Robust and Biocompatible Hybrid Matrix with Controllable

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Robust and biocompatible hybrid matrix with controllable permeability for microalgae encapsulation Bo-Bo Zhang, Li Wang, Valerie Charles, Joanna Rooke, and Bao-Lian Su ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00191 • Publication Date (Web): 08 Mar 2016 Downloaded from http://pubs.acs.org on March 9, 2016

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ACS Applied Materials & Interfaces

Robust and biocompatible hybrid matrix with controllable permeability for microalgae encapsulation

Bo-Bo Zhang a, b, *, Li Wang a, Valérie Charles a, Joanna Rooke a, Bao-Lian Su a,*

a

Laboratory of Inorganic Materials Chemistry, University of Namur, rue de Bruxelles, 61,

Namur B-5000, Belgium b

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of

Biotechnology, Jiangnan University, Wuxi 214122, PR China

Complete mailing addresses of all authors: [email protected]; [email protected]; [email protected]; [email protected]; [email protected] (*Corresponding author)

1

ACS Paragon Plus Environment


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Abstract Relying on photosynthesis, hybrid beads entrapped the microalgae Chlamydomonas reinhardtii are synthesized for sustainable production of high value metabolites. Encapsulating the microalgae requires an exquisite control of material properties, which has been achieved by modification of different composition (alginate, polycation and silica). A coating of PDADMAC avoided cell leaking indicated by OD750 value of the culture medium and the homogeneous distribution of silica prevented bead shrinkage from the strong electronic force of PDADMAC, resulting in a robust and biocompatible matrix for the cells. Besides fabricating suitable porous beads for the diffusion of expected metabolites, the permeability can be controlled in a certain degree by applying different molecular weights of PDADMAC. The hybrid Alginate+Silica/CaCl2+PDADMAC beads possessed sufficient mechanical rigidity to sheer force under constant stirring and good chemical stability to chelating agent such as sodium citrate. Moreover, the encapsulated cells exhibited excellent long-term viability and cellular functionality, which remained about 81.5% of the original value after 120-days’ encapsulation by microscopy observation and oximetry measurement. This study is not only significant for understanding the critical role of polycation and silica involved in the synthesis of hybrid beads, but also important for the real-scale bioengineering applications.

Keywords: Hybrid beads, Microalgae encapsulation, Silica, Polycation, Photosynthesis

2


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1. Introduction In recent years, researchers have turned to nature to discover the wealth of compounds that can be produced via natural processes without human intervention. Photosynthesis is one such process that has been widely believed as a greatly exploitable methodology to obtaining high value compounds such as neutraceuticals and pharmaceuticals in a sustainable fashion.1 Relying on photosynthesis process, microalgae are emerging as a natural source of sustainable chemical compounds and value-added metabolites.2-4 The model microalga Chlamydomonas reinhardtii is the best characterized species and has been poised for exploitation as an industrial biotechnology platform.5 For example, the successful expression of recombinant proteins with pharmaceutical relevance by Chlamydomonas reinhardtii has been reported recently.6 It is known that microalgae can be cultivated in open ponds and photobioreactors (PBRs). Towards the use of genetically modified strains, PBRs have more advantages than open ponds because there is no risk to the local ecosystems. However, the cultivation of microalgae and production of valuable metabolites in PBRs appears not yet to be highly efficient and sustainable in commercial scale. Encapsulating the microalgae in a porous material is one promising approach in PBRs, which can protect the microalgae against mechanical stress and adverse conditions, increase the production yield and simplify the process of separation, extraction and purification. Calcium alginate microcapsule is one of the most commonly used materials for the entrapment of a large variety of cells for diverse purposes.7 However, with the rapid development in whole-cell encapsulation for applications relating to the environment and human health, the common used matrix calcium alginate are no longer satisfied with the strict 3



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requirement in this domain.8-10 Alginate is characterized by a wide pore size distribution, making it difficult for permeability control. The high porosity structure leads to leakage of large molecules, thus limiting its use in whole cells or cell organelles encapsulation.11-13 The presence of a coating surrounding the surface of the capsule, typically with a polyelectrolyte such as poly-L-lysine (PLL) and polydiallyldimethylammonium chloride (PDADMAC), can serve as a semi-permeable barrier by retaining the cells and protecting them from environmental damage.14-16 In addition, the combination of alginate and silica through a coacervation process can ensure superior properties, which overcome the significant drawbacks when used calcium alginate alone, such as poor chemical stability, swelling, rupturing and sensitivity to the chelating agents.17 Recently, an efficient one-step coacervation process was successfully employed to synthesize hybrid alginate-silica beads entrapped the microalga Dunaliella tertiolecta.14-15 However, several critical parameters and factors for synthesis of hybrid beads still remain unrevealed, which impede our understanding on the synthesis process and its bioengineering applications. For instance, the exact role of polycation and silica, which is the fundamental and key issue for the synthesis of hybrid beads, is still unclear. Moreover, the accessible porosity of hybrid beads should be exquisitely designed not only for the protection of cells but also for the secretion of expected metabolites. Hence, a feasible approach is necessary for tuning the permeability of hybrid bead by the regulation of its composite. To tackle these issues, hybrid beads entrapped the microalgae Chlamydomonas reinhardtii are synthesized with different compositions (alginate, polycation and silica) and the characteristics of these hybrid beads are comparatively investigated in-depth. Subsequently, the mechanical and 4


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chemical stabilities of the hybrid beads under adverse conditions and the long-term viability of the entrapped cells are also discussed in this work.

2. Materials and methods 2.1 Microalgae cultivation Chlamydomonas reinhardtii (CC125) cells were grown photoheterotrophically in tris-acetate-phosphate (TAP) medium. The suspension cultures were maintained in flasks at ambient temperature under fluorescent strip lighting and transferred into fresh TAP medium at regular intervals. 2.2 Synthesis of different hybrid beads Briefly, bead formation is based on crosslinking of an alginate biopolymer and mineralization of silicic acid in combination with a coacervation process between a polycation and the silica sol, according to our previous work.15 Four different kinds of hybrid beads were synthesized via this efficient one-step process and noted as follows. Alginate/CaCl2 means the simple crosslinking of an alginate biopolymer by Ca2+. Alginate/CaCl2+PLL means adding a PLL coating surrounding the calcium alginate beads. Alginate/CaCl2+PDADMAC means adding PDADMAC outside the calcium alginate beads. Alginate+Silica/CaCl2+PDADMAC means that the beads were synthesized by crosslinking of alginate and mineralization of silicic acid in combination with a coacervation process between PDADMAC and the silica sol. 5



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Here, the procedure of the preparation of hybrid Alginate+Silica/CaCl2+PDADMAC bead was given in details while the other kinds of beads were prepared accordingly. Briefly, hybrid Alginate+Silica/CaCl2+PDADMAC beads were prepared using a silica sol obtained from sodium silicate (1.5 M, assay 25.5-28.5 %, Merck) passed over an acid ion exchange resin (Amberlite IR 120, H form, Acros). 5 mL preformed silicic acid sol was adjusted to pH 5.1 (± 0.1) with NaOH (0.5 M) and mixed with a solution of 4 g (3.75 wt. %) sodium alginate (alginic acid sodium salt, from brown algae, Aldrich) and 1 mL cell suspension of Chlamydomonas reinhardtii (CC125). This mixture was then dropped into an aqueous solution of polycation polydiallyldimethylammonium chloride (PDADMAC, 20 wt. % in H2O, Aldrich) containing CaCl2 to catalyze the polycondenzation of the silica precursor and the cross-linking and gelification of the alginate, respectively. After incubation within this mixture, the hybrid beads that formed were washed three times with distilled water prior to being transferred into culture medium. 2.3 Cell leaking measurement To analyze the extent of cell leakage, different kinds of hybrid beads were suspended in the culture medium at ambient temperature under light. The cell density in the medium was evaluated at 750 nm (OD750) with a UV/Vis spectrometer (PerkinElmer Lambda 35) at certain interval. 2.4 Photosynthetic activity: oximetry Photosynthesis can be broken down into the light dependent and light-independent reactions. Oximetry measurements are direct evidence of the continuation of photosynthetic light reactions in Chlamydomonas reinhardtii post encapsulation. In the light reaction, a 6


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chlorophyll molecule will absorb a photon and transfer an electron to a quinone molecule which initiates a cascade of events ultimately ending in photolysis in which the chlorophyll molecule will regain an electron from a water molecule releasing dioxygen (O2). Photosynthetic activity of hybrid beads containing microalgae was monitored using a Clark electrode (Pt/Ag), purchased from HansaTech, to determine oxygen concentration in a closed system. A suspension of 0.2 g hybrid beads in 1.0 mL of TAP medium, mixed with NaHCO3 (20 µL, 0.5 M, Aldrich), was placed in the chamber and atmospheric oxygen was flushed from the system by Ar and the chamber was subsequently obscured from natural light. An external light source, λ= 650 nm, 1200 µmol m-2 s-1, was used to stimulate photosynthesis. 2.5 Zeta-Potential analysis Zeta-potential of each polycation solution was measured at 25 oC in a 6 mm carbon electrode cell on a SZ-100 analyzer (HORIBA Ltd, Kyoto, Japan) by averaging three independent tests. 2.6 Microscopical observation Optical and fluorescence microscopy techniques were performed by a Nikon AZ100 multi-zoom microscope fitted with a Nikon DS-Ri1 camera to observe the morphology and size of the hybrid beads, as well as the viability of the entrapped cells. The auto-fluorescence of pigment granules (chlorophylls) found within microalgae was exploited to identify the photosynthetic activity of cells. In parallel, the viability of the cells entrapped in the hybrid bead was determined with the aid of a dye Fluorescein diacetate (FDA). Living cells can actively convert the non-fluorescent FDA into the green fluorescent compound fluorescin, a sign of viability. The hybrid beads entrapped with algal cells were cut into thin slices and 7



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incubated with FDA dye for 20 minutes at room temperature, which were subjected to sequential fluorescent observation. Morphological image and element distribution information were also performed by scanning electron microscopy equipped with energy dispersive spectroscopy (SEM-EDS, JEOL, JSM-7500F). Hybrid beads were dehydrated using ethanol baths with progressively increasing concentrations prior to supercritical drying with carbon dioxide in order to remove the water within the porous network without destroying it. 2.7 Accessible porosity analysis The accessible porosity of the hybrid beads was investigated by studying the diffusion of fluorescent probes within the beads. Five fluorescent probes (Dextran-FITC, Sigma) with different molecular weights (4 nm, 12 nm, 22 nm, 30 nm and 50 nm) were used in the experiment. Briefly, about 2.0 g of hybrid Alginate+Silica/CaCl2+PDADMAC beads were suspended in 5 mL (0.1 mg/mL) of fluorescent solution, and aliquots of each solution were analyzed at regular intervals of time. The fluorescence intensity of the different solutions was recorded with a luminescence spectrometer (PerkinElmer LS45). The intensity of the fluorescence solution without capsules was taken as the reference and this reference was analyzed each time before a measure. 2.8 Textural properties measurement Textural

properties

of

hybrid

beads

were

evaluated

through

nitrogen

adsorption-desorption measurements at -196 °C on a Micromeritics TRISTAR 3000 porosimeter. Prior to performing the analysis, the beads were dehydrated with anhydrous 8


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ethanol, dried with supercritical CO2 and degassed at 110 oC under vacuum. The average pore size was established by the Barrett-Joyner-Halenda (BJH) method. 2.9 Mechanical strength & Chemical stability measurement To evaluate the mechanical resistance of the hybrid material, every one hundred beads with different composition were placed in separated flask with 30 mL culture medium and left under constant agitation (300 rpm) with the stirrer at room temperature. The mechanical strength of different hybrid beads was evaluated by counting the intact number and calculating the integrity rate of the beads in 30 days. Generally, degradation of a Ca2+ cross-linked alginate gel occurs when immersed in a solution containing chelating agent such as citrate, lactate or EDTA. To test the chemical stability of the hybrid material, every twenty beads with different composition were placed in separated flask with 5 mL sodium citrate solution (0.1 M) with gentle stirring. The chemical stability of different hybrid beads was evaluated by observing the integrity rate of the beads in 8 hours.

3. Results and Discussion 3.1 Important role of the polycation and silica: prevent cell leaking and bead shrinkage It is well know that one of the significant drawbacks with calcium alginate encapsulation is the relatively high porosity of gel matrices and the formation of cracks with time due to dehydration. This can lead to severe leaking of encapsulated cells and the infection of contaminants like bacteria. Hence, in order to reduce and control the permeability of the 9



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capsules, the presence of a coating outside the alginate capsule is often desirable in cell encapsulation.18 To improve the ability of retaining living cells inside the capsules, two typical polycations PLL and PDADMAC were applied as the coating membrane to form alginate/polycation capsule and meanwhile alginate alone was used for comparison. As indicated by the OD750 value in Fig. 1, obvious cell leaking occurred when the microalgae cells were only encapsulated by calcium alginate. The phenomenon of cell leaking with calcium alginate beads became more serious over time and the culture medium turned green after 60-days’ incubation. In contrast, the culture medium almost kept clear after 60-days’ incubation with the capsules alginate/CaCl2+PLL and alginate/CaCl2+PADAMAC, which suggested that a coating of polycation on the capsule surface significantly prevented cells escaping into the culture medium. Moreover, the imprisoning of cells inside the beads is beneficial when a genetically modified strain is used. Hence, the presence of a polycation coating is necessary for a long-term application. Although PLL is a typical polycation that can be successfully used as a semi-permeable membrane, it has been restricted to large-scale application due to the high cost (1000 US $/g). 19

For the large-scale production of valuable metabolites by hybrid beads encapsulated

Chlamydomonas reinhardtii, the polycation PDADMAC seems a better choice to serve as the membrane coating for lowering the cost. However, as indicated in Fig. 2, the type of coating polycation greatly affected the morphological properties and average diameter of hybrid beads. For the hybrid beads prepared with Alginate/CaCl2 and Alginate/CaCl2+PLL, normal morphology and unchanged 10


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size were observed after 7 days, whereas serious shrinkage occurred with the composition of Alginate/CaCl2+PDADMAC.

More

specifically,

the

average

diameter

of

Alginate/CaCl2+PDADMAC bead dramatically decreased from 3225 µm to 2093 µm, which corresponded to 35.1% of shrinkage. The phenomenon can be well explained by the different physicochemical characteristics of polycation. Although both served as a coating material, PLL is a weak polycation while PDADMAC is a strong one, which exerts great electronic force to alginate (a polyanion) and causes obvious shrinking of the hybrid beads.20 The difference of electronic force between PLL and PDADMAC can be further characterized by their zeta potential values. As exhibited in Fig. 3, the zeta potential of PDADMAC was significantly higher than that of PLL, which induced the morphological changes of these hybrid beads. Consequently, some necessary treatments were required when PDADMAC was proposed in the large-scale preparation of hybrid beads to replace the expensive polycation PLL. In order to enhance the resistance of the encapsulating matrix, the combination of alginate with silica via a coacervation process could be a good solution in this case.8 As displayed in Fig. 2, the addition of silica in the preparation of hybrid beads exhibited superior properties, which maintained the original morphology and diameter. It suggests that silica is necessary for the formation of robust hybrid beads when a strong polycation (i.e. PDADMAC) is used. Hence, the additional mixture of mineralized silica and flexible alginate resisted the shrinkage causing by the strong electronic force of PDADMAC. Furthermore, the oxygen production was measured by oximetry at certain intervals and the results were shown in Fig. 4. The sharp decrease in oxygen production of 11



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Alginate/CaCl2+PDADMAC beads indicated the rapid loss of the viabilities of entrapped cells, corresponding to the loss of green color which also indicated in the upper site of Fig. 4. In contrast, the Alginate+Silica/CaCl2+PDADMAC beads almost preserved their cellular activity as Alginate/CaCl2 and Alginate/CaCl2+PLL. It means the combination of alginate and silica exhibits superior properties, not only extending the mechanical resistance to PDADMAC, but also providing as biocompatible matrix for the cell viability. For further elucidating the critical role of silica, the cross-section image and element distribution information of Alginate+Silica/CaCl2+PDADMAC beads were revealed by SEM-EDS analysis. As shown in Fig. 5, silica was homogeneously embedded in the hybrid matrix, supporting the whole structure of the beads. The uniform distribution of silica can prevent the shrinkage from the strong electronic force of PDADMAC, which well explains the aforementioned intact morphology and the excellent cellular activity. In addition, the EDS spectra revealed minor sodium and calcium components, as well as relatively higher peaks of oxygen that might be due to the alginate and cellular components. It is clear that a coating of PDADMAC can prevent cell leaking and the homogeneous distribution of silica can resist the strong electronic force of PDADMAC, resulting in a robust and biocompatible matrix for the entrapment of microalgae cells.

3.2 Tailoring porosity and Controllable permeability Another critical parameter when designing and fabricating cell-encapsulated hybrid beads relies on their permeability properties.21 The matrix should act like a semi-permeable membrane, which imprisons the cells and allows the diffusion of nutrients/products, whereas 12


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protects against contamination from any predatory species.8, 22 Aforementioned, the presence of a PDADMAC coating outside the alginate capsule is necessary for preventing cell leaking. However, it was likely that the PDADMAC coating also decreased the membrane porosity and limited the size of molecules diffusing into/outside the bead. Therefore, the accessible porosity of the hybrid Alginate+Silica/CaCl2+PDADMAC bead was further investigated by studying the diffusion of fluorescent probes. As outlined in Fig. 6, the hybrid beads exhibited variations in diffusion extent of fluorescent probes with different molecular weights. For the larger fluorescent probes (50 nm & 30 nm), the hybrid bead hardly demonstrated permeability due to the PDADMAC coating and silica embedding. In contrast, the smaller fluorescent probes (12 nm & 4 nm) could easily penetrate into the capsule, which corresponded to obvious fluorescence decrease in the solution. These results suggested the existence of a molecular weight cut-off for diffusion through the matrix and the accessible porosity of the beads was suitable for the diffusion of target metabolite laccase (about 10 nm). In addition, the average pore size distributions of the beads were analyzed by adsorption– desorption of nitrogen. As indicated in Fig. 7, the hybrid Alginate+Silica/CaCl2+PDADMAC beads yielded a type II isotherm according to the IUPAC classification and the major pore size was around 22 nm. Besides the synthesis of suitable porous beads for the diffusion of expected metabolites, the further investigation on fabricating a capsule with controllable permeability is significant for fundamental research as well as bioengineering applications. The concentration of the components can greatly affect the permeability of the beads. For example, increasing the 13



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concentration of alginate in the beads decreased the rate of diffusion of the protein IgG from the gel.23 Herein, the effects of several possible influencing factors were investigated by the measurement of nitrogen adsorption–desorption. As shown in Table 1, the concentration of sodium silica and PDADMAC did not exert significant impact on the average pore size. Although the homogeneous distribution of silica can prevent the shrinkage of the beads from the strong electronic force of PDADMAC and enhance the mechanical property of the hybrid beads (Fig. 2 and Fig. 4), the variation on the silica amount does not change the pore size of the silica supported matrix. However, there was a close relationship between the molecular weight of PDADMAC and the average pore size. The higher molecular weight of PDADMAC the hybrid beads used, the larger average pore size they possessed. These results indicated that the PDADMAC with a lower molecular weight induced a more compact shell of the beads, which decreased the substance diffusion into/outside the matrix. Therefore, the permeability of hybrid beads can be controlled by applying different molecular weights of PDADMAC to a certain degree.

3.3 Excellent mechanical strength and Chemical stability in adverse conditions Long-term stability of the materials is a key challenge towards real scale production.

24

The matrix used for cell encapsulation should possess sufficient mechanical rigidity to support the beads without rupture during a long period. For this point, another limitation for calcium alginate concerns its relatively weak mechanical strength. 14


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Herein, the mechanical resistance of the material was evaluated by monitoring the integrity rate of different hybrid beads in the culture medium under constant stirring at 300 rpm for 30 days. As illustrated in Fig. 8(a), the integrity rate of hybrid beads was tightly related to the composition and structure of the matrix. Interestingly, 80% of the pure calcium alginate beads still maintained intact structure, which were not as fragile as we imaged. The addition of a polycation didn’t guarantee an improvement on the mechanical strength. When PLL was used as coating agent, the hybrid beads possessed better resistance to the sheer force than pure alginate beads. However, the presence of PDADMAC not only led to obvious shrink of the beads, but also made the beads vulnerable to the stirring. Only about 30% of the Alginate /CaCl2+PDADMAC beads kept integrity after 30 days of constant stirring in the culture medium. The difference behaviors between the beads coating with PLL and PDADMAC may be due to the distinct physicochemical characteristics of these two polycation.

20

PDADMAC exerted strong electronic force to alginate and made the inner

structure of the beads fragile to the sheer force, which led to sharp decrease in the integrity rate along the time. For alleviating the adverse impact originated from PDADMAC, a distribution of silica in the bead may be beneficial for the improvement of mechanical resistance to the sheer force. As shown in Fig. 8(a), Alginate+Silica/CaCl2+PDADMAC beads exhibited the best performance among different compositions and almost all of them remained their original morphology and structure under constant stirring after 30 days. The mineralized silica which homogeneously distributed throughout the entire beads may contribute to the excellent mechanical stabilities (Fig. 5). These results were in agreement with the study of Coradin, 22 15



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in which alginate-polycation beads were coated with sodium silicate and those composite particles have greater resistance to fracture than alginate-polycation-alginate beads. Besides the mechanical strength, chemical stability is another important aspect for fabricating hybrid beads for wide application. Degradation of a Ca2+ cross-linked alginate gel often occur by removal of the Ca2+ ions with a chelating agent such as citrate, lactate or EDTA. 23 This can lead to leakage of encapsulated materials and dissolution of the alginate polymers. As shown in Fig. 8(b), a typical concentration of sodium citrate (0.1 M) was used to examine the chemical stability of different hybrid compositions. Normally, pure calcium alginate beads were completely dissolved in half an hour in the citrate solution. The presence of a coating polycation can stabilize the beads and extend the degradation time with the sodium citrate solution. However, the weak polycation PLL only lasted 2 hours before totally dissolved by sodium citrate, whereas the strong polycation PDADMAC prolonged the degradation

time

to

8

hours.

Among

the

tested

matrixes,

the

complex

Alginate+Silica/CaCl2+PDADMAC beads showed excellent resistance to the chelating agent, which still kept about 80% integrity rate after 8 hours exposure to the citrate solution. In order to further explore the mechanism for the resistance in adverse conditions, a close observation on the fine structure of the bead was necessary. Hence, the cross-section of hybrid Alginate+Silica/CaCl2+PDADMAC bead was imaged by SEM and it gave some clues to possible reasons. As depicted in Fig. 9(a), a core-shell structure was obtained in the presence of PDADMAC when fabricated the hybrid beads. The shell, which mainly composed of the 16


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polycation, was clearly distinguished from the core part of the hybrid alginate-silica bead. It can protect the core part and well explain the excellent mechanical strength and resistance in adverse conditions. Moreover, as shown in Fig. 9(b), the microalgae cells were well encapsulated in the matrix and protected from the sheer forces. Almost all of them remained their original morphology and exhibited the excellent performance as aforementioned.

3.4 Excellent biocompatibility of the matrix and Long-term viability Despite of certain progress in this field, long-term viability of the encapsulated cells remains a significant obstacle to real scale applications. A proper relation between the stability, biocompatibility, durability and diffusional properties of the encapsulation matrix will guarantee the long-term functionality of the cells, thereby allowing long-term metabolite production. 21 Herein, the viability and long-term functionality of the encapsulated cells were evaluated by microscopy observation and oximetry measurement. First of all, a thin cross-section layer of hybrid bead (Alginate-Silica/CaCl2+PDADMAC) containing entrapped microalgae was imaged and depicted in Fig. 10. The normal morphology of Chlamydomonas reinhardtii cell was observed in the bright field and most of the cells maintained intact structure in the matrix. Fluorescent field revealed that the pigment granules display a reddish auto-fluorescence by chlorophylls and it indicated that the photosynthetic activities were well preserved within the algal cells. More importantly, after staining with FDA dye, the green florescence suggested that most of the encapsulated algal cells were still alive after 120 days. Besides the microscopy observation, oximetry measurement can provide a real-time 17



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monitoring on the encapsulated cells and give more evidence on the long-term viability. As shown in Fig. 11, the encapsulated cells preserved cellular activity and continually produced oxygen for over 120 days within the culture medium. The Chlamydomonas reinhardtii cells kept a steady state in the matrix and still produced 2.3 µmol O2 per gram of hybrid bead per hour after 120-days’ encapsulation, which remained about 81.5% of the original functionality. The long-term viability and cellular functionality can be attributed to the excellent biocompatibility of the composite materials and the fine structure of the matrix, which combining major advantages of the flexible alginate and mineralized porous silica.

4. Conclusions The robust and biocompatible hybrid matrix with controllable permeability has been synthesized for Chlamydomonas reinhardtii encapsulation. A proper regulation on the composition guarantees the robust, permeability controllable, stable, biocompatible and durable matrix for cell encapsulation, thereby ensuring long-term production of valuable metabolites. At present, these exciting results are employing in the large-scale synthesis of hybrid living beads, which is promising for the photosynthetic production of high value metabolites such as recombinant protein laccase in a green and sustainable fashion.

Acknowledgement The authors thank the Région Wallonne for funding the FOTOBIOMAT project. The authors thank Luis Pinho, Emeric Danloy, Cyrille Delneuville, Julien Olivet and Corry Charlier for their technical assistance. The authors are also grateful to our partner from 18


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Université catholique de Louvain for supplying the strain Chlamydomonas reinhardtii, and partner from Université de Mons and Université de Liège for their suggestions and assistance in the framework of the FOTOBIOMAT project.

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Table 1 Average pore size of hybrid beads prepared with different compositions Hybrid beads

Values

Pore size (nm)

Concentration of sodium silica

0.3 M

22.2

0.6 M

22.6

0.9 M

23.1

0.5% (wt. %)

21.9

2.0% (wt. %)

22.6

4.0% (wt. %)

23.3

Robust and Biocompatible Hybrid Matrix with Controllable

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