Accepted Manuscript

Title: Application of Chitosan as an Adsorbent to Treat Rice

Mill Wastewater - Mechanism, Modeling and Optimization
Author: K. Thirugnanasambandham V. Sivakumar J. Prakash
Maran
PII:
DOI:
Reference:

S0144-8617(13)00487-6
http://dx.doi.org/doi:10.1016/j.carbpol.2013.05.012
CARP 7732

To appear in:
Received date:
Revised date:
Accepted date:

5-4-2013
19-4-2013
9-5-2013

Please cite this article as: Sivakumar, K. T., *thirusambath5@gmail.comV.,

APPLICATION OF CHITOSAN AS AN ADSORBENT TO TREAT RICE
MILL WASTEWATER - MECHANISM, MODELING AND OPTIMIZATION,
Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.05.012
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APPLICATION OF CHITOSAN AS AN ADSORBENT TO TREAT RICE MILL

WASTEWATER - MECHANISM, MODELING AND OPTIMIZATION

K. Thirugnanasambandhama*,V. Sivakumarb and J. Prakash Maranc

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a,b,c

TN, India.

E-mail: drvsivakumar@yahoo.com.

E-mail: prakashmaran@gmail.com.

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Department of Food Technology, Kongu Engineering College, Perundurai, Erode-638052,

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E-mail: thirusambath5@gmail.com.

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*Corresponding author

E-mail: thirusambath5@gmail.com (K. Thirugnanasambandham)

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Tel.: +91-4294-226606

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Fax: +91-4294-220087

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RESEARCH HIGHLIGHTS

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Application of chitosan for rice mill wastewater treatment.

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Response surface methodology was applied to study the process.

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Second order polynomial regression model was developed.

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Experimental values showed good agreement with predicted values. .

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FT-IR was used to find out the process mechanism.

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Abstract
The objectives of the present study is to investigate the chitosan as an adsorbent to treat

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rice mill wastewater under different process conditions such as agitation time (2 - 6 min), initial

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pH (2.5 - 6.5), chitosan dose (400 800 mg/l) and settling time (10 30 min) in order to study

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the removal efficicency of chemical oxygen demand (COD) and total suspeneded solids (TSS).

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The results showed that, all process variables have significant effect on the removal efficiencies.

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The optimum process conditions were determined (agitation time of 4 min, initial pH of 4.5,

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chitosan dose of 600 mg/l and settling time of 20 min) and showed high removal efficiencices

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(COD:98 % and TSS:95 %). FT-IR spectrophotometry was used to analysis and confirm the

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adsorption process. From the experimental data, Box-Behnken design (BBD) was used to

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develop the second order polynomial models with high coefficient of determination values

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(COD: 0.991 and TSS: 0.989).

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Keywords: Rice mill wastewater, Chitosan, Adsorption, FT-IR, Box-Behnken design.

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1. Introduction

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In recent years, clean environment is necessary for smooth living and better health of

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human beings (Pooja Vaishnav and Anupam Bala, 2012). For that fact, in last few decades,

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considerable attention has been paid to treat the industrial wastewaters, which are usually

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discharged on land or into different natural water sources such as rivers and canals. Higher

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growth rate of industrialization will increase the adverse impact on ecosystem drastically due to

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the discharge of untreated industrial wastewater, which could degradae the environment (Galvez

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et al., 2003). Among, various kinds of wastewaters, rice mill wastewater are recognized as one

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of the toxic wastewater, which contains high amount of organic and inorganic pollutants

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(Rajesh et al., 1999). Therefore, there is a critical need to treat the rice mill wastewater before

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discharged into the environment. An extensive literature survey showed that, treatment methods

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such as UASB reactor, microbial fuel cells (MFC), electrocoagulation and biological treatment

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(Rajesh et al., 1999; Manaswini et al., 2010; Pooja Vaishnav and Anupam Bala, 2012;

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Manogari et al., 2008) techniques have been employed to treat the rice mill wastewater. But,

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these treatment processes requires longer time, higher extensive set-up and high energy

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consumption. So, these techniques are not economically applicable in small community

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systems. Moreover, in these techniques addition of chemicals generates a considerable quantity

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of secondary pollutants such as solid sludge, which also pose serious environmental problems

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(Kalyuzhnyi et al., 2005). Therefore, the research interest in use of alternative treatment

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processes for treatment of rice mill wastewater without producing secondary pollutants has

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nowadays intensified.

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Chitosan is a linear copolymer of D-glucosamine and N-acetyl-D-glucosamine produced

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by the deacetylation of chitin. Chitosan have individual properties among various biopolymers

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especially the presence of primary amino groups (-NH2) and higher nitrogen content

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(Synowiecki and Al-Khateeb, 2003). The potential application of chitosan has been widely

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recognoized in various fileds such as

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ophthalmology, biotechnology, chemistry, cosmetics, textile, oenology, agriculture and

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photography (Rinaudo, M., 2006). Chitosan is also widely applied in wastewater treatment

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process, because it is environmentally friendly, non-toxic, bio-degradable, renewable and has

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the ability remove the pollutants with outstanding pollutant-binding capacities (Gregorio and

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Pierre-Marie, 2008). Chitosan has been used as an adsorbent to treat various wastewaters such

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as food processing industrial wastewaters, brewery wastewater, pulp and paper mill wastewater,

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olive oil wastewater, effluent containing metal ions and phenol derivatives (Amit and Mika,

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2009).

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biomedical engineering, pharmacy, dentistry,

However, from the extensive literature analysis, there is no research reports are available

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on the treatment of rice mill wastewater using chitosan as an adsorbent. Hence the primary

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objective of the present study has been made to investigate and optimize the individual and

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interactive effect of process variables such as agitation time, initial pH, chitosan dose and

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settling time on the pertantage removal of chemical oxygen demand (COD) and total suspended

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solids (TSS) from rice mill wastewater. The mechanism of treatment process was analyzed and

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confirmed by using fourier transform infrared (FT-IR) spectroscopy. Moreover, response

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surface methodlogy (RSM) coupled with Box-Behnken response surface design (BBD) was

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used to develop mathematical models in order to predict the response values under the

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experimental range used in this study. Response surface methodology is a collection of

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statistical techniques commonly used to understand the performance of complex systems and

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modeling the various biochemical process. This technique also be used to evaluate the relative

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significance of several affecting factors even in the presence of complex interactions between

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the independent variables (Prakash Maran et al., 2013).

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2. Materials and methods

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2.1. Raw wastewater and chemicals

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The rice mill wastewater used in this study was collected from rice processing industry

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near Erode, TamilNadu, India and were stored at 4 C prior to the experiments in order to avoid

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changes in the physico-chemical properties of wastewater. The initial conditions of rice mill

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wastewater was determined and found to be: initial pH of 4.98, COD of 2200 mg/l and TSS of

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768 mg/l respectively, which indicated the higher content of organic and inorganic pollutants.

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Hydrochloric acid (HCl) was purchased from Sigma chemicals, Mumbai. Sodium hydroxide

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(NaOH) was supplied by Merck chemicals, Chennai. All the chemicals used in this study were

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anlaytical grade. Chitosan (RM9358-100G) was purchased from HiMedia laboratories,

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Mumbai.

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2.2. Experimental method

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A conventional batch studies were carried out under different working conditions such

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as agitation time (2 - 6 min), initial pH (2.5 - 6.5) and chitosan dose (400 800 mg/l) in 250 ml

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conical flask containing 100 ml of composite rice mill wastewater. Incubator shaker equipment

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(SLM-1NC-OS-250) was used to agitate (1 min at 100 rpm followed by 3 min at 40 rpm) the

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sample containing conical flask and were allowed to settle at different settling time (10 30

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min). Then the sample was centrifuged at 6500 rpm for 15 min (Remi R-24 Centrifuge, India)

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and the supernatant was filtered through a 0.45 m filter papers and the filterate was collected

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for the analysis of chemical oxygen demand (COD) and total suspended solids (TSS). All the

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experiments were performed in triplicates and the results were presented as the mean value of

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the triplicates.

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2.3. Analytical method

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The pH of the sample was measured with the help of pH meter (Elico LI120, India), and

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pH adjustment was done using 0.1N HCl or 0.1N NaOH (Sridhar et al., 2011). The COD and

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TSS analysis were carried out by according to the procedures described by American Public

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Health Association (APHA). The removal efficiency (RE) of COD and TSS was calculated by

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using the following equation (Sridhar et al., 2011),

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c ce
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c0

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where, c0 and ce is the initial and final concentrations of COD and TSS respectively.

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2.4. Fourier Transform Infrared spectroscopy (FT-IR) analysis

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FT-IR spectra of chitosan (before and after adsorbtion process) was recorded in the

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range of 4000400 cm1 with the help of potassium bromide (KBr) pellets using a FT-IR

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spectrometer (Instrument model RX1, India).

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3. Results and discussions

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3.1. Effect of agitation time

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The agitation time is one of the important parameter, which influences the performance

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of the adsorption process significantly. To examine its effect, the experiments were carried out

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at different agitation time (2 -6 min), while keeping the other working parameters (initial pH of

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3.5, chitosan dose of 500 mg/l and settling time of 15 min) are constant and the results were

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exhibited in Fig.1a. The mechanical agitation caused more fibrillation and increases the exposed

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specific surface area for adsorption process by creating collisions between the chitosan and

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organic particles present in the effluent (Dzul Erosa et al., 2001), which could increased the

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removal efficiency of COD and TSS, while increasing the agitation time from 2-4 min. Further

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increase in agitation time could destabilize interaction (bond) between chitosan and organic

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particles and decreased the removal efficiency. So, agitation time of 4 min is considered as the

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optimum time for the higher percentage removal of COD (70.25 %) and TSS (68.02 %).

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3.2. Effect of initial pH

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The surface charge of the chitosan effectively varies with varying the pH of the

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wastewater. So, the experiments were performed at different initial pH (2.5 6.5) while keeping

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the agitation time of 4 min, chitosan dosage of 500 mg/l, settling time of 15 min as constant and

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the observation is depicted in Fig.1b. From the Fig.1b, it was observed that the percentage of

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COD and TSS removal increased with increasing initial pH upto 4.5. This is mainly due to the

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formation NH3+ ions in acidic conditions, which could adsorbs the negatively charged organic

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matters present in the wastewater significantly (Guzmanet al., 2003) and increased the removal

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efficiency of COD (77.98%) and TSS (75.24%) upto the pH of 4.5. After that, solubility of

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chitosan was decreased, while increasing the pH towards the basic condition and leads to

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decrease in removal efficiencies of COD and TSS. From the results, initial pH of 4.5 and

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agitation time of 4 min were determined as an optimum condtions for further experiments.

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3.3. Effect of chitosan dose

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The chitosan dose is a key factor to determine the pertantage removal of COD and TSS

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in rice mill wastewater. In order to evaluate its effect, experiments were performed at various

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ranges of chitosan dose (400 800 mg/l), while other parameters such as agitation time (4

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min), initial pH (4.5) and settling time (15 min) were kept at constant and the results are

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illustrated in Fig.1c. The pertantage removal of COD and TSS were increased with increasing

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chitosan dose upto 600 mg/l, further increase in chitosan dose (600 800 mg/) did not show any

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significant effect. Higher dosage of chitosan (upto 600 mg/l) can effectively react with the

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organic and inorganic pollutants present in the wastewater, due to the availabiity of more

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number reaction sites, which can increased the removal efficiency of COD (80.68%) and TSS

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(78.08%). Beyond this dosage, the adsorption process attains a equilibrium stage with the

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charged molecules of the wastewaster (Riano et al.,2012) and did not improve the efficiency of

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adsorption process. Hence, initial pH of 4.5 agitation time of 4 min and chitosan dose of 600

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mg/l were choosen as optimal conditions for upcoming experiments.

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3.4. Effect of settling time

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In the adsorption process, the settling time is an important factor which can influences

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the overall removal efficiencies of COD and TSS in wastewater treatment. To determine the

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effective settling time, experiments were done at different settling time of (10- 30 min) while

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other parameters such as agitation time (4 min), initial pH (4.5), and chitosan dose (600 mg/l)

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were kept as constant. The results indicates that, the pertantage removal of COD and TSS were

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increased with increasing settling time upto 20 min and then did not show any significant effect.

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The aggregation of flocs (formed between chitosan and colloidal particle in the wastewater)

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increases up to 20 min, in which most of the flocs are settle down, thus removal efficiencies

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increased during adsorption process (Abu Hassan et al., 2004). Finally, the pertange removal

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efficiency of COD and TSS under optimum settling time (20 min) were found to be 97.84,

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95.34 % respectively.

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3.5. FT-IR analysis

FT-IR spectra of chitosan (before and after adsorption process) was recorded in the

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range of 4000-400 cm-1 (Fig. 4) in order to the study the mechanism of adsorption process.

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From the Fig. 4a, it was found that the broad band at 3402 cm1 and the peak at 1371cm1

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indicating the presence of NH2 and O-H groups on the raw chitosan (Anirudhan and

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Radhakrishnan, 2009). Transmittance shifted from 3402 cm1 (Fig.4a) to 3118 cm1 (Fig.4b)

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and the formation of new peak at 1655 cm-1 (Fig.4b) were clearly indicated the interaction of

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chitosan with the organic pollutants present in the wastewater. This is explained by the fact that

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presence of NH2 and O-H groups in the raw chitosan can acts as the reaction sites for the

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adsorption process (Pawlak and Mucha, 2003).

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3.6.Process modelling and statistical analysis

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In this study, Box-Behnken response surface experimental design (BBD) with four

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factors at three levels was employed to investigate the interactive effect of process variables

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(agitation time (2 - 6 min), initial pH (2.5 - 6.5), chitosan dose (400 800 mg/l) and settling

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time (10 30 min)) on the removal efficiencies of COD and TSS. In addition, this experimental

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deisgn was used to develop mathematical models in order to predict the responses. Process

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variables (coded and un-coded) and their ranges were shown in Table 1. A total number of 29

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experiments (including five centre points) were established based on the method described by

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Prakash Maran et al. (2013) and the results are stated in Table 2. The results were analyzed by

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multiple regression analysis, Pareto analysis of variance (ANOVA), F-test, coefficient of

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determination (R2), adjusted coefficient of determination (adj-R2) and predicted coefficient of

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determination (pre-R2). All the statistical analyses were done with the help of Stat ease Design

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Expert 8.0.7.1 statistical software package (Stat-Ease Inc., Minneapolis, USA). After analysis of

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the experimental data, second order polynomial model (SOPM) was developed and general

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form of SOPM was given in elsewhere (Prakash Maran et al., 2013a). The developed SOPM

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were used for the construction of contour response surface plots to examine the relationships

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between independent and dependent variables.

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3.6.1. Model development and analysis

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The experimental data was analyzed by two different tests namely the sequential model

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sum of squares and model summary statistics in order to obtain suitable regression models to

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predict the responses and the results are listed in Table 3. From the Table 3, it was observed

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that, quadratic model shows high R2, adjusted-R2, predicted-R2, F-value and low p-value, when

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compared with other models (linear and interactive (2FI)). Cubic model was found to be aliased.

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Therefore the quadratic model is selected to portray the effects of process variables on the

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reponses.

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From the experimental data, two empirical models were developed to understand the

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interactive correlation between the responses and process variables (Prakash Maran et al.,

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2013b) and final developed mathematical models obtained in terms of coded factors are given

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below

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COD removal (%) = 95.84-2.32A+2.78B+0.39C+21.56D +2.25AB-1.64AC-6.50AD-8.25BC

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+1.85BD-1.84CD-19.85A2-19.51B2-5.94C2-19.84D2

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TSS removal (%) =93.86-2.38A+2.81B+0.38C+21.65D+2.18AB-1.68AC-6.56AD-8.25BC

+1.80BD-1.84CD-19.99A2-19.60B2-5.97C2-19.81D2

(3)

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Moreover, Pareto analysis of variance (ANOVA) was used to analyze the experimental

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data and significance of the developed model equations were evaluated by their corresponding

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F- value and p-values (Table 4). The higher model F- values and lower p-values of the both

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responses (COD removal and TSS removal) demonstrated that, the developed model was highly

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significant. From this results, it was concluded that, the developed mathematical models has the

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ability to describe the adsorption process very robustly (Prakash Maran et al., 2013b).

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3.6.2. Adequacy of developed models

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Generally, it is very essential to check the capability of the developed mathematical

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model to represent and describe the present treatment process conditions. In this perspective,

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construction of diagnostic plot such as predicted versus actual plot (Fig.2) was used to evaluate

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the model suitability and find out the relationship between predicted and experimental values.

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The data points lies very close to the diagonal line and it depicts a good relationship between

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experimental and predicted data (Prakash Maran et al., 2013c).

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3.6.3. Response surface analysis of process variables on responses

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From the developed models, response surface contour plots were constructed to evaluate

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the interactive effects of the independent variables on the responses and it was given in Fig.3.

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From the Fig.3, it was observed that all the combined process variables shows the significant

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effect on the percentage removal of COD and TSS in adsorption process as well as the trends

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obtained in the contour plots are closely agreed with Fig.1.

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4. Conclusions

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In this present study, removal efficiencies of chemical oxygen demand (COD) and total

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suspeneded solids (TSS) from rice mil wastewater was examined under different process

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conditions such as agitation time (2 - 6 min), initial pH (2.5 - 6.5), chitosan dose (400 800

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mg/l) and settling time (10 30 min). Under the optimum working conditions such as agitation

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time of 4 min, initial pH of 4.5, chitosan dose of 600 mg/l and settling time of 20 min shows the

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removal efficiency of > 95 % for COD and TSS respectively. Process mechanism was identified

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by using FT-IR spectrophotometry. Process modeling of the present study was done by response

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surface methodlogy (RSM) coupled Box-Behnken response surface design (BBD) with four

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factors at three levels. Second order polynomial models were developed from the experimental

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data in order to predict the responses with high correlation coefficient values. This results

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exhibited that, chitosan is found to be a suitable adsorbent to treat rice mill wastewater in terms

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of high removal efficiency of pollutants as well as protection of ecosystem by eliminating the

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creation of secondary pollutants.

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333

Sridhar, R., Sivakumar, V., Prince Immanuel, V., & Prakash Maran, J. (2012). Development of

337

model for treatment of Pulp and Paper industry bleaching effluent using response surface

338
339
340
341

Ac
ce
p

336

methodology. Environmental Progress & Sustainable Energy, 31, 558-565.

Synowiecki, J., & Al-Khateeb, N.A. (2003). Production, properties, and some new applications
of chitin and its derivatives. Critical Review of Food Science and Nutrition, 43, 145171.

342

Page 16 of 27

17
342

Table 1

343

Ranges of independent variables and their levels

Variable (unit)

-1

Initial pH (B)

2.5

4.5

6.5

Chitosan Dose (mg/l, C)

400

600

800

Settling time (min, D)

10

20

an

346

347
348

354
355
356
357
358
359
360
361
362
363
364
365
366

te
Ac
ce
p

353

349

352

30

us

345

351

ip
t

344

350

cr

Agitation time (min, A)

Levels
0

Page 17 of 27

18

Settling
time (D)
20
10
20
20
10
20
20
10
20
20
20
30
10
20
30
20
20
30
20
10
20
20
10
20
30
30
30
20
20

COD removal
(%)
76.54
36.54
59.54
60.54
30.54
55.64
65.54
44.32
70.54
95.84
80.54
73.24
45.54
95.84
68.54
95.84
70.54
89.84
95.84
38.54
68.78
45.64
40.54
58.54
82.64
95.98
84.54
95.84
75.34

TSS removal
(%)
74.38
34.28
57.62
58.62
28.34
53.02
63.56
42.34
68.48
93.86
78.56
71.26
43.28
93.86
66.54
93.86
68.34
87.86
93.86
36.48
66.38
43.28
38.56
56.38
80.66
94.28
82.54
93.86
73.24

ip
t

Chitosan
dose (C)
800
600
600
600
600
600
800
400
800
600
400
600
800
600
600
600
400
800
600
600
400
600
600
400
600
400
600
600
800

Initial
pH (B)
2.5
2.5
2.5
6.5
4.5
6.5
6.5
4.5
4.5
4.5
6.5
2.5
4.5
4.5
4.5
4.5
4.5
4.5
4.5
6.5
4.5
2.5
4.5
2.5
6.5
4.5
4.5
4.5
4.5

Ac
ce
p

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

Agitation
time (A)
4.00
4.00
2.00
2.00
2.00
6.00
4.00
4.00
6.00
4.00
4.00
4.00
4.00
4.00
6.00
4.00
6.00
4.00
4.00
4.00
2.00
6.00
6.00
4.00
4.00
4.00
2.00
4.00
2.00

te

Run

cr

BBD and their experimental results

us

368

an

Table 2

367

369
370
371
372
373

Page 18 of 27

19

Multi regression analysis of responses

Sum of
Degree of
Mean Square
Squares
freedom
Sequential model sum of squares for COD removal
Source

Mean
Linear
2FI
Quadratic
Cubic
Residual
Total

137616.73
5739.44
499.52
5531.43
87.89
22.95
149497.96

1.00
4.00
6.00
4.00
8.00
6.00
29.00

137616.73
1434.86
83.25
1382.86
10.99
3.83
5155.10

5.61
0.27
174.67
2.87

Prob > F

Remarks

0.0025
0.9459
< 0.0001
0.1078

Suggested
Aliased

5.62
0.26
160.50
3.36

0.0025
0.9465
< 0.0001
0.0787

Suggested
Aliased

an

Sequential model sum of squares for TSS removal

F Value

ip
t

375

cr

Table 3

us

374

Mean
Linear
2FI
Quadratic
Cubic
Residual

129455.73
5791.77
501.04
5566.43
99.22
22.16

1.00
4.00
6.00
4.00
8.00
6.00

Total

141436.37

29.00

4877.12

Std.Dev.

R2

Adjusted R2

Predicted R2

PRESS

Remarks

Model summary statistics for COD removal

15.9971
0.4831
Linear
17.7048
0.5251
2FI
2.8137
0.9907
Quadratic
1.9560
0.9981
Cubic

0.3969
0.2613
0.9813
0.9910

0.3411
0.1104
0.9462
0.7218

7828.91
10570.00
638.72
3305.49

Suggested
Aliased

Model summary statistics for TSS removal

16.0583
0.4834
Linear
17.7761
0.5252
2FI
2.9445
0.9899
Quadratic
1.9220
0.9981
Cubic

0.3973
0.2615
0.9797
0.9914

0.3414
0.1099
0.9416
0.7336

7890.16
10664.40
699.47
3191.71

Suggested
Aliased

te

Ac
ce
p

Source

129455.73
1447.94
83.51
1391.61
12.40
3.69

376
377
378

Page 19 of 27

20
379

Table 4

380

ANOVA table for responses

0.0001
0.0127

Sum of
squares
11859.25
67.78

11.75

0.0041

94.72

1.79
5577.87
20.25
10.74
169.05
272.25
13.69
13.54

0.23
704.53
2.56
1.36
21.35
34.39
1.73
1.71

0.6422
0.0001
0.1321
0.2635
0.0004
0.0001
0.2097
0.2120

2555.76
2468.17
228.61
2553.68
7.92
11.08
0.00

322.81
311.75
28.88
322.55

93.05

C
D
AB
AC
AD
BC
BD
CD
A2
B2
C2
D2
Residual
Lack of fit
Pure error

382
383
384

106.19
8.16

93.05

1.79
5577.87
20.25
10.74
169.05
272.25
13.69
13.54
2555.76
2468.17
228.61
2553.68
110.84
110.84
0.00

te

0.0001
0.0001
0.0001
0.0001

Mean
square
847.09
67.78

F-value

p-value

97.70
7.82

0.0001
0.0143

94.72

10.92

0.0052

1.69
5625.39
19.10
11.27
171.92
272.25
12.96
13.54

1.69
5625.39
19.10
11.27
171.92
272.25
12.96
13.54

0.20
648.81
2.20
1.30
19.83
31.40
1.49
1.56

0.6652
0.0001
0.1600
0.2733
0.0005
0.0001
0.2417
0.2319

2591.06
2490.79
231.44
2544.46
121.38
121.38
0.00

2591.06
2490.79
231.44
2544.46
8.67
12.14
0.00

298.84
287.28
26.69
293.47

0.0001
0.0001
0.0001
0.0001

an

p-value

Ac
ce
p

381

F-value

cr

Mean
square
840.74
64.59

us

Model
A

Sum of
squares
11770.39
64.59

Source

TSS removal

ip
t

COD removal

385
386
387
388

Page 20 of 27

21
389
390
FIGURE CAPTIONS

393

Fig.1 Effect of process variables on the percentage removal of COD and TSS

394

Fig.2. Adequacy of model tested for responses

395

Fig.3. Response surface contour plots for responses

396

Fig.4. FT-IR spectra of raw chitosan (a) and after treatment process (b)

398

403
404
405
406

te
Ac
ce
p

402

399

401

cr
us

an

397

400

ip
t

391
392

407
408
409
410

Page 21 of 27

22
411
412
413
Fig.1.

ip
t

414

Ac
ce
p

te

416

an

us

cr

415

417
418
419

Page 22 of 27

us

cr

ip
t

23

420

422
423
424

Ac
ce
p

te

an

421

425
426
427
428

Page 23 of 27

24
429
Fig.2.

te
Ac
ce
p

432
433

an

us

cr

ip
t

430
431

434

Page 24 of 27

25

Fig.3.

an

us

cr

ip
t

435
436
437

439
440
441
442
443
444

Ac
ce
p

te

438

Page 25 of 27

an
M
d
te

Fig.4.

Ac
ce
p

445
446
447
448
449
450
451
452
453
454
455
456
457
458

us

cr

ip
t

26

Page 26 of 27

d
te
Ac
ce
p

459
460

an

us

cr

ip
t

27

Page 27 of 27