INTRODUCTION

Hydrogels are highly hydrophilic polymers that are capable of absorbing and releasing substantial amounts of fluids because of their elastic three-dimensional networks and the presence of functional groups such as OH-, COOH-, and CONH- (^{Ahmed 2015}). The degree of swelling, absorption capacity, and permeability are qualities of hydrogels, and these abilities depend on the type of material with which the hydrogel has been manufactured (^{Bai et al. 2010}, ^{Ekebafe 2011}, ^{Asgher et al. 2012}, ^{Ahmed 2015}). Some factors can affect the degree of swelling in hydrogels, such as pH, ionic strength, temperature, and electromagnetic radiation (^{Chen and Zhao 2000}, ^{Ekebafe 2011}, ^{Ahmed 2015}). Most applications of hydrogels are focused on the field of medicine and the controlled release of drugs. Currently, research has been developed for the application of hydrogels in agriculture, for the partial release of pesticides, in bioremediation by creating fungal inoculums, and in the capture of metal ions for automotive and industrial use waters (^{Galhaup et al. 2002}, ^{Escobar et al. 2003}, ^{Farrel et al. 2013}, ^{Ahmed 2015}). Superabsorbent polymers are hydrogels that have been widely accepted for their liquid capture capacity and their wide application in various agricultural, pharmaceutical, and construction sectors, and can reach an absorption of up to 500 times their weight (^{Hublik and Schinner 2000}, ^{Kabiri and Zohuriaan-Mehr 2004}, ^{Kumar et al. 2009}, ^{Li 2010}).

Molecular immobilization is a technique that allows the industrial application of enzymes because it improves stability, activity, and handling, which translates into prolonged activity times (^{Nannipieri and Bollag 1991}, ^{Ortiz et al. 2006}, ^{Liu et al. 2009}, ^{Muñoz and Zuluaga 2009}). However, this process is carried out with polymerizations prepared at the time of the immobilization, and to date, there are no reports of using prefabricated copolymers for the immobilization of nutrient medium conditioned with enzymatic activities. Thus, this study aimed to develop an alternative to use Hydrogel 3005K4® as an immobilizing vehicle for a conditioned medium containing ligninolytic enzymes with application potential in bioremediation. The latter can be achieved thanks to the high absorption of the copolymer in liquid media and the interactions between the positive charges, specifically of the enzyme laccase. This enzyme has the most tolerance to changes in pH and temperature, generating an interaction with the negative charges of the networks formed by the acrylamide acrylate potassium copolymer. Once the copolymer absorbs the conditioned medium, it is kept in the three-dimensional network. As the copolymer dehydrates or is replaced with water, the conditioned medium containing the enzymes of interest is released.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

The absorption percentage obtained at various times of the conditioned medium showed a statistically significant correlation between the ratio of the copolymer-conditioned medium with the exposure time and copolymer particle size (p = 0.000, p = 0.000 and p = 0.026 with α = 0.05). The controls showed more efficient absorption in less time and had a lower amount of 100% copolymer absorption at 6 h at a ratio of 0.1:10 w/v copolymer-water (Table II). For both copolymer particle sizes, improved absorption was detected when 0.2 g of copolymer per 10 mL of conditioned medium was added, resulting in an absorption ratio 50 times the weight of the copolymer. This ratio is less efficient than that of the control at 0.1 g of polymer with 10 mL of water. According to the supplier, this copolymer can absorb 270 to 400 times its weight in deionized water and 150 to 270 times its weight in water with 1000 ppm of NaCl. Therefore, its absorption capacity was much lower than that reported by the supplier, although the fluid conditions were not the same. This result was attributed to the presence of ions with positive charges. Even with this absorption capacity, the costs of using this copolymer (approximately US $ 7) are below the use of other materials such as alginates (approximately US $ 170).

There are reports of absorption of various compounds, such as methylene blue, crystal violet and heavy metals or arsenic, by using specific designs of copolymers for each type of compound and manipulating the pH and temperature for their conditioning. Our experiment was conducted under pH 6.3 - 6.7 and room temperature (25 - 31 ºC) (^{Zhen and Zhang 2008}, ^{Singh et al. 2010}, ^{Villarroel et al. 2010}, ^{Snoeck et al. 2014}). To optimize the absorption processes, the conditions and compound to be absorbed must be evaluated, and a specific hydrogel or copolymer design must be assessed, which can result in considerable increases in costs. We used a single type of copolymer in different treatments to reduce costs.

In the analysis of hydration and degree of swelling, we found 150 to 300 times increased hydration and swelling in the CM0 and CM1 control treatments with respect to the treatments with the conditioned medium (Table III).

Table III shows that the degree of swelling and hydration of the copolymer were similar between the control and the samples with conditioned medium in the CM2 treatment (1 - 2 mm copolymer with conditioned medium 4736.40 ± 95.77; control with water 4678.44 ± 106.61; 2-4 mm copolymer with conditioned medium 4331.40 ± 57.15; contro with water 4484.15 ± 130.86). This result shows that this relationship is ideal to recover the conditioned medium.

In the desorption process of the medium conditioned from the hydrogel, we found that both time and the copolymer’s size had a statistically significant effect on the release of the conditioned medium (p = 0.0000, p = 0.0013, respectively, with α = 0.05). In the case of desorption in soil, only time had a statistically significant effect, with day 10 being the maximum release point (p = 0.0000 with α = 0.05.) The desorption time of the conditioned medium decreased when exposed to the soil. In the case of soil, the desorption time was slower and began on day 10. The process was constant without desorption at three days of exposure for both particle sizes. Desorption reached a maximum desorption point of < 83%, as shown in table IV.

The release of the conditioned medium contained in the hydrogel in water was determined by changing the absorbance of the water. Due to the release of the characteristic color of the conditioned medium, it was possible to determine the desorption of this medium in water. The absorbances correlated with the amount of copolymer, showing that the absorbance increased with the amount of copolymer (Table V).

In contrast, we detected that the copolymer, when in water, tended to release the conditioned medium and absorb the available water simultaneously. By this, the copolymer increased its weight by more than 70% when the ratio was 3 g of copolymer (with conditioned medium) to 10 mL of water and increased more than 85% when the ratio was 1.5 g of copolymer (with conditioned medium) to 10 mL of water (Fig. 1). From day 2, the weight of the copolymer varied between 0.02-0.4 g, showing that the release of the conditioned medium and the absorption of the water had ceased (Fig. 1).

Fig. 1 Behavior of the copolymer with conditioned medium when immersed in water (CP = copolymer 1-2 mm; CG = copolymer 2-4 mm; 10:3 = ratio of 10 mL of water to 3 g of copolymer with conditioned medium; 10:1.5 = ratio of 10 mL of water to 1.5 g of copolymer with conditioned medium). 

One possible factor that affected the percentage of absorption of the conditionate medium is the presence of cations such as Co⁺ and Mg⁺. The positive charges may compete for binding with the negative partial charges from the oxygen in the copolymer, causing interference between dipole-dipole bonds (Fig. 2). Additionally, the presence of enzymes that have a positive charge may compete with water and prevent the formation of hydrogen bonds with the molecules of the polymeric network that make up the copolymer, and can decrease their swelling and absorption capacities in a range of pH 5-9. ^{Villarroel et al. (2010)} used potentially biodegradable hydrogels obtained from polymerized and cross-linked acrylamide in the presence of the poly [3-hydroxybutyrate] biopolymer for the absorption of copper and nickel. They showed a high affinity and efficiency of absorption for copper molecules. This affinity results in a decrease in the capacity of the absorption process. During the desorption process on filter paper, enzymatic activity of laccase was detected, resulting in the characteristic green coloration of the ABTS oxidation reaction (Fig. 3).

Fig. 2 Proposed behavior of the interaction of the conditioned medium with the copolymer molecule. 

Fig. 3 Enzymatic activity of laccase released from the acrylamide and potassium acrylate copolymer on filter paper after 72 h when reacting with ABTS (1.8 mM), A) 3 h, B) 24 h, C) 48 h, D) 72 h, E) control with water only. ABTS = 2,2’-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid]. 

CONCLUSION

It has been proven that the use of copolymers for the immobilization of nutrient medium conditioned with viable enzymes is possible. The copolymer had reduced absorption capacity in the presence of the conditioned medium recovered from T. versicolor due to possible interactions between the ion-dipole or dipole-dipole forces of the copolymer and the Mg+, Cu+, and enzymes with positive charges present in the conditioned medium. We found an absorption capacity of 50 times the weight of the copolymer. In the desorption process, the conditioned medium was released into water and soil and exposed to the environment, and we found that the copolymer had an affinity for water under the condition tested. Laccase activity was seen visually during desorption on filter paper. However, the initial and final enzyme units still need to be determined due to the conditions of the assay. These results help to consider whether this mixture could help to release the enzymes during a bioremediation process and at the same time, to keep the humidity on the plant.