificial medium (MD1)

Elements N–P–K (mg/l) Artificial medium (mg/l)

(Fig.2) Antioxidant capacity and carotene yield in MD1,

MD4 were almost twice higher than those in MD5, MD6,

which were 50 % and [4 lg/ml respectively

Discussion

Low cost medium for various algae based on fertilizers

have been investigated and shown to be potential for lab

and large scale cultivation of various groups of algae

(Garcı´a-Gonza´lez et al 2003; Simental and

Sa´nchez-Sa-avedra 2003) However using direct local resources

regarding low cost chemicals and natural seawater should

be more practical and at least one step closer for industrial

development With the abundance of natural seawater in

Vietnam, it provides a great opportunity to develop large

scale cultivation of Dunaliella (and other algae) for various application such as feed in aquaculture, food, medical uses, and even biofuel (Takagi et al 2006; Chen et al 2011), which all together promoted us to initiate this study From all media devised in this study, Dunaliella adapted and started to grow after 3 days However, it performed best in the MD4 medium of seawater enriched with N–P–K fertilizer This was revealed through biomass, cell density and chlorophyll a data significantly at P \ 0.05 (Fig.1), as all cultures reached stationary phase after 2 weeks and started collapsing/dead phase which are common due to nutrient limit or inhibitory metabolites releasing from algal culture (Brown et al.1993; Sergio and Rubens2010; Chen

et al 2011; Tran et al unpublished data) Growth of Du-naliella in MD6 medium [which was used in open culti-vation by Garcı´a-Gonza´lez et al (2003)], and in MD5

Fig 1 Cell density (a), biomass (b) and chlorophyll a (c) of Dunaliella bardawil grown in six different media (MD1–MD6)

Fig 2 Antioxidant activity (a) and total carotene (b) of D salina grown in six different media

medium (which was similar to MD4 except there was no

N–P–K), was much slower, which are not appropriately

considered for Dunaliella cultivation The success of MD4

is underscored by the presence of N–P–K and the absence

of Tris–HCl, a high cost component comprising over 90 %

of media MD1 and MD2 The biomass in MD2 and MD3

dropped after 12 days which were earlier than MD1 and

MD4; this could be probably that the algae were stressed

under imbalance ratio of nutrients due to one of the nutrient

components was additionally high coming from seawater

and other nutrient(s) simultaneously run out Presumingly,

growth of Dunaliella in these media (MD1, MD4) is

sim-ilar, but with a cost lower than 90 % in MD4, this makes

the productivity of MD4 90 % higher than MD1 (Tables3,

4) Currently this new MD4 medium is being used more

successfully in our laboratory for most marine strains from

isolation to cultivation than other artificial media such as BG11, F/2, BBM, CM (Tran et al., unpublished data), which we recommended to use this medium for other marine algae as well The effectiveness of this new med-ium is probably due to non-defined minerals and micro-nutrients present in the natural seawater, which we predicted when devising the medium basing on the mini-mal amount of components of the artificial medium (MD1)

In addition, higher concentration of phosphate, Cu and the presence of three hormones (lNAA, bNOA, GA3) in N–P–

K which were not present in artificial MD1 medium (Table2, in bold) may have further stimulatory effects on Dunaliella growth Though the new medium (MD4) was much successful compared with other media regarding low cost and higher productivity, it was not optimized for biomass and pigments production yet Further study on feeding Dunaliella with different nitrogen, phosphate salts

to sustain and optimize continuous growth for optimal biomass, antioxidant and b-carotene production are on progress in our lab, which will be applied for pilot and large scale cultivation in climate conditions of Vietnam

Acknowledgments The authors are grateful for the funding of Nafosted (Funding number: 106.16-2011.31) and the funding of HCM National University to carry out this research (Funding number: B2012-28-02/HD-DHQT-QLKH) The authors are also grateful to Dr Jeurgen Polle at Department of Biology, Brooklyn college for pro-viding Dunaliella salina DCCBC15 for the study and his valuable advices The authors would like to thank anonymous reviewers of this manuscript for correction and improvement.

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Table 3 The cost of chemicals using in artificial medium

Chemicals Gram/1 l medium Cost VND/1 l medium

NaCl 87.75 4,914

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Tris–HCl 9.48 37,920

KNO3 0.6675 93.45

MgSO4 1.86 260.4

CaCl2 0.06615 5.9535

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FeCl3 9.75 9 10-4 0.0741

EDTA 8.76 9 10-3 1.99728

H3BO3 6.975 9 10 -4 0.062775

MnCl2 9.45 9 10 -5 0.014175

ZnSO4 8.16 9 10-6 9.792 9 10-4

CuCl2 3.834 9 10-6 1.1502 9 10-3

Na2MoO4 3.6 9 10-5 0.09

CoCl2 3.567 9 10-6 5.7072 9 10-3

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