evaluation of an alternative source of dextran as a phase forming polymer for aqueous two-phase...

Biochemical Engineering Journal 21 (2004) 241–252

Evaluation of an alternative source of dextran as a phase formingpolymer for aqueous two-phase extractive system

Sanjoy Ghosha,1, R. Vijayalakshmib, T. Swaminathana,∗a Department of Chemical Engineering, Indian Institute of Technology, Madras, Chennai 600036, India

b Department of Chemical Engineering, A.C. College of Technology, Anna University, Chennai 600025, India

Received 26 March 2004; received in revised form 21 July 2004; accepted 21 July 2004

Abstract

Aqueous two-phase system has already been established to be powerful tool for biomolecule purification, and is increasingly gainingimportance as an alternative to conventional extractive fermentation methods. High cost of dextran is the main inhibition in such effortand hence in this paper a much cheaper source of dextran is evaluated as successful phase forming polymer and compared with standardmicrocrystalline dextran T500. Other polymer used for both the cases was polyethylene glycol (PEG). An exhaustive study has been carried toc nd partitionp ntent andd tions. Ther antageously,b©

K

1

ptoboTtsgjc

t

6

fer-tran.ion

sibil-siveextranrscts,d ashasemadelf. Aex-

rce ofue-thatgramtranf the

1d

ompare all its properties such as phase composition properties, polymer properties, gel-forming properties, rheological property aroperty. Various fundamental properties of polymer such as critical temperature, specific rotation, intrinsic viscosity, glucose cory weight were estimated for this new dextran source. Finally, cost analysis was carried out and tallied for both the dextran fracesults of these studies show that significant cost reduction can be achieved without much effect on the process, rather more advy replacing high cost microcrystalline dextran T500 with relatively cheaper new dextran T40 source.2004 Elsevier B.V. All rights reserved.

eywords:Aqueous two-phase; Dextran; Liquid–liquid extraction; Downstream processing; Polymer; Fermentation

. Introduction

Extractive fermentation using aqueous two-phase systemsrovide a promising alternative to conventional fermenta-

ion processes for simultaneous production and purificationf biomolecules[1–7]. This not only helps to obtain theiomolecule in a cell free system, but also increases the ratef product formation and minimizes product inhibition[8].hough, aqueous two-phases can be obtained by using any

wo mutually incompatible hydrophilic polymers[9], the re-earchers got maximum success using dextran/polyethylenelycol (PEG)/water two-phase system[10]. However, the ma-

or disadvantage of using such system seems to be the highost of highly purified fractionated dextran[11]. Maximum

∗ Corresponding author. Tel.: +91 44 2257 8222; fax: +91 44 2257 0509.E-mail addresses:[email protected] (S. Ghosh),

[email protected] (T. Swaminathan).1 Present address: Centre for Biotechnology, Anna University, Chennai00 025, India. Tel.: +91 44 2235 0772; Fax: +91 44 2235 0299.

literature data for purification, isolation and extractivementation are available with such kind of expensive dexIf this method is to be extended for industrial exploitata more economic substitute must be found[11]. In searchof alternatives, scientists have tried to explore the posity of substituting this expensive dextran with less expenpolymers. Some researchers have tried to use crude dor hydrolyzed dextran[11]. Unfortunately, all such polymeor different fractions of dextran fail in one or other aspeand highly purified fractionated dextran always remainepivotal element of all success as far as aqueous two-psystems are concerned. In this study, efforts have beento replace this high cost dextran instead the polymer itsemedical product that is generally used as blood volumetender (plasma substitute) was used as alternative soudextran. This is available in solution form (10%, w/v aqous solution in normal saline). This source of dextranwas used here is much cheaper, costing only Rs. 6 percompared to Rs. 50.00 per gram of microcrystalline dexT500 and hence, a significant decrease in the cost o

369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
oi:10.1016/j.bej.2004.07.005

242 S. Ghosh et al. / Biochemical Engineering Journal 21 (2004) 241–252

Nomenclature

List of symbolsA2 second virial coefficient (m3 mol kg−2)C concentration of polymer in w/w basis

(kg kg−1)Cb solute concentration in the bottom phase

(kg m−3)Ct solute concentration in the top phase (kg m−3)kb boltzmann’s constant (J mol−1)k′ coefficient of Huggin’s viscosity constantK partition coefficient of the solutelog abbreviation for logarithm to the base 10m mass of the samples drawn from the phases

(kg m−3)M molecular weight of the solute in Bronstedt

equation (Dalton)Mn number average molecular weight (Dalton)Mw weight average molecular weight (Dalton)New dextran dextran sample with average molecular

weight 40000 DaltonPEG abbreviation of polyethylene glycolR universal gas constant (J mol−1 K−1)Rs. Indian currency, full form rupeesStandard dextran dextran sample with average molec-

ular weight 500000 DaltonT absolute temperature (K)v volume of the phase sample (m3)

Greek symbolsα optical rotation of dextran sampleη viscosity of the sample (cp)ηb viscosity of the bottom phase of the phase sys-

tem (cp)ηin intrinsic viscosity (cp)η0 viscosity of water (cp)ηsp specific viscosityηt viscosity of the top phase of the phase system

(cp)λ correlation factor with respect to interactions

between solute and environment given in Bron-stedt equation

π osmotic pressure of the polymer sample (atm)ρ density of the phase sample (kg m−3)ρb density of the bottom phase (kg m−3)ρt density of the top phase (kg m−3)�ρ difference in density of the bottom phase and

top phase (kg m−3)

system was expected. In this piece of work, efforts have beenmade to study all aspects that could influence the prospectof exploiting this new source of dextran to replace the well-established fractionated microcrystalline dextran T500. Spe-

cial attention was given on the partition coefficients that gen-erally decrease upon decrease in dextran molecular weight[12]. Enough focus has been given to study the viscosityand rheological properties in comparison to dextran T500fraction, as there is tendency to increase the viscosity withtime where low molecular weight dextran shows faster ten-dency to increase the viscosity with time[11]. This prop-erty was studied keeping in view of exploring the prospectof this source of dextran for continuous extractive fermen-tation where the process is to continue for several hourswithout interruption. Finally, some calculations have beenmade to show the cost effectiveness in comparison to conven-tional dextran T500. Work has been on to exploit this dex-tran most efficiently for continuous extractive fermentation of2,3-butanediol using the organismKlebsiella oxytocaNRRLB199.

2. Materials and methods

2.1. The chemicals used

The polymers used were dextran T40, dextran T500 andPEG 6000. Dextran T40 (10%, w/v aqueous solution in nor-mal saline, available in 500 ml bottle) is available under thet careL 2,3-b (St.L archL alsw , In-d sub-s hurF sti-t Dr.V

2

wingw caseo 500)a auseo uredb y in-v thena d at3 haseb f thef anal-y dex.T theo latesc of as n andi en

rade name of Plasmex, manufactured by Core Healthtd., Gujrat, India. Dextran T500 and analytical gradeutanediol were obtained from Sigma Chemicals Co.ouis, MO, USA). PEG 6000 a product of Sisco Reseaboratory Pvt. Ltd., Bombay, India. All other chemicere procured from S.D.Fine-Chem Ltd., Boisar 401501ia. The enzyme chitinase (Sigma Chemicals Co.) and itstrate chitin (Sigma Chemicals Co.) were a gift by Dr. Artelse of Biochemical Engineering Laboratory of this In

ute. Greseofulvin (Sigma Chemicals Co.) was a gift from. Venkata Dasu.

.2. Binodal curve and phase compositions

Several phase systems were prepared in the folloay. Calculated amounts of PEG, plasmex solution (inf dextran 40000) or dextran (in the case of dextran Tnd water were weighed into a separating funnel. Becf their high viscosity, the polymer solutions are measy weight instead of volume. The contents was mixed berting the funnel for several times. The funnels werellowed to settle for 24 h. Temperature was maintaine0◦C through out. The samples were collected from top py pipette and from bottom phase through the outlet o

unnel. The dextran and PEG content of samples weresed by measuring both optical rotation and refractive inhe dextran concentration was obtained directly fromptical rotation value (using a standard curve that correoncentration and optical rotation). The refractive indexolution depends on both dextran and PEG concentratios known to be additive[12]. The PEG concentration was th

S. Ghosh et al. / Biochemical Engineering Journal 21 (2004) 241–252 243

calculated by using standard curve of dextran and PEG thatcorrelates their concentration and refractive index values. Adetail method is described by Albertsson[12]. This top andbottom phase compositions (%w/w dextran/%w/w PEG) arenecessarily be the points on the phase separation curve, andhence, these are plotted on a graph to obtain a smooth best-fitted curve which is known as binodal curve. The tie-linesare the straight line joining the corresponding total, top andbottom phase composition.

2.3. Measurement of optical rotation

The optical rotation of the phase samples were measuredusing a Digital Polarimeter (Jasco DIP-370, Japan Spec-troscopy Company Ltd.) using a cell length of 10 cm. Thetemperature was maintained constant at 25◦C (unless other-wise stated).

2.4. Measurement of refractive index

The refractive index of the phase sample was estimatedby HPLC (CT0-10A) method using RID-6A detector (Shi-madzu, Japan). A liquid flow rate of 1.0 ml min−1 was chosenand 5�l of samples was injected. Temperature maintainedconstant at 30◦C.

2

itived er-l usingE

ρ

w sa

2

one( V-I A0 keptc as0

2

forb oticp t 090G theu asur-i nt at3

2.8. Determination of polymer properties

Dextran T500 sample was dried at 50◦C in a desiccator for24 h in presence of anhydrous CaCl2 for dry mass estimation.The glucose content was measured by DNS method[13]. Thedextran T40 was available in solution form and so, dry masswas not estimated but, the glucose content was measured bythe same method. The intrinsic viscosity (ηin) was measuredusing the followingEq. (2) [14]:

ηsp

C= ηin + k′η2

inC (2)

whereηsp is the specific viscosity defined by theEq. (3)andk′ is Huggin’s constant:

ηsp = η − η0

η0(3)

η is the viscosity of the polymer solution at the given polymerconcentrations (C). η0 is the viscosity of water at the giventemperature. Viscosity (η) was measured by the method de-scribed earlier. Whereas,η0 value was taken from the standardchart[15]. The weight average molecular weight (MW) wasdetermined by theEq. (4) [16]using the intrinsic viscosityvalue:

ηin = 2.43× 10−3M0.42w (4)

T du

w nt,Tiw tions( theo cularw

P panyL5

2 rw

lesw mem theni idity[

2

rentd from

.5. Density measurement

A fixed volume samples were weighed in a highly sensigital balance, Mettler AE240 (Mettler Instrument, Switz

and), and then the density of the sample was calculatedq. (1):

= m

v(1)

hereρ is the density of the sample andmandvare the masnd volume of the sample, respectively.

.6. Viscosity measurement

All viscosity measurements were carried out using cCP-40) and plate Brook field Digital viscometer, Model DI (Brookfield Engineering Laboratory Inc., Stoughton, M2072, USA). During measurement the temperature wasonstant at room temperature (30◦C) and sample amount w.5 g in all measurements.

.7. Osmotic pressure measurement

Solutions of five different concentrations (1–5%)oth the dextran fractions were prepared and their osmressures were measured using osmometer (OsmomaONOTEC GmbH, Berlin). Two ml sample was placed inpper chamber keeping 0.5 ml water (solvent) in the me

ng chamber. The temperature was maintained consta0◦C.

,

he number average molecular weight (Mn) was estimatesing the followingEq. (5) [17]:

π

RTC= 1

Mn+ A2C (5)

hereπ is the osmotic pressure,R is universal gas constais absolute temperature,C is polymer concentration andA2

s second virial constant. A straight line ofπ/RTCversusCas plotted in both the cases for five different concentra

1–5%) of dextran and the inverse of the intercept fromrdinate was calculated for getting number average moleeight of the respective dextran fractions.The optical rotation (α) was measured at 27◦C in a Digital

olarimeter (Jasco DIP-370, Japan Spectroscopy Comtd.) using a cell length of 10 cm (C = 1, 0.01 g ml−1) at78 nm wave length.

.9. Measurement of critical temperature and moleculaeight determination

Five percent (w/v) solution of different dextran sampere mixed in water–methanol mixture (40.5% by voluethanol) at a very low temperature. Temperature was

ncreased slowly. The temperature at the onset of turb18] was noted. This is known as critical temperature.

.10. Gel-forming properties

Two aqueous two-phase systems from the two diffeextran (dextranT500 and dextranT40) sources, one


each, were prepared. The aqueous two-systems that had cho-sen to compare gel-forming properties were 6.5% (w/w)dextran T500/7.0% (w/w) PEG 6000/water and 7.0% (w/w)dextranT40/7.5% (w/w) PEG 6000/water. The bottom phaseswere separated and analysed by the viscometer as statedabove. Unless otherwise mentioned in the specific cases thepH and temperature was maintained at 7.0 (0.1 M phosphatebuffer) and 30◦C.

2.11. Rheological properties

To study the rheological property 8% (w/v) volume sam-ple for both dextran sources were made and in dextran T500sample 0.9% (w/v) of sodium chloride was added to com-pensate sodium chloride already present in the other dextransource. The solution temperature was maintained at 30◦C.

2.12. Partition coefficient measurement

2.12.1. 2,3-ButanediolA pure (analytical grade) 2,3-butanediol was added (10 mg

per gram of total phase system) while making the aqueoustwo-phase preparation. The mixture was then mixed well andallowed to settle. The pH and temperature of the phase systemwas set at 7.0 (0.1 M phosphate buffer) and 30◦C, respec-t ses.T ed byg n En-g s thec opak∼ iento dingt ttomp

2os-

p ousp asr r[ n re-s .75).

ded( ak-i wella hases 0r thep e wase

llenc pH4 5 mlo 7f i-n gent

to the reaction mixture. A control was taken where the en-zyme was deactivated by adding 1 ml of potassium sodium(+)-tartrate reagent before the commencement of incubation(as perSection 3.4). One milliliter of dinitrosalicylic acidreagent was added to the reaction mixture and the same waskept in a boiling water bath for 5 min. The reaction mixturewas then cooled to 30◦C. Seven milliliter of distilled wa-ter was added to each tube to make up the final volume to10 ml. The mixture was centrifuged at 5000 rpm for 5 minand the intensity of color (formed due to the reaction ofN-acetyl-d-glucosamine released with dinitrosalicylic acid) wasmeasured at 540 nm on a spectrophotometer (UV–vis spec-trophotometer, UV-1601PC, Shimadzu, Japan). The partitioncoefficient of chitinase was then obtained by dividing thespectrophotometer reading (optical density) of the sampledrawn from top phase by that of the sample drawn from cor-responding bottom phase.

2.12.3. GriseofulvinA pure (analytical grade) griseofulvin was added (0.4 mg

per gram of total phase system) while making the phasepreparation. The mixture was then mixed well and allowedto settle. The pH and temperature of the phase system was setat 7.0 (0.1 M phosphate buffer) and 30◦C, respectively. Thesamples were then drawn from both the phases. The amounto d fol-l

rredta stop-p sion.4 min,a eredt wasa d at2 thea red at2 ciento ho-t romt dingb

3

3

in-c rtainc Thea tion inb fer-m mer,i eouss ting

ively. The samples were then drawn from both the phahe 2,3-butanediol content in each phase was estimatas chromatography (Nucon Gas Chromatograph, Nucoineers, New Delhi) method. Nitrogen gas was used aarrier gas. The flame ionisation detector (FID) and Por

q column was used for analysis. The partition coefficf 2,3-butanediol for each system was calculated by divi

he peak area (in mV s) of the top phase to that of the bohase.

.12.2. Chitinase enzymeOne gram of chitin was added to 10 ml of 85% orthoph

horic acid. The mixture was stirred to make a gelatinaste and was stored at 0◦C for 24 h. Gelatinous mixture weprecipitated into an excess of cold (15◦C) distilled wate19]. It was made to paste using pestle mortar and theuspended in 200 ml of Na-acetate buffer (50 mM, pH 4

A pure (analytical grade) chitinase enzyme was ad100 U of enzyme per gram of total phase system) while mng the phase preparation. The mixture was then mixednd allowed to settle. The pH and temperature of the pystem was set at 7.0 (0.1 M phosphate buffer) and 3◦C,espectively. The samples were then drawn from bothhases. The content of chitinase enzyme in each phasstimated following the method described below.

A tube containing reaction mixture 0.55 ml of acid swohitin (5 g l−1), suspended in 50 mM Na-acetate buffer,.75), 0.30 ml of acetate buffer (50 mM, pH 4.75) and 0.1f phase (top or bottom) solution was incubated at 4◦C

or 1 h in unstirred condition[20]. The reaction was termated by adding 1 ml of potassium sodium (+)-tartrate rea

f griseofulvin present in each phase was then assayeowing Holbrook method[21].

0.25 g of phase (top or bottom) solution was transfeo a stoppered test tube. To this 0.2 g of anhydrous Na2SO4nd 0.5 ml of methanol was added. The test tubes wereered and shaken vigorously to obtain an even disper.5 ml of ethyl acetate was then added, shaken for 2nd filtered. 0.25 ml of filtrate was transferred to stopp

est tube. To this 0.25 ml of 2N methane sulphonic aciddded and mixed thoroughly. The mixture was incubate0◦C for 30 min. 4.5 ml of methanol was then added andbsorbance against the appropriate blank was measu66 nm using a spectrophotometer. The partition coeffif griseofulvin was then obtained by dividing the spectrop

ometer reading (optical density) of the sample drawn fop phase by that of the sample drawn from corresponottom phase.

. Results and discussion

.1. Binodal curve and phase composition property

Dextran and PEG are the two well-known hydrophilicompatible polymers. Their aqueous mixture above ceritical concentration results in two-phase formation.queous two-phase systems enjoy tremendous applicaiotechnology for purification, isolation and extractiveentation processes. Dextran, a natural hydrophilic poly

s having a very long and coiled structure and its aquolution in crude form becomes very viscous precipita


Table 1Comparison of phase compositions of both the aqueous two-phase systems for different dextran (new and standard) sources

Sample source Total composition (%w/w) Top phase composition (%w/w) Bottom phase composition (%w/w)

Dextran PEG Water Dextran PEG Water Dextran PEG Water

New dextran 7.0 7.5 85.5 2.06 10.68 87.26 15.94 2.68 81.38Standard dextran 6.5 7.0 86.5 0.60 9.26 90.14 15.30 3.94 80.76

Fig. 1. Comparison of binodal curves of two dextran sources (new dextranT40 and standard dextran T500) using PEG 6000 as the other polymer forphase preparation Conditions Temperature 30◦C, pH 7.0, 0.1 M phosphatebuffer.

severe mass transfer problem in the extractive fermentationprocesses. On the other hand, microcrystalline dextran T500(standard dextran) is very expensive though it enjoys manysuccesses in the scientific study. A new source of dextran(dextran T40, commercial name plasmex, will be called asnew dextran) is thus tried in order to over come this problem.

The binodal curves for phase separation for these two dex-tran sources, standard dextran and new dextran, were firstconstructed separately using PEG 6000 as the other polymerfor both the cases. Two-phase systems can be characterized bits unique phase diagram (binodal curve), which contains theequilibrium phase compositions for the system. To facilitatecomparison, binodal curve using new dextran was over laidon the phase diagrams using standard dextran (Fig. 1). Fromthe diagrams this is very clear that the standard dextran/PEG6000/water system requires lesser phase composition forphase separation compared to new dextran/PEG/water sys-

Table 2Comparison of physico-chemical parameters of both the aqueous two-phase

Samples source Dextran (%w/w) PEG (%w/w) Volume ratio (Vt/Vb)

New dextran 7.0 7.5 1.7 7Standard dextran 6.5 7.0 1.5 .2

tem. Alternatively, the critical phase composition in case ofphase equilibrium curve is lesser with standard dextran thenthat with new dextran T40. The binodal curve using new dex-tran is much elevated compared to the other binodal curvesuggesting the presence of shorter molecules (less averagemolecular weight)[9] in this source of dextran comparedto standard dextran. This observation confirms the manufac-turer’s claim. Albertsson[8] had studied such systems veryextensively and the results are published in his book. Similarbehavior, including him, was also observed by all workersin this area[22–24]. The Binodal curve separates one phasearea from two-phase area. Any phase composition below thiscurve leads to single phase whereas two-phase systems can behad choosing any mixture composition above this line. Suchmixture composition separates into two phases on settling.The top phase is rich with PEG while the bottom with dex-tran. This top and bottom phase compositions necessarily arethe two points on the equilibrium curve and the straight linejoining them which passes through the mixture compositionpoint is known as tie-line and the length (%w/w) betweentop and bottom phase compositions for a given total mixturecomposition is known as tie-line length[8].

Few phase systems were tried with both the polymers toidentify two equivalent phase systems, one from each system,which were comparable. The phase compositions, top andb -a withint and1 , re-s 1.5,r latedt vol-u therpr otht ever,t bot-t wasm quiteo withh ma-t ly by

y

systems for different dextran (new and standard) sources

Tie-line length (%w/w) ρt/ρb �ρ (kg m−3) ηt (cp) ηb (cp)

16.02 0.96 42.4 3.93 15.15.63 0.95 56.4 2.88 91

ottom, of such two systems are given inTable 1. The equivlent phase systems were chosen based on their equal (

he range of experimental error) tie-line lengths (16.025.63 %, w/w) for new dextran and standard dextranpectively) and their comparable volume ratios (1.7 andespectively for new and standard dextran). The calcuie-line lengths and volume ratio (defined as the ratio ofme of top phase to that of the bottom phase) along with ohysico-chemical parameters is given inTable 2. The densityatios (ratio of density of top phase to that of bottom) in bhe cases were equal (0.96 and 0.95, respectively) howhe difference of densities (42.4 and 56.4, respectively) ofom and top phases were not the same. The differenceore in the case of standard dextran system. This isbvious because the bottom phase of the two systemsigher molecular weight dextran fraction does have more

erial content for the same volume. This happens simp


exclusion principle[8]. A very small difference in viscosityof the top phase (3.93 and 2.88, respectively) was observed.This can be explained by the fact that the presence of higheramount of dextran in the top phase (2.06%) in the new dex-tran phase system than in the standard dextran system (0.6%).Viscosity of the bottom phase of theses two systems were dif-fering widely and this phenomenon can not be attributed tothe amount of dextran or total polymer content in the bottomphase. Rather reverse is observed (Table 1). This is takingplace because of the molecular weight difference of dextran.This is known fact that higher the molecular weight of a poly-mer the longer is the chain length and the higher the degree ofcoiling. The viscosity in aqueous solution is the result of boththe properties of a polymer described above[22]. This highviscosity in the bottom phase (91.2 cp) in the case of standarddextran phase system may pose threat to the mass transfer andmixing problem during extractive fermentation. Whereas, theuse of new dextran as phase forming polymer may prove ad-vantageous because viscosity in the bottom phase (in extrac-tive fermentation with aqueous two-phase system, cells aregenerally partitioned into dextran rich bottom phase whereasthe product strips into top phase) is relatively less (15.7 cp).

3.2. Polymer property

andc twod r(a her em.M ularw attv is-p xtrans newdt yt same( hichct eightslp dex-t ois-t d dryw e of

TC dard) f

T Mn )

S 1×N 1.×

literature value (dry weight can be 92% or more). There wasno need to determine the dry weight value for new dextran as itis available in solution form. However, dextran content in thesolution was estimated and it was found to be 10.5% (w/v),which was slightly higher than the supplier’s value (10.0%,w/v). The glucose content was measured for both dextranfractions and it was found to be zero. Aqueous solution of boththe samples was then sterilized (at 121◦C for 20 min) and theglucose content was estimated. The values were found to bebelow detectable range. This suggests that both the dextrando not get hydrolised very easily which suggests that there isno breakage of�-1,6-glucosidic bond, which is the primarystructure of a dextran molecule. The visosity at higher temper-atures were found to be little less which may be due to partialbreakage of most labile�-1,3 bond (where branching occurs)that causes lowering of viscosity value at higher temperature[27]. Of course, it regains its previous value at bringing backthe temperature to room temp (30◦C) again. This was studiedbecause it is required to sterilize the medium containing thedextran to exploit this aqueous two-phase system for simulta-neous fermentation and product separation. Optical rotationvalues for both the polymers were found to be almost thesame (188.87 and 190.22, respectively for new and standarddextran). This eventually suggests the same degree of purityprevail in both the dextran sources and very much supportst sim-i ragem of ap ) forb an isi eci dia-g ent,up re ine mit-i ofc ture,wd n( uresw as am-p thent -l lta r new

A table (Table 3) is presented here which summarizesompares all the important polymer properties of theseextran sources. The significance of weight (Mw) and numbeMn) average molecular is note worthy. The quantityMw islways greater thanMn, except for monodisperse system. Tatio Mw/Mn is a measure of polydispersity of the syst¯w is particularly sensitive to the presence of high moleceight species, whereasMn is influenced more by species

he lower end of the molecular weight distribution[25]. Thealues presented inTable 3suggest that the degree of polydersity of both the dextran sources is same for both deources. There is no risk of end-group cleavage for theextran source as theMw is much less than 105, above which

he cleavage chances are more[26]. The value supplied bhe manufacturer of standard dextran was almost thesupplied value was 2 and value determined was 1.9), wonfirms the manufacturers claim. HighMw value for bothhe cases suggest the presence of very high molecular wpecies. However,Mw value for new dextran (2.5× 104) wasess than that of standard dextran (3.2× 105) which hints theresence of lesser molecular weight species in the new

ran than in the case of standard dextran fraction. The mure content value for standard dextran was very less aneight was found to be 94.8, which is well within the rang

able 3omparison of polymer properties for both the dextran (new and stan

ypes of dextran Critical temperature (◦C) α ηin

tandard dextran 28.5 190.22 0.50ew dextran 15.0 188.87 0.17a A new dextran was available in solution form.

ractions

Mw Mw/Mn Dry mass Glucose (%w/w

.7105 3.2× 105 1.9 94.8 0.06104 2.5× 104 1.6 –a 0.0

he literature data (197 for pure standard dextran) underlar conditions. The intrinsic viscosity (depends on ave

olecular weight of a polymer), fundamental propertyolymer, was determined (0.17 and 0.50, respectivelyoth the dextran sources. The value for standard dextr

n agreement with literature data (0.52).Fig. 2 presents thorrelation of critical temperature in◦C and log (Mw). This

s also a fundamental property of a polymer. A phaseram using the mixture of polymer, solvent and non-solvsually termed as ternary mixture, can be drawn[22] and theosition of the binodal curve, along which two-phases aquilibrium, depends upon the molecular weight; the li

ng critical point at finite molecular weight is the analogritical temperature, popularly known as Flory temperahere polymer solvent interactions are zero[26]. Literatureata were collected[11] for different fractions of dextraT70, T110, T500 and T2000) and their critical temperatere plotted against log (Mw). This plot necessarily wastraight line. The critical temperatures of our dextran sles (new and standard dextran) were determined and

heir critical temperature values against log (Mw) were overaid on the same graph (Fig. 2) for comparison. The criticaemperature value for standard dextran (28.5◦C) is in goodgreement with the literature data where as, the same fo


Fig. 2. Observation of the critical temperatures of two dextran sources (newdextran T40 and standard dextran T500) in a ternary mixture (5% dextran inwater-methanol mixture, 405% by volume methanol). Data for dextran T70,T110, T500 and T2000 were collected from literature[11].

dextran (15.0◦C) fell on the same straight line if the line isextrapolated. This demonstrates the similarity in behavior ofthis new source of dextran with standard dextran with the dif-ference in molecular weight only[28–30]. Alternatively, themolecular weight of both the dextran sources can be calcu-lated and compared with the corresponding values obtainedby the other method (Mw by intrinsic viscosity method). Thecorresponding values in both the cases were calculated tobe in excellent agreement and this undoubtedly validates ourresults further.

3.3. Gel-forming property

The study of gel-forming properties for any polymer in so-lution is necessary as the structural property of the polymerplays the pivotal roll. Dextran is a polysaccharide of glucoseunits linked by�-1,6-glucosidic bonds. This applies to themain chain as well as to the side chains. The side chains arelinked to the main chains by�-1,3 branching. The interactionis possible through intermolecular hydrogen bonds betweenthe regularly spaced 3-hydroxyl groups, when six or moreglycosidic groups are�-1,6 linked[31]. The gelation prop-erty depends on the energy-state (temperature), the degreeof hydration (water content) and the concentration and typesof other ions present in the mixture. The dextran is generallyh -t ighlyc theo tionr

As the polymerisation proceeds the amount of gel increasesat the expense of sol, and the mixture rapidly transforms froma viscous liquid to an elastic material of infinite viscosity. Atthis state all water molecules present in solution bounds anda gel is formed. An important feature of the onset of gelationis the low number average molecular weight (Mn) of the mix-ture at gel point, where its weight average molecular weight(Mw) becomes infinite[25].

The top phase of an aqueous two-phase system is rich inlow molecular weight PEG and viscosity is also very muchless compared to high molecular weight dextran rich bottomphase. So, special focus was given to study the highly viscouslower phase. Polymerization of dextran in alkaline solutionis known to accelerate in the presence of phosphate[33]. Thepresence of PEG in small amount in the bottom phase alsoaccelerates the process of gel formation. In the two-phasesystem, the phosphate ions distribute preferentially causingthe building up of phase potential which is one of the vi-tal reasons for charged molecules (e.g. Proteins) to separatepreferentially to one of the two-phases[9,34].

The flow character of the dextran rich bottom phase isgiven inFig. 3. The bottom phases are drawn from two equiv-alent (on the basis of same tie-line length and volume ratio,Table 2) aqueous two-phase systems one for new dextran andthe other for standard dextran at 30◦C including 0.1 M phos-p twod os imingf en-t da con-t ffectiw wasc easedw sheert enonw samev viousl f thea redc eer.T theb ndardd hilicp artiali tionm xylg geni tra-t omesl vis-c er eta sam-p aso rease

ighly hydrated in aqueous solution[32] and if the water conent decreases, which may occur when the solution is honcentrated or available water is more tightly bonded tother molecules present in the mixture, the polymerisaeaction starts by interacting through hydroxyl groups[31].

hate buffer. The results of pH effect on flow property ofextran fractions are given inFig. 3 for pH 5.5 and 7.0. Ntudy was made in the alkaline zone as the study was aor extractive fermentation of 2,3-butanediol. The fermation of 2,3-butanediol byK. oxytocastarts at pH 6.8 ans fermentation proceeds, pH decreases and is finally

rolled at pH 5.5 during continuous operation. So, the es studied at two extremes of the pH values. From theFig. 3here the effect was studied keeping the pH at 5.5, thislear that the rate of increase of shear stress is decrith the increase in shear rate. So, the phenomenon of

hinning or pseudoplasticity was observed. This phenomas observed to be reversible as viscosity regains thealue when the sheer rate was brought back to the preower value. Shear thinning results from the tendency opplied force to disturb the long chains from their favoonformation, causing elongation in the direction of shhis effect is more prominent in the sample drawn fromottom phase of the new dextran system than in the staextran sample. Though, dextran is a nonionic hydropolymer, the presence of phosphate ion may result in p

onization and hence, the initiation of further polymerisaay start under this condition. In the acidic pH hydroroups of dextran may come away and react with hydro

on present in the solution to form water. That way concenion of the polymer decreases and hence, viscosity becess. At higher pH, in the alkaline range, the increase inosity is expected with the increase of shear rate. Kronl., [11] also observed the same behavior in the dextranles. At pH 7.0 (Fig. 3) almost no increase of viscosity wbserved in standard dextran sample whereas little dec


Fig. 3. Comparison of flow characteristic of the bottom phases derived from aqueous two-phases generated from two dextran sources (new dextran T40 andstandard dextran T500) at pH 5.5 and 7.0. Systems: 7.0% new dextran T40 + 7.5% PEG 6000 and 6.5% standard dextran T500 + 7.0% PEG 6000. Conditions:30◦C, 0.1 M phosphate buffer.

in viscosity was observed in the case of new dextran sample.This is evident in the figure from the steeper nature of curves(higher slope) at pH 7.0 compared to slopes of the curves atpH 5.5. However, both the bottom phases behaved almost inthe same way at both the pH values tested. The ionizationdegree of a gel is its key swelling parameter. Even at low im-purity level, ionized groups in a hydrogel network can cause asignificant increase in the swelling degree. Since the numberof ionogenic groups actually dissociated is a function of pH,the swelling degree of such systems is also a function of pH.Furthermore, the swelling caused by ionic groups is quite sen-sitive to ionic strength, in contrast to swelling caused by theinherent hydrophobicity of the polymer[35,36] and hence,the intermolecular forces is much more pronounced at pH 7.0then that at pH 5.5.

Hydrogel properties vary widely by the presence of dif-ferent monomers and cross linkers and their amounts. Thedegree of swelling of a hydrogel reduces by the presenceof a hydrophobic monomer to the formulation. Conversely,swelling degree increases by copolymerisation with the in-clusion of some hydrophilic or an ionic monomer or by the re-duction of their amounts. The presence of another hydrophiliccopolymer such as PEG in small amount influences the gelformation. Addition of salts such as NaCl, KCl, Na2SO4,K2HPO4 and KH2PO4 also leads to the gel formation, onec of the

anions in the sequence HPO4−2 > SO4

−2 > Cl−1. But the ef-fect of PEG and salt together on gel formation is much morepronounced than their individual effect. TheFig. 4 presentsthe effect of amount of phosphate in the dextran rich bottomphase for both the dextran systems. The samples were drawnfrom the bottom phases of the same two-phase system as de-scribed earlier. The pH and temperature was kept constantat 7.0 and 30◦C, respectively for both the systems whereas,the phosphate concentration was varied from 0.1 to 0.6 M. Inthis range, the viscosity of both the samples increased steadilywith the increase in phosphate concentration till 0.3 M phos-phate (approximately) for both the cases, However, the rate ofincrease of viscosity was found to be more with new dextransample then that with standard dextran sample. The corre-sponding samples with no potassium phosphate the viscositywere 15.7 and 91.2 cp respectively for new and standard dex-tran respectively. Whereas, addition of 0.1 M potassium tothe phase media the corresponding values were 265.7 and291 cp respectively, this behavior continued at higher phos-phate concentration also. This can be explained by the factthat the lesser molecular weight dextran (new destran) haslesser number of branching per molecule. The higher molec-ular weight fractions (standard dextran) are more randomlycoiled and also have higher proportion of longer chain permolecule and hence, the possibility of more intra-chain inter-a , a
an see a direct correspondence in the salting out effect ction increases[17]. After 0.3 M phosphate concentration


Fig. 4. Comparison of effect of phosphate on flow property of the bottomphases derived from aqueous two-phases generated from two dextran sources(new dextran T40 and standard dextran T500). Systems: 7.0% new dextranT40 + 7.5% PEG 6000 and 6.5% standard dextran T500 + 7.0% PEG 6000.Conditions: 30◦C, pH 7.0, 0.1 M phosphate buffer. K2HPO4 and KH2PO4

were used in equal proportion.

relatively slower increase in viscosity was observed with theincrease in phosphate concentration in both the cases. Thisis clearly a case of ion saturation and thereby cessation ofpolymerisation reaction. The increase in the exclusion vol-ume may not also be ruled out as the cause of observed depen-dence of the gel formation on the concentration of phosphate.If the samples were left for longer time without any distur-bance they go for complete gelation. However, this effect wasnot studied in detail in this piece of work.

The time dependence of both the bottom phases was alsostudied and the result is presented inFig. 5. The study showsreversible-time dependent changes in viscosity when sheeredat constant stress. The time dependence behaviour of bothsamples resembles rheopectic fluids. The higher the molecu-lar weight gelling occurs at lower pace then that for the lowermolecular weight fraction. The same reason as is given inearlier case (Fig. 4) can be put forward to explain the phe-nomenon. The phenomenon was also observed by Kroner etal. [11]. However, the viscosity values for both the polymersat all time were comparable and not much difference wasnoticed.

3.4. Rheological property

From the study of the rheological property, presented inF velyl . Thev frac-t se of

Fig. 5. Comparison of time dependence on gel-forming property of the bot-tom phase derived from aqueous two-phases generated from two dextransources (new dextran T40 and standard dextran T500). Systems: 7.0% newdextran T40 + 7.5% PEG 6000 and 6.5% standard dextran T500 + 7.0%PEG 6000. Conditions: 30◦C, pH 7.0, 0.1 M phosphate buffer.

new dextran sample. New dextran seems to have more idealflow property (Newtonian type) then standard dextran. Un-like other samples this sample only contained dextran andNaCl (0.9%, w/v). There was no presence of copolymerslike PEG or strong ions like phosphate that influences the

F urces( n T40i pera-t

ig. 6, this is clear that the new dextran sample relatiess viscous in comparison to standard dextran sampleery less sheer thinning property was observed in T500ion whereas, no such behaviour was observed in ca

ig. 6. Comparison of the rheological property of the two dextran sonew dextran T40 and standard dextran T500). System: 8% new dextran normal saline and 8% standard dextran T500 with 0.9% NaCl. Temure: 30◦C.


gelling property very strongly. So, this observation, partic-ularly when the samples were made differently for two dif-ferent polymer fractions, clearly and strongly supports ourprevious findings. Sodium chloride (0.9%, w/v) was addedexternally to standard dextran sample as new dextran samplealready contain the same amount of sodium chloride so thatthe samples may be compared.

3.5. Partition property

Polymer/polymer phase systems containing a high amountof water (65–90%) provide a gentle environment for biolog-ical materials and also enable the distribution of very labilesubstances while retaining their biological activity. This isdue to the stabilising effect of the phase forming polymers,especially that of PEG[9]. The partition of proteins and cellparticles in such systems is governed by the interactions ofthese substances with their environment in the phases. Theseinteractions are based on electrostatic, hydrophobic and vander Waals forces, leading to an overall partition with minimalenergy content of the system[9].

However, a pure thermodynamically controlled partitionapplies strictly only to dissolve molecules, especially forlarge particle sedimentation and other kinetic phenomenacontribute to the observed partition resulting in a time andob con-d ribedb

G

P be-t rtitionc

K

C ses,r lutei thec

es-t ondi-t nc mingp luew 500a herm -e t oft redt poly-m rved,f de-

Table 4Comparison of partition coefficient data of both the aqueous two-phase sys-tems for different dextran (new and standard) sources

Products Partition coefficient (K) Yield (%)

2,3-Butanediol New dextran 1.05 65Standard dextran 1.07 62

Griseofulvin New dextran 1.9 77Standard dextran 2.22 77

Chitinase New dextran 0.9 61Standard dextran 0.86 56

Systems were same in all cases. Systems: 7.0% new dextran T40/7.5% PEG6000/water and 6.5% standard dextran T500/7.0% PEG 6000/water at roomtemperature (30◦C), pH 7.0 with 0.1 M phosphate buffer.

viation is also observed in literature, for example, in the caseof isoleucyl-tRNA-synthetase[37]. For 2,3-butanediol thisvalue is almost equal to unity in both the phase systems.This behavior can be predicted from the already establishedliterature. The small molecules like 2,3-butanediol havingless interaction with the phases and hence, distributes moreevenly between the phases[9,38]. The most encouraging re-sults were found in the calculation of percentage yield value.This value is higher in case of phase system with new dextranas phase forming polymer than standard dextran in the casesof 2,3-butanediol (65 and 62%, respectively) and chitinaseenzyme (61 and 56%, respectively) whereas, same (77% inboth cases) in case of griseofulvin antibiotic.

It is known that the presence of potassium phosphate con-centration in the phase mixture influences the partition ofmolecules in aqueous two-phase system[9]. The effect ofpotassium phosphate concentration on 2,3-butanediol parti-tion is presented inFig. 7. It has been observed that the parti-tion coefficient increases with the increase of total potassiumconcentration in the phase mixture in both the cases. How-ever, the increment was observed to be less in both cases inthe range (potassium phosphate concentration) tested. Thiscan be explained by the fact that the 2,3-butanediol is anuncharged molecule and hence, does not come under the in-fluence of increasingly higher electro-potential difference be-t nts ofp . Theo duet thatt titionc r (int w ands

3

two-p ands e-c tran.T dardd igma

peration dependent distribution[26,28,29]. The distributionehaviour of particles, which is governed under identicalitions by their surface property and charges, can be descy partition ratio[8,9,12]:

= amount in top phase

total amount in the system− amount in the top phase(6)

artition of biological molecules generally takes placeween both the phases and can be described by the paoefficient according to Nernst equation,Eq. (7):

= Ct

Cb(7)

t andCb are the concentrations in the top and bottom phaespectively. Under identical conditions partition of sos influenced by the molecular weight, net charge andonformation of the solute molecule.

Partition coefficient for solute and biomolecules wereimated with these two-phase systems under identical cion and presented inTable 4for comparison. The partitiooefficient values using new dextran as the phase forolymer were comparable in all respect. A little higher vaas observed in the case of griseofulvin with dextran Ts the phase forming polymer. This is mainly due to higolecular weight of the dextran used[2]. Often, partition cofficient increases with the increase in molecular weigh

he dextran[8]. However, this effect is not very high compao the phase system using dextran T40 as one of theers. Deviation from this general behavior is also obse

or example, partition coefficient of chitinase. The same

ween the phases created by increasingly higher amouotassium phosphate content in the total phase mixturebserved little increment in partition coefficient may be

o exclusion effect. The most interesting notable point ishe effect of potassium phosphate concentration on paroefficient of 2,3-butanediol was observed to be similahe range tested) in both the phase systems (using netandard dextran).

.6. Cost analysis

The comparison chart of cost analysis for the aqueoushase system using two different source of dextran (newtandard) is given inTable 5. This analysis was needed bause it gives us more logic to use the new source of dexhe calculations were done in Indian currency. The stanextran price was taken on the basis of 100 g pack of S


Table 5Comparison of cost analysis data of both the aqueous two-phase systems for different dextran (new and standard) sources

Dextransource

Phase system Cost of dextran(Rs.)

Cost of PEG(Rs.)

Cost ofbuffer (Rs.)

Total cost(Rs.)

Dextran cost as thepercentage of total cost

Dextran (%w/w) PEG (%w/w)

New 7.0 7.5 42.00 3.41 1.32 46.73 90Standard 6.5 7.0 331.50 3.08 1.32 335.90 99

Calculations are based upon: (1) 100 g pack for standard dextran, (2) 500 ml bottle for new dextran, (3) 500 g pack for PEG, and (4) 500 g packs of K2HPO4

and KH2PO4. Prices are taken in Indian currency (Rs.). All the prices are for preparation of 100 g of phase systems.

Fig. 7. Comparison of effect of phosphate on partition coefficient of 2,3-butanediol on the aqueous two-phase system. Systems: 7% new dextran T40+ 7.5% PEG 6000 and 6.5% standard dextran T500 + 7.0% PEG 6000.Conditions: temperature 30◦C, pH 7.0, 0.1 M phosphate buffer. K2HPO4

and KH2PO4 were used in equal proportion.

chemicals in Indian currency (rupees). No import duty waslevied on the price. All other chemicals were procured fromthe Indian Chemical manufacturers. The price of new dextranwas calculated on the basis of 500 ml bottle. Other chemicalprices were calculated on the basis of 500 g pack. No kinds oftaxes were added on any of the chemicals. The list price onlywas taken for cost analysis. If we closely look into the table,we will find the total price for phase formation is only about13.9% with the new dextran compared to standard dextran.Conversely, the phase formation cost with standard dextran is719% of that with new dextran. For new dextran phase sys-tem, the dextran cost is comparatively cheaper (90%) thanwith standard (99%). So, this is clear fromTable 5that dex-tran costs only dominate the phase formation cost and hence,searching for cheaper source of dextran was important if weare to exploit the enormous advantages of aqueous two-phasesystem. Considering only the dextran cost, it reveals that thecost is only 12.7% with new dextran compared to standarddextran. This is also clear fromTable 5that the prices of otherchemicals for phase separation are negligible. This gives us

Fig. 8. Comparison of cost of phase formation of the aqueous two-phasesystems from two dextran sources (new and standard). Conditions: for stan-dard dextran cost is based on 100 g pack, for new dextran cost is based on500 ml bottle available in the market, phosphate buffer cost was based on500 g pack. All the prices were taken in Indian currency (Rs.).

more justification to use new dextran as the phase polymerthan standard dextran.

The same conclusion can be drawn fromFig. 8that givesthe cost curve for the phase systems with new and standarddextran. The line with standard dextran is placed much higherthan that with new dextran. In this case the tie-line length iskept constant and the volume ratio is changed in both thecases. It is observed that with the increase in volume ratio theprice of the phase system comes down in both the cases. Thisis because it is required to add less and less amounts of dextranto increase the volume ratio higher and higher keeping the tie-line length constant. Since, the price of dextran dominatesthe phase formation cost, the price of the whole system alsocomes down.

4. Conclusion

Aqueous two-phase systems enjoy lots of advantages overother methods of separation and extractive fermentation.There are many successes reported in the literature to prove


its excellence, however, this suffers from one major disad-vantage, that is, the high cost of dextran that mainly restrictsits use in the industry. In this study, we have tried to comparethe new source of dextran with the popularly used standarddextran. As this source of dextran is much cheaper, comparedto other source, an exhaustive study has been carried to com-pare almost all necessary parameters and properties to estab-lish it against the standard dextran. No major set back wasobserved in any of the parameters that have been comparedwith the standard dextran, rather in some cases some advan-tage was observed with new dextran. Finally, the compara-tive results of cost analysis boosts up our claim that this newdextran source can successfully and fruitfully be exploitedfor isolation and separation of biomolecules in general and2,3-butanediol in particular with aqueous two-phase systemmore economically without compromising on in any prop-erty. So, this present study proves undoubtedly that relativelycheap aqueous solution of new dextran has all potential toreplace standard microcrystalline dextran as a phase formingpolymer of the aqueous two-phase system for isolation andpurification purposes.

Acknowledgement

Authors like to acknowledge the help forwarded by Prof.N merl thee bra-m en-t V.V lp atd

R

ng.

ng.

d-mic

29

icrob.

les,

[9] P-A. Albertsson, Partition of Cell Particles and Macromolecules,second ed., Wiley, New York, 1971.

[10] A.D. Diamond, J.T. Hsu, Aqueous two-phase systems forbiomolecule separation, Adv. Biochem. Eng. Biotechnol. 47 (1992)89.

[11] K.H. Kroner, H. Hustedt, M.-R. Kula, Evaluation of crude dextran asphase forming polymer for the extraction of enzymes in aqueous two-phase systems in largescale, Biotechnol. Bioeng. 24 (1982) 1015.

[12] Dextran – The Versatile Biopolymer, Information booklet, Pfeiferand Langen, 1985.

[13] G.L. Miller, Use of dinitrosalicilic acid reagent for determination ofreducing sugar, Anal. Chem. 31 (1959) 426.

[14] M.L. Huggins, The viscosity of dilute solutions of long chainmolecules IV. Dependence on concentration, J. Am. Chem. Soc. 64(1942) 2716.

[15] R.B. Bird, E.S. Stewart, E.N. Lightfoot, Transport Phenomena, Wi-ley, New York, 1994.

[16] A.B. Uppsala, Dextran fractions, Pharm. Inf. 9 (1977), available fromPharmacia Fine Chemicals A.B., Uppsala, Sweden.

[17] A. Kumar, R.K. Gupta, Fundamentals of Polymer Science, McGraw-Hill, Singapore, 1998.

[18] M. Wales, P.A. Marshall, S.G. Weissberg, J. Polym. Sci. 10 (1952)229.

[19] J. Monreal, E.T. Reese, The chitinase of Serratia marcescens, Can.J. Microbiol. 15 (1969) 689.

[20] A. Kapat, S.K. Rakshit, T. Panda, Parameter optimization of chitinhydrolysis byTrichoderma harizianumchitinase under assay condi-tions, Bioprocess. Eng. 14 (1996) 275.

[21] A. Holbrook, F. Bailey, G.M. Bailey, A rapid method for the de-termination of griseofulvin in fermentation broth, J. Phar. Pharmcol.

[[[[ ew

[ em.

[ and

[ ain

[ sity

[ their

[[ .[[[[ Gel-

g. 76

[ 36

[ angen,

.R. Neelakantan and Dr. Susy Varughese of high polyaboratory for extending the lab facilities for some ofxperiments. Authors are indebted to Prof. K.K. Balasuanian of Chemistry department for providing instrum

al facility. They also like to thank Dr. Arthur Felse, Dr.enkata Dasu and Dr. B. Sobhana Babu for their kind heifferent times.

eferences

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evaluation of an alternative source of dextran as a phase forming polymer for aqueous two-phase...

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