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Experimental optimization of SC-CO2 extraction of carotenoids from Dunaliella salina

Experimental optimization of SC-CO2 extraction of carotenoids from Dunaliella salina
Experimental optimization of SC-CO2 extraction of carotenoids from Dunaliella salina

J.of Supercritical Fluids 121(2017)89–95

Contents lists available at ScienceDirect

The Journal of Supercritical

Fluids

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /s u p f l

u

Experimental optimization of SC-CO 2extraction of carotenoids from Dunaliella salina

Seyed Reza Pour Hosseini,Omid Tavakoli ?,Mohammad Hossein Sarrafzadeh

School of Chemical Engineering,College of Engineering,University of Tehran,Tehran 14176,Iran

a r t i c l e

i n f o

Article history:

Received 28March 2015

Received in revised form 9November 2016Accepted 10November 2016

Available online 11November 2016

Keywords:SC-CO 2

Extraction

Microalgae Dunaliella salina Carotenoids Chlorophyll

a b s t r a c t

An experimental design procedure (based on the response surface method,RSM)was used to investigate the effect of pressure and temperature as operating parameters on the supercritical carbon dioxide (SC-CO 2)extraction of carotenoids from Dunaliella salina https://www.wendangku.net/doc/6c17872369.html,ing RSM method,11experiments were performed in random order and empirical correlation was developed.Results indicated that the effect of pressure on yield of extraction is much more than temperature.The most appropriate operating condition to obtain the best extraction yield of carotenoids was found at 400bar and 55?C (115.43?g/g dry microalgae).The recovery of carotenoids at optimum condition with supercritical extraction was half (47%)of the solvent extraction yield.The highest carotenoids/chlorophylls ratio (11.09)was obtained at 300bar and 30?C which show the selectivity of this technology.Under these operating conditions the separation and puri?cation of the two extracted pigments facilitated and a higher selectivity is obtained.

?2016Elsevier B.V.All rights reserved.

1.Introduction

Over 70%of earth is covered with water and the dominant groups of organisms in such aquatic area are seaweeds and microal-gae [1].Microalgae are eukaryotic photosynthetic microorganisms which can be used to produce high value compounds and can be seen in all aquatic ecosystems,lands and even in places where other plants cannot grow (such as the desert,sea coast,etc).Moreover,due to the simple structure of a single cell or multiple cells,microal-gae can be quickly grown in dif?cult environmental conditions such as high or low temperature,light oxidation,high salinity,osmotic pressure and exposure to ultraviolet light.

Dunaliella salina is unicellular and photosynthetic green algae with two equal ?agella that belong to the phylum chlorophyta,order volvocales and family polyblepharidaceae.The cell shapes are ovoid,ellipsoid or spherical and varies in different growth con-ditions and light intensity [2].The cells size varies from 5to 25?m in length and from 3to 13?m in wide.Although cells reproduc-tion are mainly by cell division,sexual reproduction by isogamy does occur occasionally [2].Dunaliella cells are able to accumulate large amount of ?-carotene when grown under selective stress con-ditions including high salt concentration,high light intensity and nitrogen starvation [3,4].These cells contain various pigments such

?Corresponding author.

E-mail addresses:otavakoli@ut.ac.ir ,otavakoli@https://www.wendangku.net/doc/6c17872369.html, (O.Tavakoli).

as chlorophyll a,chlorophyll b ,?-carotene,violaxantine,neoxan-thin,zeaxanthin and lutein [5].Carotenoids are one of the most important groups of natural pigments that found in a wide range of natural sources.They are conjugated polyprenoids compounds and classi?ed into two main groups,a)carotenes which composed from carbon and hydrogen atoms,b)xanthophylls that also have oxygen atoms in their structures.Carotenoids show antioxidant activity [6–8]and prevent illnesses like cancer and cellular aging [9,10].Consumption of these compounds by humans is very useful there-fore development of an ef?cient extraction technique is necessary to separate these compounds from natural resources.

Traditionally,extraction of various bioactive compounds from natural sources has been performed using several solvents at differ-ent conditions [11–16].Conventional solvent extraction methods not only are time consuming and require multiple extraction and puri?cation steps but also need large amounts of organic sol-vents,which are often expensive and potentially harmful.Although use of ultrasound assisted extraction reduces the extraction time [17],environmental impact and consumer acceptability still exist.Supercritical carbon dioxide extraction represents various opera-tional advantages.The advantage in using carbon dioxide includes simpler,faster,and more ef?cient extraction and avoids the con-sumption of large amounts of organic solvents.Utilizing this method does not need a unique separation step to recovery of our products,so the extraction factors such as cost and time are reduced [18].Furthermore,the use of carbon dioxide does not give rise to excessive heating,which usually has a negative effect

https://www.wendangku.net/doc/6c17872369.html,/10.1016/j.sup?u.2016.11.006

0896-8446/?2016Elsevier B.V.All rights reserved.

90

S.R.Pour Hosseini et al./J.of Supercritical Fluids 121(2017)

89–95

Fig.1.Microalgae Dunaliella salina ;a)powdered microalgae,b)SEM photo of microalgae (500×)and c)SEM photo (2500×).

on the thermolabile compounds [19].Application of this tech-nique for extraction of carotenoids from higher plants [20–23]and marine microalgae such as:Chlorella vulgaris [24–27],Spir-ulina sp.[28,29],Haematococcus pluvialis [30,31],Nannochloropsis sp.[19,32,33],Synechococcus sp.[32,34],Scenedesmus sp.[35,36]and Dunaliella salina [5,26,32]has been widely studied in the last decade.

The aim of this work was to investigate the effect of temper-ature and pressure on extraction of carotenoids from Dunaliella salina using supercritical CO 2based on RSM experimental design.An empirical equation was developed to predict the yield of carotenoids in the extraction operating range using Design Expert 7.0.0and optimized by ANOVA analysis.

2.Materials and methods

2.1.Raw material and sample preparation

The raw material employed in this experiment was the microalga Dunaliella salina of Persian Gulf which kindly provided from Qeshm Sina Microalgae Company (Iran,Qeshm Island).The biomass was spray-dried at 180?C and the moisture content reaches 0.93%.Sample was stored under vacuum condition in dark-ness at 4?C prior to the extraction experiments.Fig.1illustrates the photo of powdered microalgae and SEM photo with two magni?-cations.

2.2.Chemicals

Extractions were performed with high purity carbon dioxide (Food grade >99.9%purity)which purchased from Roham Gas (Iran).The extracted compounds kept under nitrogen (99.99%)which obtained from Roham Gas (Iran).Methanol (Merck,99.5%)was used as the storage solvent for the extracts.

2.3.Supercritical ?uid extraction procedure

The aim of this study was to determine the in?uence of pressure and temperature,by several experimental runs at 100–500bar and 40–60?C,for the extraction of carotenoids and chlorophyll from Dunaliella salina powder using supercritical carbon dioxide method.SC-CO 2extraction experiments were carried out in a supercriti-cal extraction apparatus purchased from TST (Taiwan Supercritical Technology Co.,Ltd.,Taiwan)included a booster pump (model PM-10000B)to introduce carbon dioxide in to the extractors.This device consists of two extraction chamber,which is used in par-allel.When the extraction process in a chamber comes to an end,the extraction process can be continued with the other chamber by separating the ?rst one from the circuit.Extractors were heated by hot air in an oven (model OV-SCF).The apparatus is provided schematically in Fig.2.

In this research 5.0g of the Dunaliella salina biomass,which had previously been homogenized with 80.0g glass bead (to enhance CO 2transfer and avoid caking)was loaded to the extraction cell of 250ml.The cell was left 15min in oven to reach the operating temperature.CO 2cylinder valve was opened and the pump was loaded with carbon dioxide (MFC ?mass ?ow controller-was used to measure the CO 2mass rate).The pump started pressurizing CO 2and the vessel was then loaded with CO 2.After the cell reaches to the desired pressure,allowed the system 100s to reach stable con-dition.When the system had attained a balanced state,the metering valve was opened up from the thermostatically controlled restric-tor (at 70?C)until a constant ?ow of 3lit/min (1atm &25.7?C)was achieved.Extraction was carried out during 90min as optimum residence time (data are not shown).

The extracts were collected in a sampler tube.After the extrac-tion process was complete,the extracts were dissolved in methanol (10ml)and stored at 4?C under nitrogen gas with the absence of light until subsequent analysis.

2.4.Experimental design

The effects of pressure and temperature on the extraction of carotenoids were evaluated applying Response Surface Method-ology (RSM).The pressure and temperature were systematically manipulated in the range of 100–500bar and 30–60?C,respec-tively.To do this,11experiments were designed and carried out in randomized run order.Table 1shows the experimental design with the experimental levels of the independent variables (factors)along with the results obtained by analysis of the extracts.

The experimental data were processed with the software pack-age of Design-Expert version 7.0.0(2005,Stat-Ease,Inc.)in order to obtain empirical equations.This relation has an ability to pre-dict the extraction yields of the carotenoids in Dunaliella salina ,as a function of operating pressure and temperature.

After performing the speci?ed experiments,different models have been investigated to optimize the extraction process.For ver-ifying these models two parameters;regression and p-value,have been applied [40].Models which have regression higher than 95%are acceptable.That means,the difference between experimental and estimated data is less than%5.Another parameter is p-value which has been applied for the selected model and coef?cients to study the signi?cance of each parameter in the equation.p-value less than 0.05indicates that model terms are signi?cant.When a factor has a p-value smaller than 0.05it in?uences in the process in a signi?cant way for a con?dence level of 0.95.If the p-value is greater than 0.05,the possibility of existence of another coef?cients in equation is greater than%95that means the provided param-eter cannot be signi?cant.If there are many insigni?cant model terms (not counting those required to support hierarchy),model reduction may improve your model.To evaluate the authenticity of predictions,different models including ?rst-order and quadratic

S.R.Pour Hosseini et al./J.of Supercritical Fluids121(2017)89–95

91

Fig.2.Schematic diagram of the supercritical CO2extraction apparatus:1.gas cylinder;2.check valve;3.water cooler;https://www.wendangku.net/doc/6c17872369.html,pressed air;5.?lter;6.booster pump;7,8. manometers;9,10.CO2inlet valves;11,12.extractors;13,14.purge valve;15,16.outlet valve;17,18.?ow metering valve;19.sampler;20.rotameter;21.heat exchanger.

Table1

Carotenoids and chlorophylls yields obtained for an extraction time of90min.

Experimental Run Pressure

[bar]

Temperature

[?C]

Yield of carotenoids

[?g/gr dry weight

microalgae]

Yield of chlorophylls

[?g/gr dry weight

microalgae]

Carotenoids/

Chlorophylls

11004513.974 4.334 3.224 24003573.42316.168 4.541 33004557.12110.894 5.244 42005561.608 6.5599.393 540055115.43532.681 3.532 63004095.6719.8399.724 73006073.81916.686 4.424 82003530.291 4.450 6.808 930030101.8359.18311.090 105004582.64117.914 4.613 113005061.16313.234 4.622 Methanol Extraction245.738917.9570.268

were applied to the experimental data gathered in this study[41]. The use of the latter brings the bene?t of being able to investigate the interaction between changing variables.If the adequacy of the used model is questionable,which is the case where p-value takes quantities more than0.05,the use of reduced model or third-order models is recommended[42].

2.5.Methanol extraction

Solvent extraction was used to compare with supercritical extraction method.Solvent extraction was carried out by Soxhlet apparatus with2g of microalgae powder and150ml of methanol at8h.After the extraction,the solvent was collected and stored at 4?C with the exclusion of light under nitrogen gas to refrain the oxidation prior to subsequent analysis.

2.6.Analysis method

The total concentrations of carotenoids and chlorophylls were determined by measuring the absorbance of the samples using UV-1800Spectrophotometer(Shimadzu,Japan).The equation proposed by Wellburn[37]was used for the determination of carotenoid and chlorophyll concentrations in the samples of Dunaliella salina.This equation has more parameters than other equations presented in the literature and allows determining of the chlorophyll b contained in the samples.

The concentration of total carotenoids was calculated using the following equation:

C totalcarotenoids

?g/ml

=1000A470

?1.63C a?104.96C b

221

(1) where A470is the absorbance at470nm,and C a and C b are the concentrations of chlorophyll a and b calculated by:

C a

?g/ml

=16.72A665.2?9.16A652.4(2) C b

?g/ml

=34.09A652.4?15.28A665.2(3) A665.2and A652.4are the absorbance values at665.2nm and 652.4nm,respectively.

3.Result and discussion

The yields of the carotenoid and chlorophyll extractions are shown in Table1.The carotenoid/chlorophyll ratios are also shown. These values were obtained for an extraction time of90min and5g biomass for the different extraction pressure and temperature.

3.1.Analysis of experimental design

Regression and p-value are considered in order to investigate the accuracy of the models provided by software for carotenoids extraction.After studying the different equations,quadratic equa-tions could not cover all the data.In the best condition the quadratic model(reversed)provided regression of90%.Hence cubic equation

92S.R.Pour Hosseini et al./J.of Supercritical Fluids121(2017)89–95

Table2

The analysis of variance of the process for carotenoid extraction with supercritical carbon dioxide.

Term p-value Coef?cient Model0.0051

A-Pressure0.0010?9.63324E-004 B-Temperature0.3288+0.016479

AB0.1695?2.73130E-005 A20.0033+3.87819E-006

B20.2764?2.62476E-004 A2B0.0477?7.14232E-008 AB20.0273+8.12477E-007

was used for carotenoids extraction and can provide regression of 99%.Also“Adjusted R-squared”was determined and compare with quadratic equation.Increase in“Adjusted R-squared”(from85%to 97%)means that adding cubic terms were signi?cant.The results from the analysis of the experimental design including p-value and each parameters coeffcient are summarized in Table2.

As shown in this table for p-value;A(pressure),A2,A2B and AB2 are signi?cant model terms.Conversly,B(temperature),AB and B2 are insigni?cant parameters and illustrated the important in?uence of pressure in this process.

3.2.Effect of pressure

Fig.3shows3D graph and contour plot of pressure,tempera-ture and extraction yield as well as cook’s distance of experimental run.From Fig.3a,b can be observed that increasing pressure at low temperatures initially enhanced the yield of extraction and reduced it over the pressure of300bar.This behavior can be attributed to double effects:by increasing the pressure subsequently the density and the solvating power of the supercritical carbon dioxide increase and its diffusion coef?cient decreases.It can be said that increas-ing in the density has a positive impact on the extraction yield,but the positive impact does not compensate the decrease in the dif-fusivity and?nally the yield of extraction reduced.This procedure continues until temperatures close to45?C.Due to the very high diffusivity of CO2by increasing temperature,the extraction rate reduction will be less than low temperature means that increase in diffusivity at temperatures above45?C has enhanced the extraction yield.Although the diffusivity is reduced by increasing pressure and density,a high percentage of diffusivity reduction compen-sated with solvent density.The research done by some researchers showed the similar phenomenon on mass transfer and diffusivity of supercritical CO2extraction process[38,39].

3.3.Effect of temperature

As shown in Fig.3,two opposite phenomena observed by increasing temperature at constant pressure.By increasing tem-perature at low pressure,yield of the carotenoid extraction initially decreased and then increased.In other words,by increasing tem-perature at constant pressure,the density of carbon dioxide is reduced and increase in diffusivity of the?uid does not compensate this reduction.But at temperature above45?C,increase in diffusiv-ity is the dominant effect and compensates the density reduction.

Conversely,at pressures higher than300bar,increase in tem-perature lead to a sharp increase in extraction yield till45?C and above this temperature the extraction yield decreases with a slightly lower slope.In general the interaction of temperature and pressure before and after the pressure of300bar is in contrast to each other.

In Table3the effects of temperature and pressure on the extrac-tion of carotenoids from microalgae are summarized.As shown the dominant effect of temperature(less than and above45C)and pres-sure(less than and above300bar)on density and diffusivity and their in?uences on extraction yield could give supportive informa-tion for optimum performance of this process.

3.4.Empirical correlation

Empirical correlations were obtained using the experimental data and parameters obtained from RSM design.This correlation relates to the variables that in?uence in the extraction process of carotenoids with supercritical carbon dioxide and illustrate as Eq.

(4)expression:

1

Y

=?0.091111?

9.63324×10?4×P

+(0.016479×T)?

2.73130×10?5×P×T

+

3.87819×10?6×P2

?

2.62476×10?4×T2

?

7.14232×10?8×P2×T

+(8.12477×10?7×P×T2

(4)

where Y is the yield of extracted carotenoids expressed in?g carotenoids per g dry weight of microalga,T is the temperature (?C)and P is the pressure(bar).

Using this relation and software,the optimal condition for extraction of carotenoids was achieved at a pressure of459.76bar and temperature of50.07?C.

In Table4experimental values and the values estimated by the empirical equation are compared.As presented the maximum extraction yield was found at400bar and55C for both exper-imental(115.435?g/g dry microalgae)and theoretical amount (134.41?g/g dry microalgae).The cook’s distance of Fig.3c also shows the accuracy of model.As shown the data of experimental run1is irrelevant since is not affected by pressure and temperature (based on the contour plot of Fig.3b).

https://www.wendangku.net/doc/6c17872369.html,parison between supercritical carbon dioxide extraction and methanol extraction

The maximum extraction yield of carotenoids by SC-CO2 (115.435?g/g dry biomass)was obtained at400bar and55?C is

Table3

Effects of temperature and pressure on the extraction of carotenoids from microalgae(?=negative,+=positive).

Temperatures higher than45?C Temperatures less than45?C

Pressures less than300bar Increasing temperature at constant pressure Dominant effect Density Reduction Difusivity Enhancement

Extraction yield?+

Increasing pressure at constant temperature Dominant effect Density Enhancement Density Enhancement

Extraction yield++

Pressures higher than300bar Increasing temperature at constant pressure Dominant effect Difusivity Enhancement Density Reduction

Extraction yield+?

Increasing pressure at constant temperature Dominant effect Difusivity Reduction Density Enhancement

Extraction yield?+

S.R.Pour Hosseini et al./J.of Supercritical Fluids121(2017)89–9593

Fig.3.Estimated yields of carotenoid extraction with supercritical carbon dioxide using the empirical correlation,(a)3D-surface plot,(b)Contour plot and(c)Cook’s Distance plot.

94S.R.Pour Hosseini et al./J.of Supercritical Fluids121(2017)89–95 Table4

Comparison of experimental and estimated values by the empirical relationship.

Extraction conditions Extraction yield of dunaliella salina

Pressure(bar)Temperature(C)Experimental amount

(?g/g dry microalgae)Estimated amount

from empirical model

(?g/g dry microalgae)

%Yield difference

1004513.97414.140 1.17 2003530.29131.455 3.70 2005561.60847.81022.39 30030101.82580.03721.39 3004095.67180.02516.35 3004557.12179.97128.57 3005061.16380.02523.57 3006073.81980.1287.87 4003573.42354.64125.58 40055115.435134.40911.88 5004582.64189.0717.22

almost half of solvent(methanol)extraction of245.74?g/g dry biomass(Table1).However,the Carot/Chlor ratio that obtained through methanol extraction(0.268)was much lower than super-critical carbon dioxide(11.09).This is not unexpected as methanol is a good solvent for the extraction of polar compounds such as chlorophyll.This suggests that the supercritical extraction process is more selective than the conventional one and should facilitate the separation of the two extracted pigments.

4.Conclusion

In the operating parameter range selected in these experiments the highest carotenoids extraction yield was obtained at pressure of400bar and temperature of55?C.It is not advisable to increase the temperature as thermal degradation of the extracts may occur. Due to the low polarity of carotenoids,supercritical carbon diox-ide is a suitable solvent for the extraction of carotenoids and gives more selective extraction process for such compounds.In the best condition,selectivity of11.09obtained at300bar and30?C.In SC-CO2extraction,the pressure with a minimum amount of p-value is the most effective parameter in this process.

Acknowledgements

This research was partially supported by the Qeshm Sina Microalgae Co.,Iran and authors are thankful to Mr.Khalajbabaei for his valuable support to provide some facilities and materials. References

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————————————————————————————————作者:————————————————————————————————日期: 2

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