- Email: [email protected]
Evaluation of the efficiency of natural coagulant obtained by ultrafiltration of common bean seed extract in water turbidity removal
Ecological Engineering 49 (2012) 48–52
Contents lists available at SciVerse ScienceDirect
Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Short communication
Evaluation of the efficiency of natural coagulant obtained by ultrafiltration of common bean seed extract in water turbidity removal ´ Mirjana G. Antov ∗ , Marina B. Sˇ ciban, Jelena M. Prodanovic´ University of Novi Sad, Faculty of Technology, Blvd. Cara Lazara 1, 21000 Novi Sad, Serbia
a r t i c l e
i n f o
Article history: Received 9 March 2012 Received in revised form 6 July 2012 Accepted 10 August 2012 Available online 28 September 2012 Keywords: Natural coagulant Ultrafiltration Coagulation activity Organic load Common bean seed
a b s t r a c t Efficiency of natural coagulant obtained by ultrafiltration of common bean (Phaseolus vulgaris) seed extract in water turbidity removal was experimentally evaluated. Prepared ultrafiltration fractions have shown differences in behavior regarding pH values and dosages at which they expressed their highest efficiency in turbidity removal. The highest obtained coagulation activities were at pH 9.5 with crude extract and fraction containing molecules with molecular weight less than 10 kDa, 50.6% and 49.1%, respectively. The organic load that remained in water after treatment was reduced when natural coagulant processed by ultrafiltration instead of crude extract was applied. Moreover, fraction with molecules between 10 kDa and 30 kDa, decreased chemical oxygen demand by 9% relative to blank at conditions this fraction expressed its highest coagulation activity. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Water question is surely one of the main factors that are involved in the human development considering its influence on human lives. Among the new techniques for water and wastewater treatment there is the use of natural coagulants, aiming at a better quality of treated water by reducing the use of chemicals. Namely, coagulation/flocculation step which is essential process in the treatment of both surface water and industrial wastewater, includes removal of dissolved organic species and turbidity from water most commonly via addition of conventional chemicalbased coagulants – alum, ferric chloride and synthetic organic polymers. While the effectiveness of these chemicals as coagulants is well-recognized (Edzwald, 1993; Kang et al., 2003) there are, nonetheless, disadvantages associated with their usage such as ineffectiveness in low-temperature water (Haaroff and Cleasby, 1988), relatively high procurement costs, detrimental effects on human health, production of large sludge volumes and the fact that they significantly affect pH of treated water. There is also strong evidence linking aluminum-based coagulants to the development of Alzheimer’s disease in human beings (Flaten, 2001). It is therefore desirable to replace these chemical coagulants with plant-based coagulants to counteract the aforementioned drawbacks.
∗ Corresponding author. Tel.: +381 21 485 3647; fax: +381 21 450 413. E-mail address: [email protected] (M.G. Antov). 0925-8574/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ecoleng.2012.08.015
Thus, in water treatment, the use of natural coagulants could be an option with many advantages over chemical agents, particularly the biodegradability, low toxicity and low residual sludge production (Narasiah et al., 2002). Biopolymers may be of great interest since they are natural low-cost products, characterized by their environmental friendly behavior and usually have large number of surface charges that increase the efficiency of the coagulation process. These advantages are especially augmented if the plant from which the coagulant is extracted is indigenous to local community. In recent numerous studies variety of plant materials has been reported as a source of natural coagulants (Raghuwanshi ´ et al., 2009) et al., 2002; Diaz et al., 1999; Miller et al., 2008; Sˇ ciban but the most studied is Moringa oleifera whose efficiency has been approved for turbidity removal (Ndabigengensere and Narasiah, 1998; Okuda et al., 2001; Ghebremichael et al., 2006) as well as antimicrobial properties (Ghebremichael et al., 2005). During the course of plants’ screening program in our laboratory, crude extract from common bean (Phaseolus vulgaris) seed was applied for water turbidity removal while partially purified coagulant has shown high coagulation efficiency followed by low organic load (Antov et al., 2010). Seed of common bean has food grade nature and contains no oil, so, delipidation step is not necessary which is beneficial for both economic and environmental reasons. Considering this common bean is a promising source of natural coagulant for water treatment. The objective of the study was to experimentally evaluate the efficiency of different fractions prepared by ultrafiltration of common bean seed extract in water turbidity removal. Membrane
M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52
49
separation technique was applied instead of isolation and purification multiple steps procedure to examine its suitability for the processing of natural coagulant. Coagulation efficiencies of prepared fractions were measured at different pH values and dosages, and have been compared to the one of crude extract. In addition, suitability of prepared natural coagulants was tested in respect of organic load that remained in water after treatment.
stirred at 200 rpm for 1 min. The mixing speed was then reduced to 60 rpm and was kept for 30 min. Then, the suspensions were left to allow sedimentation. After 1 h of sedimentation, upper clarified liquid was collected and residual turbidity was measured. The residual turbidity of sample was RTS . The same coagulation test was performed with no coagulant as the blank. The residual turbidity in the blank was RTB . Coagulation activity was calculated as:
2. Materials and methods
Coagulation activity (%) =
2.1. Extraction of active components from common bean seed The locally obtained common bean (P. vulgaris) dry seed was ground to a fine powder by using a laboratory mill and sieved through 0.4 mm sieve. The fraction with particle size less than 0.4 mm was used in experiments. Ten grams of seed powder was suspended in 1 L of NaCl water solution (0.5 mol/L). The suspension was stirred using a magnetic stirrer for 10 min to accomplish extraction and then filtered through a rugged filter paper (Macherey-Nagel, MN 651/120) to obtain filtrate – crude extract of active components. 2.2. Preparation of fractions of natural coagulant by ultrafiltration
RTB − RTS · 100 RTB
(1)
2.5. Analytical methods Protein concentration was measured according to Bradford (1976) with bovine serum albumin as standard. Turbidity was measured using a turbidimeter (WTW TURB 550/550 IR) and it was expressed in nephelometric turbidity units (NTU). Chemical oxygen demand was determined according to Standard Methods (APHAAWWA-WEF, 1998). All experiments were run in duplicate (the accuracy is considered to be ±5%) and the mean value is presented herein. 3. Results and discussion 3.1. Ultrafiltration of common bean seed extract
The coagulation active components from common bean extract were further processed by ultrafiltration in Amicon stirred cell (model 8200, MilliporeTM ) with polyethersulfone membranes (Biomax, MilliporeTM ) having cut-off 10,000 and 30,000 Da. Ultrafiltration was performed under constant pressure of inert gas (2.5 bar) and gentle magnetic stirring (30 rpm) at constant room temperature (21 ◦ C). The flow rate through membranes with 10,000 and 30,000 Da cut-off was 13.5 and 35 mL/h, respectively. Fractionation of components was conducted using membranes in consecutive manner – ultrafiltration through lower cut-off membrane was followed by the one through membrane having higher used cut-off. In both steps ultrafiltration was running until volume of retentates reached 10% of initial volume. After each ultrafiltration step, volumes of retentates were adjusted to value of initial volume with 0.5 mol NaCl/L. Permeate obtained by ultrafiltration with membrane cut-off 10,000 was assigned as the 1st fraction, fraction containing components having approximate molecular weight between 10,000 and 30,000 Da was assigned as the 2nd fraction, and retentate from ultrafiltration with 30,000 cut-off as the 3rd fraction. 2.3. Preparation of turbid water Turbid water for coagulation tests was prepared by adding 1 g kaolin to 1 L tap water. The suspension was stirred for 1 h to achieve uniform dispersion of kaolin particles, and then it was allowed to remain for 24 h for completing hydration of the particles. This suspension was used as the stock suspension. Turbid water having 35 nephelometric turbidity units (NTU) was prepared by diluting of stock suspension to 1000 mL tap water just before the coagulation test. The initial pH of the synthetic water was adjusted to 9.0, 9.5 or 10.0 with 1 mol/L NaOH, in accordance to previous investigations ´ et al., 2005) just before experiments. (Sˇ ciban 2.4. Coagulation test Coagulation activity of fractions of natural coagulant prepared by ultrafiltration as well as crude extract was evaluated in jar tester VELP model FC6S. Samples were added to the beakers at different dosages (from 0.125 to 2.5 mL/L turbid water) and the content was
Our previous investigations have shown that proteins from common bean seed expressed coagulation activity (Antov et al., 2010). This was the reason for crude extract and fraction prepared by ultrafiltration to be characterized by measuring protein concentration although it is clear that they contained numerous water soluble components from the seed as well (Morales-de Leon et al., 2007). Results of ultrafiltration regarding protein concentration revealed that majority of extracted proteins was in the third fraction, 0.47 mg/mL, which mostly contained molecules having molecular weight above 30 kDa, while the concentration of proteins in the second fraction with molecules between approximately 10 kDa and 30 kDa was the lowest one, amounting 0.04 mg/mL. Protein concentration in crude extract and the first fraction were 0.86 mg/mL and 0.36 mg/mL, respectively. On the base of these results it can be calculated that content of proteins in the 1st and the 3rd ultrafiltration fractions amounted about 40% and 55% of total extracted protein, respectively. It is known that common bean (P. vulgaris) seed is a valuable source of protein which content is approximately 20–30%. The storage globulin phaseolin represents more than half of the total protein content in the seed and it is a trimer with subunit Mw ∼ 50 kDa, while albumin, prolamin and glutelin proteins represent the other seed protein fractions. According to obtained results distribution of proteins in fractions prepared by ultrafiltration corresponded in great extent to common bean proteins’ characteristics regarding both their molecular weights and content in seed (Morales-de Leon et al., 2007; Osborn et al., 1988; Montoya et al., 2010). 3.2. Turbidity removal in model water Efficiency of prepared ultrafiltration fractions in turbidity removal was investigated in model water at three pH values starting from the turbidity 35 NTU. Coagulation tests were conducted with different dosages of tested fractions and crude extract, and obtained results are shown in Fig. 1. Values of pH were chosen in accordance to our previous results which revealed the highest efficiency of natural coagulant from common bean seed in removal ´ of turbidity from synthetic water was attained at pH 9–11 (Sˇ ciban
50
M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52
60
60
Crude extract 50
pH 9.0 pH 9.5 pH 10.0
40
Coagulation activity (%)
Coagulation activity (%)
50
st
1 fraction
30
20
10
0
40
30
20
10
0 0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
Dosage (mL/L)
1.5
2.0
2.5
2.0
2.5
Dosage (mL/L)
60
60
2
nd
rd
fraction
3 fraction 50
Coagulation activity (%)
50
Coagulation activity (%)
1.0
40
30
20
10
40
30
20
10
0
0 0.0
0.5
1.0
1.5
2.0
2.5
Dosage (mL/L)
0.0
0.5
1.0
1.5
Dosage (mL/L)
Fig. 1. Coagulation activities of crude extract from common bean seed and fractions prepared by ultrafiltration in relation to coagulant dosages and at different pH values.
et al., 2005). Residual turbidity of the blank varied slightly within investigated pH range and its mean value was 23.4 ± 0.6 NTU. Crude extract from common bean seed as well all ultrafiltration fractions have shown differences in behavior when they were evaluated for turbidity removal regarding pH and dosages at which they expressed the highest obtained coagulation activities as well as the values of this highest activities. These differences might be explained by differences in kind and characteristics of biomolecules as well as by their content in fraction and crude extract. Proteins as well as other extracted molecules separated by ultrafiltration differ not only in molecular weight but e.g. in pI which may additionally affect coagulation as consequence of their charge at certain pH. However, it can be noticed that the higher efficiencies in turbidity removal generally were obtained at higher investigated pH values and that pH 9 was the least appropriate for common bean seed molecules to express their coagulation action. Crude extract has attained its highest coagulation activity, slightly above 50%, at dosage 1 mL/L and pH 9.5. In comparison to this result, highest obtained coagulation activities crude extract has reached at pH 9 and 10 were about 2.4 and 1.5 times lower, respectively. The highest obtained efficiency in turbidity removal
resulting in coagulation activity just below 50%, the 1st fraction has shown also at pH 9.5 but at double dosage in comparison to crude extract. In addition, at pH 10 this fraction containing compounds having molecular weight below 10,000 Da has also showed high coagulation activity in comparison to other results, 47.6% with relatively low dosage 0.5 mL/L. In comparison to crude extract and other two ultrafiltration fraction, the highest coagulation activity of the 2nd fraction was the lowest one, 45.3%. It was achieved at the highest investigated pH value, pH 10, which appeared to be the most appropriate for molecules contained in this fraction to act as turbidity removing agents. However, at the same conditions, this fraction exhibited the best performance regarding COD removal (see Table 1). As for the 3rd fraction containing biomolecules above 30 kDa and having the highest protein concentration, maximum of coagulation activity was recorded at pH 9.5 with relatively small volume of fraction added to turbid water, 0.25 mL/L. In comparison to this result, the highest coagulation activities this fraction has shown at pH 9 and pH 10 were about 2.4 and 1.4 times lower, respectively, which were achieved at higher dosage, 1 mL/L.
M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52 Table 1 COD values in water treated by crude extract from common bean seed or fractions prepared by ultrafiltration at conditions expressing their highest coagulation activities (COD for blank 2.02 mg O2 /L). Natural coagulant
Crude extract 1st fraction 2nd fraction 3rd fraction
Dosage (mL/L) 1.0 9.5 pH Coagulation activity (%) 50.6 COD (mg O2 /L) 3.74
2.0 9.5 49.1 3.33
1.0 10.0 45.3 1.84
0.25 9.5 47.7 2.32
Evaluation of crude extract of common bean and ultrafiltration fraction for turbidity removal revealed that the most efficient was the crude extract. Although lower than that of crude extract, the highest coagulation activities of all fractions were close to this highest obtained result. Efficiency of crude extract and fraction prepared by ultrafiltration in turbidity removal were lower than those for natural coagulants from the most efficient M. oleifera and other plant materials (Ndabigengensere and Narasiah, 1998; Diaz et al., 1999; Okuda et al., 2001; Sanchez-Martin et al., 2010). However, this lower efficiency can be also perceived from the aspect that it was evaluated at low water turbidity and considering that M. oleifera is also not efficient coagulant for low-turbidity water (Lea, 2010).
51
yielding in fractions that are efficient in turbidity removal giving, at the same time, diminished organic load.
4. Conclusion Evaluation of fractions obtained by ultrafiltration of crude extract of common bean seed for their suitability for turbidity removal revealed that they were efficient in it although their coagulation activities were slightly lower that of crude extract. Obtained fractions brought reduced organic load into the water after the treatment in comparison to crude extract or even diminished it in comparison to blank. According to these results ultrafiltration appeared to be promising technique for processing crude extract of common bean seed in respect of its simplicity in comparison to multistep isolation and purification procedures.
Acknowledgment The financial support from Ministry of Education and Science, Republic of Serbia (Project No. 31002) is greatly acknowledged.
3.3. Organic load in treated water
References
The crude extracts which are used as natural coagulants contain biomolecules and inorganic substances which may be released in water leading to increased COD (Ghebremichael et al., 2006). Besides, the use of natural coagulants may increase the organic load in water which may result in increased microbial activity (Ndabigengensere and Narasiah, 1998; Okuda et al., 2001). In addition, the organic matter might consume additional chlorine or other disinfectants in the water treatment plant and can acts as a precursor of byproducts during the disinfection process. In order to solve these problems and minimize the value of unnecessary organic material which might adversely affect quality of the water, purification of the active components is usually recommended (Okuda et al., 2001; Ghebremichael et al., 2006; Antov et al., 2010). Organic matter in water before and after coagulation tests was measured to establish the increase in organic load when crude extract and ultrafiltration fractions were added to turbid water at dosages exhibiting their highest coagulation activity. Results presented as relative increase in COD in comparison to blank (Table 1) have shown that the increase in organic matter that remained in water after coagulation was the highest when crude extract was used. Substantially lower relative increase in COD in comparison to that for crude extract was caused by the use of the 3rd fraction. Contrary to that, when the 2nd fraction was used for turbidity removal, COD was even decreased, relatively to blank, by 9%. Obtained results might be explained by lower proteins concentration in tested fraction because of their fractionation by ultrafiltration but also by fractionation of other compounds from crude sample which caused reduced organic load in comparison to crude extract. As for the 2nd fraction, it might be suggested that its composition as well as conditions for the removal of turbidity regarding pH and dosage were beneficial resulting in even lower chemical oxygen demand in comparison to blank. Obtained results might be promising in respect of obtaining natural coagulant that is suitable for application in turbidity removal regarding the problem of organic load. Instead of complicated and demanding multistep procedures of isolation and purification of coagulation active components which can be expensive and have low yield, a membrane separation technique can be applied
´ ´ N., 2010. Proteins from common bean (Phaseolus Antov, M., Sˇ ciban, M., Petrovic, vulgaris) seed as a natural coagulant for water turbidity removal. Bioresour. Technol. 100, 2167–2172. APHA-AWWA-WEF, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, American Water Works Association, Water Federation, Washington, DC, USA. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Diaz, A., Rincon, N., Escorihuela, A., Fernandez, N., Chacin, F., Forster, C.F., 1999. A preliminary evaluation of turbidity removal by natural coagulants indigenous to Venezuela. Process Biochem. 35, 391–395. Edzwald, K., 1993. Coagulation in drinking water treatment: particles, organics and coagulants. Water Sci. Technol. 27, 21–35. Flaten, T.P., 2001. Aluminium as a risk factor in Alzheimer’s disease, with emphasis on drinking water. Brain Res. Bull. 55, 187–196. Ghebremichael, K.A., Gunaratna, K.R., Dalhammar, G., 2006. Single-step ion exchange purification of the coagulant protein from Moringa oleifera seed. Appl. Microbiol. Biotechnol. 70, 526–532. Ghebremichael, K.A., Gunaratna, K.R., Henriksson, H., Brumer, H., Dalhammar, G., 2005. A simple purification and activity assay of the coagulant protein from Moringa oleifera seed. Water Res. 39, 2338–2344. Haaroff, J., Cleasby, J.L., 1988. Comparing aluminum and iron coagulants for in-line filtration of cold waters. J. Am. Water Works Assoc. 80, 168–175. Kang, M., Kamei, T., Magara, Y., 2003. Comparing polyaluminium chloride and ferric chloride for antimony removal. Water Res. 37, 4171–4179. Lea, M., 2010. Bioremediation of turbid surface water using seed extract from Moringa oleifera Lam. (Drumstick) tree. Curr. Protoc. Microbiol. 16, pp. 1G.2.1–1G.2.14. Miller, S., Fugate, E., Craver, V.O., Smith, J., Zimmerman, J., 2008. Toward understanding the efficacy and mechanism of Opuntia spp. as a natural coagulant for potential application in water treatment. Environ. Sci. Technol. 42, 4274–4279. Montoya, C.A., Lalles, J.-P., Beebe, S., Leterme, P., 2010. Phaseolin diversitu as a possible strategy to improve the nutritional value of common beans (Phaseolus vulgaris). Food Res. Int. 43, 443–449. Morales-de Leon, J., Vazquez-Mata, N., Torres, N., Gil-Zenteno, L., Bressani, R., 2007. Preparation and characterization of protein isolate from fresh and hardened beans (Phaseolus vulgaris L.). J. Food Sci. 72, 96–102. Narasiah, K., Vogel, S.A., Kramadhati, N.N., 2002. Coagulation of turbid waters using Moringa oleifera seeds from two distinct sources. Water Sci. Technol. 2, 83–88. Ndabigengensere, A., Narasiah, K.S., 1998. Quality of water treated by coagulation using Moringa oleifera seeds. Water Res. 32, 781–791. Okuda, T., Baes, A.U., Nishijima, W., Okada, M., 2001. Isolation and characterization of coagulant extracted from Moringa oleifera seed by salt solution. Water Res. 35, 405–410. Osborn, T.C., Burow, M., Bliss, F.A., 1988. Purification and characterization of arcelin seed protein from common bean. Plant Physiol. 86, 399–405.
52
M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52
Raghuwanshi, P.K., Mandloi, M., Sharma, A.J., Malviya, H.S., Chaudhari, S., 2002. Improving filtrate quality using agrobased materials as coagulant aids. Water Qual. Res. J. Can. 37, 745–756. Sanchez-Martin, J., Gonzalez-Velasco, M., Beltran-Heredia, J., 2010. Surface water treatment with tannin-based coagulants from Quebracho (Schinopsis balansae). Chem. Eng. J. 165, 851–858.
ˇ ´ ´ B., 2009. Removal of water turbidity by Sˇ ciban, M., Klaˇsnja, M., Antov, M., Skrbi c, natural coagulants obtained from chestnut and acorn. Bioresour. Technol. 100, 6639–6643. ´ ´ J., 2005. Investigation of coagulation activity Sˇ ciban, M., Klaˇsnja, M., Stojimirovic, of natural coagulants from seeds of different leguminose species. APTEFF 36, 81–87.
Contents lists available at SciVerse ScienceDirect
Ecological Engineering journal homepage: www.elsevier.com/locate/ecoleng
Short communication
Evaluation of the efficiency of natural coagulant obtained by ultrafiltration of common bean seed extract in water turbidity removal ´ Mirjana G. Antov ∗ , Marina B. Sˇ ciban, Jelena M. Prodanovic´ University of Novi Sad, Faculty of Technology, Blvd. Cara Lazara 1, 21000 Novi Sad, Serbia
a r t i c l e
i n f o
Article history: Received 9 March 2012 Received in revised form 6 July 2012 Accepted 10 August 2012 Available online 28 September 2012 Keywords: Natural coagulant Ultrafiltration Coagulation activity Organic load Common bean seed
a b s t r a c t Efficiency of natural coagulant obtained by ultrafiltration of common bean (Phaseolus vulgaris) seed extract in water turbidity removal was experimentally evaluated. Prepared ultrafiltration fractions have shown differences in behavior regarding pH values and dosages at which they expressed their highest efficiency in turbidity removal. The highest obtained coagulation activities were at pH 9.5 with crude extract and fraction containing molecules with molecular weight less than 10 kDa, 50.6% and 49.1%, respectively. The organic load that remained in water after treatment was reduced when natural coagulant processed by ultrafiltration instead of crude extract was applied. Moreover, fraction with molecules between 10 kDa and 30 kDa, decreased chemical oxygen demand by 9% relative to blank at conditions this fraction expressed its highest coagulation activity. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Water question is surely one of the main factors that are involved in the human development considering its influence on human lives. Among the new techniques for water and wastewater treatment there is the use of natural coagulants, aiming at a better quality of treated water by reducing the use of chemicals. Namely, coagulation/flocculation step which is essential process in the treatment of both surface water and industrial wastewater, includes removal of dissolved organic species and turbidity from water most commonly via addition of conventional chemicalbased coagulants – alum, ferric chloride and synthetic organic polymers. While the effectiveness of these chemicals as coagulants is well-recognized (Edzwald, 1993; Kang et al., 2003) there are, nonetheless, disadvantages associated with their usage such as ineffectiveness in low-temperature water (Haaroff and Cleasby, 1988), relatively high procurement costs, detrimental effects on human health, production of large sludge volumes and the fact that they significantly affect pH of treated water. There is also strong evidence linking aluminum-based coagulants to the development of Alzheimer’s disease in human beings (Flaten, 2001). It is therefore desirable to replace these chemical coagulants with plant-based coagulants to counteract the aforementioned drawbacks.
∗ Corresponding author. Tel.: +381 21 485 3647; fax: +381 21 450 413. E-mail address: [email protected] (M.G. Antov). 0925-8574/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ecoleng.2012.08.015
Thus, in water treatment, the use of natural coagulants could be an option with many advantages over chemical agents, particularly the biodegradability, low toxicity and low residual sludge production (Narasiah et al., 2002). Biopolymers may be of great interest since they are natural low-cost products, characterized by their environmental friendly behavior and usually have large number of surface charges that increase the efficiency of the coagulation process. These advantages are especially augmented if the plant from which the coagulant is extracted is indigenous to local community. In recent numerous studies variety of plant materials has been reported as a source of natural coagulants (Raghuwanshi ´ et al., 2009) et al., 2002; Diaz et al., 1999; Miller et al., 2008; Sˇ ciban but the most studied is Moringa oleifera whose efficiency has been approved for turbidity removal (Ndabigengensere and Narasiah, 1998; Okuda et al., 2001; Ghebremichael et al., 2006) as well as antimicrobial properties (Ghebremichael et al., 2005). During the course of plants’ screening program in our laboratory, crude extract from common bean (Phaseolus vulgaris) seed was applied for water turbidity removal while partially purified coagulant has shown high coagulation efficiency followed by low organic load (Antov et al., 2010). Seed of common bean has food grade nature and contains no oil, so, delipidation step is not necessary which is beneficial for both economic and environmental reasons. Considering this common bean is a promising source of natural coagulant for water treatment. The objective of the study was to experimentally evaluate the efficiency of different fractions prepared by ultrafiltration of common bean seed extract in water turbidity removal. Membrane
M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52
49
separation technique was applied instead of isolation and purification multiple steps procedure to examine its suitability for the processing of natural coagulant. Coagulation efficiencies of prepared fractions were measured at different pH values and dosages, and have been compared to the one of crude extract. In addition, suitability of prepared natural coagulants was tested in respect of organic load that remained in water after treatment.
stirred at 200 rpm for 1 min. The mixing speed was then reduced to 60 rpm and was kept for 30 min. Then, the suspensions were left to allow sedimentation. After 1 h of sedimentation, upper clarified liquid was collected and residual turbidity was measured. The residual turbidity of sample was RTS . The same coagulation test was performed with no coagulant as the blank. The residual turbidity in the blank was RTB . Coagulation activity was calculated as:
2. Materials and methods
Coagulation activity (%) =
2.1. Extraction of active components from common bean seed The locally obtained common bean (P. vulgaris) dry seed was ground to a fine powder by using a laboratory mill and sieved through 0.4 mm sieve. The fraction with particle size less than 0.4 mm was used in experiments. Ten grams of seed powder was suspended in 1 L of NaCl water solution (0.5 mol/L). The suspension was stirred using a magnetic stirrer for 10 min to accomplish extraction and then filtered through a rugged filter paper (Macherey-Nagel, MN 651/120) to obtain filtrate – crude extract of active components. 2.2. Preparation of fractions of natural coagulant by ultrafiltration
RTB − RTS · 100 RTB
(1)
2.5. Analytical methods Protein concentration was measured according to Bradford (1976) with bovine serum albumin as standard. Turbidity was measured using a turbidimeter (WTW TURB 550/550 IR) and it was expressed in nephelometric turbidity units (NTU). Chemical oxygen demand was determined according to Standard Methods (APHAAWWA-WEF, 1998). All experiments were run in duplicate (the accuracy is considered to be ±5%) and the mean value is presented herein. 3. Results and discussion 3.1. Ultrafiltration of common bean seed extract
The coagulation active components from common bean extract were further processed by ultrafiltration in Amicon stirred cell (model 8200, MilliporeTM ) with polyethersulfone membranes (Biomax, MilliporeTM ) having cut-off 10,000 and 30,000 Da. Ultrafiltration was performed under constant pressure of inert gas (2.5 bar) and gentle magnetic stirring (30 rpm) at constant room temperature (21 ◦ C). The flow rate through membranes with 10,000 and 30,000 Da cut-off was 13.5 and 35 mL/h, respectively. Fractionation of components was conducted using membranes in consecutive manner – ultrafiltration through lower cut-off membrane was followed by the one through membrane having higher used cut-off. In both steps ultrafiltration was running until volume of retentates reached 10% of initial volume. After each ultrafiltration step, volumes of retentates were adjusted to value of initial volume with 0.5 mol NaCl/L. Permeate obtained by ultrafiltration with membrane cut-off 10,000 was assigned as the 1st fraction, fraction containing components having approximate molecular weight between 10,000 and 30,000 Da was assigned as the 2nd fraction, and retentate from ultrafiltration with 30,000 cut-off as the 3rd fraction. 2.3. Preparation of turbid water Turbid water for coagulation tests was prepared by adding 1 g kaolin to 1 L tap water. The suspension was stirred for 1 h to achieve uniform dispersion of kaolin particles, and then it was allowed to remain for 24 h for completing hydration of the particles. This suspension was used as the stock suspension. Turbid water having 35 nephelometric turbidity units (NTU) was prepared by diluting of stock suspension to 1000 mL tap water just before the coagulation test. The initial pH of the synthetic water was adjusted to 9.0, 9.5 or 10.0 with 1 mol/L NaOH, in accordance to previous investigations ´ et al., 2005) just before experiments. (Sˇ ciban 2.4. Coagulation test Coagulation activity of fractions of natural coagulant prepared by ultrafiltration as well as crude extract was evaluated in jar tester VELP model FC6S. Samples were added to the beakers at different dosages (from 0.125 to 2.5 mL/L turbid water) and the content was
Our previous investigations have shown that proteins from common bean seed expressed coagulation activity (Antov et al., 2010). This was the reason for crude extract and fraction prepared by ultrafiltration to be characterized by measuring protein concentration although it is clear that they contained numerous water soluble components from the seed as well (Morales-de Leon et al., 2007). Results of ultrafiltration regarding protein concentration revealed that majority of extracted proteins was in the third fraction, 0.47 mg/mL, which mostly contained molecules having molecular weight above 30 kDa, while the concentration of proteins in the second fraction with molecules between approximately 10 kDa and 30 kDa was the lowest one, amounting 0.04 mg/mL. Protein concentration in crude extract and the first fraction were 0.86 mg/mL and 0.36 mg/mL, respectively. On the base of these results it can be calculated that content of proteins in the 1st and the 3rd ultrafiltration fractions amounted about 40% and 55% of total extracted protein, respectively. It is known that common bean (P. vulgaris) seed is a valuable source of protein which content is approximately 20–30%. The storage globulin phaseolin represents more than half of the total protein content in the seed and it is a trimer with subunit Mw ∼ 50 kDa, while albumin, prolamin and glutelin proteins represent the other seed protein fractions. According to obtained results distribution of proteins in fractions prepared by ultrafiltration corresponded in great extent to common bean proteins’ characteristics regarding both their molecular weights and content in seed (Morales-de Leon et al., 2007; Osborn et al., 1988; Montoya et al., 2010). 3.2. Turbidity removal in model water Efficiency of prepared ultrafiltration fractions in turbidity removal was investigated in model water at three pH values starting from the turbidity 35 NTU. Coagulation tests were conducted with different dosages of tested fractions and crude extract, and obtained results are shown in Fig. 1. Values of pH were chosen in accordance to our previous results which revealed the highest efficiency of natural coagulant from common bean seed in removal ´ of turbidity from synthetic water was attained at pH 9–11 (Sˇ ciban
50
M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52
60
60
Crude extract 50
pH 9.0 pH 9.5 pH 10.0
40
Coagulation activity (%)
Coagulation activity (%)
50
st
1 fraction
30
20
10
0
40
30
20
10
0 0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
Dosage (mL/L)
1.5
2.0
2.5
2.0
2.5
Dosage (mL/L)
60
60
2
nd
rd
fraction
3 fraction 50
Coagulation activity (%)
50
Coagulation activity (%)
1.0
40
30
20
10
40
30
20
10
0
0 0.0
0.5
1.0
1.5
2.0
2.5
Dosage (mL/L)
0.0
0.5
1.0
1.5
Dosage (mL/L)
Fig. 1. Coagulation activities of crude extract from common bean seed and fractions prepared by ultrafiltration in relation to coagulant dosages and at different pH values.
et al., 2005). Residual turbidity of the blank varied slightly within investigated pH range and its mean value was 23.4 ± 0.6 NTU. Crude extract from common bean seed as well all ultrafiltration fractions have shown differences in behavior when they were evaluated for turbidity removal regarding pH and dosages at which they expressed the highest obtained coagulation activities as well as the values of this highest activities. These differences might be explained by differences in kind and characteristics of biomolecules as well as by their content in fraction and crude extract. Proteins as well as other extracted molecules separated by ultrafiltration differ not only in molecular weight but e.g. in pI which may additionally affect coagulation as consequence of their charge at certain pH. However, it can be noticed that the higher efficiencies in turbidity removal generally were obtained at higher investigated pH values and that pH 9 was the least appropriate for common bean seed molecules to express their coagulation action. Crude extract has attained its highest coagulation activity, slightly above 50%, at dosage 1 mL/L and pH 9.5. In comparison to this result, highest obtained coagulation activities crude extract has reached at pH 9 and 10 were about 2.4 and 1.5 times lower, respectively. The highest obtained efficiency in turbidity removal
resulting in coagulation activity just below 50%, the 1st fraction has shown also at pH 9.5 but at double dosage in comparison to crude extract. In addition, at pH 10 this fraction containing compounds having molecular weight below 10,000 Da has also showed high coagulation activity in comparison to other results, 47.6% with relatively low dosage 0.5 mL/L. In comparison to crude extract and other two ultrafiltration fraction, the highest coagulation activity of the 2nd fraction was the lowest one, 45.3%. It was achieved at the highest investigated pH value, pH 10, which appeared to be the most appropriate for molecules contained in this fraction to act as turbidity removing agents. However, at the same conditions, this fraction exhibited the best performance regarding COD removal (see Table 1). As for the 3rd fraction containing biomolecules above 30 kDa and having the highest protein concentration, maximum of coagulation activity was recorded at pH 9.5 with relatively small volume of fraction added to turbid water, 0.25 mL/L. In comparison to this result, the highest coagulation activities this fraction has shown at pH 9 and pH 10 were about 2.4 and 1.4 times lower, respectively, which were achieved at higher dosage, 1 mL/L.
M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52 Table 1 COD values in water treated by crude extract from common bean seed or fractions prepared by ultrafiltration at conditions expressing their highest coagulation activities (COD for blank 2.02 mg O2 /L). Natural coagulant
Crude extract 1st fraction 2nd fraction 3rd fraction
Dosage (mL/L) 1.0 9.5 pH Coagulation activity (%) 50.6 COD (mg O2 /L) 3.74
2.0 9.5 49.1 3.33
1.0 10.0 45.3 1.84
0.25 9.5 47.7 2.32
Evaluation of crude extract of common bean and ultrafiltration fraction for turbidity removal revealed that the most efficient was the crude extract. Although lower than that of crude extract, the highest coagulation activities of all fractions were close to this highest obtained result. Efficiency of crude extract and fraction prepared by ultrafiltration in turbidity removal were lower than those for natural coagulants from the most efficient M. oleifera and other plant materials (Ndabigengensere and Narasiah, 1998; Diaz et al., 1999; Okuda et al., 2001; Sanchez-Martin et al., 2010). However, this lower efficiency can be also perceived from the aspect that it was evaluated at low water turbidity and considering that M. oleifera is also not efficient coagulant for low-turbidity water (Lea, 2010).
51
yielding in fractions that are efficient in turbidity removal giving, at the same time, diminished organic load.
4. Conclusion Evaluation of fractions obtained by ultrafiltration of crude extract of common bean seed for their suitability for turbidity removal revealed that they were efficient in it although their coagulation activities were slightly lower that of crude extract. Obtained fractions brought reduced organic load into the water after the treatment in comparison to crude extract or even diminished it in comparison to blank. According to these results ultrafiltration appeared to be promising technique for processing crude extract of common bean seed in respect of its simplicity in comparison to multistep isolation and purification procedures.
Acknowledgment The financial support from Ministry of Education and Science, Republic of Serbia (Project No. 31002) is greatly acknowledged.
3.3. Organic load in treated water
References
The crude extracts which are used as natural coagulants contain biomolecules and inorganic substances which may be released in water leading to increased COD (Ghebremichael et al., 2006). Besides, the use of natural coagulants may increase the organic load in water which may result in increased microbial activity (Ndabigengensere and Narasiah, 1998; Okuda et al., 2001). In addition, the organic matter might consume additional chlorine or other disinfectants in the water treatment plant and can acts as a precursor of byproducts during the disinfection process. In order to solve these problems and minimize the value of unnecessary organic material which might adversely affect quality of the water, purification of the active components is usually recommended (Okuda et al., 2001; Ghebremichael et al., 2006; Antov et al., 2010). Organic matter in water before and after coagulation tests was measured to establish the increase in organic load when crude extract and ultrafiltration fractions were added to turbid water at dosages exhibiting their highest coagulation activity. Results presented as relative increase in COD in comparison to blank (Table 1) have shown that the increase in organic matter that remained in water after coagulation was the highest when crude extract was used. Substantially lower relative increase in COD in comparison to that for crude extract was caused by the use of the 3rd fraction. Contrary to that, when the 2nd fraction was used for turbidity removal, COD was even decreased, relatively to blank, by 9%. Obtained results might be explained by lower proteins concentration in tested fraction because of their fractionation by ultrafiltration but also by fractionation of other compounds from crude sample which caused reduced organic load in comparison to crude extract. As for the 2nd fraction, it might be suggested that its composition as well as conditions for the removal of turbidity regarding pH and dosage were beneficial resulting in even lower chemical oxygen demand in comparison to blank. Obtained results might be promising in respect of obtaining natural coagulant that is suitable for application in turbidity removal regarding the problem of organic load. Instead of complicated and demanding multistep procedures of isolation and purification of coagulation active components which can be expensive and have low yield, a membrane separation technique can be applied
´ ´ N., 2010. Proteins from common bean (Phaseolus Antov, M., Sˇ ciban, M., Petrovic, vulgaris) seed as a natural coagulant for water turbidity removal. Bioresour. Technol. 100, 2167–2172. APHA-AWWA-WEF, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, American Water Works Association, Water Federation, Washington, DC, USA. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Diaz, A., Rincon, N., Escorihuela, A., Fernandez, N., Chacin, F., Forster, C.F., 1999. A preliminary evaluation of turbidity removal by natural coagulants indigenous to Venezuela. Process Biochem. 35, 391–395. Edzwald, K., 1993. Coagulation in drinking water treatment: particles, organics and coagulants. Water Sci. Technol. 27, 21–35. Flaten, T.P., 2001. Aluminium as a risk factor in Alzheimer’s disease, with emphasis on drinking water. Brain Res. Bull. 55, 187–196. Ghebremichael, K.A., Gunaratna, K.R., Dalhammar, G., 2006. Single-step ion exchange purification of the coagulant protein from Moringa oleifera seed. Appl. Microbiol. Biotechnol. 70, 526–532. Ghebremichael, K.A., Gunaratna, K.R., Henriksson, H., Brumer, H., Dalhammar, G., 2005. A simple purification and activity assay of the coagulant protein from Moringa oleifera seed. Water Res. 39, 2338–2344. Haaroff, J., Cleasby, J.L., 1988. Comparing aluminum and iron coagulants for in-line filtration of cold waters. J. Am. Water Works Assoc. 80, 168–175. Kang, M., Kamei, T., Magara, Y., 2003. Comparing polyaluminium chloride and ferric chloride for antimony removal. Water Res. 37, 4171–4179. Lea, M., 2010. Bioremediation of turbid surface water using seed extract from Moringa oleifera Lam. (Drumstick) tree. Curr. Protoc. Microbiol. 16, pp. 1G.2.1–1G.2.14. Miller, S., Fugate, E., Craver, V.O., Smith, J., Zimmerman, J., 2008. Toward understanding the efficacy and mechanism of Opuntia spp. as a natural coagulant for potential application in water treatment. Environ. Sci. Technol. 42, 4274–4279. Montoya, C.A., Lalles, J.-P., Beebe, S., Leterme, P., 2010. Phaseolin diversitu as a possible strategy to improve the nutritional value of common beans (Phaseolus vulgaris). Food Res. Int. 43, 443–449. Morales-de Leon, J., Vazquez-Mata, N., Torres, N., Gil-Zenteno, L., Bressani, R., 2007. Preparation and characterization of protein isolate from fresh and hardened beans (Phaseolus vulgaris L.). J. Food Sci. 72, 96–102. Narasiah, K., Vogel, S.A., Kramadhati, N.N., 2002. Coagulation of turbid waters using Moringa oleifera seeds from two distinct sources. Water Sci. Technol. 2, 83–88. Ndabigengensere, A., Narasiah, K.S., 1998. Quality of water treated by coagulation using Moringa oleifera seeds. Water Res. 32, 781–791. Okuda, T., Baes, A.U., Nishijima, W., Okada, M., 2001. Isolation and characterization of coagulant extracted from Moringa oleifera seed by salt solution. Water Res. 35, 405–410. Osborn, T.C., Burow, M., Bliss, F.A., 1988. Purification and characterization of arcelin seed protein from common bean. Plant Physiol. 86, 399–405.
52
M.G. Antov et al. / Ecological Engineering 49 (2012) 48–52
Raghuwanshi, P.K., Mandloi, M., Sharma, A.J., Malviya, H.S., Chaudhari, S., 2002. Improving filtrate quality using agrobased materials as coagulant aids. Water Qual. Res. J. Can. 37, 745–756. Sanchez-Martin, J., Gonzalez-Velasco, M., Beltran-Heredia, J., 2010. Surface water treatment with tannin-based coagulants from Quebracho (Schinopsis balansae). Chem. Eng. J. 165, 851–858.
ˇ ´ ´ B., 2009. Removal of water turbidity by Sˇ ciban, M., Klaˇsnja, M., Antov, M., Skrbi c, natural coagulants obtained from chestnut and acorn. Bioresour. Technol. 100, 6639–6643. ´ ´ J., 2005. Investigation of coagulation activity Sˇ ciban, M., Klaˇsnja, M., Stojimirovic, of natural coagulants from seeds of different leguminose species. APTEFF 36, 81–87.