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Proteins from common bean (Phaseolus vulgaris) seed as a natural coagulantfor potential application in water turbidity removal

Mirjana G. Antov *, Marina B. ciban, Nada J. PetrovicFaculty of Technology, University of Novi Sad, Blvd. Cara Lazara 1, 21000 Novi Sad, Serbia

a r t i c l e i n f o

Article history:Received 30 July 2009Received in revised form 2 November 2009Accepted 5 November 2009Available online 30 November 2009

Keywords:

Natural coagulantCommon beanIon-exchangeCoagulation activity

a b s t r a c t

The ability of coagulation active proteins from common bean (Phaseolus vulgaris) seed for the removal ofwater turbidity was studied. Partial purification of protein coagulant was performed by precipitationwith ammonium sulphate, dialysis and anion exchange chromatography. Adsorption parameters forion-exchange process were established using dialysate extract. Results revealed that the highest valuesof the adsorbed protein were achieved in 50 mmol/L phosphate buffer at pH 7.5 and the maximumadsorption capacity was calculated to be 0.51 mg protein/mL matrix. Partially purified coagulant at initialturbidity 35 NTU expressed the highest value of coagulation activity, 72.3%, which was almost 22 timeshigher than those obtained by crude extract considering applied dosages. At the same time, the increasein organic matter that remained in water after coagulation with purified protein coagulant was morethan 16 times lower than those with crude extract, relatively to its content in blank.

2009 Elsevier Ltd. All rights reserved.

1. Introduction

Coagulation/flocculation as a step in water treatment processesis applying for removal of turbidity in raw water that comes fromsuspended particles and colloidal material. Materials that are usedin this stage of water treatment can be inorganic coagulants, syn-thetic organic polymers or coagulants from natural sources. Alu-minium sulfate (alum) is a common coagulant globally used inwater treatment. In spite of its undoubtfull effusiveness in turbid-ity removal, alum increases concerns towards ecotoxicological im-pact when introduced into the environment as post-treatmentsludge having large volumes. Regarding the application of syn-thetic polymers, the presence of residual monomers is undesirablebecause of their neurotoxicity and strong carcinogenic properties(Mallevialle et al., 1984).

A part of possible solution of these problems might be develop-

ment of new coagulants, preferably from natural and renewablesources, which have to be safe for human health as well asbiodegradable. Because their production relies on local materials,renewable resources and food grade plant material, and is rela-tively inexpensive, it can contribute to achieving sustainable watertreatment technologies. By using natural coagulants considerablesavings in chemicals and sludge handling cost may be achievedalong with production of readily biodegradable and less volumi-nous sludge that amounts only 2030% that of alum treatedcounterpart (Narasiah et al., 2002).

In recent numerous studies variety of plant materials as asource of natural coagulants has been reported (Raghuwanshiet al., 2002; Diaz et al., 1999; Miller et al., 2008) but the most stud-ied is Moringa oleifera whose efficiency has been reported for tur-bidity removal (Ndabigengensere and Narasiah, 1998; Okudaet al., 2001a; Ghebremichael et al., 2006) as well as antimicrobialproperties (Ghebremichael et al., 2005). Apart of all previouslymentioned preferences of using natural coagulants instead of syn-thetic ones, major disadvantage of their application as crude ex-tracts in water treatment is an increase of organic matter inwater. This complicates further processing and adversely affectswater quality but could be overcome by purification of the coagu-lant (Ghebremichael et al., 2006).

During the course of plants screening program in our labora-tory (ciban et al., 2005, 2009), crude extract from common bean(Phaseolus vulgaris) seed showed the ability to act as a natural

coagulant. Seed from common bean as potential source of coagu-lant for water treatment would be promising considering its foodgrade nature. Moreover, it offers a few advantages over M. oleiferaseed because of no oil present in it there is no need for extractionby organic solvents thus avoiding delipidation step which is bene-ficial for both economic and environmental reasons.

The objective of the study was partial purification of thecoagulation active components extracted from common bean seed.Optimal conditions for ion-exchange chromatographic purificationof coagulant protein regarding the process of adsorption wereestablished. In addition, application of the partially purifiedcommon bean coagulant was evaluated as well as its suitabilityin comparison to crude extract regarding organic load.

0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2009.11.020

* Corresponding author. Tel.: +381 21 485 3647; fax: +381 21 450 413.E-mail address: mantov@uns.ac.rs (M.G. Antov).

Bioresource Technology 101 (2010) 21672172

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2. Methods

2.1. Extraction of active component from common bean seed

The locally obtained common bean (P. vulgaris) dry seed wasground to a fine powder by using a laboratory mill and sievedthrough 0.4 mm sieve. The fraction with particle size less than

0.4 mm was used in experiments. Fifty grams of seed powderwas suspended in 1 L of 0.5 mol NaCl/L water. The suspensionswere stirred using a magnetic stirrer for 10 min to accomplishextraction and then filtered through a rugged filter paper (Mache-rey-Nagel, MN 651/120) to obtain filtrates crude extracts of ac-tive component.

2.2. Precipitation of active component

The coagulation active component from common bean wasfurther processed by precipitation and dialysis. Crude extractswere saturated to 80% by addition of (NH4)2SO4 and centrifugedat 4000g (5804-R, Eppendorf) for 10 min. Precipitate was redis-solved in 10 mmol/L appropriate buffer (i.e. some of the buffers

listed in the following text) and dialysed overnight at 4 Cagainst Millipore water in dialysis bag with molecular cut-off1214 kDa.

2.3. Adsorption studies

Adsorption studies were conducted using dialysate extractsobtained above in series of buffers in batch ion-exchange (IEX)experiments with AmberliteTM IRA 900 Cl (Rohm and Haas) asmatrix. AmberliteTM IRA 900 Cl is a macroreticular polystyrenetype 1 strong base anion exchange resin containing quarternaryammonium groups whose shipping weight is 700 g/L and total ex-change capacityP 1.00 eq/L (Cl form). In order to find the opti-mum pH of the buffer for the adsorption, dialysate extract wasdiluted in universal Britton and Robinson (I) buffer having pH from7 to 9 with an increment of increase of pH 0.5. The choice of thebuffering substance was made by measuring the amount of boundprotein in phosphate, TrisHCl or ammonium acetate buffer at pH7.5. The effect of ionic strength of the buffer on the adsorption ofactive compounds to anion exchange resin was evaluated by vary-ing concentration of phosphate buffer (10, 25, 50, and 100 mmol/L)at pH 7.5.

In order to estimate optimum volume of IEX matrix, adsorptionexperiments were carried out by adding 0.33 mg dialysate extractin phosphate buffer (50 mmol/L, pH 7.5) to different volumes of thematrix ranging from 0.33 mL to 1.0 mL.

2.4. Kinetic studies and adsorption isotherm

Kinetics of adsorption was studied using 3.3 mg dialysate ex-tract in 10 mL phosphate buffer (50 mmol/L, pH 7.5). Protein solu-tion was added to 10 mL IEX matrix and mixed in magnetic stirrerat 100 rpm. Samples were collected in certain time intervals, cen-trifuged immediately and supernatants were analysed for proteincontent. Blanks were carried out without matrix to check out ifany measurable loss of protein came out from reasons other thanits adsorption to matrix.

Adsorption capacity of the matrix was estimated using 1.786.44 mg dialysate extract in 10 mL phosphate buffer (50 mmol/L,pH 7.5). Protein solution were added to 10 mL of IEX matrix andmixed at 100 rpm for 90 min at room temperature. After that un-

adsorbed protein concentration was measured and amount of ad-sorbed protein was calculated from a mass balance.

Maximum adsorption capacity of the matrix and the dissocia-tion constant of the adsorption were determined form the Lang-muir adsorption model (Faust and Aly, 1987):

Ceqe

1

b Xm

CeXm

; 1

where Ce is the concentration of protein in solution in equilibrium

(mg/mL); qe is the amount of protein adsorbed per volume of adsor-bent (mg/mL); b is a constant that is related to the enthalpy ofadsorption (mL/mg) and Xm is the maximum adsorption capacity(mg/mL).

2.5. Purification of active component

Dialysate extract was loaded onto column (10 mm 150 mmglass column) packed with 10 mL of AmberliteTM IRA-900 Cl previ-ously equilibrated with 50 mol/L phosphate buffer, pH 7.5. Activecomponents were eluted from resin by linear gradient of ionicstrength of NaCl solution from 0 to 1 mol/L at a flow rate 1 mL/min. Protein content and coagulation activity of fractions (2 mL)were determined.

2.6. Preparation of turbid water

Turbid water for coagulation tests was prepared by adding 1 gkaolin to 1 L tap water. The suspension was stirred for 1 h toachieve uniform dispersion of kaolin particles, and then it was al-lowed to remain for 24 h for completing hydration of the particles.This suspension was used as the stock suspension. Turbid waterwith 50 mg/L kaolin (about 35 nephelometric turbidity units NTU) was prepared by diluting 50 mL of stock suspension to1000 mL tap water just before the coagulation test. The initial pHof the synthetic water was adjusted to 9.0 with 1 mol/L NaOH solu-tion, in accordance with previous investigations (Okuda et al.,2001a; ciban et al., 2005).

2.7. Coagulation test

Coagulation activity of the fraction eluted from column as wellas crude extract was evaluated in jar tester VELP, model FC6S. Sam-ples were added to the beakers at different dosages (0.5, 1.0 or2.0 mL/L turbid water) and the content was stirred at 200 rpmfor 2 min. The mixing speed was then reduced to 80 rpm andwas kept for 30 min. Then, the suspensions were left to allow sed-imentation. After 1 h of sedimentation, an aliquot of 100 mL ofclarified sample was collected from the top of the beaker and resid-ual turbidity was measured. The residual turbidity of sample wasRTS. The same coagulation test was performed with no coagulantas the blank. The residual turbidity in the blank was RTB. Coagula-

tion activity was calculated as:

Coagulation activity % RTB RTS

RTB 100: 2

2.8. Analytical methods

Protein concentration was measured according to Bradford(1976) with bovine serum albumin as standard. Turbidity wasmeasured using a turbidimeter (TURB 550 IR) and it was expressedin nephelometric turbidity units (NTU). The amount of organicmatter released from common bean seed crude extract and par-tially purified protein were determined as chemical oxygen de-mand (COD) according to Standard Methods (APHA, 1998).

All experiment were run in duplicate (the accuracy is consid-ered to be 5%) and the mean value is presented herein.

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3. Results and discussion

Our previous investigations showed that the highest values ofprotein concentration and coagulation activity were achievedwhen protein coagulant from common bean seed was extractedby 0.5 mol/L NaCl in water (Antov et al., 2007). In the current studythe coagulation active components were precipitated by ammo-

nium sulphate and dialysed, and dialysate extract was used inadsorption studies in order to maximise results of purification pro-cess of common bean coagulant (CBC).

3.1. Adsorption parameters

According to the literature, isoelectric points of proteins from P.vulgaris are near to pH 4.5 (Belitz et al., 2004; Morales-de Leonet al., 2007). Cloud point test (data not shown) conducted in ourlaboratory indicated that isoelectric point (pI) of our dialysate ex-tract of common bean seed was between pH 4 and 5.5. So, the ef-fect of pH of buffer on the adsorption of dialysate extract on anionresin was studied in batch experiments within pH range 79 bymonitoring the amount of protein bound to the matrix (Fig. 1a).

Results revealed that in the investigated pH range percentage ofadsorbed protein was varied in narrow range from 75.6% to84.6%. Maximum of protein adsorption was achieved, as expected,at the highest investigated pH 9 considering the fact that net sur-face charge of proteins increases with the increase in distance frompH to pI. However, because the portions of adsorbed protein at pH7.5 (84%) and pH 9 (84.6%) were just slightly different and also con-sidering the following elution step, further experiments were con-ducted at pH 7.5.

In the next experiments, several buffering substances weretested at pH 7.5 in order to find the most appropriate one for theadsorption of dialysate extract to IEX matrix. The highest percent-age of protein, 80.7%, was bound to matrix in the presence of phos-phate ions (Fig. 1b). So, the effect of ionic strength of buffer on theadsorption was monitored by measuring the amount of proteinbound to matrix in phosphate buffer within concentration range10100 mmol/L at pH 7.5. With an increase of buffer ionic strengthamount of adsorbed protein increased and the highest value wasobserved with 50 mmol/L phosphate buffer (Fig. 1c). However, at100 mmol/L amount of adsorbed protein was decreased whichwas indicated by increased concentration of protein in un-boundfraction. This result can be explained by the increased competitiontoward adsorption sites at IEX matrix between protein-ions andbuffer-ions when they are present in higher concentrations(Scopes, 1994).

Estimate of optimum volume of IEX matrix that is required for

adsorption and purification of protein extracted from commonbean was based on the experiments conducted with constantamount of dialysate extract but varying volumes of matrix (datanot shown). Results revealed that increase in matrix volume from0.33 mL to 1 mL led to an increase in percentage of adsorbed pro-tein from 50% to nearly 85% added protein, respectively, and thatthe highest investigated volume of matrix was sufficient for

0

20

40

60

80

100

7 7.5 8 8.5 9

pH

Adsorbed

protein(%)

0

20

40

60

80

100

Phosphate

buffer

Tris-HCl buffer Ammonium

acetate buffer

Adsorbedprotein

(%)

0

20

40

60

80

100

10 25 50 100

Cbuffer (mmol/L)

Adsorbedprotein(%)

(a)

(b)

(c)

Fig. 1. The effect of (a) pH, (b) buffering substance and (c) concentration of

phosphate buffer on the adsorption of dialysate extract from common bean seed onanion exchange matrix AmberliteTM IRA 900 Cl.

0 20 40 60 80 100 1200.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Adsorbedprotein(mg/mLresin)

Time (min)

Fig. 2. Adsorption kinetics of dialysate extract from common bean seed on anionexchange matrix AmberliteTM IRA 900 Cl at 50 mmol/L phosphate buffer, pH 7.5.

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adsorption of 0.28 mg protein/mL matrix. Higher efficiency of pro-tein adsorption at higher matrix volume can be attributed to thegreater number of active sites at matrix which increases the prob-ability of the adsorption of protein molecules (under condition thatmatrix was not overloaded like in these experiments, see Fig. 3) asit was already found for M. oleifera protein coagulant (Ghebremich-ael et al., 2006).

3.2. Kinetics of adsorption and equilibrium parameters

The kinetics of adsorption of proteins from dialysate extract onmatrix was studied during 2 h at room temperature and simulta-neously blanks without matrix were carried out. Experiments con-

firmed that changes in protein concentration came out only as aconsequence of its adsorption to IEX matrix. The rate of proteinadsorption was high in the first 10 min and after that incrementalincrease started to decline (Fig. 2). The time needed to reach theequilibrium was estimated to 6090 min when maximal valuesof adsorbed protein were determined.

Equilibrium parameters were determined from the Langmuirisotherm model considering the smooth continuous time courseof protein adsorption (Fig. 2) that suggests the formation of proteinmonolayer on surface. From a linear relationship Ce/qe vs. Ce (Fig. 3)the maximum adsorption capacity was calculated to be 0.51 mgprotein/mL matrix. It should be noticed that this result was basedon dialysate extract and not on the purified protein. Obtained valuefor

Xmis significantly lower than those for coagulant protein from

M. oleifera (Ghebremichael et al., 2006) even considering that dif-ferent IEX matrixes were used. This might be also explained bythe differences in molecular weight of coagulation active proteinsfrom P. vulgaris and M. oleifera. Namely, it is estimated that coagu-lant protein from M. oleifera has molecular weight 6.5 kDa (Ghe-bremichael et al., 2005) i.e. 13 kDa for dimer (Ndabingengensereet al., 1995) while Mw of subunit of major storage protein (whichis trimer) in common bean seed is $50 kDa (Belitz et al., 2004;Montoya et al., 2008). It is known fact that small macromoleculeshave higher adsorption per unit of weight (volume) of adsorbent(Kilduf et al., 1996). In addition, larger molecules can bind onlyto the surface of the ion exchanger particles, so capacity for theseis very low (Scopes, 1994).

3.3. Purification of coagulation active components

The chromatogram of dialysate extract on anion exchange ma-trix is shown in Fig. 4. Dialysed extract (containing 3.3 mg protein)was loaded to column and then bound proteins were eluted by lin-ear gradient NaCl. Coagulation activity of each fraction was deter-mined at a dosage 1 mL/L turbid water. Results revealed existenceof several protein peaks that were not completely separated fromeach other but coincided in some extent with the highest mea-sured coagulation activities in fractions. Flocs formed during thecoagulation test of fractions eluted from 0.375 mol/L to0.875 mol/L NaCl were visible to the naked eye (12 mm) duringthe slow mixing part of the process. The presence of several protein

as well as coagulation active peaks might be explained by the het-erogeneity of protein sample from common bean seed. The fractionhaving the highest coagulation activity 72.3% was eluted by0.875 mol/L NaCl. This fraction containing partially purified com-mon bean coagulant (CBC) was further analysed in coagulationstudy.

R2

= 0.9675

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.05 0.1 0.15 0.2 0.25 0.3

Ce (mg/mL)

C

e/qe

Fig. 3. Linearized form of Langmuir model for adsorption of dialysate extract fromcommon bean seed on anion exchange matrix Amberlite TM IRA 900 Cl.

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

0.00

0.05

0.10

0.15

0

20

40

60

80

100

0.0

0.2

0.4

0.6

0.8

1.0

Coagulationactivity(%)

Proteinconcentration(mg/mL)

V(mL)

CNaCl

(mol/L)

Fig. 4. Elution diagram of dialysate extract from common bean seed from anion exchange matrix Amberlite TM IRA 900 Cl.

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3.4. Coagulation study

The primary goal of coagulation/flocculation is turbidity re-moval and this was investigated at conditions regarding initial tur-bidity 35 NTU according to Okuda et al. (2001b). Results ofcoagulation test of crude extract and the fraction containing puri-fied CBC, that had protein concentrations 0.73 mg/mL and0.081 mg/mL, respectively, in relation to coagulant dosage areshown in Fig. 5a. The optimal dosage which was the minimumone led to the lowest residual turbidity had the equal value forcrude extract and fraction with partially purified CBC andamounted 1 mL/L turbid water. Calculated on the base of proteinconcentration, the optimal coagulation dosage of purified CBCwas 0.081 mg/L turbid water which expressed the highest coagula-tion activity 72.3%. Starting from the same initial turbidity, crudeextract at its optimal dosage 0.73 mg/L turbid water exhibitedthe highest efficiency in turbidity removal which resulted in coag-ulation activity 30%. Hence, calculated on the base of protein whichwas added in turbid water, the highest obtained coagulation activ-ity of partially purified CBC was almost 22 times higher than thatof crude extract.

3.5. Organic load in treated water

The crude extracts which are used as natural coagulants containbiomolecules and inorganic substances which may be released inwater leading to increased COD (Ghebremichael et al., 2006). Be-

sides, the use of natural coagulants may increase the organic loadin water which may result in increased microbial activity (Ndabig-engensere and Narasiah, 1998; Okuda et al., 2001a). In addition,the organic matter might consume additional chlorine in the watertreatment plant and can acts as a precursor of byproducts duringthe disinfection process. Thus, purification of the active compo-nents is essential to minimize the value of unnecessary organic

material which might adversely affect quality of the water.Organic matter in water before and after coagulation tests wasmeasured to establish the increase in organic load when crude ex-tract and the partially purified CBC were added to turbid water atthe dose exhibiting the highest coagulation activity. Resultsshowed that the increase in organic matter that remained in waterafter coagulation was 5.9 and 0.35 mg O2/L when crude extract andpurified CBC acted as coagulants, respectively, in comparison to itscontent in blank (Fig. 5b). More than 16 times lower organic loadincrease in water after turbidity removal by purified CBC in com-parison to crude extract was in accordance with lower protein con-centration in tested fraction (9 times) but additionally it might beas well a consequence of the diminished load of other organic com-pounds from crude sample. Chemical oxygen demand value whenpartially purified CBC was applied was in the range of those onesobtained when purified fractions of M. oleifera protein coagulantwere applied for turbidity removal (Ghebremichael et al., 2006).In addition, it should be stressed that COD of water treated withthe partially purified CBC with applied coagulation dose was belowthe maximum admissible concentration according to Europeandirective which is 5 mg/l (Commission Directive 98/83/EC, 1998).

4. Conclusion

Coagulation activity of the protein from common bean seed ob-tained after optimization of purification procedure and the in-crease in organic load were about 22 times higher and more than16 times lower, respectively, than that of crude extract. Purifica-

tion of natural coagulant from common bean seed has no delipida-tion step, that is usual for coagulants from oily seed, which makeits application in water turbidity removal potentially more eco-nomical and environmental friendly.

Acknowledgement

The financial support from Ministry of Science and Technologi-cal Development, Republic of Serbia (Project No. 20064) is greatlyacknowledged.

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