Immobilized microbial cells have been used extensively in various laboratory-scale, industrial, biotechnological and environmental technology applications. Many different carriers, especially natural polymers (e.g. algal polysaccharides – agar, agarose, alginate, carrageenan) and synthetic polymers (polyacrylamide, polystyrene, polyurethane) were successfully applied for the encapsulation of microbial cells (Cassidy et al., 1996). Among them, chitosan (a linear polysaccharide composed of randomly distributed β-(1,4)-linked d-glucosamine and N-acetyl-d-glucosamine) has been used several times (Aguilar-May and Sanchez-Saavedra, 2009; Li et al., 2007; Odaci et al., 2009). Chitosan is a cheap, biocompatible, hydrophilic, mechanically stable biopolymer containing reactive functional groups suitable and accessible for chemical modification. The immobilization of cells on chitosan carriers can usually be performed in a simple way.
Magnetic carriers enable simple magnetic separation of immobilized cells (Safarik and Safarikova, 2007, 2009). Magnetic chitosan derivatives have been already prepared and used for the immobilization of various enzymes (Ju et al., 2012; Peniche et al., 2005; Wu et al., 2009; Yang et al., 2010) or the isolation of target biologically active compounds, such as lectins (Safarik et al., 2010). In many cases, complicated procedures for magnetic chitosan particle preparations are used which are not applicable for large-scale synthesis.
In this paper we describe a simple procedure for the immobilization of Saccharomyces cerevisiae cells onto microwave-synthesized magnetic chitosan microparticles, which can be prepared in an extremely simple, one-pot procedure. The results show that immobilized yeast cells can be successfully used for sucrose hydrolysis, hydrogen peroxide decomposition and the adsorption of important xenobiotics, e.g. dyes.
Materials and methods
Chitosan [medium molecular weight (ca. 400 000), 75–85% deacetylated] was from Fluka, while glutaraldehyde was obtained from Sigma. Iron(II) sulphate heptahydrate, sodium hydroxide, hydrogen peroxide, sucrose and common chemicals were from Lach-Ner, Czech Republic. S. cerevisiae cells (compressed baker's yeast) were obtained from a food shop. A domestic microwave oven (700 W, 2450 MHz, type 0205, Eta, Czech Republic) with a rotary plate was used for the preparation of the magnetic carrier.
Microwave-assisted preparation of magnetic chitosan microparticles
Microwave-synthesized magnetic chitosan microparticles were prepared as follows: Chitosan (1 g) was dissolved in 250 ml 5% v/v acetic acid solution under stirring with a mechanical overhead stirrer (300 rpm; RZR 2041, Heidolph, Germany). After dissolution of the chitosan, 250 ml water was added, followed by 100 ml 3.6% w/v solution of FeSO4 . 7H2O. Then 10% w/v NaOH was added dropwise under intense stirring (1000 rpm) until the pH of the suspension was at least 10 and a dark precipitate was formed; 250 ml fractions of the suspension were transferred into 1000 ml beakers and submitted to 10 min microwave treatment at the maximum power (700 W). The magnetically responsive chitosan microparticles formed were repeatedly washed with water (Pospiskova and Safarik, 2013).
Yeast cells immobilization
The microwave-synthesized magnetic chitosan microparticles were cross-linked and activated, using glutaraldehyde, before immobilization of yeast cells. 400 mg magnetic particles (wet weight) were mixed with 20 ml 5% v/v solution of glutaraldehyde in a capped Falcon tube and shaken at room temperature at 20 rpm for 3 h on an automatic rotator (Multi Bio RS-24, Biosan, Latvia). Then the particles were magnetically separated and washed with distilled water. Cross-linked and activated chitosan particles were mixed with a suspension of 3g S. cerevisiae cells in 30 ml distilled water (measured pH = 5.7) and shaken for 2 h at 18 rpm at room temperature. Finally, the magnetic particles with immobilized yeast cells were washed with distilled water until no free cells were detected in the supernatant (absorbance measurement at 600 nm). This immobilized biocatalyst was stored at 4 °C in 0.9% w/v NaCl.
Sucrose hydrolysis by immobilized yeast cells
Study of sucrose hydrolysis by immobilized yeast cells, their operational and storage stabilities and time dependence of substrate hydrolysis were performed as described recently (Pospiskova et al., 2013).
Hydrogen peroxide degradation by immobilized yeast cells
Study of hydrogen peroxide degradation by immobilized yeast cells (effects of various amounts of magnetic biocatalyst) was performed as described recently (Safarik et al., 2008). Briefly, different amounts of magnetic biocatalyst were incubated for 1 h in 50 ml reaction medium (0.15 m NaCl and 0.05 m CaCl2) containing hydrogen peroxide at an initial concentration of 300 mm.
Adsorption of dyes by immobilized yeast cells
The adsorption of safranin O and crystal violet dyes was performed as described recently (Safarik et al., 2007). Briefly, 0.4 ml of the suspension of immobilized yeast cells (the volume of the settled adsorbent was 0.1 ml) was mixed with 4.8 ml water in a 15 ml test tube. Then 0.01–2.0 ml stock water solution (1 mg/ml) of a tested dye was added and the total volume of the suspension was made up to 10.0 ml with water. The suspension was mixed for 3 h at room temperature. Then the magnetic yeast cells were separated from the suspension using a magnetic separator and the clear supernatant was used for the spectrophotometric measurement. The concentration of free (unbound) dye in the supernatant (Ceq) was determined from the calibration curve. The amount of dye bound to the unit amount of the adsorbent (qeq) was calculated using the following formula, taking into account that the dry weight of 1 ml sedimented immobilized yeast cells was 0.035 g:
where Cinit is the initial concentration of dye used in the experiment.
Results and discussion
An extremely simple, inexpensive, one-pot, microwave-assisted procedure has been used for the preparation of magnetically responsive chitosan microparticles with typical diameters of 10–200 µm (Figure 1A). The production of these particles is rapid, and they can be easily removed using an appropriate magnetic separator.
The magnetic chitosan microparticles activated with glutaraldehyde efficiently captured S. cerevisiae yeast cells by the simple mixing of cells and magnetic microparticles under defined conditions and a relatively short interaction time (2 h at room temperature). Washing out of the unbound cells was satisfactory. Optical microscopy images clearly showed the binding of yeast cells onto the surface of magnetic chitosan particles (Figure 1B). The yeast cells thus immobilized exhibited the same viability as native cells after vital staining with methylene blue (data not shown).
In the absence of glucose repression, the S. cerevisiae yeast chosen as the model microorganism for this study expressed invertase enzymatic activity (β-fructofuranosidase; EC 126.96.36.199) in the periplasmic space. Invertase catalyses the hydrolysis of the disaccharide sucrose into the monosaccharides glucose and fructose; this is why magnetically modified yeast cells were used as a whole-cell biocatalyst for sucrose hydrolysis. After six cycles of sucrose hydrolysis, the immobilized yeast retained 95% of the initial invertase activity (Figure 2A). The biocatalyst was very stable during 1 month of storage at 4 °C in saline (Figure 2B). Figure 2C shows sucrose hydrolysis in time (performed for 5 h), while Figure 2D illustrates the dependence of sucrose hydrolysis on the initial concentration of sucrose after 20 min reaction time. Figure 2E demonstrates the dependence of sucrose hydrolysis on the amount (wet weight) of magnetically responsive biocatalyst used for the reaction. Potential leaching of cells from the magnetic material was tested by turbidity measurement at 600 nm; the leaching was negligible during a 1 month period and had no significant impact on the enzyme activity of the biocatalyst (Figure 2B). No extracellular invertase activity was detected during the same period.
Immobilized yeast cells also enabled efficient degradation of hydrogen peroxide, due to the presence of intracellular catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase; EC 188.8.131.52). Figure 2F shows the dependence of hydrogen peroxide decomposition on the amount of wet biocatalyst used.
Microbial cells can also be efficiently used as adsorbents for the removal of organic and inorganic xenobiotics. Yeast cells immobilized on magnetic chitosan microparticles adsorbed two important dyes belonging to different dye classes, viz. safranin O (safranin group) and crystal violet (triphenylmethane group). The adsorption was completely caused by the yeast cells, not by the chitosan carrier (Figure 3). The experimental data, analysed by means of both linear and non-linear regression, using SigmaPlot 2000 software (SPSS Inc., USA), fitted well to the Langmuir isotherm equation, usually expressed as:
where qeq (expressed in mg/g) is the amount of the adsorbed dye/unit mass of immobilized yeast cells and Ceq (expressed in mg/l) is the unadsorbed dye concentration in solution at equilibrium. Qmax is the maximum amount of the dye/unit mass (mg/g) of immobilized yeast cells to form a complete monolayer on the surface, bound at high dye concentration, and b is a constant related to the affinity of the binding sites (expressed in l/mg).
This allowed the calculation of the maximum adsorption capacities of the magnetic biocomposite, which are very important parameters describing the adsorption process: the values 99 ± 5 and 69 ± 3 mg/g dry biosorbent for safranin and crystal violet, respectively, were calculated using the linear regression, while the values 111 ± 6 and 68 ± 3 mg/g dry biosorbent were calculated using non-linear regression. The values of r2 (goodness of fit) were 0.986 for both dyes in linear regression. These maximum adsorption capacities are quite high and fully comparable with other described magnetic biosorbents used for the same purpose (Safarik and Safarikova, 2007).
This study demonstrated a fast, easy and inexpensive method for the preparation of magnetically responsive chitosan microparticles with immobilized yeast cells, and their use as a biocatalyst and/or biosorbent. Microwave-assisted synthesis of magnetic chitosan microparticles is very simple, and their interaction with S. cerevisiae cells leads to the formation of magnetically responsive biocomposites. These magnetic whole-cell biocatalysts retain their intracellular enzymatic activities and have very good stability; they can be used for sucrose hydrolysis by intracellular invertase and for hydrogen peroxide degradation by intracellular catalase. In addition, the prepared biocomposite can be used as an efficient adsorbent for the removal of xenobiotics.
This research was supported by the Grant Agency of the Czech Republic (Project No. 13-13709S), the Ministry of Education of the Czech Republic (Research Project Nos LD13023, CZ.1.05/2.1.00/03.0058 and CZ.1.07/2.4.00/31.0189) and the COST Action FA0907–BIOFLAVOUR.