Silica Hydrogels as Entrapment Material for Microalgae

Despite being a promising feedstock for food, feed, chemicals, and biofuels, microalgal production processes are still uneconomical due to slow growth rates, costly media, problematic downstreaming processes, and rather low cell densities. Immobilization via entrapment constitutes a promising tool to overcome these drawbacks of microalgal production and enables continuous processes with protection against shear forces and contaminations. In contrast to biopolymer gels, inorganic silica hydrogels are highly transparent and chemically, mechanically, thermally, and biologically stable. Since the first report on entrapment of living cells in silica hydrogels in 1989, efforts were made to increase the biocompatibility by omitting organic solvents during hydrolysis, removing toxic by-products, and replacing detrimental mineral acids or bases for pH adjustment. Furthermore, methods were developed to decrease the stiffness in order to enable proliferation of entrapped cells. This review aims to provide an overview of studied entrapment methods in silica hydrogels, specifically for rather sensitive microalgae.


Introduction
Microalgae are a diverse group of unicellular organisms including pro-and eukaryotes, freshwater and marine organisms, living individually and in chains or groups. They contain high-value products such as pigments, polyunsaturated fatty acids, vitamins, and polysaccharides, while biodiesel and biohydrogen represent low-value products [1][2][3][4][5][6]. Consequently, they are considered a promising renewable feedstock for the biotechnological production of food, feed, fine and bulk chemicals, and biofuels. Furthermore, they sequester atmospheric CO 2 and can be produced throughout the year without arable land being required [6][7][8]. Although the first commercialization attempts of microalgal products were undertaken in 1942 [9], the first success was reported in 1957 with Chlorella and Spirulina as "health food" [10]. However, microalgal production competes with chemical synthesis and biotechnological production with other organisms [1,8]. For this reason, microalgal production focuses on niche markets at the moment. Additionally, limited knowledge about costs on their cultivation and processing at commercial scale is available, and the technology for commercialization is still challenging [4,8]. Consequently, many small-and medium-sized companies disappeared shortly after their foundation [3].
The major obstacle for the commercialization of microalgal products is the high cost of production due to slow growth rates, costly media and photobioreactors, problematic downstream processing, rather low cell densities, and high risk of contaminations [8,11].
A solution is provided by using immobilized microalgae. In this way, cells are effectively separated from the liquid phase, which allows for cultures with high cell densities in comparison with free cells overcoming the disadvantage of slow growth rates. Consequently, harvesting is simplified as well, and the costs of downstream processing are reduced because of the physical separation of the microalgal biomass from the product. Additionally, the separation of biomass and liquid phase enables continuous processes The resulting silanolate-cation reacts with another silanolate group to a s bond through separation of an oxonium ion according to Equation (3).
As the hydrolysis degree increases, the hydrolysis and the condensation rate d [58]. For this reason, monomers and terminal groups are preferable in hydrolyze [59,60]. In total, the hydrolysis rate is greater than the condensation rate. As a resu hardly branched chains are generated, which grow to 2-3 nm before a network and gelation starts [60].
Under alkaline conditions, a hydroxide ion attacks the silicon via a nucleoph stitution. Subsequently, an alkoxide ion separates on the opposite site. Analogou acid catalysis, an inversion of the silicon's tetrad structure occurs (see Equation (4 The condensation at alkaline conditions starts with the deprotonation of the group via separation of water (Equation (5)).
(1) At the same time, condensation starts with a protonation of the silanol group according to Equation (2). preparation of silica gels is given. In this section, three different routes based on alkoxysilanes, aqueous silicates, and aminosilanes are compared. Second, the consequences for the biocompatibility of the silica hydrogels are pointed out and the reported efforts to increase the biocompatibility are described. Third, the applied methods for microalgae entrapment are summarized.

Sol Synthesis with Alkoxysilanes
Conventional precursors for sol-gel methods are alkoxysilanes such as tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS). In contrast to aqueous silicates (see the next paragraph), the condensation occurs while hydrolysis has not yet been completed.
Under acidic conditions, hydrolysis starts with protonation of the alkoxy group, making it easier for water to attack the silicon and the alcohol demerged by a nucleophilic substitution. The tetrad structure of the precursor is inverted in the meantime (see Equation (1)).

Si
At the same time, condensation starts with a protonation of the silanol group according to Equation (2). (2) The resulting silanolate-cation reacts with another silanolate group to a siloxane bond through separation of an oxonium ion according to Equation (3). (3) As the hydrolysis degree increases, the hydrolysis and the condensation rate decrease [58]. For this reason, monomers and terminal groups are preferable in hydrolyzed form [59,60]. In total, the hydrolysis rate is greater than the condensation rate. As a result, long, hardly branched chains are generated, which grow to 2-3 nm before a network is built and gelation starts [60].
Under alkaline conditions, a hydroxide ion attacks the silicon via a nucleophilic substitution. Subsequently, an alkoxide ion separates on the opposite site. Analogous to the acid catalysis, an inversion of the silicon's tetrad structure occurs (see Equation (4)). (4) The condensation at alkaline conditions starts with the deprotonation of the silanol group via separation of water (Equation (5)). (2) The resulting silanolate-cation reacts with another silanolate group to a siloxane bond through separation of an oxonium ion according to Equation (3).
Polymers 2022, 14, x FOR PEER REVIEW 3 of 25 preparation of silica gels is given. In this section, three different routes based on alkoxysilanes, aqueous silicates, and aminosilanes are compared. Second, the consequences for the biocompatibility of the silica hydrogels are pointed out and the reported efforts to increase the biocompatibility are described. Third, the applied methods for microalgae entrapment are summarized.

Sol Synthesis with Alkoxysilanes
Conventional precursors for sol-gel methods are alkoxysilanes such as tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS). In contrast to aqueous silicates (see the next paragraph), the condensation occurs while hydrolysis has not yet been completed.
Under acidic conditions, hydrolysis starts with protonation of the alkoxy group, making it easier for water to attack the silicon and the alcohol demerged by a nucleophilic substitution. The tetrad structure of the precursor is inverted in the meantime (see Equation (1)).

Si
At the same time, condensation starts with a protonation of the silanol group according to Equation (2). (2) The resulting silanolate-cation reacts with another silanolate group to a siloxane bond through separation of an oxonium ion according to Equation (3). (3) As the hydrolysis degree increases, the hydrolysis and the condensation rate decrease [58]. For this reason, monomers and terminal groups are preferable in hydrolyzed form [59,60]. In total, the hydrolysis rate is greater than the condensation rate. As a result, long, hardly branched chains are generated, which grow to 2-3 nm before a network is built and gelation starts [60].
Under alkaline conditions, a hydroxide ion attacks the silicon via a nucleophilic substitution. Subsequently, an alkoxide ion separates on the opposite site. Analogous to the acid catalysis, an inversion of the silicon's tetrad structure occurs (see Equation (4)). (4) The condensation at alkaline conditions starts with the deprotonation of the silanol group via separation of water (Equation (5)).

(3)
As the hydrolysis degree increases, the hydrolysis and the condensation rate decrease [58]. For this reason, monomers and terminal groups are preferable in hydrolyzed form [59,60]. In total, the hydrolysis rate is greater than the condensation rate. As a result, long, hardly branched chains are generated, which grow to 2-3 nm before a network is built and gelation starts [60].
Under alkaline conditions, a hydroxide ion attacks the silicon via a nucleophilic substitution. Subsequently, an alkoxide ion separates on the opposite site. Analogous to the acid catalysis, an inversion of the silicon's tetrad structure occurs (see Equation (4)). preparation of silica gels is given. In this section, three different routes based on alkoxysilanes, aqueous silicates, and aminosilanes are compared. Second, the consequences for the biocompatibility of the silica hydrogels are pointed out and the reported efforts to increase the biocompatibility are described. Third, the applied methods for microalgae entrapment are summarized.

Sol Synthesis with Alkoxysilanes
Conventional precursors for sol-gel methods are alkoxysilanes such as tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS). In contrast to aqueous silicates (see the next paragraph), the condensation occurs while hydrolysis has not yet been completed.
Under acidic conditions, hydrolysis starts with protonation of the alkoxy group, making it easier for water to attack the silicon and the alcohol demerged by a nucleophilic substitution. The tetrad structure of the precursor is inverted in the meantime (see Equation (1)).

Si
At the same time, condensation starts with a protonation of the silanol group according to Equation (2). (2) The resulting silanolate-cation reacts with another silanolate group to a siloxane bond through separation of an oxonium ion according to Equation (3). (3) As the hydrolysis degree increases, the hydrolysis and the condensation rate decrease [58]. For this reason, monomers and terminal groups are preferable in hydrolyzed form [59,60]. In total, the hydrolysis rate is greater than the condensation rate. As a result, long, hardly branched chains are generated, which grow to 2-3 nm before a network is built and gelation starts [60].
Under alkaline conditions, a hydroxide ion attacks the silicon via a nucleophilic substitution. Subsequently, an alkoxide ion separates on the opposite site. Analogous to the acid catalysis, an inversion of the silicon's tetrad structure occurs (see Equation (4)).

(4)
The condensation at alkaline conditions starts with the deprotonation of the silanol group via separation of water (Equation (5)). The condensation at alkaline conditions starts with the deprotonation of the silanol group via separation of water (Equation (5)).
The resulting silanolate anion reacts with another silanol group to a siloxane bond, analogous to acid conditions. Likewise, the reaction is supposed to follow an SN2 mechanism (Equation (6)). (6) In contrast to the acid catalysis, at alkaline conditions, the condensation rate is higher than the hydrolysis rate. Furthermore, both rates increase with an elevated degree of hydrolysis, and therefore highly branched clusters develop [59][60][61]. At alkaline conditions and high molar water to silica ratios, colloids develop that grow via Ostwald ripening until they are stabilized by their surface loading (Stöber process) [58]. The size of the colloids depends on the solvent and the ratio of solvent to water [62]. The impact of the conditions on the structure is schematically displayed in Figure 1.

Sol Synthesis with Aqueous Silicates
Aqueous silicates such as sodium silicate belong to the conventional precursor of the sol-gel method and are therefore well known [58,61,[63][64][65]. After dilution of the precursor in water, monomers of silicic acid develop that rapidly condense. The monomers polymerize to particles by maximization of the Si-O-Si bonds and minimization of terminal hydroxyl groups. For this reason, a ring formation initially occurs, to which further monomers are added, resulting in a three-dimensional particle. Subsequently, the particles grow by Ostwald ripening. The particles' size depends on the pH and the presence of salts [58]. This is schematically displayed in Figure 2. The resulting silanolate anion reacts with another silanol group to a siloxane bond, analogous to acid conditions. Likewise, the reaction is supposed to follow an S N 2 mechanism (Equation (6)).
The resulting silanolate anion reacts with another silanol group to a siloxane bond, analogous to acid conditions. Likewise, the reaction is supposed to follow an SN2 mechanism (Equation (6)). (6) In contrast to the acid catalysis, at alkaline conditions, the condensation rate is higher than the hydrolysis rate. Furthermore, both rates increase with an elevated degree of hydrolysis, and therefore highly branched clusters develop [59][60][61]. At alkaline conditions and high molar water to silica ratios, colloids develop that grow via Ostwald ripening until they are stabilized by their surface loading (Stöber process) [58]. The size of the colloids depends on the solvent and the ratio of solvent to water [62]. The impact of the conditions on the structure is schematically displayed in Figure 1.

Sol Synthesis with Aqueous Silicates
Aqueous silicates such as sodium silicate belong to the conventional precursor of the sol-gel method and are therefore well known [58,61,[63][64][65]. After dilution of the precursor in water, monomers of silicic acid develop that rapidly condense. The monomers polymerize to particles by maximization of the Si-O-Si bonds and minimization of terminal hydroxyl groups. For this reason, a ring formation initially occurs, to which further monomers are added, resulting in a three-dimensional particle. Subsequently, the particles grow by Ostwald ripening. The particles' size depends on the pH and the presence of salts [58]. This is schematically displayed in Figure 2. In contrast to the acid catalysis, at alkaline conditions, the condensation rate is higher than the hydrolysis rate. Furthermore, both rates increase with an elevated degree of hydrolysis, and therefore highly branched clusters develop [59][60][61]. At alkaline conditions and high molar water to silica ratios, colloids develop that grow via Ostwald ripening until they are stabilized by their surface loading (Stöber process) [58]. The size of the colloids depends on the solvent and the ratio of solvent to water [62]. The impact of the conditions on the structure is schematically displayed in Figure 1.
The resulting silanolate anion reacts with another silanol group to a siloxane bond, analogous to acid conditions. Likewise, the reaction is supposed to follow an SN2 mechanism (Equation (6)). (6) In contrast to the acid catalysis, at alkaline conditions, the condensation rate is higher than the hydrolysis rate. Furthermore, both rates increase with an elevated degree of hydrolysis, and therefore highly branched clusters develop [59][60][61]. At alkaline conditions and high molar water to silica ratios, colloids develop that grow via Ostwald ripening until they are stabilized by their surface loading (Stöber process) [58]. The size of the colloids depends on the solvent and the ratio of solvent to water [62]. The impact of the conditions on the structure is schematically displayed in Figure 1.

Sol Synthesis with Aqueous Silicates
Aqueous silicates such as sodium silicate belong to the conventional precursor of the sol-gel method and are therefore well known [58,61,[63][64][65]. After dilution of the precursor in water, monomers of silicic acid develop that rapidly condense. The monomers polymerize to particles by maximization of the Si-O-Si bonds and minimization of terminal hydroxyl groups. For this reason, a ring formation initially occurs, to which further monomers are added, resulting in a three-dimensional particle. Subsequently, the particles grow by Ostwald ripening. The particles' size depends on the pH and the presence of salts

Sol Synthesis with Aqueous Silicates
Aqueous silicates such as sodium silicate belong to the conventional precursor of the sol-gel method and are therefore well known [58,61,[63][64][65]. After dilution of the precursor in water, monomers of silicic acid develop that rapidly condense. The monomers polymerize to particles by maximization of the Si-O-Si bonds and minimization of terminal hydroxyl groups. For this reason, a ring formation initially occurs, to which further monomers are added, resulting in a three-dimensional particle. Subsequently, the particles grow by Ostwald ripening. The particles' size depends on the pH and the presence of salts [58]. This is schematically displayed in Figure 2. At acidic pH, the solubility of a particle is low, and therefore the particles grow up to 2-4 nm before they are connected first to chains and afterward to networks. These networks spread in the aqueous medium before they finally gel. If the pH is lower than 2, formation and aggregation of particles occur at the same time. After the particles have reached a size of 2 nm, the Ostwald ripening stops, and a network is formed from these small particles. In contrast, at alkaline conditions, the solubility of the particles is higher. Furthermore, the condensed particles are charged, and thus they repel each other. For this reason, an enhanced growth via Ostwald ripening instead of a connection of the particles occurs. In the absence of salts, aggregation is lacking and a stabilized sol is developed [58,64].
At a pH greater than 2, condensation starts with the deprotonation of the silanol group by a hydroxide ion (Equation (7)). Already condensed species are more likely ionized.
Afterward, the silanolate anion reacts with another silanol group via separation of a hydroxide ion (Equation (8)). In comparison, the first reaction (Equation (7)) occurs faster than the second (Equation (8)) [58]. At acidic pH, the solubility of a particle is low, and therefore the particles grow up to 2-4 nm before they are connected first to chains and afterward to networks. These networks spread in the aqueous medium before they finally gel. If the pH is lower than 2, formation and aggregation of particles occur at the same time. After the particles have reached a size of 2 nm, the Ostwald ripening stops, and a network is formed from these small particles.
In contrast, at alkaline conditions, the solubility of the particles is higher. Furthermore, the condensed particles are charged, and thus they repel each other. For this reason, an enhanced growth via Ostwald ripening instead of a connection of the particles occurs. In the absence of salts, aggregation is lacking and a stabilized sol is developed [58,64].
At a pH greater than 2, condensation starts with the deprotonation of the silanol group by a hydroxide ion (Equation (7)). Already condensed species are more likely ionized. At acidic pH, the solubility of a particle is low, and therefore the particles grow up to 2-4 nm before they are connected first to chains and afterward to networks. These networks spread in the aqueous medium before they finally gel. If the pH is lower than 2, formation and aggregation of particles occur at the same time. After the particles have reached a size of 2 nm, the Ostwald ripening stops, and a network is formed from these small particles. In contrast, at alkaline conditions, the solubility of the particles is higher. Furthermore, the condensed particles are charged, and thus they repel each other. For this reason, an enhanced growth via Ostwald ripening instead of a connection of the particles occurs. In the absence of salts, aggregation is lacking and a stabilized sol is developed [58,64].
At a pH greater than 2, condensation starts with the deprotonation of the silanol group by a hydroxide ion (Equation (7)). Already condensed species are more likely ionized.
Polymers 2022, 14, x FOR PEER REVIEW 6 of 25 (8) During acid catalysis, growth according to Equation (8) occurs preferably with highly condensed and less condensed species, while during base catalysis, the charged condensed species repel each other. This is why at a pH greater than 7, the addition of monomers is favored [58].
During the condensation at a pH smaller than 2, the addition of a proton leads to a partially positively charged intermediate according to Equation (9).
The intermediate reacts with another silanol group via separation of an oxonium ion (Equation (10)).

Sol Synthesis with Aminosilanes
In the case of the precursor tetra(n-propylamino)silane as a representative of tetra(alkoxyamino)silanes, investigations on the ammonolysis and the subsequent condensation to NSi3 and HNSi2 networks are reported [66,67]. It was observed that the precursor as well as the occurring by-product n-propylamine function as a base and therefore cause the autocatalysis of the precursor [68]. Furthermore, Si-N bonds show a smaller bond energy of 437.1 ± 9.9 kJ/mol in comparison to Si-O bonds of 799.6 ± 13.4 kJ/mol [69]. For this reason, the reactivity of tetra(n-propylamino)silane is greater than that of alkoxysilanes such as TEOS [70]. As a consequence of the autocatalysis and the smaller bond energy, the precursor reacts to the addition of water with a rapidly formed white precipitation that was identified as (SixOy)z [70]. This reaction (Equation (11)) takes place at the interface of the tetra(n-propylamino)silane emulsion droplets. (11) The developed (SixOy)z agglomerates and the emulsion droplets of the precursor form a turbid dispersion in the aqueous medium. The increasing alkalinity based on the developed by-product causes a fragmentation of the agglomerates according to Equation (12) and dilutes in the aqueous solution. The resulting polydisperic sol is transparent and displays particles comparable with basic catalyzed TEOS gels [70].
The described development of a particulate sol in aqueous media at basic conditions is displayed in Figure 3. During acid catalysis, growth according to Equation (8) occurs preferably with highly condensed and less condensed species, while during base catalysis, the charged condensed species repel each other. This is why at a pH greater than 7, the addition of monomers is favored [58].
During the condensation at a pH smaller than 2, the addition of a proton leads to a partially positively charged intermediate according to Equation (9).
Polymers 2022, 14, x FOR PEER REVIEW 6 of 25 (8) During acid catalysis, growth according to Equation (8) occurs preferably with highly condensed and less condensed species, while during base catalysis, the charged condensed species repel each other. This is why at a pH greater than 7, the addition of monomers is favored [58].
During the condensation at a pH smaller than 2, the addition of a proton leads to a partially positively charged intermediate according to Equation (9). (9) The intermediate reacts with another silanol group via separation of an oxonium ion (Equation (10)).

Sol Synthesis with Aminosilanes
In the case of the precursor tetra(n-propylamino)silane as a representative of tetra(alkoxyamino)silanes, investigations on the ammonolysis and the subsequent condensation to NSi3 and HNSi2 networks are reported [66,67]. It was observed that the precursor as well as the occurring by-product n-propylamine function as a base and therefore cause the autocatalysis of the precursor [68]. Furthermore, Si-N bonds show a smaller bond energy of 437.1 ± 9.9 kJ/mol in comparison to Si-O bonds of 799.6 ± 13.4 kJ/mol [69]. For this reason, the reactivity of tetra(n-propylamino)silane is greater than that of alkoxysilanes such as TEOS [70]. As a consequence of the autocatalysis and the smaller bond energy, the precursor reacts to the addition of water with a rapidly formed white precipitation that was identified as (SixOy)z [70]. This reaction (Equation (11)) takes place at the interface of the tetra(n-propylamino)silane emulsion droplets.
The developed (SixOy)z agglomerates and the emulsion droplets of the precursor form a turbid dispersion in the aqueous medium. The increasing alkalinity based on the developed by-product causes a fragmentation of the agglomerates according to Equation (12) and dilutes in the aqueous solution. The resulting polydisperic sol is transparent and displays particles comparable with basic catalyzed TEOS gels [70]. (12) The described development of a particulate sol in aqueous media at basic conditions is displayed in Figure 3.
The intermediate reacts with another silanol group via separation of an oxonium ion (Equation (10)).
Polymers 2022, 14, x FOR PEER REVIEW 6 of 25 (8) During acid catalysis, growth according to Equation (8) occurs preferably with highly condensed and less condensed species, while during base catalysis, the charged condensed species repel each other. This is why at a pH greater than 7, the addition of monomers is favored [58].
During the condensation at a pH smaller than 2, the addition of a proton leads to a partially positively charged intermediate according to Equation (9). (9) The intermediate reacts with another silanol group via separation of an oxonium ion (Equation (10)).

Sol Synthesis with Aminosilanes
In the case of the precursor tetra(n-propylamino)silane as a representative of tetra(alkoxyamino)silanes, investigations on the ammonolysis and the subsequent condensation to NSi3 and HNSi2 networks are reported [66,67]. It was observed that the precursor as well as the occurring by-product n-propylamine function as a base and therefore cause the autocatalysis of the precursor [68]. Furthermore, Si-N bonds show a smaller bond energy of 437.1 ± 9.9 kJ/mol in comparison to Si-O bonds of 799.6 ± 13.4 kJ/mol [69]. For this reason, the reactivity of tetra(n-propylamino)silane is greater than that of alkoxysilanes such as TEOS [70]. As a consequence of the autocatalysis and the smaller bond energy, the precursor reacts to the addition of water with a rapidly formed white precipitation that was identified as (SixOy)z [70]. This reaction (Equation (11)) takes place at the interface of the tetra(n-propylamino)silane emulsion droplets.
The developed (SixOy)z agglomerates and the emulsion droplets of the precursor form a turbid dispersion in the aqueous medium. The increasing alkalinity based on the developed by-product causes a fragmentation of the agglomerates according to Equation (12) and dilutes in the aqueous solution. The resulting polydisperic sol is transparent and displays particles comparable with basic catalyzed TEOS gels [70].
The described development of a particulate sol in aqueous media at basic conditions is displayed in Figure 3.

Sol Synthesis with Aminosilanes
In the case of the precursor tetra(n-propylamino)silane as a representative of tetra(alkoxyamino)silanes, investigations on the ammonolysis and the subsequent condensation to NSi 3 and HNSi 2 networks are reported [66,67]. It was observed that the precursor as well as the occurring by-product n-propylamine function as a base and therefore cause the autocatalysis of the precursor [68]. Furthermore, Si-N bonds show a smaller bond energy of 437.1 ± 9.9 kJ/mol in comparison to Si-O bonds of 799.6 ± 13.4 kJ/mol [69]. For this reason, the reactivity of tetra(n-propylamino)silane is greater than that of alkoxysilanes such as TEOS [70]. As a consequence of the autocatalysis and the smaller bond energy, the precursor reacts to the addition of water with a rapidly formed white precipitation that was identified as (Si x O y ) z [70]. This reaction (Equation (11)) takes place at the interface of the tetra(n-propylamino)silane emulsion droplets.
During acid catalysis, growth according to Equation (8) occurs preferably with highly condensed and less condensed species, while during base catalysis, the charged condensed species repel each other. This is why at a pH greater than 7, the addition of monomers is favored [58].
During the condensation at a pH smaller than 2, the addition of a proton leads to a partially positively charged intermediate according to Equation (9).
The intermediate reacts with another silanol group via separation of an oxonium ion (Equation (10)).

Sol Synthesis with Aminosilanes
In the case of the precursor tetra(n-propylamino)silane as a representative of tetra(alkoxyamino)silanes, investigations on the ammonolysis and the subsequent condensation to NSi3 and HNSi2 networks are reported [66,67]. It was observed that the precursor as well as the occurring by-product n-propylamine function as a base and therefore cause the autocatalysis of the precursor [68]. Furthermore, Si-N bonds show a smaller bond energy of 437.1 ± 9.9 kJ/mol in comparison to Si-O bonds of 799.6 ± 13.4 kJ/mol [69]. For this reason, the reactivity of tetra(n-propylamino)silane is greater than that of alkoxysilanes such as TEOS [70]. As a consequence of the autocatalysis and the smaller bond energy, the precursor reacts to the addition of water with a rapidly formed white precipitation that was identified as (SixOy)z [70]. This reaction (Equation (11)) takes place at the interface of the tetra(n-propylamino)silane emulsion droplets.
The developed (SixOy)z agglomerates and the emulsion droplets of the precursor form a turbid dispersion in the aqueous medium. The increasing alkalinity based on the developed by-product causes a fragmentation of the agglomerates according to Equation (12) and dilutes in the aqueous solution. The resulting polydisperic sol is transparent and displays particles comparable with basic catalyzed TEOS gels [70].
The described development of a particulate sol in aqueous media at basic conditions is displayed in Figure 3. The developed (Si x O y ) z agglomerates and the emulsion droplets of the precursor form a turbid dispersion in the aqueous medium. The increasing alkalinity based on the developed by-product causes a fragmentation of the agglomerates according to Equation (12) and Polymers 2022, 14, 1391 7 of 24 dilutes in the aqueous solution. The resulting polydisperic sol is transparent and displays particles comparable with basic catalyzed TEOS gels [70].
The developed (SixOy)z agglomerates and the emulsion droplets of the precursor form a turbid dispersion in the aqueous medium. The increasing alkalinity based on the developed by-product causes a fragmentation of the agglomerates according to Equation (12) and dilutes in the aqueous solution. The resulting polydisperic sol is transparent and displays particles comparable with basic catalyzed TEOS gels [70]. (12) The described development of a particulate sol in aqueous media at basic conditions is displayed in Figure 3.

(12)
The described development of a particulate sol in aqueous media at basic conditions is displayed in Figure 3.

Gel Synthesis
Entrapped cells are affected by the gel time due to sedimentation of the cells and delay until the hydrogel can be covered with cultivation medium to supply nutrients [41]. The gel time is specified as the time until the gel point is reached, which in turn is defined as the time when an infinite, spanning polymer or aggregate first appears [71]. Consequently, the gel time is a function of the hydrolysis and condensation rate and therefore depends on the pH value as well [58,72]. In case of alkoxysilanes, the rate of hydrolysis is linearly proportional to the concentration of acid and base [72]. Below the isoelectric point, i.e., pH 2, the rate of hydrolysis is large compared to the rate of condensation. Consequently, at large molar water/silica ratios (H2O:Si > 4), the hydrolysis will be complete at an early stage of the reaction [58]. At intermediate pH conditions, i.e., between pH 3 and pH 8, the rate of condensation increases with the pH value, while the rate of hydrolysis goes through a minimum at approximately neutral pH. As a consequence, hydrolysis is rate-limiting for gelation at these pH conditions [58,72]. Above pH 7, condensation occurs by nucleophilic displacement reactions via SiOanions, preferentially between protonated and deprotonated acidic species [58]. For this reason, the rate of condensation decreases with increased pH due to mutual repulsion.
In the case of diluted aqueous silicates, hydrolysis is immediately and fully completed at all pH conditions. Hence, the gel time solely depends on the condensation time. Around pH 1.5-3, sols display a maximum stability, and therefore the longest gel time. The rate of condensation is proportional to the concentration of protons. Between pH 2 and 7, the condensation rate is proportional to the concentration of hydroxide ions. Consequently, the gel time decreases with the pH, with a minimum at pH 5-6. Above pH 7, the particles are charged and therefore mutually repulsive. For this reason, the particles grow, but no gelation can be observed. The addition of salts lowers the ionic charge on particles, and therefore a gelation is possible and the gel time decreases [58,64]. The relative reaction rates of hydrolysis and condensation as well as the gel time are displayed in Figure 4.

Gel Synthesis
Entrapped cells are affected by the gel time due to sedimentation of the cells and delay until the hydrogel can be covered with cultivation medium to supply nutrients [41]. The gel time is specified as the time until the gel point is reached, which in turn is defined as the time when an infinite, spanning polymer or aggregate first appears [71]. Consequently, the gel time is a function of the hydrolysis and condensation rate and therefore depends on the pH value as well [58,72]. In case of alkoxysilanes, the rate of hydrolysis is linearly proportional to the concentration of acid and base [72]. Below the isoelectric point, i.e., pH 2, the rate of hydrolysis is large compared to the rate of condensation. Consequently, at large molar water/silica ratios (H 2 O:Si > 4), the hydrolysis will be complete at an early stage of the reaction [58]. At intermediate pH conditions, i.e., between pH 3 and pH 8, the rate of condensation increases with the pH value, while the rate of hydrolysis goes through a minimum at approximately neutral pH. As a consequence, hydrolysis is ratelimiting for gelation at these pH conditions [58,72]. Above pH 7, condensation occurs by nucleophilic displacement reactions via SiO − anions, preferentially between protonated and deprotonated acidic species [58]. For this reason, the rate of condensation decreases with increased pH due to mutual repulsion.
In the case of diluted aqueous silicates, hydrolysis is immediately and fully completed at all pH conditions. Hence, the gel time solely depends on the condensation time. Around pH 1.5-3, sols display a maximum stability, and therefore the longest gel time. The rate of condensation is proportional to the concentration of protons. Between pH 2 and 7, the condensation rate is proportional to the concentration of hydroxide ions. Consequently, the gel time decreases with the pH, with a minimum at pH 5-6. Above pH 7, the particles are charged and therefore mutually repulsive. For this reason, the particles grow, but no gelation can be observed. The addition of salts lowers the ionic charge on particles, and therefore a gelation is possible and the gel time decreases [58,64]. The relative reaction rates of hydrolysis and condensation as well as the gel time are displayed in Figure 4.  Besides the gel time-the time until nutrients can be added to the entrapped cellsthe diffusion rate of nutrients and therefore the gel structure affects survival, growth, and productivity of entrapped cells. The gel structure, more precisely the pore size and its distribution, depends on the gel formation process [61]. Three gel formation processes are mainly distinguished, i.e., polymeric, cluster, and colloidal gel formation ( Figure 5).  Besides the gel time-the time until nutrients can be added to the entrapped cells-the diffusion rate of nutrients and therefore the gel structure affects survival, growth, and productivity of entrapped cells. The gel structure, more precisely the pore size and its distribution, depends on the gel formation process [61]. Three gel formation processes are mainly distinguished, i.e., polymeric, cluster, and colloidal gel formation ( Figure 5).  Besides the gel time-the time until nutrients can be added to the entrapped cellsthe diffusion rate of nutrients and therefore the gel structure affects survival, growth, and productivity of entrapped cells. The gel structure, more precisely the pore size and its distribution, depends on the gel formation process [61]. Three gel formation processes are mainly distinguished, i.e., polymeric, cluster, and colloidal gel formation ( Figure 5).  Polymeric gel formation occurs with alkoxysilanes as precursor at low molar water to silica ratios (H 2 O: Si ≤ 5) and acid catalyzed conditions. The linear or randomly branched polymers entangle and form additional branches resulting in gelation.
In contrast, at high molar water to silica ratios and/or base catalyzed conditions, the highly branched clusters do not interpenetrate before gelation and thus behave like discrete species. Gelation occurs by linking together clusters similar to colloidal gel formation [61].
Colloidal gel formation is a commonly known process based on aqueous precursors. As briefly described in the previous chapter, gelation of aqueous colloidal particles occurs only at pH values below 7 or in the presence of salts. When two particles collide, neutral and protonated silanol groups on the surface of the particles condense to form Si-O-Si linkages.
In the presence of soluble silica or monomers, the particles are cemented together [64].

Biocompatibility
The biocompatibility of silica hydrogels is limited for three main reasons: First, alkoxysilane precursors such as TMOS or TEOS are poorly soluble in water, and therefore the conventional sol synthesis applies organic solvents, e.g., the alcohol that is already released during the hydrolysis.
Second, osmotic stress is generated by the addition of acids or bases as catalysts to increase the reaction speed during hydrolysis and condensation and to modify the molecular structure of the resulting hydrogel. Further ions are added by adjusting the pH of the sol and therefore of the resulting hydrogel with mineral bases or acids.

Conventional Sol-Gel Method for the Entrapment of Insensitive Biological Material
For these reasons, the entrapment by the conventional sol-gel method is limited to biological material that tolerates the applied solvents, the released by-products, and the applied acids or bases for catalysis and pH adjustment. The first pioneering studies reported the entrapment of Saccharomyces cerevisiae by Carturan et al. in 1989 [38] and of the alkaline phosphatase by Braun et al. in 1990 [77]. In both cases, the entrapped biological material tolerates the applied and the released alcohol. Hence, the entrapped enzyme as well as the yeast cells were reported to show activity; however, this was reduced to free biocatalysts. This is why the synthesis conditions were assumed to be in principle biocompatible by these authors.
The biocompatibility of silica hydrogels was increased by omitting alcohol as a solvent on the basis of the observation that the alcohol released during hydrolysis sufficiently increased the solubility of the precursor [78].

Reduction of Released By-Products and Avoidance of Increased Ion Concentrations
As reviewed by Coradin and Livage [39], the limitation of biocompatibility due to the release of the alcohol as a by-product during hydrolysis can be reduced by different methods (see Figure 6): mixing water and precursor in a high ratio [79]; -modification of the precursor with biocompatible alkoxides, e.g., to poly(glyceryl silicate) [80]; -application of the precursor from the gas phase during which the alcohol evaporates and contact with the entrapped material is avoided (Biosil method) [54,55,[81][82][83][84]; -dip-coating of a carrier in order to create a thin layer of a few micrometers from which the released alcohol can evaporate quickly from the close proximity of the entrapped biological material [38,85,86]; -evaporation of the alcohol released from the sol before the biological material is added [44,55,76,[87][88][89][90][91].
evaporation of the alcohol released from the sol before the biological material is added [44,55,76,[87][88][89][90][91]. Another method to avoid organic solvents is the application of aqueous silicates as precursors for synthesis. On the one hand, the disadvantages compared to alkoxysilanes originate in the limitation of the precursor concentration and the pH as well as in the reactions that are difficult to control. The latter is caused by the mixture of oligomers, while alkoxysilanes exist in the solution as monomers. On the other hand, the advantage of aqueous silicates originates in the metal ions that are released as by-products. Since they occur naturally, some microorganisms show a tolerance towards them [39]. For the entrapment of cells that are sensitive to the induced osmotic stress, the cations are removed via strongly acidic ion exchangers [43,44,76,[92][93][94][95][96] (see Figure 6).
Another possibility to avoid organic solvents as well as acids and bases as catalysts arose from the novel silica precursor tetra(n-propylamino)silane, which exhibits higher reactivity when compared to the use of alkoxysilanes [70]. Another method to avoid organic solvents is the application of aqueous silicates as precursors for synthesis. On the one hand, the disadvantages compared to alkoxysilanes originate in the limitation of the precursor concentration and the pH as well as in the reactions that are difficult to control. The latter is caused by the mixture of oligomers, while alkoxysilanes exist in the solution as monomers. On the other hand, the advantage of aqueous silicates originates in the metal ions that are released as by-products. Since they occur naturally, some microorganisms show a tolerance towards them [39]. For the entrapment of cells that are sensitive to the induced osmotic stress, the cations are removed via strongly acidic ion exchangers [43,44,76,[92][93][94][95][96] (see Figure 6).
Another possibility to avoid organic solvents as well as acids and bases as catalysts arose from the novel silica precursor tetra(n-propylamino)silane, which exhibits higher reactivity when compared to the use of alkoxysilanes [70].
In order to enable cell growth, a two-step method has been developed. Here, cells were entrapped in a biopolymer hydrogel that was afterward entrapped in silica hydrogels [111,117,119,126,139].
Besides biopolymers, the synthetic polymer polydiallyldimethylammonium chloride (PDADMAC) was utilized for the production of composites: cells were entrapped in an alginate-silica composite by dropping a mixture of cells, sodium alginate, and a silica precursor into a mixture of calcium chloride and PDADMAC. As a result, a double network of calcium alginate and silica hydrogel with a shell of PDADMAC was created [96,[131][132][133], in which cell growth was enabled [131].

Entrapment of Microalgae in Alkoxysilanes
Alkoxysilanes, mostly TEOS and rarely TMOS, have been applied for the entrapment of the green microalgae Chlorella vulgaris, Haematococcus pluvialis, and Chlamydomonas reinhardtii, as well as the cyanobacteria Anabaena and Synechocystis sp. Applied TEOS concentrations of 5 wt % to 22 wt % released ethanol in concentrations of 0.92 to 4.24 mol/L, while applied TMOS concentration of 6.87-11.70 wt % released 4.6-7.88 mol/L methanol. In order to reduce the detrimental effect of the released alcohols, dip-coating of a carrier to create a thin layer [75] as well as evaporation of alcohols were applied [46,52,53,74,76]. In all reviewed studies, sol-gel synthesis was acid catalyzed, resulting in barely branched chains and a polymeric gel formation (see Sections 2.1 and 2.4). Where necessary for increased biocompatibility and for induction of gelation, the pH was adjusted with NaOH, KOH, or phosphate buffer (see Table 1). In one study, a polyether-modified polysiloxane enhanced the mechanical stability, which led to decreased cell loss of the thin layer [75].
In order to further increase the biocompatibility, glycerol, sorbitol, and/or polyethylene glycol were added to the gels. Addition of sorbitol or glycerol stabilized cell vitality of Haematococcus pluvialis, also upon astaxanthin extraction with solvents [75]. While glycerol had a negligible effect on hydrogen production in wild-type Synechocystis sp. cells and hindered hydrogen production in the mutant cells, polyethylene glycol 400 improved hydrogen production. Finally, hydrogen production was observed for 5 days, similar to free cells [46].
In comparison with silica hydrogels prepared via other routes, photosynthetic activity of Synechocystis sp. was diminished and less stable in TEOS-derived silica hydrogels [46,76]. Similar observations were reported for the microalga Chlamydomonas reinhardtii [52,53].  [163] Besides additives, one study investigated the application of the alkoxide precursor tetrakis(2-hydroxyethyl)orthosilicate with the more biocompatible by-product glycolic acid. The precursor was applied in a concentration of 12 wt % to immobilize the microalga Porphyridium purpureum. For the sol synthesis, no additional acid or base was necessary. The gelation occurred upon mixing with the cells at pH 6. The entrapped cells showed a stable viability at elevated temperatures as well as pigment fluorescence over two weeks. Hence, the potential application as whole-cell biosensor for aqueous contaminants was demonstrated [163].

Entrapment of Microalgae in Aqueous Silicates
A possibility to avoid the inhibitory by-products of alkoxysilanes is the application of aqueous silicates such as potassium, lithium, or sodium silicates. Here, the metal ions occur as by-products. Commonly applied sodium silicate concentrations of 1.06-19 wt % resulted in sodium ion concentrations of 0.64 to 18.8 mol/L [41,42,97,98,124,129,130,164,165]. It has already been applied for the entrapment of the microalgae Chlorella vulgaris, Dictosphaerium chlerelloides, Scenedesmus intermedius, Scenedesmus sp., Mesotaenium sp., and Cyanidium caldarium as well as the euglenoid Euglena gracilis and the cyanobacteria Anabaena flos-aqua, Synechococcus sp., and Cyanothece [41,97,98,124,129,130].
In all reviewed studies (see Table 2), the sol synthesis was base catalyzed, and the pH was adjusted with HCl, resulting in colloidal gel synthesis (see Sections 2.2 and 2.4). In most of the studies, the colloidal suspension of nanoparticulate silica, i.e., LUDOX ® , was added to the sol in order to increase the silica content and reinforce the gel. However, one study indicates that the colloidal silica adsorb on the cell wall, forming a crust that potentially blocks active transport sites and limits cell activity of C. caldarium cells. Moreover, the authors argued that the nanoparticles are small enough to be potentially internalized [42]. This effect seems to be species-specific, as other microalgae and cyanobacteria show cell activity in LUDOX ® -reinforced silica hydrogels [97,98,124,129,130,164,165]. It was discussed that observed harmful effects of elevated LUDOX ® concentrations from 1 to 3 mol/L on entrapped Anabaena flos-aquae could be caused by an increased Young's modulus [130].
In order to prevent osmotic shock induced by the upcoming sodium ions, glycerol was added to the sol or to the cells before mixing with the sol in some studies [41,42,97,98,124]. Furthermore, minimizing cracks in the gel microstructure and improving mechanical properties have been discussed [98,124]. However, while in some cases glycerol slows down the rate of degradation of the photosynthetic pigments within the cyanobacteria cells and consequently preserves the viability [41], in other cases, no effect of glycerol on chlorophyll fluorescence was observed [98,124], and for some organisms and strains, the addition of glycerol is not even biocompatible [42,97]. The observed detrimental effect of glycerol was attributed to reduced surface area and pore volume, hence closing pores that potentially reduce diffusion of nutrients [41].
The reviewed studies aimed at whole-cell biosensors for aqueous contaminants on the basis of the fluorescent properties of photosynthetic pigments or presented the results as a first step toward important biotechnological applications, such as biofuels and (secondary) metabolites, however, without giving insights on specific products. All studies reported the viability of cells, while a proliferation was only observed for Synechococcus. However, proliferation was limited to two generations, which the authors attributed to space limitations in the silica hydrogel [41,97].

Entrapment of Microalgae in Aqueous Silicates with Metal Ion Removal
In order to further increase biocompatibility, sodium ions can be removed with an ion exchanger before the cells are added to the sol, resulting in the so-called "low-sodium route." Consequently, higher precursor concentrations, i.e., 4.8-25 wt %, were applied in comparison to 1.06-19 wt % without ion removal (see the previous paragraph). By applying an ion exchanger, the sol acidifies, causing acid catalysis. However, when LUDOX ® is added to the sol in order to strengthen the gel analogous to the previously described sodium silicate-based gels, the sol turns alkaline again. Therefore, the pH was adjusted with HCl. With this method, two strains of the cyanobacterium Synechococcus sp. have been entrapped. While the addition of the nanoparticles has a beneficial effect on the viability of the cells over time, the mechanical stability was enhanced upon depletion of LUDOX ® . In fact, the gel prepared with the medium low in sodium ions for the freshwater Synechococcus strain was reported to liquefy more quickly than the gel prepared with the salt-water medium for the marine strain. This indicates the need of sodium ions for gel formation in presence of silica nanoparticles.
Since the sodium ions of the silicate precursor were removed via an ion exchanger and LUDOX ® was omitted, glycerol was no longer necessary to prevent osmotic stress of the cells. With this method, entrapped cyanobacteria cells produced oxygen for 17 weeks [94]. Similarly, the addition of glycerol and polyethylene glycol was observed to be detrimental toward entrapped Synechocystis sp. cells. Nevertheless, cells entrapped in silica hydrogels prepared via the "low-sodium route" displayed a higher vitality than entrapped in TEOSderived hydrogels. Furthermore, photosynthetic activity was reported for 8 weeks in aqueous silica gels compared to 6 weeks in TEOS-based hydrogels [76].
As an alternative to LUDOX ® , SiO 2 nanopowder was added to the sol to strengthen the gel. The pH of the sol remained acidic upon addition of the nanopowder, and thus the pH was adjusted with KOH. In contrast to LUDOX ® , the aggregates of the nanopowder were too large to be internalized within the cell and were shown to maintain the amount of viable Cyanidium caldarium, Chlorella vulgaris, and Botryococcus braunii cells and their activity in the hydrogel. Furthermore, the nanopowder not only delayed liquefaction of the gels, but also increased diffusion. It was discussed to be caused by the created void pockets around the silica aggregates found in close proximity to the cells. Microalgae entrapped via this method showed oxygen production for 75 days [43,44].
Despite the improvement of the viability by removing the sodium ions, cell growth was still limited, which was again attributed to space limitations [43]. The application of the low-sodium route together with the addition of chitosan as well as pH adjustment with tris(hydroxymethyl)aminomethane allowed entrapment of viable and growing Chlamydomonas reinhardtii [52,53].
In all reviewed studies, the authors aimed at enabling viability and activity for important biotechnological applications, such as biofuels and (secondary) metabolites with simultaneous CO 2 mitigation (see Table 3).

Entrapment of Microalgae in Aminosilane-Based Silica Hydrogels
The precursor tetra(n-propyl amino)silane, an aminosilane precursor, is reported to autocatalyse. The emerged sol displays particles comparable with basic catalyzed TEOS gels (see Section 2.3).
This precursor has already been employed for the entrapment of C. reinhardtii without morphological changes of entrapped cells. However, the quantum yield of photosystem II and the oxygen consumption rate were drastically reduced, and an oxygen production was not observed. The investigation stopped observation after 2 h after entrapment [70]. The by-product of this precursor, i.e., n-propylamine, can act analogously to the herbicide atrazine [166][167][168][169]. Analogous to the low-sodium route for aqueous silicates, n-propylamine can be removed via ion exchanger. Hydrogels prepared via this low-propylamine route enabled the entrapment of photosynthetically active and growing Chlamydomonas reinhardtii cells (see Table 4) [52,53].
Viability of entrapped cells for several weeks or even months as well as c growth of entrapped green microalgae has been reported when Chlorella vulgaris, Pseudokirchneriella subcapitata, and Chlamydomonas reinhardtii were entrapped via a two-step method: in the first step, the cells were entrapped in calcium alginate beads, which were entrapped in silica hydrogels in a second step. As silica precursor for the second step, either TEOS with the evaporation of released alcohol [126,127], in one case in combination with a diamine-functionalized silane [127], or sodium silicate with the addition of LUDOX ® was applied [125,128,170]. Mostly, the authors envisioned the application of the entrapped cells as whole-cell biosensor for aqueous contaminants (see Table 5).

Conclusions
Despite being a promising entrapment material due to the high transparency and biological, chemical, mechanical, and thermal stability, silica hydrogels still display a limited biocompatibility caused by the by-products that are released during hydrolysis of the precursors, by pH adjustment of the sol with mineral bases and by the stiffness of the corresponding hydrogel.
As a closer look at microalgae entrapment in the previous chapter reveals, the development of biocompatible silica hydrogels focused on the precursor selection, the choice of acid or base for pH adjustment, and the addition of other (bio)polymers. While pigment stability, photosynthetic activity, bioactivity, and cell growth have been investigated, the transparency and stiffness of the hydrogel have rarely been reported.
On the basis of the given overview, an in-depth understanding of how the sol and hydrogel synthesis as well as the resulting structures affect the viability, activity, and proliferation capability of entrapped microalgae cells is still needed.
Future developments of biocompatible silica hydrogels for the entrapment of sensitive, photosynthetically active microorganisms may include the synthesis of novel silica precursors that release non-toxic by-products or by-products that even improve viability and/or growth of the entrapped cells. Further organic and inorganic compounds may be screened for their potential application as plasticizers of silica hydrogels in order to decrease stiffness, improve abrasion resistance, and increase the growth capacity of entrapped cells.