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Chitosan-modiied Cellulose as Adsorbent to Collect and Reuse Nitrate from Groundwater Christie Jiang ABSTRACT Nitrate pollution of water systems in the United States continues to increase, presenting hazards to humans and the environment. To remove this extremely soluble ion contributed largely by synthetic agricultural fertilizers, a cost-eicient and resource-eicient method, adsorption, has great potential compared to other options. In this study, chitosan, which becomes protonated in acidic solution, was combined with cellulose derived from cardboard. his combination of polymers yields a positively charged surface to attract nitrate. Batch studies revealed chitosanmodiied cellulose to improve adsorption capacity from 0.3356 to an average 3.124 milligrams of NO3- per adsorbent mass. Using the Langmuir isotherm, linear regression was performed to describe the adsorption characteristic. Based on the it, efective adsorption increases as more adsorbent is present, producing a relationship between adsorption site availability and resulting concentration due to increased aggregate charge and attraction to nitrate ions. Desorption was evaluated, with chitosan-modiied cellulose releasing .294 â€“ 4.7% of adsorbed amounts, indicating possibility of slow-release fertilizer use as the organic polymers decompose in soil. Compared to related materials, the investigated adsorbent had more environmentally friendly and adsorptive properties, as well as simpler production. Larger scale studies and optimization of the cellulose-chitosan ratio will improve further upon this research.
Introduction From 1988 to 2004, the proportion of wells in the United States exceeding the national limit of nitrate concentration increased from 16 to 21 percent. Wells account for about 15 percent of the public water supply; this translates to over 3 percent of the population drinking water containing excess nitrate . Nitrates can cause medical complications if consumed as well as undesirable phenomena in the environment such as eutrophication . he increasing presence of nitrates in fresh water wells is a growing concern; managing the nitrogen cycle has been named one of fourteen â€œGrand Challengesâ€? by the National Academy of Engineering . Recent comprehensive assessments on nitrate pollution have also sparked media attention related to nitrate pollution of groundwater, shown to afect the quality of life of whole communities at a time . he United States Environmental Protection Agency (USEPA) has set the Maximum Contaminant Level, or MCL, to 10 mg/L nitrate as nitrogen .Recent trends indicate increasing levels of nitrates. As part of the nitrogen cycle, nitrate emerges from both direct input to the soil and conversion from other nitrogen-based compounds, such as ammonia. Nitrate becomes toxic when converted to nitrite in the human body, leading to medical problems such as methemoglobinemia, or blue baby syndrome, as well as higher risk for thyroid cancer. One study of a primarily farming-based community has reported health complications that may have stemmed from unusually high nitrate concentrations in the local water. he same report concluded that 96 percent of nitrate pollution in the area had come from agricultural sources . Concentrations in the rest of the nation show no signs of stabilizing as agricultural demands along with fertilizer input continue to rise (Figure 1).
Figure 1. Jagged line shows historical and sampled data concerning input from fertilizer. Plotted points indicate the increase of number of wells over the MCL, a trend set to increase along with nitrogen input . Nitrate Removal Techniques he complexity of soil and water processes and the variety of substances involved make it diicult to pinpoint methods to begin uncovering the most efective means of water remediation. Current large-scale methods of decontamination are typically incomplete in removal, unreliable, or dependent on high amounts of resources or power, and are therefore costly . Nitrate removal presents an even more challengingproblem; because it is extremely soluble, basic precipitation and iltration techniques are inefective. Current technologies for treating nitrate-contaminated water include ion exchange, reverse osmosis, and electrodialysis . hese and other techniques are generally expensive and Volume 2 | 2012-2013 | 33
Street Broad Scientific not suiciently efective, often complicating processes and factors involved. Of those listed, ion exchange presents the least cost and technology-intensive option, but it involves releasing some other like-charged substance as the pollutant is taken in. Consideration of Nitrate Absorption Recent studies suggest that an even simpler and more efective technique, adsorption, or attraction to the surface of a material, ofers great potential . Because nitrates readily leach from soil and dissolve completely in water, extensive research has and continues to be done to ind adsorbents that ofer an environmentally friendly, reusable, and cost-eicient solution to lessening the problem of nitrate pollution. his makes it much more desirable, but still no feasible nitrate adsorbent has been found. Due to the promising premise of nitrate adsorption as the future primary method of decontamination, a variety of materials have been investigated. hough there is no doubt that engineered substances, such as activated materials and altered clays, could be very efective nitrate adsorbents as well, this would be counterproductive as adding new materials would potentially cause even more problems to be addressed. In a review detailing advances made in phosphorus removal, it is suggested that, ideally, the pollutants removed would be able to be used as raw materials for fertilizer . In the case of nitrates, if this were accomplished, a sustainable cycle would be achieved as it would not be necessary to exploit new resources. Among natural adsorbent possibilities, categories include carbon-based sorbents, natural sorbents, biosorbents, waste materials, and miscellaneous . he wide range indicates widespread uncertainty on the topic as to what an ideal adsorbent would entail. But one very common waste product that was not listed as having been researched is paper, which is present in signiicant volumes and accessible for studies. Waste paper comes in many forms and in large volumes, making it an attractive possibility. After saturation, it could be repurposed for agricultural use. In addition, it ofers potential for both physical and chemical modiication. A variety of other adsorbents have been investigated, and paper could be a viable option, having comparable surface area, texture, and chemically unreactive composition. Paper is relevant to previously mentioned materials not only in terms of being environmentally friendly but also in terms of composition and properties. he characteristics of a desirable adsorbent are signiicant surface area and volume on and in which the target substance can collect. Cellulose, the main component of paper, has been experimentally determined as one with high values for desirable adsorbent characteristics as compared to other similar ibers. Using Brunauer, Emmett, and Teller (BET) theory which determines physical adsorption on a surface, and related isotherms, surface area of 0.45 square meters per gram and total micropore volume of 0.50 cubic millimeters per gram of cellulose were determined . Although 34 | 2012-2013 | Volume 2
REsEaRch these values are not comparable to those of, for example, activated carbon or organoclays, which have values greater by several orders of magnitude, they are suicient to indicate potential for improvement and use as adsorbents. Cellulose has a slightly negative surface charge due to outer hydroxyl groups (Figure 2.a) that would repel likecharged negative nitrates, and as a polymer it is not strong enough to induce ion exchange. herefore modiication of surface charge is necessary. Chitosan, a natural polymer of dried crab and shrimp shell matter, can become positively charged when its outer amino groups (Figure 2.b) are protonated, which suggests potential for nitrate removal as it has been reviewed for efective removal of the negative chromate ion . (a)
Figure 2. (a) Structure of cellulose unit with hydroxyl groups, (b) structure of chitosan unit with hydroxyl groups and amino groups . To this end, a need for efective and green nitrate removal technology is evident, and the proposed nitrate adsorption method may hold great potential. his study aimed to develop an efective adsorbent of nitrate based on the principles of simplicity and sustainability from cellulose and chitosan and evaluate the material for its properties and possibility of repurposing.
Materials and Methods Preparation of Cellulose Adsorbent A plain used corrugated cardboard box was chosen to be the source of the cellulose base for the adsorbent. he wide availability of cardboard and often minimal ink coverage made it an ideal candidate for the study. he cardboard was cut to approximately centimeter square pieces and broken down to a pulp by soaking in water. Prior to proceeding, in order to evaluate any possible chemicals already involved in the cardboard, precipitation tests were done. Small drops of AgNO3; HNO3 and (NH4)2MoO4; BaCl2; and NaOH were added to test for chlorides, phosphates, sulfates, and ammonium, respectively. Precipitation was minimal for all tests, indicating low concern for interfering and polluting ions. After soaking, the cellulose samples were then processed in a blender into pulp. he pulp was dried on a iberglass screen, and inal particle size after drying was
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REsEaRch reduced to around 40 mm3 volume and 68 mm2 surface area. Practical grade chitosan from Sigma-Aldrich was used to make a 1% chitosan by mass and 1% acetic acid by volume solution . he protonated chitosan amino groups from -NH2 to -NH3+ in the presence of acid made it soluble as well as positively charged. he mixture was stirred at 80째C until the chitosan was dissolved completely, forming a viscous solution. After it was cooled, the same soaking and pulping procedure for cardboard was used as had been for the water pulped cellulose. Dried chitosan-modiied cellulose formed a thinner and more brittle sheet, so inal particle size was reduced to the same amount of approximately 68 mm2 surface area but only 24 mm3 volume. Preparation and Measurement of Nitrate Adsorbent Because potassium is another common chemical used in conjunction with nitrates in fertilizer, solid crystal KNO3 was used throughout the experiment to make the standard nitrate solutions to be relatively realistic to actual environmental situations. Units of nitrate as nitrogen were used for concentration. he concentration of 50 mg/L NO3 as N was made and used for batch studies. A Vernier Nitrate Ion-Selective Electrode (ISE) probe was used to measure nitrate as nitrogen concentration and calibrated by voltages from standards of 100 and 1 mg/L NO3 as N. In all concentration and mass values of nitrate, the units used were in NO3 as N. LoggerPro software was used to collect data from the probe. Batch Studies Adsorption experiments were primarily carried out using batch studies. To generalize the characteristic of the plain pulped cellulose, approximately 0.2 grams of adsorbent were added per 50 milliliters of equal concentration nitrate solution in a beaker for each sample. An individual magnetic stirrer for each beaker was set to 600 rpm, and samples were stirred for one hour. Samples from each beaker were then centrifuged and left for more contact time for at least 48 hours. hey were then centrifuged once again. Before data collection, ammonium sulfate ionic strength adjuster (ISA), 2M (NH4)2SO4, was added in the ratio 2:100 to the sample volume to reduce possible measurement interference from other content in the sample. During collection, the ISE was held in place in each centrifuge tube for at least one minute for values to equilibrate. Samples from batch studies needed to be centrifuged before measurement so that the ISE would only come in contact with solution and not pieces of adsorbent. Overall, the contact time for samples was maintained to be approximately 48 hours as described previously, but variations of centrifuge procedures were tested for efectiveness. hree methods, denoted as CB1, CB2, and CB3 (where CB stands for cardboard) to indicate use of plain cardboard, were used. Samples using CB1 method were centrifuged
immediately after mixing but left alone for the remainder of the time. Samples using CB2 were not centrifuged until after the 48 hours, and those using CB3 were centrifuged both immediately and after sitting, as done originally. To compare unmodiied and modiied cellulose, batch studies were run with all nitrate samples originating from one 500 milliliter lask to ensure standardization. 0.2 grams of both kinds of adsorbent were used per 50 milliliters of solution. As before, all samples were stirred, centrifuged, left, and centrifuged again. ISA was added before measurement. Adsorption Isotherms To characterize the adsorptive behavior of cardboard cellulose with chitosan, the ratio of adsorbent-to-adsorbate was varied by running batch studies once again and changing adsorbent dosage. Adsorbent masses of 0.1002, 0.1992, 0.3007, 0.4992, and 0.6994 g were used. he relationship of dosage and adsorptive capacity to inal concentration was analyzed by applying two major adsorption models, as done in published adsorption analyses . he Freundlich isotherm is the most basic adsorption isotherm and describes the amount of adsorbate per adsorbent as a function of the resulting solution concentration. he isotherm is deined as
and the linear form as
where x is mass of adsorbate adsorbed per mass of adsorbent, C is solution concentration after adsorption, and K and 1/n are constants. he Langmuir isotherm takes some more speciic assumptions into consideration. In particular, the Langmuir isotherm hypothesizes uniform monolayer capacity for the adsorbent, or equal capability of all sites to adsorb. Also included is the assumption that adsorbed molecules do not interact or deposit on each other. he isotherm is deined as
and the linear form as
where x and C are the same as in the Freundlich isotherm, and xm and K are constants. In particular, xm denotes the maximum x in a monolayer of adsorbate on adsorbent. he R2 value for the linear it of each model was considered, and the better it used to evaluate the adsorptive behavior of the cellulose with chitosan.
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Street Broad Scientific Desorption Experiment for Reuse Adsorbent pieces contained in samples from the comparison of unmodiied and modiied cellulose were dried on iberglass screen. If the pieces were to be added back into the ground as fertilizer, the characteristic of nitrate leaching out would be important. In order to examine possible signiicance in reusability, the adsorbents were placed in separate beakers of 6 mL of water. After three days of soaking, the nitrate as nitrogen concentration of the water was measured as a representation of desorption, to be compared to the original amounts adsorbed by the particles and considered for efectiveness of nitrate reuse.
REsEaRch adsorbed per gram adsorbent.
Results and Discussion Efect of Centrifuge Methods he data collected directly from the nitrate probe produce plots of concentration versus time (Fig. 3), which cannot be easily directly used. For the study of the efect of centrifuge methods, as in subsequent studies, several steps were taken to obtain a more useful form. he mean concentration was calculated for each sample, but only considering the latter 75% of the data for each sample. hat is, the values measured from the irst 25% of total time for each sample were disregarded to allow the nitrate ISE to reach stability. In particular, measurements related to the previously mentioned CB1, CB2, and CB3 methods were analyzed. Once singular data points associated with each sample contained within each set of data were determined, they were then used in their respective analyses.
Figure 4. Average concentrations of solutions using different centrifuge methods.
Table 1. Comparison data for centrifuge methods. he data indicates CB3 as the most efective procedure because centrifuging samples multiple times may improve adsorption, as the adsorbent and the nitrate that has already been collected on it are physically separated from the solution to decrease homogeneous desorption while still allowing for extended contact time. CB3 was used for all subsequent batch studies. hough of these methods CB3 produced the greatest adsorptive result, a ratio of approximately 1 milligram adsorbed per gram of adsorbent leaves much room for improvement.
Figure 3. Format of raw data when measuring concentrations (mg/L NO3) for centrifuge methods CB1, CB2, and CB3 using ISE. Comparison he average concentrations for samples run with no CB, CB1, CB2, and CB3 are shown in Fig. 4. To appropriately compare the methods, the concentrations were converted to mass of nitrate as nitrogen to then evaluate milligrams 36 | 2012-2013 | Volume 2
Figure 5. Average concentrations of solutions with unmodiied and modiied CB. Error bar indicates standard deviation of concentrations with chitosanmodiied CB.
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Table 2. Speciic data for unmodiied and modiied cellulose comparison.
Table 3. Data for isotherm analysis, using various CB dosages.
Table 4. Modiied values for linear it. * Outlier not used in isotherm analysis.
Table 5. Adsorption isotherm values.
Figure 6. (a) Linear Freundlich it, with Log(C) along x-axis, Log(x) along y-axis, and R-squared = .7917; (b) linear Langmuir it, with 1/C along x-axis, 1/x along y-axis, and R-squared = .9596, indicating a better it statistically. Volume 2 | 2012-2013 | 37
Street Broad Scientific Efect of Chitosan Modiication Multiple samples of both unmodiied and modiied cellulose were run using initial concentration taken from one lask of 50 mg/L NO3 solution. he results (Figure 5) indicate the addition of chitosan to greatly improve nitrate uptake. A typical, standard sample of unmodiied cardboard is compared to samples with chitosan content, showing that an over 10% reduction in concentration is possible. he relative strength of cellulose adsorbent with chitosan is greater than that of just cellulose. he attractive nature of a positive chitosan surface charge as compared to the negative nitrate ions is likely the explanation for the improvement. he standard deviation of 3.48 mg/L NO3 in post adsorption concentrations is due to the non-uniformity of samples. he inal sample of cardboard with chitosan produced a signiicantly higher concentration reading though it was taken from the same batch in the same beaker as other samples. As samples were transferred from beakers to centrifuge tubes, the distribution of cardboard pieces throughout solution was not homogeneous, causing the adsorbent-to-adsorbate ratio to be varied during the extended contact time. But even with such variations in amount of adsorbent, a signiicantly higher proportion of nitrate was adsorbed with the presence of chitosan, ranging from 13.495 to 32.087% as compared to plain cardboard which in this case adsorbed 2.713% of total nitrate. Adsorption Isotherms Tables 3 and 4 provide data produced given ive different amounts of cellulose and one standard sample. he dosage of 0.1002 g produced values that were ultimately not used in the isotherms due to the uncertainty of transferring batch solutions to centrifuge tubes, where the low mass of adsorbent would lend to less certain homogeneity in mixture. Data indicated this uncertainty, as the value contributed an extreme outlier. Linear its were performed for Freundlich (Equation 1.2) and Langmuir (Equation 2.2) isotherms, depicted in Figures 6.a and 6.b, respectively. he linear regression values are given in Table 5, corresponding to the respective notation. he Langmuir it was better suited to the data as given by its R-squared value of .9596 as opposed to that of the Freundlich it, .7917. he Freundlich isotherm is empirical, involving only the concentration and two constants, and therefore too simple to appropriately model the data. Interrelated constants and more parameters likely made the Langmuir isotherm more mathematically appropriate for the data. Because the Langmuir regression exhibited a better linear it, the values were taken and placed into the original equation (2.1), giving
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which has a modiied form and is shown graphically in Figure 7. However, the maximum adsorbate per site, xm, value is lower than the plotted x values, so it appears that, in this case, the data does not follow Langmuir assumptions. From the raw data, it was clear that concentration generally decreased as more adsorbent was added. However, the isotherms compare sites illed per adsorbent and resulting concentration, not just amount of adsorbent and concentration. In an ideal Langmuir it, increasing concentrations would indicate increased proportion of adsorbate per adsorbent as more sites on the adsorbent would be available to be used. But the data presented suggests an inverse proportionate relationship instead. For the chitosan-modiied cellulose, surface area and volume did not necessarily increase proportionately with mass as they were lat pieces as opposed to rough particles. herefore it would be inaccurate to suppose that the amount of nitrate adsorbed would decrease accordingly with increasing adsorbent mass due to more open adsorption sites. Instead, the it seems to suggest the cellulose with chitosan become more and more apt to adsorb as more adsorbent is available and attractive (with greater aggregate charge) to the nitrate, rather than having a set number of sites that compete with each other.
Figure 7. Inverse proportionate relationship between C, resulting concentration after adsorption, on the x-axis and x, amount adsorbed per mass adsorbent, on y-axis given by Langmuir isotherm. Desorption for Reuse To evaluate the possibility of re-releasing the adsorbed nitrate into the ground by reusing adsorbent as fertilizer material, desorption was measured for used samples of plain and chitosan-modiied cardboard cellulose. he plain adsorbent exhibited much higher desorption proportions than the modiied adsorbent, in part due to the much less ibrous and more rigid structure of the latter. Total adsorbed mass and adsorbent mass were used from Table 3 to calculate desorption values. A standard concentration was measured from the water used to soak the samples to calculate amount desorbed.
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Table 6. Desorption of used samples. he extreme polarity in desorption rates indicates a signiicant diference in the properties of the cellulose after modiication. hough this experiment were originally run to identify promising desorption rates for nitrate re-release in soil, the low desorption rates in fact ensure another desirable quality. he behavior of plain cardboard would suggest only very temporary adsorption because if left in solution for extended periods of time, the nitrate would leach back out. Cardboard with chitosan, on the other hand, would attract and keep adsorbed nitrate. Once added to soil, decomposition of the adsorbent as a whole would slowly release nitrate, due to the organic nature of both cellulose and chitosan, allowing for various practical applications. Comparison to Other Absorbents he maximum mass of nitrate adsorbed per mass adsorbent (“x”) compared to existing data for other adsorbents indicates the signiicance of this study in the context of all nitrate adsorption experiments. he maximum “x” was not found through Freundlich or Langmuir because the characteristic of the chitosan-modiied cellulose adsorbent did not follow the basic assumptions of the isotherms. hus considering the highest “x” values in multiple batch studies, including the adsorption study, it appears that 0.200 g adsorbent per 50 mg/L NO3 as N solution caused the most eicient adsorption and gives the closest estimate to xm. Published papers cover a multitude of techniques and materials tested for nitrate adsorption. But many of these involve synthesis (chitosan beads, carbon cloth, organoclays) or addition of chemicals (HCl activation, dimethylamine), some of which are reactive (epichlorohydrin, which forms a carcinogen in water). Such adsorbents would not be fairly compared to the adsorbents in this research, which use existing waste and only adds a natural polymer. herefore only xm values for adsorbents comparable in either principle or materials were considered for comparison.
Untreated natural waste products exhibit lower adsorption potential, while synthesized chitosan beads show immense adsorptive strength. Combining these two situations gives the slightly improved capacity of cellulose with chitosan. Desorption data is unknown for these materials, but it is unlikely that chitosan hydrobeads, which are less cost-eicient, more complex to form, and involve no recycled materials, would be reused in a fertilizer. herefore, compared with the possibly reusable adsorbents mentioned in published work, the adsorbent produced in this research has considerable adsorptive strength, as well as further implications for environmental sustainability.
Conclusion Cellulose waste in the form of cardboard was successfully characterized as an adsorbent for aqueous nitrate. Modiication with chitosan improved cellulose adsorption in a standardized experiment from adsorbing 2.713% to an average of 24.97% of nitrate in solution. An adsorption isotherm study was then performed, but ofered inconclusive results for describing the mechanism of the adsorbent, though a trend was identiied. Low rates of desorption were determined for the cellulose with chitosan, which suggests the possibility of slow-release fertilizer use in application. Minimal desorption in solution is also promising in that adsorbed material will remain on the adsorbent even if exposed to water for extended amounts of time. he maximum determinable adsorption capacity from the study was compared to values in published work. Only research involving natural materials, such as bamboo and straw, was considered, as those with added chemicals would increase cost environmental complications. he cardboard and chitosan adsorbent made in this study exhibited more eicient adsorptive behavior than such published work. High adsorptive capacity of synthesized chitosan beads was also considered, as it suggests improved chitosan and cellulose integration could improve adsorption as well. Positive implications for the use of chitosan-modiied cellulose include decreasing paper waste, minimizing addition of environmental hazards, reuse as fertilizer, and appreciable adsorptive ability. Column studies have been planned to be carried out to scale up the research. he results of such research would then indicate possibility of using the cellulose pieces in water iltration systems, groundwater and well treatment, or integration into other processes. A wide range of applications exist, especially because the chitosan-modiied cellulose shows considerable aptitude to adsorbing nitrate.
Table 7. Adsorptive capacity xm of published adsorbents versus that of this study. *Average of all x values given batch of 0.200 g CB per standard solution. Volume 2 | 2012-2013 | 39
Street Broad Scientific Acknowledgements I would irst like to thank Dr. Myra Halpin for guidance and inspiration through the Research in Chemistry program at the North Carolina School of Science andMathematics (NCSSM) which provided me with lab space. Dr. Halpin sparked my interest in environmental science, speciically nitrate pollution, and helped me develop the project. I would also like to thank Dr. Monique Williams from NCSSM for supervising the project for several weeks. Next, I would like to thank Dr. Martin Hubbe and Dr. David Genereux from the North Carolina State University for answering questions I had for them on adsorption studies, materials, and groundwater treatment. I also have many thanks to my peers in the Research in Chemistry program as they also provided invaluable encouragement and input throughout the project. Last but not least, I would like to thank my family for support throughout this venture as I approached the project largely independently.
References  Dubrovsky, N.M., and P.A. Hamilton (2010). Nutrients in the Nation’s Streams and Groundwater: National Findings and Implications. U.S. Geological Survey Fact Sheet 2010-3078, 6.  Sparks, D. L. (1995). Environmental Soil Chemistry. San Diego, CA: Academic Press. National Academy of Engineering (2008). Manage the nitrogen cycle. NAE Grand Challenges for Engineering. Retrieved September 25, 2012 from http://www.engineeringchallenges.org/cms/8996/9132.aspx.  Holbrook, S. (2012). Farming Communities Facing Crisis Over Nitrate Pollution, Study Says. Food & Environment Reporting Network. Retrieved September 25, 2012 from http://thefern.org/2012/03/farming-communities-facing-crisis-over-nitrate-pollution-study-says/.  United States Environmental Protection Agency (2012). Drinking Water Contaminants. Retrieved September 25, 2012 from http://water.epa.gov/drink/contaminants/index.cfm. Bhatnagar, A., & Sillanpää, M. (2011). A review of emerging adsorbents for nitrate removal from water. Chemical Engineering Journal, 168, 2, 493-504.  de-Bashan, L.E., & Bashan, Y. (2004). Recent advances in removing phosphorus from wastewater and its future use as fertilizer (1997–2003). Water Research, 38, 19, 4222-4246.  Bismarck, A., Aranberri-Askargorta, I., Springer, J., Lampke, T., Wielage, B., Stamboulis, A., Shenderovich, I. & Limbach, H. (2002). Surface Characterization of Flax, Hemp and Cellulose Fibers; Surface Properties and the Water Uptake Behavior. Polymer Composites, 23, 5. 40 | 2012-2013 | Volume 2
REsEaRch  Hubbe, M.A., Hasan, S. H., & Ducoste, J. J. (2011). Cellulosic Substrates for Removal of Pollutants from Aqueous Systems: A Review. 1. Metals. BioResources 6, 2161-2287.  Royal Society of Chemistry. Cellulose and Chitosan chemical structures. ChemSpider. Retrieved on September 25, 2012 from http://www.chemspider.com/ChemicalStructure.26943876.html and http://www.chemspider. com/Chemical-Structure.2342878.html?rid=b656acee9c8e-4951-9c69-480347c7db87  Urreaga, J.M., & de la Orden, M.U. (2006). Chemical interactions and yellowing in chitosan-treated cellulose. European Polymer Journal, 42, 10, 2606-2616.  Okeola, O. F. & Odebunmi, E. O. (2010). Comparison of Freundlich and Langmuir Isotherms for Adsorption of Methylene Blue by Agrowaste Derived Activated Carbon. Advances in Environmental Biology, 4, 329-335.  Mizuta, K., Matsumoto, T., Hatate, Y., Nishihara, K., & Nakanishi, T. (2004). Removal of nitrate-nitrogen from drinking water using bamboo powder charcoal. Bioresource Technology, 95, 3, 255-257.