Bioremoval of Antibiotics by Using Biodegradable Hydrogel Beads from Aqueous Solutions
Tenzing Japhe1, Roshanna Paulsingh1, Kwonil Ko1, Jaehoung Hong1, Abel E. Navarro1*
1Science Department, Borough of Manhattan Community College, City University of New York, NY, USA.
Elimination and proper disposal of pollutants of emerging concern are a major challenge in environmental remediation. On the
Numerous studies are being carried out to achieve the preparation and characterization of new materials for the elimination of organic pollutants from contaminated water. Naturally occurring adsorbents are potential candidates for this application as they are biodegradable and inexpensive. There are diverse types of polymeric particles that are commonly used in the removal of organic pollutants (i.e. drugs and antibiotics) from solutions. According to their size, they can be classified as micro- and nanoparticles. Microparticles are polymeric spheres whose size ranges from 1 to 250μm (ideal diameter is <125μm). Within this group, we can include microcapsules, which are vesicular systems where the pollutant is confined in a cavity and surrounded by a single polymeric membrane; and the microspheres that are matrix systems in which the pollutant is dispersed in the particle. On the other hand, nanoparticles are polymeric systems with smaller sizes (<1μm) .
Concern for democracy and participation is quite crucial to the current practice of environmental education (EE) and education for sustainable development (ESD). Recent publications in EE and ESD journals emphasize the need to reflect on implicit normativity of education, reject the anthropocentrism vs. ecocentrism dichotomy in favor of more plural ethics approaches  and caution educators not to preach pre-determined values  (One of the prominent dilemmas discussed in journals specialized in EE (e.g. Environmental Education Research, The Journal of Environmental Education and Canadian Journal of Environmental Education) and ESD (e.g. International Journal of Sustainability in Higher Education and Journal of Education for Sustainable Development) is that between open, plural or democratic education on the one hand and goal-oriented, instrumental, education for sustainability on the other hand [6-13].
The polymers that are used for the preparation of these particles are macromolecules formed by hundreds or thousands of functional units, named monomers. These polymers can be synthetic, artificial and/or natural. These last years, natural polymers or biopolymers have attracted the scientific interest in biotechnology , where biopolymers are utilized to protect cells and tissue or as encapsulating and delivery agents of several drugs and biologically active molecules.
An ideal polymer should possess these characteristics to succeed as an adsorbent: biodegradable, mechanically resistant, chemically stable and low toxicity .
Amongst the broad number of natural polymers that are used for pollutant adsorption, this study uses alginate and chitosan. Chitosan is a lineal biopolymer that can be produced by the partial deacetylation of chitin by alkaline hydrolysis at high temperatures. Chitosan has several applications due to its biocompatibility and low toxicity [4,5]. Perhaps one of the most important advantages of using chitosan in several applications is its high water solubility in acidic conditions. This is crucial, because pollutant adsorption can be tuned by simply adjusting the pH of the medium where the micro- or millisphere is utilized. Polyalginate are chains of alginic acid, a natural polysaccharide that is formed by lineal chains of α-L-guluronic and
The chemical structures of chitosan and alginate polymers are shown in Figure 1. As observed, both biopolymers share the same glucose-like scaffolds. This particular similarity to glucose makes them biodegradable and avoids any secondary contamination by the accumulation of these materials. Figure 1 also displays the presence of important functional groups in both polymers. Chitosan has an amino group that can become positively charged (NH3+) in response to acidic pH values. On the other hand, polyalginic acid shows carboxyl groups that are pH-dependent and can become negatively charged at relatively high pH values (pH higher than 3)  .
Figure 1. Chemical structure of chitosan (left) and polyalginic acid (right)
The American Environment Protection Agency (US-EPA) has listed antibiotics and other pharmaceutical products as top priority pollutants of emerging concern [11,12]. The prevalence of these substances in wastewater puts in danger not only the aquatic life, but also contributes to the development of microbial resistance . Enrofloxacin (En) and Penicillin G (Pe) were chosen as our target antibiotics due to their widespread use in the population. En is prescribed for the treatment of infection in animals. Pe is typically given intravenously or intramuscularly in humans. Chemically speaking, En and Pe belong to different antibiotic categories. En is a fluoroquinolone with different functional groups like carboxyl, amine, aromatic and ketone . Conversely, Pe is a β-lactam with carboxyl, thioether, amide and aromatic as the most important functional groups .
Figure 2. Chemical structure of the antibiotics Enrofloxacin (left) and Penicillin G (right)
In this article, we shall address the following questions: Should we uphold democratic practices in education, allowing for a plurality of opinions on the problems and causes of unsustainability? Should we teach for sustainability, and what type of sustainability should we choose – social, economic, ecological, or all of them at the same time? Should EE/ESD courses reflect on these social and/or environmental concerns, should they actually teach – and even advocate – one course of action over the other? Are students ‘rational, self-managing, self-promoting’ agents able to ‘make informed choices and manifest endless possibilities’, and are they ‘equally positioned to recognize, mobilize and consolidate productive or successful choices’?  Regardless of whether one is examining a particular dimension of sustainability or sustainability as a whole, these ‘successful choices’ become crucial in issues ranging from climate change to biodiversity loss.
For this study, alginate (AB) and chitosan (CH) hydrogel beads were prepared and tested as potential adsorbents for these antibiotics. The industrial application of these beads in envisioned in the large scale. Experimental conditions such as pH, mass of dry beads, antibiotic concentration, salinity, presence of ionic and molecular interferences, and time were explored to maximize the uptake of En and Pe. The relevance of this research goes beyond the encapsulating properties of AB and CH. In the short term, these hydrogels could be used for the treatment of residual waters of pharmaceutical industries. Most of the available literature focuses on the development of materials as drug delivery agents [2-4], but few studies have addressed the potential application of these materials in environmental remediation.
Materials and Methods
Reagents and solutions
Preparation of the hydrogel beads
Alginate beads (AB) were prepared by a protocol that is available elsewhere . In summary, sodium alginate (reagent grade, Fisher Scientific) was dissolved in deionized water and left under magnetic stirring overnight for its complete dissolution (a thick and dense solution was obtained). In the meantime, a calcium chloride (reagent grade, Fisher Scientific) solution (0.2M) was also prepared with deionized water under magnetic stirring. Upon complete dissolution of both reagents, the alginate solution was added drop by drop into the calcium chloride solution by using a peristaltic pump. Alginate beads (AB) were immediately observed upon contact of both solutions. Finally, AB were rinsed and suspended in deionized water and finally stored in the refrigerator at 4°C degrees. This technique allows obtaining millispheres of AB a diameter of 2mm.
Chitosan beads (CH) were prepared following a commonly used protocol . It consists on the dissolution of chitosan in a mixture of 10mL of acetic acid in 240mL of deionized water. Chitosan took about 24h to totally dissolve in the solution, and generated a highly viscous solution. In a separate plastic container, a 2.5M solution of NaOH was prepared and kept under magnetic stirring. Finally the chitosan/acetic acid solution was added drop by drop into the NaOH solution using a peristaltic pump. CH beads were formed by neutralization of the acetic acid in NaOH. Chitosan beads were also rinsed, suspended and stored, using a procedure similar to AB.
Determination of the dry/wet mass ratio of the beads AB and CH hydrogel beads have high water content. These samples are highly porous and need water to maintain their structure and porosity. Prior adsorption experiments, the dry/ wet mass ration of both beads was obtained to report actual dry mass values (dry alginate or chitosan). To do so, the masses of wet beads were recorded and then placed in an oven at 60°C for drying (close to 12h). Higher temperatures were not used to avoid organic decomposition. Then, the masses of dry beads were taken and compared to those of the wet beads. A linear calibration curve was constructed to correlate the wet and dry masses.
%ADS = (Ci — Ceq) * 100/Ci (1)
where Ci and Ceq are the initial and final concentrations of En and Pe in mg/L, respectively.
Characterization of the Encapsulating biopolymers
Results and Discussion
Previous reports demonstrate that the pKa value of polymeric alginic acid is 3.0 . This means that at pH values higher than
Figure 3. Effect of the initial solution pH on the bioremoval of En (left) and Pe (right) onto AB and CH.
Figure 4. Effect of the mass of beads on the adsorption of En (left) and Pe (right) onto AB and CH.
Figure 5. Effect of antibiotic concentration on the adsorption of En (left) and Pe (right) onto AB and CH.
Figure 6. Salinity Effect on the adsorption of En with AB (top left), En with CH (top right), PE with AB (bottom left) and Pe with CH (bottom right).
Therefore, the adsorption of En onto AB could be mostly driven by electrostatic interactions between the carboxylate groups of AB and the protonated amines of En. In addition to these results, CH shows a very low adsorption due to the same reason. At low pH values, the surface of CH is positively charged (on the amino groups) and En is also positively charged, inhibiting their attraction.In the case of Pe a similar behavior is observed. AB shows ahigh adsorption at pH 2 (close to 100%). Surprisingly, higher pH values show zero adsorption. This could also be explained by electrostatic interactions. At pH 2, the surface of AB is neutral and according to the structure of Pe (Figure 2) at that pH, Pe is also neutral. However, the presence of several polar groups in Pe (amides and carboxyl) can potentially form dipole-dipole interactions and hydrogen bonds with the hydroxyl
Mass effect A cost-effective mindset not only involves the elimination of antibiotics, but also the minimization of the amount of biopolymers that are used. For this purpose, different masses of AB and CH were put in contact with En and Pe solution to determine the minimum mass of the beads to remove the maximum or a reasonably high adsorption percentage. According
Figure 7. Effect on metal ions on the adsorption of En (top left) and PE (top right) and PEG on the adsorption of En (bottom left) and Pe (bottom right).
Figure 8: Time-dependence of the adsorption of En (left) and Pe (right) onto AB and CH.
Figure 9. SEM analyses of AB beads (top left), AB+En (top center), AB+Pe (top right) and CH beads (bottom left), CH+En (bottom center), and CH+Pe (bottom right)
Effect of the presence of other ionic and covalent interferences The bioremoval of antibiotics from solutions can also be disrupted by the presence of other solutes like other metal ions and surfactants (covalent compounds). In this test, En and Pe were mixed with a lead ions and a surfactant, polyethyleneglycol (PEG) to investigate the effect of these substances on the adsorption. Results are shown in Figure 7, indicating that lead (II) ions have a small effect on the adsorption of En for both biopolymers. Conversely, Pe shows a decrease in the adsorption at increasing doses of Pb ions. These results suggest that En targets different adsorption sites than Pb ions and Pe competes for the same adsorption sites with Pb ions for both biopolymers. The purpose of adding a crowding agent such as PEG aims to increase the molecular traffic in the solution, making the adsorption sites less accessible for the antibiotics. Due to the limited solubility and relatively larger sizes, En and Pe decrease their adsorption on AB and CH in the presence of PEG. Once again, the effect is more intense with Pe, due to its reduced water solubility.
Cite this article: Navarro A E. Bioremoval of Antibiotics by Using Biodegradable Hydrogel Beads from Aqueous Solutions. J J Environ Sci. 2015. 1(1): 002.