Biotechnol. frog. 1990, 6, 2-6
2
ARTICLES Chitosan-Poly(acry1ic acid): Mechanism of Complex Formation and Potential Industrial Applications Visith Chavasit and J. Antonio Torres* Department of Food Science and Technology, Oregon State University, Corvallis, Oregon 97331-6602
The food industry is interested in polyelectrolytic coagulants of natural origin for the clarification of food beverages and the recovery of colloidal and dispersed particles from processing waste streams. This paper discusses potential industrial applications of recent findings on polymer complex formation obtained with a chitosanpoly(acry1ic acid) model system. Process recommendations could be made on the basis of the ionic strength, pH, and charged group concentration of the fluid to be treated. Ionic strength does not affect the complex formation process. The amount of chitosan in the complex formed is controlled by the solution pH. The mechanism of complex formation indicates that pH measurements could be used to monitor the coagulation process.
Introduction Chitosan consists of unbranched chains of &(1-.4)-2amino-2-deoxy-D-glucanresidues (Figure 1). It is obtained by deacetylation of chitin, which is present in marine invertebrates, insects, fungi, and yeasts.' Chitosan is also found in various fungi.zg Thus, chitin and chitosan are, at least to some small extent, part of our food supply. Chitin and chitosan are obtained industrially from shellfish-processing waste (Bioshell Products, Albany, O R Protan,Inc., Raymond, WA; Kyowa Oil and Fat, Tokyo, Japan; Kyokuyo Co., Tokyo, Japan). The total annual global estimates of accessible chitin amount to 150 X lo6 kga4+ Despite the quantitative importance of chitin and chitosan, only limited attention has been given to its applications. This is especially true for food applications.' As reviewed by Knorr,' the three key future applications of chitosan in the food industry are its use (1)as a flocculation agent, (2) as a functional food ingredient, and (3) as a new polymer for the formation of a matrix with unique properties. The use of chitosan to prepare edible coatings which control diffusion of preservatives applied on food surfaces has recently been examined in our l a b ~ r a t o r y .The ~ complex formation process between chitosan and polyanions, which could be used to design improved systems for the recovery of proteins and other bioproducts, has also been the subject of research in our laboratory.' During the past decade, increasing attention has been given to polyelectrolytic coagulants of natural origin to aid the separation of colloidal and dispersed particles from food-processing waste^.^"^ Chitosan, the polycationic carbohydrate polymer,has been found to be particularly effective in aiding the coagulation of protein from food-processing Examples reported in the literature of biomass recovery from food-processing waste have ranged from 70% to 97%.' Undoubtedly, it is possible to find synthetic polymers that perform as well or better than chitosan. The difference is that it should be possible to use chitosan-coagulated byproducts recovered from food-processing waste as a feed ingredient.
Chitusan toxicity studies with animal models have shown no physiological effects.'3914 For example, chitosanprotein complexes containing up to 5% chitosan fed to rats for 6 weeks resulted in insignificant differences in growth rate, blood, or liver compared to control groups. This is 10-20 times the levels expected in feeding coagulated byproducts to animals.15 However, current U.S. regulations allow only up to 0.1% chitosan in livestock feed.16 Treatment of the effluent from food-processing plants is another approved use in the U.S.17 Chitosan for direct use in foods has already been approved in Japan,"where it is incorporated into several products, including noodles and breadsticks. European beer and wine makers use it as a clarifying agent.17"' Polyelectrolyte complex formation between chitosan and polyanions such as alginates,lg esterified algin a t e ~ , sodium '~ (carboxymethyl)cellulose,20heparin,'l and acidic glycosaminoglycansz2has been previously reported. In this paper, we review the potential applications of model studies conducted in our laboratory to characterize the effect of pH, ionic strength, and mixing ratio on chitosan-poly(acry1ic acid) complex formation. Poly(acry1ic acid) has the experimental advantage of its very simple structure (Figure 1). Particular attention is given to industrial food processes such as beverage clarification, wastewater treatment, and biomass recovery from foodprocessing waste. The formation and potential industrial applications of chitosan-alginate coacervate capsules has been recently reviewed by Daly and Knorr.lg Such capsules are mechanically strong and stable in a wide pH range.lQ Information on the mechanism formation process for chitosan-poly(acry1ic acid) complexes shows that chitosan-poly(acry1ic acid) complexes could also be used for microencapsulation purposes as well. Materials a n d Methods Materials. Chitosan (CHI, Lot 5112A) was purchased from Bioshell Inc., Albany, OR. To obtain a higher purity material, it was first dissolved in 0.1 N HC1, filtered through a medium-porosity fritted disk Buchner-
8756-7938/90/3006-0002$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers
Biofechnol. Pmg., 1990,Vol. 6, No. 1
3
a. TURBIDITY MEASUREMENTS opH-3 ~ p H = 4
0 2.0
QoQo
L
U
m m
U
0.0 0.0
I b.
CHITOSAN
CH2
0.2
INSOLUBLE
0.4
0.6
COMPLEX
0.8
opH=3
- CH -
n
POLYACRYLIC ACID Figure 1. Molecular structures on chitin, chitosan, and poly-
(acrylic acid).
0.0
0.2
0.4
0.6
0.8
M I X I N G RATIO. [ A / ( A t B > I type filtration funnel, reprecipitated with NaOH, rinsed with deionized water, and finally freeze-dried. The molecular weight of CHI (220000) was determined at 25 "C with a Cannon-Fenske viscometer and following the procedures reviewed by Kien~le-Sterzer.~~ CHI was dissolved in a solution of 27.5 g of NaCl in 1000 mL of 1% acetic acid. The molecular weight of poly(acry1ic acid) (PAA, Aldrich, Milwaukee, WI) was estimated to be 202 000 using dioxane as the solvent.24 Complex Formation. CHI (0.1 g) and PAA (0.1 g) were dissolved in 100 mL of HC1 and 100 mL of NaCl solutions, respectively. The ionic strength, 0.025-0.300, was varied by adjusting the concentration of the HC1 and NaCl solutions. No complex can be formed at pH 2.s Nagasawa et al.25have shown that at this pH the PAA does not have a charge density sufficiently high to form a complex with chitosan. Since chitosan is insoluble at pH values higher than 6, experiments could be conducted only in the pH 3-6 range. The pH of both reactants was adjusted by using HC1 or NaOH solution. The pH was measured with a combination pH electrode (Ross Model 81550) and read to 0.001 pH unit on a microprocessor pH/millivolt meter (Orion Model 811). The amounts of added pH-adjusting solutions were recorded to determine the final reactant concentrations. Reactant solutions with equal pH values were mixed in 5-mL increments in volumetric proportions (milliliters of CH1:milliliters of PAA) ranging from 0:40 to 40:O. A mixing ratio (MR) was defined as
A A+B where A = weight of chitosan/MW of chitosan monomer and B = weight of poly(acry1ic acid)/MW of poly(acrylic acid) monomer. The mixture was shaken vigorously and left for 15 min before measuring the turbidity in a Varian DMS 80 UV/ visible spectrophotometer (absorbance at 420 nm). MR=-
Figure 2. Complex formation as a function of polymer mixing ratio and initial pH (ionic strength = 0.3). (a) Turbidity measurements (420 nm). (b) Insoluble complex weight.
Complex Analysis. The insoluble complex was separated by centrifugation at 34800g for 40 min. The pellet was twice resuspended in distilled water and then recentrifuged. The washed complex was finally freeze-dried and weighed. The pH of the supernatant was recorded, and the CHI concentration was measured by using the Nessler reagent method.26 A material balance was used to calculate the amount of PAA left in the supernatant.
Results and Discussion Turbidity is a simple indicator for complex formation but cannot always be used to quantitate the amount of complex formed. Some complex formation conditions result in sedimentation and lower the expected turbidity of the mixture. For instance, measurements of mixtures at pH 5 (ionic strength = 0.3) show two turbidity maxima (MR = 0.56 and 0.30, Figure 2a) while missing the true maximum measured by insoluble complex formation (MR = 0.41, Figure 2b). This observation highlights how easily a complex can be removed from the solution and explains why one of the most promising chitosan industrial applications is its use as a natural flocculating agent. However, as noted by Chavasit et a1.: future model studies are needed to characterize these chitosan-poly(acrylic acid) complexes. Another problem of turbidity determinations is that they are affected by particle size. As will be shown later, the complex composition (chitosan-to-poly(acry1ic acid) ratio) is a function of the pH of the solution. Thus, it can be expected that the complex size will depend upon the solution pH. The amount of complex formed at a given initial pH was the same for all ionic strength values (0.025-0.300) used in this study (Figure 3). This finding has practical
4
Biotechnol. Prog., 1990,Vol. 6, No. 1
l
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Figure 3. Complex formation as a function of polymer mixing ratio, initial pH, and various ionic strengths. (a) Initial pH = 3. (b) Initial pH = 4. ( c ) Initial pH = 5. (d) Initial pH = 6.
value since the ionic concentration of industrial waste streams can vary widely. Measurements of pH have been used to investigate the complex formation mechanism and are confirmed by quantitative and IR analyses.' At pH 3, 4, and 5, the degree of ionization of chitosan is about 1.0, 0.95, and 0.85, re~pectively.~~ A t the same conditions, the degree of ionization of poly(acry1ic acid) is about 0.1,0.2, and 0.5, re~ p e c t i v e l y .In ~ ~other words, in the 3-5 pH range, most of the CHI amine groups are in the NH3+ form, while most of the PAA carboxyl groups are in the COOH form. This sug ested the following complex formation mechanism:1
+ HOOC (CHI) + (PAA) NH;
--
NH;-OOC
+ Hf
-
(complex) pH1 (1) At pH = 6, the degree of ionization of chitosan is reduced to about 0.6,23 while that of PAA is about 0.8;25i.e., most of the amine groups are in the NH2 form, while most of the PAA carboxyl groups are in the COO- form. This suggested the following complex formation mechanism:' +H+
NH,
+ -0OC -,NH;-OOC
-
-
(CHI) + (PAA) (complex) pHf (2) Equation 1suggests that complex formation a t low initial pH values should lower the supernatant pH, while eq 2 suggests that the opposite behavior should be observed a t high initial pH values. Supernatant pH determinations were consistent with this expected behavior (Figure 4). The complex formation effect on supernatant pH suggests that pH measurements could be used in industrial processes to monitor the flocculation rate.
The supernatant pH changes are not only a function of the initial pH conditions and the amount of complex formed but also of the buffering properties of the excess reactant left in the supernatant. Due to differences in the CHI and PAA buffering capacities, their amount present in the supernatant affects the change in the supernatant pH. For example, at initial pH 4 conditions, the supernatant pH values after formation of the same complex amount (20 mg) were different depending upon the mixing ratio condition (pH = 3.95 for MR 0.08 and pH = 3.6 for MR 0.58). This was due to the difference in the excess reactant remaining in the supernatant. A similar situation was observed for initial pH 6 conditions (Figure 4b). The effect of pH on the mixing ratio for maximum insoluble complex formation was also studied using the analysis data for the supernatant fraction (Figure 5). The thick vertical lines indicate the MR for maximum insoluble complex formation, MR,, = 0.12, 0.29, 0.42, and 0.55 for initial reaction mixture pH = 3,4,5, and 6, respectively (Figure 2). Composition analysis of the supernatant after complex formation showed that at each of these MR,,, and pH value combinations the solution contains only trace amounts of CHI and PAA. A t MR # MR,,,, the solution assays showed an excess of one of the reactants, which increased in the direction indicated by the arrows, and only trace amounts of the other reagent (Figure 5). The observation that no excess reactants were left in the supernatant a t the mixing ratio for maximum insoluble complex formation has particular significance for applications such as beverage clarification. It would facilitate the approval of regulatory agencies for these applications since only trace amounts of chitosan would be
-
-
5
Biotechnol. frog., 1990, Vol. 6,No. 1 50
77
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MIXING R A T I O . [ f l / ( f l t E ) l Figure 4. Confirmation of complex formation mechanism: supernatant pH measurements. (a) Initial pH = 4. (b) Initial pH = 6. (0) Insoluble complex; (A)supernatant pH.
-
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Conclusions Polyelectrolytic coagulants of natural origin, such as chitosan, should facilitate beverage clarification processes and the recovery of colloidal and dispersed particles from processing waste streams. Furthermore, an understanding of the complex formation process can be used to identify process control strategies (e.g., monitoring supernatant pH values). Initial pH conditions determine the composition of the recovered byproducts. This information could be used to obtain byproducts with desirable properties. Future studies will be conducted to further characterize the chitosan-poly(acry1ic acid) complex. The physical and chemical stability, the rheological properties, and the charge density of chitosan-polyanionic complexes need to be quantified. Of particular interest would be the analysis of the interaction of these complexes with proteins and polysaccharides of industrial interest.
3 -I
u
remain in solution. This finding suggests that pH adjustment could be used to control the chitosan concentration of the coagulated byproducts to be recovered from food-processing wastes. This would be particularly valuable if the objective is to use these recovered byproducts as an animal feed ingredient.
u
7 g
I .o
MIXING RATIO, (A/AtBl Figure 5. Confirmation of complex formation mechanism: analysis of supernatant composition.
left in the solution while achieving a high level of clarification. However, for feed applications of chitosan-coagulated solids derived from wastewater, the major concerns of regulatory agencies are the amount of chitosan in the feed as well as other substances possibly derived from the wastewater. These include metals, pesticides, and any other possible hazardous substance of concern to animal and human toxi~ologists.~~ Further analysis of Figure 5 suggests that the complex composition a t a given pH is constant and equal to the MR,,, value for that pH condition. Excess reactants
(1) Knorr, D. Use of chitonous polymers in food. Food Technol. 1984, 38, 85. (2) Austin, P. R.; Brine, C. J.; Castle, J. E.; Zikakis, J. P. Chitin: New facets of research. Science 1981, 212, 749. (3) Rudall, K. M. Chitin and its association with other molecules. J . Polym. Sci. 1969, 28, 83. (4) Swanson, G. R.; Dudley, E. G.; Williamson, K. J. The use of
fish and shellfish wastes as fertilizers and feedstuffs. In Handbook of Organic Waste Conversion; Bewick, M. W. M., Ed.; Van Nostrand Reinhold: New York, 1980. (5) Revah-Moiseev, S.; Carroad, A. Conversion of enzymatic hydrolysateofshellfishwastechitintosingle-cell protein. Biotechnol. Bioeng. 1981,23, 1067. (6) Allan, G. G.; Fox, J. R.; Kong, N. A critical evaluation of the potential sources of chitin and chitosan. In Proceedings of the 1st National Conference on ChitinlChitosan; Muzzarelli, R. A. A., Pariser, E. R., Eds.; MIT Sea Grant Program: Cambridge, MA, 1978. (7) Vojdani, F.; Torres, J. A. Potassium sorbate permeability of polysaccharide films: chitosan, methylcellulose and hydroxypropyl methylcellulose. J. Food Proc. Eng., in press. (8) Chavasit, V.; Kienzle-Sterzer, C.; Torres, J. A. Formation and characterization of an insoluble polyelectrolyte complex: chitosan-polyacrylic acid. Polym. Bull. (Berlin) 1988, 19, 223. (9) Green, J. H.; Kramer, A. Food processing waste management; AVI Publishing Co.: Westport, CT, 1979. (10) Kargi, F.; Shuler, M. L. An evaluation of various flocculants for the recovery of biomass grown on poultry waste. In Agricultural Wastes;Miles, T. R., Ed.; Applied Science Publishers Ltd.: London, 1980. (11)Bough, W. A. Chitosan-a polymer from seafood waste for use in treatment of food processing wastes and activated sludge. Process Biochem. 1976,11, 13. (12) Fugita, T. Recovery of proteins. Japan Patent No. 01,633, 1972. (13) Arai, K.; Kinumari, T.; Fujita, T. On the toxicity of chitosan. Bull. Tokai Reg. Fish. Lab. 1968, No. 56, 889. (14) Landes, D. R.; Bough, W. A. Effects of chitosan-a coag-
ulating agent for food processing wastes- in diet of rats on growth and liver and blood composition. Bull. Environ. Contam. Toxicol. 1976, 15, 555. (15) Bough, W. A.; Landes, D. R. Treatment of food processing wastes with chitosan and nutritional evaluation of coagulated by-products. In Proceedings of the 1st International
6
Biotechnol. Prog., 1990, Vol. 6, No. 1
Conference on ChitinlChitosan; Muzzarelli, R. A. A., Pariser, E. R., Eds.; MIT Sea Grant Program: Cambridge, MA, 1978. (16) Publication PLI-002, Revision 8/17/87; Protan, Inc.: Commack, NY, 1987. (17) Hart, R. Stable protein foams: a new technology with new uses. Prep. Foods 1989,158 (6), 71. (18) Fisher, D. Shell game. Food Bus. 1989, May 22, 27. (19) Daly, M. M.; Knorr, D. Chitosan-alginate complex coacervate capsules: Effects of calcium chloride, plasticizers, and polyelectrolytes on mechanical stability. Biotechnol. Prog. 1988, 4, 76. (20) Fukuda, H. Polyelectrolyte complexes of sodium carboxymethylcellulose with chitosan. Makromol. Chem. 1979, 180, 1631-1633. (21) Kikuchi, Y.; Noda, A. Polyelectrolytic complexes of heparin with chitosan. J. Appl. Polym. Sci. 1976, 20, 2561. (22) Hirano, S.;Mizutani, C.; Yamaguchi, R.; Miura, 0. Forma-
tion of the polyelectrolyte complexes of some acidic glycosaminoglycans with partially N-acylated chitosans. Biopolymers 1978,17,805-810.
(23) Kienzle-Sterzer, C. A. Hydrodynamic behavior of a cat-
ionic polyelectrolyte. Ph.D. Thesis, Massachusetts Institute of Technology, Cambridge, 1984. (24) Sutterlin, N. Concentration dependence of the viscosity of dilute polymer solutions Huggins and Schulz-Blasehke coefficients. In Polymer Handbook, 2nd ed.; Brandrup, J., Immergut, E. H., Eds.; John Wiley & Sons: New York, 1975. (25) Nagasawa, M.; Murase, T.; Kondo, K. Potentiometric titration of stereoregular polyelectrolytes. J. Phys. Chem. 1965, 69, 4005. (26) Lang, C. A. Simple microdetermination of Kjeldahl nitrogen in biological materials. Anal. Chem. 1958, 30, 1692. (27) Bough, W. A. Personal communication, Mid-America Dairymen, Inc., Springfield, MO, 1989. Accepted September 1, 1989. Registry No. Chitosan-poly(acry1ic acid), 114539-82-1.
2
ARTICLES Chitosan-Poly(acry1ic acid): Mechanism of Complex Formation and Potential Industrial Applications Visith Chavasit and J. Antonio Torres* Department of Food Science and Technology, Oregon State University, Corvallis, Oregon 97331-6602
The food industry is interested in polyelectrolytic coagulants of natural origin for the clarification of food beverages and the recovery of colloidal and dispersed particles from processing waste streams. This paper discusses potential industrial applications of recent findings on polymer complex formation obtained with a chitosanpoly(acry1ic acid) model system. Process recommendations could be made on the basis of the ionic strength, pH, and charged group concentration of the fluid to be treated. Ionic strength does not affect the complex formation process. The amount of chitosan in the complex formed is controlled by the solution pH. The mechanism of complex formation indicates that pH measurements could be used to monitor the coagulation process.
Introduction Chitosan consists of unbranched chains of &(1-.4)-2amino-2-deoxy-D-glucanresidues (Figure 1). It is obtained by deacetylation of chitin, which is present in marine invertebrates, insects, fungi, and yeasts.' Chitosan is also found in various fungi.zg Thus, chitin and chitosan are, at least to some small extent, part of our food supply. Chitin and chitosan are obtained industrially from shellfish-processing waste (Bioshell Products, Albany, O R Protan,Inc., Raymond, WA; Kyowa Oil and Fat, Tokyo, Japan; Kyokuyo Co., Tokyo, Japan). The total annual global estimates of accessible chitin amount to 150 X lo6 kga4+ Despite the quantitative importance of chitin and chitosan, only limited attention has been given to its applications. This is especially true for food applications.' As reviewed by Knorr,' the three key future applications of chitosan in the food industry are its use (1)as a flocculation agent, (2) as a functional food ingredient, and (3) as a new polymer for the formation of a matrix with unique properties. The use of chitosan to prepare edible coatings which control diffusion of preservatives applied on food surfaces has recently been examined in our l a b ~ r a t o r y .The ~ complex formation process between chitosan and polyanions, which could be used to design improved systems for the recovery of proteins and other bioproducts, has also been the subject of research in our laboratory.' During the past decade, increasing attention has been given to polyelectrolytic coagulants of natural origin to aid the separation of colloidal and dispersed particles from food-processing waste^.^"^ Chitosan, the polycationic carbohydrate polymer,has been found to be particularly effective in aiding the coagulation of protein from food-processing Examples reported in the literature of biomass recovery from food-processing waste have ranged from 70% to 97%.' Undoubtedly, it is possible to find synthetic polymers that perform as well or better than chitosan. The difference is that it should be possible to use chitosan-coagulated byproducts recovered from food-processing waste as a feed ingredient.
Chitusan toxicity studies with animal models have shown no physiological effects.'3914 For example, chitosanprotein complexes containing up to 5% chitosan fed to rats for 6 weeks resulted in insignificant differences in growth rate, blood, or liver compared to control groups. This is 10-20 times the levels expected in feeding coagulated byproducts to animals.15 However, current U.S. regulations allow only up to 0.1% chitosan in livestock feed.16 Treatment of the effluent from food-processing plants is another approved use in the U.S.17 Chitosan for direct use in foods has already been approved in Japan,"where it is incorporated into several products, including noodles and breadsticks. European beer and wine makers use it as a clarifying agent.17"' Polyelectrolyte complex formation between chitosan and polyanions such as alginates,lg esterified algin a t e ~ , sodium '~ (carboxymethyl)cellulose,20heparin,'l and acidic glycosaminoglycansz2has been previously reported. In this paper, we review the potential applications of model studies conducted in our laboratory to characterize the effect of pH, ionic strength, and mixing ratio on chitosan-poly(acry1ic acid) complex formation. Poly(acry1ic acid) has the experimental advantage of its very simple structure (Figure 1). Particular attention is given to industrial food processes such as beverage clarification, wastewater treatment, and biomass recovery from foodprocessing waste. The formation and potential industrial applications of chitosan-alginate coacervate capsules has been recently reviewed by Daly and Knorr.lg Such capsules are mechanically strong and stable in a wide pH range.lQ Information on the mechanism formation process for chitosan-poly(acry1ic acid) complexes shows that chitosan-poly(acry1ic acid) complexes could also be used for microencapsulation purposes as well. Materials a n d Methods Materials. Chitosan (CHI, Lot 5112A) was purchased from Bioshell Inc., Albany, OR. To obtain a higher purity material, it was first dissolved in 0.1 N HC1, filtered through a medium-porosity fritted disk Buchner-
8756-7938/90/3006-0002$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers
Biofechnol. Pmg., 1990,Vol. 6, No. 1
3
a. TURBIDITY MEASUREMENTS opH-3 ~ p H = 4
0 2.0
QoQo
L
U
m m
U
0.0 0.0
I b.
CHITOSAN
CH2
0.2
INSOLUBLE
0.4
0.6
COMPLEX
0.8
opH=3
- CH -
n
POLYACRYLIC ACID Figure 1. Molecular structures on chitin, chitosan, and poly-
(acrylic acid).
0.0
0.2
0.4
0.6
0.8
M I X I N G RATIO. [ A / ( A t B > I type filtration funnel, reprecipitated with NaOH, rinsed with deionized water, and finally freeze-dried. The molecular weight of CHI (220000) was determined at 25 "C with a Cannon-Fenske viscometer and following the procedures reviewed by Kien~le-Sterzer.~~ CHI was dissolved in a solution of 27.5 g of NaCl in 1000 mL of 1% acetic acid. The molecular weight of poly(acry1ic acid) (PAA, Aldrich, Milwaukee, WI) was estimated to be 202 000 using dioxane as the solvent.24 Complex Formation. CHI (0.1 g) and PAA (0.1 g) were dissolved in 100 mL of HC1 and 100 mL of NaCl solutions, respectively. The ionic strength, 0.025-0.300, was varied by adjusting the concentration of the HC1 and NaCl solutions. No complex can be formed at pH 2.s Nagasawa et al.25have shown that at this pH the PAA does not have a charge density sufficiently high to form a complex with chitosan. Since chitosan is insoluble at pH values higher than 6, experiments could be conducted only in the pH 3-6 range. The pH of both reactants was adjusted by using HC1 or NaOH solution. The pH was measured with a combination pH electrode (Ross Model 81550) and read to 0.001 pH unit on a microprocessor pH/millivolt meter (Orion Model 811). The amounts of added pH-adjusting solutions were recorded to determine the final reactant concentrations. Reactant solutions with equal pH values were mixed in 5-mL increments in volumetric proportions (milliliters of CH1:milliliters of PAA) ranging from 0:40 to 40:O. A mixing ratio (MR) was defined as
A A+B where A = weight of chitosan/MW of chitosan monomer and B = weight of poly(acry1ic acid)/MW of poly(acrylic acid) monomer. The mixture was shaken vigorously and left for 15 min before measuring the turbidity in a Varian DMS 80 UV/ visible spectrophotometer (absorbance at 420 nm). MR=-
Figure 2. Complex formation as a function of polymer mixing ratio and initial pH (ionic strength = 0.3). (a) Turbidity measurements (420 nm). (b) Insoluble complex weight.
Complex Analysis. The insoluble complex was separated by centrifugation at 34800g for 40 min. The pellet was twice resuspended in distilled water and then recentrifuged. The washed complex was finally freeze-dried and weighed. The pH of the supernatant was recorded, and the CHI concentration was measured by using the Nessler reagent method.26 A material balance was used to calculate the amount of PAA left in the supernatant.
Results and Discussion Turbidity is a simple indicator for complex formation but cannot always be used to quantitate the amount of complex formed. Some complex formation conditions result in sedimentation and lower the expected turbidity of the mixture. For instance, measurements of mixtures at pH 5 (ionic strength = 0.3) show two turbidity maxima (MR = 0.56 and 0.30, Figure 2a) while missing the true maximum measured by insoluble complex formation (MR = 0.41, Figure 2b). This observation highlights how easily a complex can be removed from the solution and explains why one of the most promising chitosan industrial applications is its use as a natural flocculating agent. However, as noted by Chavasit et a1.: future model studies are needed to characterize these chitosan-poly(acrylic acid) complexes. Another problem of turbidity determinations is that they are affected by particle size. As will be shown later, the complex composition (chitosan-to-poly(acry1ic acid) ratio) is a function of the pH of the solution. Thus, it can be expected that the complex size will depend upon the solution pH. The amount of complex formed at a given initial pH was the same for all ionic strength values (0.025-0.300) used in this study (Figure 3). This finding has practical
4
Biotechnol. Prog., 1990,Vol. 6, No. 1
l
CD
E
3
DH =
5
I
o
u m
1
501
a.
=
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=
0.025
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= =
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=
0.100
=
0.150
0.050
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.
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rn
z
z
c (
c (
0' 0.0
0.2
0.8
0.6
0.4
MIXING RATIO.
-1
0' 0.0
I
[A/(AtB>I
Fn
c. pH = 5 O A 0
I I
= =
0.050 0-025 0.100
1
CD
I
0.2
MIXING RATIO.
o
E
0.4
A
I I
= 0.025 =
0.e
0.8
CA/(AtB>I d. pH
=
6
0.050
0
i.0
0.2
0.8
0.4
MIXING RATIO.
0.8
CA/(AtB>l
i.0
0.2
0.4
MIXING RATIO.
0.8
0.e
CA/(AtB>l
Figure 3. Complex formation as a function of polymer mixing ratio, initial pH, and various ionic strengths. (a) Initial pH = 3. (b) Initial pH = 4. ( c ) Initial pH = 5. (d) Initial pH = 6.
value since the ionic concentration of industrial waste streams can vary widely. Measurements of pH have been used to investigate the complex formation mechanism and are confirmed by quantitative and IR analyses.' At pH 3, 4, and 5, the degree of ionization of chitosan is about 1.0, 0.95, and 0.85, re~pectively.~~ A t the same conditions, the degree of ionization of poly(acry1ic acid) is about 0.1,0.2, and 0.5, re~ p e c t i v e l y .In ~ ~other words, in the 3-5 pH range, most of the CHI amine groups are in the NH3+ form, while most of the PAA carboxyl groups are in the COOH form. This sug ested the following complex formation mechanism:1
+ HOOC (CHI) + (PAA) NH;
--
NH;-OOC
+ Hf
-
(complex) pH1 (1) At pH = 6, the degree of ionization of chitosan is reduced to about 0.6,23 while that of PAA is about 0.8;25i.e., most of the amine groups are in the NH2 form, while most of the PAA carboxyl groups are in the COO- form. This suggested the following complex formation mechanism:' +H+
NH,
+ -0OC -,NH;-OOC
-
-
(CHI) + (PAA) (complex) pHf (2) Equation 1suggests that complex formation a t low initial pH values should lower the supernatant pH, while eq 2 suggests that the opposite behavior should be observed a t high initial pH values. Supernatant pH determinations were consistent with this expected behavior (Figure 4). The complex formation effect on supernatant pH suggests that pH measurements could be used in industrial processes to monitor the flocculation rate.
The supernatant pH changes are not only a function of the initial pH conditions and the amount of complex formed but also of the buffering properties of the excess reactant left in the supernatant. Due to differences in the CHI and PAA buffering capacities, their amount present in the supernatant affects the change in the supernatant pH. For example, at initial pH 4 conditions, the supernatant pH values after formation of the same complex amount (20 mg) were different depending upon the mixing ratio condition (pH = 3.95 for MR 0.08 and pH = 3.6 for MR 0.58). This was due to the difference in the excess reactant remaining in the supernatant. A similar situation was observed for initial pH 6 conditions (Figure 4b). The effect of pH on the mixing ratio for maximum insoluble complex formation was also studied using the analysis data for the supernatant fraction (Figure 5). The thick vertical lines indicate the MR for maximum insoluble complex formation, MR,, = 0.12, 0.29, 0.42, and 0.55 for initial reaction mixture pH = 3,4,5, and 6, respectively (Figure 2). Composition analysis of the supernatant after complex formation showed that at each of these MR,,, and pH value combinations the solution contains only trace amounts of CHI and PAA. A t MR # MR,,,, the solution assays showed an excess of one of the reactants, which increased in the direction indicated by the arrows, and only trace amounts of the other reagent (Figure 5). The observation that no excess reactants were left in the supernatant a t the mixing ratio for maximum insoluble complex formation has particular significance for applications such as beverage clarification. It would facilitate the approval of regulatory agencies for these applications since only trace amounts of chitosan would be
-
-
5
Biotechnol. frog., 1990, Vol. 6,No. 1 50
77
I
a. INITIAL pH-4
i n E
-4.1
pH=3.95
2 n -1 I d 4
I
m
X
Lu
JJ
J
- 3 . 8 cJ
x 0 u
0
n
3 D
r
I
Lu
\
\
,
J
m
r
n
Literature Cited
0
Ln
z
I/
1 0.2
0.0
0.4
501
t
1
I
-
-
0.8
0.6
n
I
b. INITIAL pH=6
= I
-6.5 I)
n
U J
E
40 -
A
3
\
"\'
Lu
:
a
-8.3 0
3 D
E 0
u
-8.1
:I
/
0
0 '
=
;D
-5.9
I) -I
U
r(
I/
I
oa'
0.2
0.0
0.4
'5.7 0.8
0.8
L
MIXING R A T I O . [ f l / ( f l t E ) l Figure 4. Confirmation of complex formation mechanism: supernatant pH measurements. (a) Initial pH = 4. (b) Initial pH = 6. (0) Insoluble complex; (A)supernatant pH.
-
K1 10.12-MR
3 ITRflCEl
1
I
TRACE
A /
CHI
TRACE
mox
I PAR g m
m
1'
fJ7
CHI fJ7
1 4
H
z5 H
- CHI
TRACE -
0.0
*
0.2
,0.42=MRmax
0.4
0.e
TRACE
0.8
PAA
Conclusions Polyelectrolytic coagulants of natural origin, such as chitosan, should facilitate beverage clarification processes and the recovery of colloidal and dispersed particles from processing waste streams. Furthermore, an understanding of the complex formation process can be used to identify process control strategies (e.g., monitoring supernatant pH values). Initial pH conditions determine the composition of the recovered byproducts. This information could be used to obtain byproducts with desirable properties. Future studies will be conducted to further characterize the chitosan-poly(acry1ic acid) complex. The physical and chemical stability, the rheological properties, and the charge density of chitosan-polyanionic complexes need to be quantified. Of particular interest would be the analysis of the interaction of these complexes with proteins and polysaccharides of industrial interest.
3 -I
u
remain in solution. This finding suggests that pH adjustment could be used to control the chitosan concentration of the coagulated byproducts to be recovered from food-processing wastes. This would be particularly valuable if the objective is to use these recovered byproducts as an animal feed ingredient.
u
7 g
I .o
MIXING RATIO, (A/AtBl Figure 5. Confirmation of complex formation mechanism: analysis of supernatant composition.
left in the solution while achieving a high level of clarification. However, for feed applications of chitosan-coagulated solids derived from wastewater, the major concerns of regulatory agencies are the amount of chitosan in the feed as well as other substances possibly derived from the wastewater. These include metals, pesticides, and any other possible hazardous substance of concern to animal and human toxi~ologists.~~ Further analysis of Figure 5 suggests that the complex composition a t a given pH is constant and equal to the MR,,, value for that pH condition. Excess reactants
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ulating agent for food processing wastes- in diet of rats on growth and liver and blood composition. Bull. Environ. Contam. Toxicol. 1976, 15, 555. (15) Bough, W. A.; Landes, D. R. Treatment of food processing wastes with chitosan and nutritional evaluation of coagulated by-products. In Proceedings of the 1st International
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