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Extraction, purification and concentration of partially saturated canthaxanthin

from Aspergillus carbonarius

D. Krupa a,b,1, E. Nakkeeran a,1, N. Kumaresan c, G. Vijayalakshmi c, R. Subramanian a,*

a Department of Food Engineering, Central Food Technological Research Institute, CSIR, Mysore 570 020, Indiab Department of Biotechnology, PSG College of Technology, Coimbatore 641 004, Indiac Department of Food Microbiology, Central Food Technological Research Institute, CSIR, Mysore 570 020, India

a r t i c l e i n f o

Article history:

Received 30 December 2009

Received in revised form 23 April 2010

Accepted 27 April 2010

Available online 26 May 2010

Keywords:

Aspergillus carbonarius

Partially saturated canthaxanthin

Extraction

Nanofiltration

Purification

a b s t r a c t

A mutant Aspergillus carbonarius produces partially saturated canthaxanthin (PSC; C40H62O2) during sub-

merged fermentation. The pigment was extracted from dried biomass using various organic solvents and

purified using nanofiltration (NF) and nonporous membranes. Particle size had a great influence; PSC

extractability from fines fraction of biomass (75105 lm) was 1.5-fold higher compared to the coarsefraction (850920lm) in ethanol. Amongthe four solvents, hexaneexhibited the highest PSC extractabil-ity of 5.83 mg/g and purity of 32 mg/g. On a relative scale, the extraction performance of hexane, acetone,

methanol and ethanol were in the order 100, 16.1, 7.5 and 5.4. An assessment based on enrichment factor

and permeate flux revealed notable performance with NF-250 membrane in ethanol extract followed by

NF-200 and NF-GKSS membranes in methanol extract. These results suggested the suitability of hexane

for extraction followed by alcohol phase purification and concentration employing NF. Accordingly, a PSC

purity of 206 mg/g was achieved.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

Colours are one of the important aesthetic properties of food

and colouring of foods has been an age old practice. This practice

has increased many folds with the invention of synthetic colou-

rants largely owing to their good stability and colouring ability

(Pattnaik et al., 1997). Though synthetic colours dominate the mar-

ket, even among the permitted synthetic pigments, some of them

may be toxic, carcinogenic or may cause severe damage to vital or-

gans (Duran et al., 2002). This has given rise to a strong interest in

natural colouring alternatives.

Carotenoids are a group of pigments of varying tints from red to

yellow and possess significant biological activities. Irrespective of

their biological functions, selected carotenoids are widely used as

food colourants and additives in feeds (Pandey and Pandey,

2008). Industrial interest is gradually shifting away from the yel-

low carotenoids such as b-carotene and lutein towards more valu-

able orange-red keto-carotenoids owing to the beneficial effects

these compounds exert on various disease conditions (Bhosale

and Bernstein, 2005). Canthaxanthin and astaxanthin are the two

major keto-carotenoids and canthaxanthin is the precursor of asta-

xanthin. Canthaxanthin has been reported to be an effective anti-

oxidant (Palozza and Krinsky, 1992) and has important

applications in nutraceutical, cosmetic, food and feed industries

(Zhu et al., 2009). Canthaxanthin is abundant in marine sources

such as crustaceans, fishes and microalgae, and other biological

sources include fungi and bacteria (Krupa, 2009). However, cantha-

xanthin is currently produced only synthetically on an industrial

scale (Bhosale and Bernstein, 2005), for which at present no cheap

commercially exploitable plant or animal sources exist. Even

though this carotenoid can be synthesized chemically, synthesis

of nature identical enantiopure compound is difficult (Ernst,

2002). In this context, microorganisms have an advantage over

other natural sources, as the fermentation process is highly manip-

ulable to achieve higher growth rate and greater cell density (Das

et al., 2007) without posing any serious production limitations in

terms of space and time. In our earlier studies attempts were made

towards production optimization of partially saturated canthaxan-

thin (PSC; C40H62O2) from Aspergillus carbonarius (Sanjay et al.,

2007) and its characterization as 11, 12, 13, 14, 15, 11 0, 120, 130,

140, 150 decahydro b, b carotene-4,40 dione (Kumaresan et al.,

2008). Canthaxanthin is lipophilic, and so is accumulated in the cell

membrane, lipid bilayer, or in lipid deposits accumulated within

some oleaginous organisms, hence requires purification before

their indented applications.

Pigment extraction from microbial sources is generally carried

out employing organic solvents. A wide range of polar and non-po-

0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2010.04.093

* Corresponding author. Tel.: +91 821 251 3910; fax: +91 821 251 7233.

E-mail address: subbu@cftri.res.in (R. Subramanian).1 These authors contributed equally and are listed in alphabetical order.

Bioresource Technology 101 (2010) 75987604

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lar solvents either individually, sequentially or in combination

have been employed for optimal extraction of carotenoids from al-

gae, fungi and bacteria with and without pretreatment to biomass

(Meckel and Kester, 1980; Hejazi et al., 2002; Park et al., 2007;

Papaioannou et al., 2008). Ultrasonic- and HCl-assisted extractions,

as well as supercritical CO2 extraction have also been attempted for

carotenoids extraction from microbial sources (Gu et al., 2008;

Krichnavaruk et al., 2008; Thana et al., 2008). These studies re-

vealed that the method of extraction and solvent to be employed

depends on the nature of carotenoid as well as biomass produced

by the typical microbial source. In the present study, extraction

of PSC produced byA. carbonarius was carried out using various or-

ganic solvents to improve the extractability as well as purity.

Membrane processing offers several advantages over conven-

tional downstream processing methods such as chromatographic

separations and it has been demonstrated that they are convenient

and easy to scale-up in the purification and concentration of pec-

tinase (Devi and Appu Rao, 1996; Singh et al., 1999). However,

there are only a few attempts towards processing carotenoids ex-

tracts using membranes. Sarrade et al. (1998) employed supercrit-

ical CO2 extraction followed by NF to extract and purifyb-carotene

from a model carrot oil (sunflower oil and b-carotene) and natural

extract of carrot seed. Two types of NF membranes of$1 nm pore

size, namely, inorganic membrane-T (TiO2) and organic mem-

brane-TN (TiO2 coated with an organic layer, Nafion) were used.

The results indicated that the membrane selectivity for b-carotene

could vastly differ depending on the nature of the active surface of

the membrane as well as the individual system. Tsui and Cheryan

(2007) employed ultrafiltration (UF) and NF for the purification

and concentration of xanthophylls in ethanol extracts of corn. UF

eliminated ethanol-soluble proteins (zein) and other large solutes

from the extract. NF membranes with 200300 Da MWCO exhib-

ited almost total rejection of xanthophylls and a 300 Da NF mem-

brane displayed greater solvent stability indicating its suitability

for concentration and solvent recycle in the process. Subramanian

et al. (2001) made attempts to understand the actual rejection

mechanism of carotenoids in vegetable oils by a nonporous mem-brane (NP). In model systems, the membrane rejected lutein by

60% in the absence of added surfactants, whereasb-carotene rejec-

tion was only 18% under similar conditions. The hydroxyl groups

present in lutein make it more polar compared tob-carotene which

explained the higher rejections of lutein in the model system and

oxygenated carotenoids in the crude vegetable oils by the hydro-

phobic membrane. These membranes rejected 96% of chlorophyll

from model oil containing added chlorophyll (Kondal Reddy

et al., 2001).

In the present investigation, the influence of particle size of

ground biomass and type of solvent were studied towards greater

extraction of PSC. Further, various NF and NP membranes were

evaluated for the purification and concentration of PSC in biomass

extracts of A. carbonarius in polar and non-polar solvents used asextractants.

2. Methods

2.1. Chemicals

Peptone, yeast extract and agar-agar were purchased from M/s

Hi-Media, Mumbai, India. Corn flour was procured from the local

market. Potassium dihydrogen phosphate, L-dextrose and oxalic

acid used for the estimation of inorganic phosphates, carbohy-

drates and total acids were of analytical grade and purchased from

M/s Sisco Research Laboratories, Mumbai, India. All the other ana-

lytical and laboratory grade chemicals and solvents were procuredfrom reputed companies in the country.

2.2. Organism

A mutantA. carbonarius, deposited at the culture collection cen-

tre of the Food Microbiology Department, CFTRI under the acces-

sion number UV 10046, was used in this study. The inoculum

was developed in corn flour medium (4% corn flour, 0.5% diammo-

nium hydrogen phosphate and 0.5% ammonium dihydrogen phos-

phate; pH 5.5) for 48 h at 30 C and transferred to the production

medium that had an identical composition as that of inoculum

medium while maintaining pH at 3.0 with 250 mM citratephos-

phate buffer. In order to enhance aeration, baffled Erlenmeyer

flasks were used which were incubated for 48 h at 30 C ona rotary

shaker (200 rpm). The harvested culture broth was filtered through

muslin cloth and the biomass obtained was dried in a lyophilizer

(Lyodryer LT5B, Lyophilsation Systems, Bangkok, Thailand).

2.3. Sieving

The lyophilized biomass was pulverized in a mixer grinder

(Slimline, 0.5 l capacity, 500 W, 18,000 rpm; M/s Chhaya Indus-

tries, Valsad, India) for 2 min. The ground biomass was then sub-

jected to successive sieving starting from large to small sieves

with 920, 850, 710, 350, 250, 180, 150, 105 and 75 lm openings,to get various defined range of size fractions. Five different size

fractions (850920, 350710, 180250, 105150 and 75105 lm)were used in the extraction experiments.

2.4. Determination of percentage extinction coefficient of PSC in

different solvents

2.4.1. Purification of PSC by thin layer chromatography

Pigment from biomass was extracted by mixing 2 g of biomass

with 100 ml absolute ethanol under stirring at 250 rpm for 1 h at

room temperature (28 2 C). The ethanol extract obtained was

concentrated in a flash evaporator at 40 C (Rotavapor R205, Bchi

Labortechnik, Postfach, Switzerland). The concentrate was then

spotted on a preparative thin layer chromatography (TLC) silicaplate (Merck, Darmstadt, Germany) and developed in a chamber

saturated with isooctane:acetone:diethyl ether (3:1:1, v/v/v) at

room temperature. The major yellow spot (PSC) from the TLC plate

was scrapped, eluted into ethanol and centrifuged at 2200g for

5 min to remove silica gel. The PSC obtained was concentrated in

the flash evaporator and subjected to further purification by re-

peated TLC. The purity of the PSC was established by High Perfor-

mance Liquid Chromatography on a C18 Supelco Discovery Column

(isocratic elution using methanol and acetone (95:5, v/v) at 1 ml/

min) (Kumaresan et al., 2008).

2.4.2. Determination of percentage extinction coefficient

A known amount of PSC (9.04 103 lg) was dissolved in a

known volume (1 ml) of specific solvent (methanol, ethanol, ace-tone and hexane). The kmax and corresponding absorbance values

of PSC in the above solvents were determined after suitable dilu-

tion using a UVvis spectrophotometer (UV-160A, Shimadzu Sci-

entific Instruments, Kyoto, Japan). Then the percentage extinction

coefficient of PSC in each of the solvent was determined from the

following relation.

Percentage extinction coefficient 1=g=100 ml=cm

Absorbance at kmax 10

Concentration of PSC mg=ml Path length cm

2.5. Extraction of PSC

PSC extraction from biomass was carried out using methanol,ethanol, acetone and hexane. To 1 g of dried biomass, 20 ml of sol-

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vent was added and stirred at 250 rpm for 1 h at roomtemperature

(2 8 2 C). After stirring, the extract was separated from biomass

by centrifugation at 2200g for 15 min and PSC concentration in

the supernatant was determined spectrophotometrically. The par-

tially extracted biomass (centrifuged solids) was subjected to re-

peated extraction (with 10 ml of respective solvent each time) to

maximize extraction till the concentration of PSC in the extract

(supernatant) reached to610% of the first extract.

2.6. Membranes

The composite NF membranes (MPF-34 and MPF-44) of MWCO

200 and 250 Da were procured from M/s Koch Membrane Systems,

Massachusetts, USA. The MPF-34 (NF-200) is a hydrophilic mem-

brane and not recommended to exposure to organic solvents. The

MPF-44 (NF-250) is also a hydrophilic membrane but claimed to

be resistant to various organic solvents (hexane, acetone and alco-

hols) and water, by the manufacturer. In addition, a laboratory-

made NF membrane (NF-GKSS) developed by GKSS Forschungszen-

trum, Geesthacht, Germany was also used. This membrane too is

hydrophilic in nature and resistant to various organic solvents

(acetone, alcohols and ether) and water. Hydrophobic NP mem-brane (NTGS 2200) with silicon as active layer and polyimide as

support layer (Nitto Denko, Osaka, Japan) is resistant to hexane

and was obtained from the National Food Research Institute, Tsu-

kuba, Japan. The NF and NP membranes were cut into circular discs

(7.5 cm diameter with 32 cm2 effective area) and fitted in the self-

stirred membrane cell in such a way that active surface comes into

contact with the feed material.

2.7. Membrane filtration system

Experiments were conducted at room temperature (28 2 C)

and 2 MPa pressure under nitrogen atmosphere. To minimize con-

centration polarization effect, the contents in the cell were stirredon a magnetic stirrer (800 rpm). The unit was operated in batch

mode by charging the cell with 100 ml of PSC extract. The experi-

mental run was stopped upon achieving the desired volume con-

centration ratio (VCR) of 5. Flux corresponded to the actual

measurement for the experimental run. Pure solvent fluxes were

also measured before processing each of the PSC extract.

Membranes were cleaned with the respective solvents for 1 h after

the experimental run. The near complete recovery of original sol-

vent flux was the criterion followed for membrane reuse after

cleaning.

2.8. Estimation of PSC

The optical density of PSC extracts obtained with methanol, eth-anol, acetone and hexane were measured at their respective kmax(414, 415.5, 412.5 and 410 nm) in the UVvis spectrophotometer

and the PSC concentration was calculated using the respective per-

centage extinction coefficient.

2.9. Estimation of total carbohydrates, inorganic phosphate and total

acids

Total carbohydrates were determined by the phenolsulphuric

acid method using dextrose as standard (Rao and Pattabiraman,

1989). Inorganic phosphate content was determined using potas-

sium dihydrogen phosphate as standard (Ames, 1966). Total acids

were determined as titrable acidity using 0.1 N sodium hydroxide(Ranganna, 2003).

2.10. Performance parameters

The performance of the membrane process was expressed as

percentage observed rejection (Ro) of each component. Ro was

determined, assuming that it was constant for each batch experi-

ment (Cheryan, 1998). Other performance parameters of mem-

brane process (NF and NP) were determined as follows.

Ro % 100 lnCR=CF

lnVF=VR1

Volume concentration ratio VCR VF

VR2

Recovery % CR VRCF VF

100 3

Elimination % CF VF CR VR

CF VF

100 4

where CF, CP and CR are the PSC concentration (lg/ml) or carbohy-drate content (mg/ml) or phosphate salts (mg/ml) or total acids

(N) in feed, permeate and retentate, respectively. VF, VP and VR are

the volume of feed, permeate and retentate (ml), respectively.

All extraction experiments were carried out in triplicate and

membrane runs in duplicate and the mean values are reported.

3. Results and discussion

3.1. Influence of particle size on PSC extractability from biomass

The yield of target compound is very important in any extrac-

tion process, more so in the case of valuable compounds such as

carotenoids because it has a direct bearing on the production eco-

nomics. The various defined range of size fractions of dried bio-

mass were subjected to stepwise extraction with ethanol until

the concentration of PSC in the extract reached to610% of the first

extract. The amount of PSC recovered from these five size fractions

is plotted against various stages of extraction and the volume of

solvent used (Fig. 1). It clearly showed that smaller the particle sizeor in other words greater the surface area, greater was the extract-

ability for a given volume of solvent. These results revealed that

particle size had an inverse influence on PSC recovery. Further, as

the volume of solvent employed increased, the amount of PSC ex-

tracted increased for all the five size fractions used in the study

(Fig. 1). The recovery of PSC was 1.223 mg from 1 g of coarse frac-

tion of dried biomass during the first step of extraction with 20 ml

ethanol which improved to 1.694 mg by 28% after four successive

steps with 50 ml ethanol. The corresponding recoveries with the

fines fraction were 1.861 and 2.261 mg, respectively. The cumula-

tive improvement in PSC extraction during second, third and fourth

steps were 4%, 10% and 6%, respectively. These results revealed that

recovery could be improved by $85% by optimizing the extraction

conditions. The stepwise extraction basically indicated the extentto which the extraction process should be carried out. It also

helped to optimize the volume of solvent required in the extraction

process. Subsequent studies were made only with very fine (75

105lm) and coarse (850920lm) fractions of ground biomassto assess the comparative extraction performance with polar and

non-polar solvents.

3.2. Percentage extinction coefficient of PSC in extraction solvents

The extractability of PSC varies with different solvents. There-

fore percentage extinction coefficient of PSC in methanol, ethanol,

acetone and hexane were determined to quantify the actual

amount of PSC extracted (Table 1). The percentage extinction coef-

ficient and absorbance at kmax at a particular concentration of PSCwere greater in polar solvents compared to the most non-polar sol-

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vent, hexane (Table 1) suggesting that the solubility of PSC was

greater in polar solvents which is rather expected considering

the molecular structure of PSC (Kumaresan et al., 2008).

3.3. Solvent extraction of PSC

Extractability of PSC was studied with fines and coarse fractions

of biomass using polar and non-polar solvents with a view to en-

hance its recovery. Step-wise extractions were carried out with

methanol, acetone and hexane as described earlier with ethanol.

3.3.1. Purity of biomass extracts

The biomass contains the media components such as carbohy-

drates, salts and acids besides the target compound, PSC. Theseimpurities were co-extracted along with PSC during extraction

with polar and non-polar solvents. Membrane lipids would also

potentially be extracted by more non-polar solvents. Extracts of

polar solvents contain higher amounts of hydrophilic impurities

while extracts of non-polar solvent would contain substantial

amounts of lipophilic impurities. Accordingly, relatively larger

amounts of carbohydrates, salts and acids were found in polar sol-

vent extracts owing to their solubility in the order of solvent polar-

ity(Table 2). Comparatively these impurities were present in much

lower amounts in the hexane extract leading to a higher degree of

purity of extracted PSC (32 mg/g of total soluble solids). However,

hexane extract is likely to contain substantial amounts of mem-

brane lipids owing to its hydrophobic nature.

3.3.2. Solvent selection for PSC extraction

Hexane exhibited the highest PSC extractability among the var-

ious solvents studied (Fig. 2). The extractability of hexane was 2.1-

fold higher than ethanol in the first step of extraction with coarse

fraction of biomass. The increase in extractability was higher in

subsequent steps with increased volume of solvent employed;

amount of PSC extracted with hexane was 3.2-fold higher than eth-

anol with coarse fraction after four steps of extraction. The maxi-

mum amount of PSC extracted with hexane was 5.825 mg/g of

fines fraction of biomass while the maximum amount extracted

with the conventionally employed ethanol was only 2.261 mg/g

of biomass. The extraction performance was expressed as a prod-

uct of extractability and extraction purity of PSC. Accordingly the

normalized performance with hexane was 619-fold higher thanpolar solvents (Table 3). The extractability of methanol and ace-

tone fell between ethanol and hexane, methanol being closer to

ethanol (Fig. 2 and Table 3).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

15 20 25 30 35 40 45 50 55

850-920 m

350-710 m

180-250 m

105-150 m

75-105 mAmountofPSC(mg/gof

drybiomass)

Volume of ethanol (ml)

Fig. 1. Amount of PSC extracted in ethanol from various size fractions of ground biomass.

Table 1

Percentage extinction coefficient of PSC in various organic solvents.

Solvent kmax(nm)

Absorbance at

kmax ()

Percentage extinction coefficient

(1/(g/100 ml)/cm)

Methanol 414.0 0.706 781

Ethanol 415.5 0.806 891

Acetone 412.5 0.717 793

Hexane 410.0 0.236 261

Concentration of PSC used for estimation was 9.04 103 lg/ml.

Table 2

Carbohydrate, salt and acid contents in various solvent extracts of biomass from A. carbonarius.

Solvent CHO (g/l) Inorganic phosphates (g/l) Total acids (N)

Fines fraction Coarse fraction Fines fraction Coarse fraction Fines fraction Coarse fraction

Methanol 0.485 0.044 0.389 0.069 0.591 0.018 0.551 0.093 0.0664 0.0003 0.0566 0.0003

Ethanol 0.367 0.036 0.315 0.042 0.094 0.006 0.063 0.007 0.0326 0.0003 0.0296 0.0003

Acetone 0.412 0.134 0.417 0.047 0.048 0.013 0.044 0.017 0.0096 0.0003 0.0088 0.0003

Hexane 0.033 0.008 0.020 0.014 0.002 0.000 0.003 0.002 0.0025 0.0000 0.0017 0.0000

All the experiments were carried out in triplicate (n = 3) and samples were analyzed in triplicate (n = 3). 1:50 extract (1 g biomass and 50 ml solvent); CHO: carbohydrates;fines fraction: 75105 lm; coarse fraction: 850920 lm.

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The chemical formula of a specific carotenoid influences its rel-

ative polarity. For instance lutein containing two hydroxyl groups

is more polar than b-carotene while PSC with two keto (carbonyl)

groups in its structure is less polar than lutein but more polar than

b-carotene. Extractability depends on the solvating power and abil-

ity of the solvent to permeabilize the cell wall (Leshchev and Sin-

kevich, 2003; Park et al., 2007). The maximum extractability of PSC

was obtained with hexane which was nearly 2.22.6-fold highercompared to the polar solvents used in the study. The solubility

of PSC is expected to be greater in polar solvents compared to hex-

ane as reflected in the differences in percentage extinction coeffi-

cients (Table 1). Therefore, higher extractability in hexane could

be attributed to the greater permeabilizing ability of hexane in dis-

rupting the cell walls and membrane (lipid) layers to effectively re-

lease the PSC from the biomass. Interestingly, there was no

significant difference between the extractability of PSC with fines

and coarse fractions of biomass with hexane, suggesting elimina-

tion of size reduction step in the process (Fig. 2). Although fineness

of particle generally increases the extractability it could lead to

other operational problems such as fouling and flooding of the

extractor. Also, size reduction greatly increases the amount of en-

ergy used for the process. These results suggest that the penetrat-ing power of hexane played a more important role during the

extraction of PSC than the particle size and surface area as well

as the matching polarity of solvent. The purity of PSC obtainedwith

hexane extract was 32 mg/g of soluble solids and attempts were

made to improve the purity further by employing membrane

processing.

3.4. Membrane processing of biomass extracts

Membrane processing of various solvent extracts of biomass

was attempted with three different hydrophilic NF membranes

(NF-200, NF-250 and NF-GKSS) and a hydrophobic NP membrane(NTGS 2200) to improve the purity of PSC while simultaneously

aiming for higher recovery. In addition, membranes were chosen

to aid in concentrating the target compound in the product stream

(retentate). Solvent compatibility of these membranes was tested

before the experimental runs. The laboratory-made NF-GKSS

membrane was claimed to be resistant to various organic solvents

(acetone, alcohols and ether) (Zwijnenberg et al., 1999) and was

actually resistant to all the solvents including hexane used in the

study. The NF-250 membrane is claimed to be resistant to various

organic solvents by the manufacturer but was found to be not sta-

ble with hexane, as was also reported by other researchers (Van

der Bruggen et al., 2002). The NF-200 membrane is not recom-

mended to exposure to organic solvents but remained stable with

methanol and ethanol. The NTGS 2200 membrane was stable onlywith hexane.

3.4.1. Selectivity for PSC

The selectivity of NF and NP membranes was assessed for PSC

and other known impurities in four different solvent phases and

the results are presented in Table 4. NF-GKSS membrane exhibited

the highest rejection of 89% and recovery of 84% PSC in methanol

extract among the various combinations of membrane and extract

studied. This membrane gave the highest rejection of PSC in meth-

anol, acetone and hexane extracts compared to other membranes

while NF-250 membrane gave the highest rejection with ethanol

extract. The experimental runs with three different NF membranes

with ethanol and methanol extracts revealed their contrasting

behaviour in terms of PSC rejection. Bhanushali et al. (2002) re-ported the solute permeation characteristics of Sudan IV (384 Da

0

1

2

3

4

5

6

7

10 20 30 40 50 60 70

Methanol -CF

Methanol -FF

Ethanol -CF

Ethanol -FF

Acetone -CF

Acetone -FF

Hexane -CFHexane -FF

AmountofPSC(mg/gofd

rybiomass)

Volume of solvent (ml)

Fig. 2. Amount of PSC extracted from coarse and fines fractions of biomass in various organic solvents. CF coarse fraction (850920lm); FF fines fraction (75-105 lm).

Table 3

Extraction performance of solvents for PSC from biomass (fines fraction) of A.

carbonarius.

Solvent Extractability

(mg/g)

Extraction purity

(mg/g)

Normalized extraction

performance ()

Methanol 2.605 0.095 5.28 0.15 7.5

Ethanol 2.261 0.084 4.29 0.13 5.4

Acetone 3.170 0.096 9.59 0.35 16.1

Hexane 5.825 0.099 31.96 0.40 100

Extraction purity= Ratio of PSC concentration to total soluble solids.

Normalized extraction performance ExtractabilityExtraction purityany solventExtractabilityMaxExtraction purityMax

100:

All theexperiments were carried outin triplicate (n = 3) andsamples were analyzed

in triplicate (n = 3). Extractability= Amount of PSC extracted (mg of PSC from 1 g of

dried biomass) with 50 ml of solvent.

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organic dye) in polar and non-polar solvent medium using hydro-

phobic (PDMS) and hydrophilic (aromatic polyamide) NF mem-

branes. The rejection of Sudan IV in n-hexane medium was $25%

while in methanol it was $10% with the hydrophobic membrane.

The direction of separation reversed with the hydrophilic mem-

brane (86% in methanol and 43% in n-hexane). Factors such as sol-

ute-membrane interactions and solute charge/conformation could

play a role in the actual rejection behaviour of specific solute in

various solvent systems (varying in polarity) by different mem-

branes (hydrophobic and hydrophilic).

3.4.2. Removal of impurities

The membrane selectivity towards PSC with respect to removal

of impurities such as carbohydrates, salts and acids during mem-

brane processing of various solvent extracts are shown in Table 4.

In the case of hexane extract, carbohydrate and phosphate salt con-

tents were below detectable level suggesting very high membrane

selectivity; but actual values could not be determined. Among the

three polar solvent extracts, the highest selectivity towards re-

moval of carbohydrates was exhibited by NF-250 membrane with

both ethanol and methanol extracts. A very high selectivity for theremoval of phosphate salts was observed only with the NF-250

membrane, but only with the ethanol extract (Table 4). The estima-

tion of acids includes citric acid (from the media) and other organic

acids, as well as hydrolyzed fatty acids of membrane lipids. There-

fore, their solubility differed to varying degrees in different sol-

vents. The acids present in the hexane extract could be mainly

the free fatty acids that were extracted due to their polarity (Ta-

ble 2). NF-GKSS membrane exhibited an excellent selectivity to-

wards removal of acids during processing acetone extract

(Table 4). Bhosle et al. (2005) and Zwijnenberg et al. (1999) re-

ported good selectivity for deacidification by NF-GKSS membrane

while processing model systems containing triglycerides, FFA and

acetone. All the other combinations were virtually ineffective to-

wards removal of acids. The selectivity of NF process employing

an organic solvent is rather complicated with several factors such

as solubility and diffusivity of permeating components and their

solubility in solvent and membrane material playing vital role.

3.4.3. Performance assessment in solvent phase

The purity as well as recovery of the target compound and the

process productivity determines the selection of a process step

during downstream processing. These selection parameters havea direct bearing on the process economics. Membrane processing

with various extracts exhibited that the PSC enrichment was high-

est with NF-GKSS membrane in methanol extract (4.2-fold), fol-

lowed by the same membrane in acetone extract (3.2-fold), NF-

200 membrane in methanol extract and NF-250 membrane in eth-

anol extract (Table 5). Though the purity was higher in hexane and

acetone extracts, the enrichment factor or flux was much lower.

However, the membrane performance, taking into account both

membrane productivity and enrichment factor, demonstrated that

only NF-250 membrane in ethanol extract, NF-200 membrane in

methanol extract and NF-GKSS membrane in methanol extract

could be considered (Table 5) for further evaluation. Taking this

into consideration along with the superior extraction performance

of hexane, extraction with hexane followed by reconstitution in a

polar solvent before membrane processing was examined to im-

prove the overall performance.

3.5. Overall performance of extraction and membrane purification

Hexane extract of PSC was desolventized and reconstituted in

methanol and ethanol for further processing with NF membranes.

The overall extraction and purification performance in terms of

enrichment factor, process flux and purity for the three select pro-

cess combinations are presented in Table 6. All the three combina-

tions exhibited greater improvement in purity (Table 6) compared

to processing in a single solvent system (extraction and purifica-

tion in the same solvent phase) (Table 5). NF-200 membrane

exhibited the highest purity of 206 mg/g of total soluble solids fol-

lowed by NF-GKSS membrane (167 mg/g of total soluble solids) in

Table 4

Rejection of PSC and impurities during membrane processing of A. carbonarius biomass extractsa.

Solvent

Membrane

Methanol Ethanol Acetone Hexane

Ro (%) Selectivity Ro (%) Selectivity Ro (%) Selectivity Ro (%) Selectivity

CHOb Saltsc Acidsd CHOb Saltsc Acidsd CHOb Saltsc Acidsd CHOb Saltsc Acidsd

NF-200 64.9 1.15 1.29 1.36 8.5 0.49 1.10 0.59 NS NS NS NS NS NS NS NS

NF-250 45.3 2.44 1.21 1.01 60.3 2.46 3.54 1.26 32.1 1.09 0.66 0.53 NS NS NS NS

NF-GKSS 89.0 1.03 1.06 0.97 19.7 0.59 0.55 0.69 61.8 1.31 0.87 16.20 42.7 ND ND 0.40NP-NTGS 2200 NS NS NS NS NS NS NS NS NS NS NS NS 22.1 ND ND 0.79

a Ro Rejectionof PSC; NS notstable; ND notdetermined;volume concentration ratio (VCR):five-fold;valuesare expressedas mean of duplicate(n = 2) measurements.b Membrane selectivity of PSC with respect to CHO.c Membrane selectivity of PSC with respect to phosphate salts.d Membrane selectivity of PSC with respect to total acids.

Table 5

Membrane performance during processing biomass extracts.

Solvent

Membrane

Methanol Ethanol Acetone Hexane

EF

()

Flux

(l/m2h)

MP

()

Purity

(mg/g)

EF

()

Flux

(l/m2h)

MP

()

Purity

(mg/g)

EF

()

Flux

(l/m2h)

MP

()

Purity

(mg/g)

EF

()

Flux

(l/m2h)

MP

()

Purity

(mg/g)

NF-200 2.8 33.3 94.6 3.88 1.2 20.0 23.0 4.08 NS NS NS NS NS NS NS NS

NF-250 2.1 35.4 73.3 6.38 2.6 38.5 101.6 11.27 1.7 52.8 88.7 22.88 NS NS NS NS

NF-GKSS 4.2 20.0 83.8 12.11 1.4 50.0 68.5 4.98 3.2 5.0 16.2 38.12 2.0 1.0 2.0 46.84

NP-NTGS 2200 NS NS NS NS NS NS NS NS NS NS NS NS 1.4 50.0 71.5 16.88

MP membrane performance (notional) = EF Flux.

Purity Ratio of PSC concentration to total soluble solids (retentate).

NS not stable; VCR: five-fold.Values are expressed as mean of duplicate (n = 2) measurements. EF enrichment factor = Ratio of PSC concentration in retentate to feed.

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methanol extract. These results revealed that lipophilic impurities

(lipids) co-extracted along with PSC in hexane are eliminated to a

greater extent during membrane processing in a polar solvent

phase. The overall performance expressed as a product of enrich-

ment factor, flux and purity of PSC was also higher with NF-200

membrane (86.5) followed by NF-GKSS membrane (40.2) in meth-

anol extract.

4. Conclusions

Extraction with hexane exhibited much higher recovery of PSC

compared to the three more polar solvents probably owing to its

permeabilizing ability, virtually eliminating the necessity for size

reduction. The results revealed the suitability of a non-polar sol-

vent (hexane) for extraction followed by simultaneous purification

and concentration employing a suitable NF membrane in a polar

solvent (methanol) for obtaining greater purity and recovery of

PSC from A. carbonarius. Considering the potential application of

the proposed approach, it is desirable to conduct scale-up studies

and evaluate its performance as a primary purification process

for high purity products.

Acknowledgements

E. Nakkeeran and N. Kumaresan thank CSIR, New Delhi, India,

for the award of fellowship. H. Nabetani at NFRI, Japan provided

the nonporous membrane. K. Ebert at GKSS Forschungszentrum,

Germany provided the laboratory-made NF membrane. S. Umesh

Kumar, S. Divakar and K. Anbarasu at CFTRI and J. Jayapriya, T.K.

Raja and V. Ramamurthy at PSG College of Technology provided

valuable advice.

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Table 6

Membrane processing of reconstituted PSC extract (hexane) in polar solvents.

Solvent Membrane EF () Flux (l/m2h) Purity (mg/g) Overall performance ()

Methanol NF-200 4.4 33.3 206.0 86.5

Methanol NF-GKSS 4.2 20.0 167.0 40.2

Ethanol NF-250 2.6 38.5 51.0 14.6

Overall performance EFFluxPurityany solventEFmaxFluxmaxPuritymax

100.

Values are expressed as mean of duplicate (n = 2) measurements. For abbreviations see Table 5; VCR: five-fold.

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