Photobiological hydrogen production from Synechocystis sp. PCC 6803 encapsulated in silica sol–gel

Abstract

Hydrogen production was measured from viable wild-type and M55 mutant cells of Synechocystis sp. PCC 6803 encapsulated in silica sol–gel derived from tetraethoxysilane, tetramethoxysilane, and a mixture of tetraethoxysilane with methyltriethoxysilane. Glycerol and polyethylene glycol were used as additives in concentrations up to 14 wt.% to increase porosity and pore connectivity and to reduce stress on the encapsulated cells. Mixtures were evaluated for hydrogen production in a high-throughput screening assay, and samples were imaged with environmental scanning electron microscopy. Compositions showing improved H₂ production were selected for further study via quantitative analysis of H₂ production in GC vial assays. H₂ production activity was monitored for up to 5 days and H₂ production from encapsulated cells was observed at levels comparable to or exceeding cells suspended in liquid media.

Introduction

Synechocystis sp. PCC 6803 has been extensively studied since it was first isolated in 1968 and is a well characterized model organism for photosynthesis. The genome is fully sequenced and annotated and the organism is highly transformable. PCC 6803 has been of particular interest recently because, in addition to possessing a full complement of photosynthetic capabilities, it contains a bidirectional [Ni–Fe] hydrogenase enzyme capable of producing molecular hydrogen (H₂) from the reduction of protons under certain metabolic conditions [1], [2], [3], [4], [5]. A photobiological means of producing H₂ from water offers an attractive option as a sustainable source of this increasingly valuable fuel [6]. However, many issues must be resolved before this process can become a practical, widespread, and economically feasible source of H₂.

One approach to achieving increased and sustained levels of biosolar H₂ production from phototrophic microorganisms may be to encapsulate viable cells in a solid silica matrix. Encapsulation within silica sol–gel is a relatively new technique for encapsulating viable cells, first shown in 1989 with Saccharomyces cerevisiae cells by Carturan et al. [7], and has been successfully demonstrated with a variety of biological components including enzymes and proteins [8], [9], [10], [11], bacteria [12], [13], [14], yeast [7], [15], [16], plant cells [17], [18], mammalian cells [19], and phototrophic cyanobacteria [20]. The matrix creates a solid framework that provides structural, thermal and chemical stability to the encapsulated cultures. Under the right conditions, this can prolong cell life for many months [21], protect cells from contamination, prevent outgrowth, and increase the production of secondary metabolites such as H₂. The matrix is also transparent, allowing for the encapsulation of phototrophs, making this approach useful for biosensors that employ fluorescence or, in this case, for photobiological production of H₂.

H₂ production in this species generally has been reported to occur in two situations: at a low, steady rate in the dark as the cells break down accumulated glycogen anaerobically; and during a transient burst of H₂ production activity at the initial onset of photosynthesis with light exposure [2]. The predicted photobiological pathway of H₂ production is illustrated in Fig. 1 and can be summarized as follows: Oxygenic photosynthesis is initiated at photosystem II (PSII) where water is split into protons and electrons with the release of molecular oxygen. The protons are used to create a potential gradient across the thylakoid membrane enabling the production of ATP from ADP and phosphate. The electrons enter the electron transport chain where they proceed through a series of transport molecules and proteins, including plastoquinone, cytochrome b₆f, and plastocyanin, to photosystem I (PSI). The excited electrons are finally used to produce reducing equivalents in the form of NADPH, which are used in the Calvin Cycle for carbon fixation and other metabolic reactions. Excess reducing equivalents can also be used by the hydrogenase enzyme to produce gaseous H₂. The hydrogenase enzyme reportedly is sensitive to oxygen, with activity being drastically reduced or completely halted during oxygenic photosynthesis. Under any conditions, reductant availability also can limit H₂ production. The reported substrate for the hydrogenase is two molecules of NADPH, which are oxidized to NADP⁺ to produce one molecule of H₂ [2]. NADPH supplies reductant to numerous metabolic pathways, however, which compete with the hydrogenase. These two challenges have been considered to be the primary obstacles to improved H₂ production in this species.

Researchers have taken various approaches to overcome these two challenges. Two general approaches include genetic manipulation, such as altering the hydrogenase enzyme to confer some degree of added oxygen tolerance or other enhanced activity [22], or metabolic approaches [23], which manipulate the environment of the cells in order to enhance H₂ production through improved reductant availability. For example, Antal and Lindblad showed that sulfur deprivation can be used to promote more than a four-fold increase in H₂ production from Synechocystis in a methane atmosphere [24]. Burrows has shown that optimizing nutrient concentrations can yield nearly a 150-fold increase in H₂ production compared to sulfur deprived cells, after conditioning the cells in media optimized for H₂ production [25]. Encapsulation may also be regarded as a metabolic manipulation since it will alter cell metabolism and potentially increase reductant availability for H₂ production.

Sol–gel processing is an empirically well characterized materials science technique used for fabricating metal oxide thin films, optoelectrical devices, monoliths for separations, and other specialty applications. The process is generic and can be used with numerous transition metals and semi-metal elements to make amorphous or crystalline metal oxides after heat treatment. The fundamentals of sol–gel processing are described in the seminal text of Brinker and Scherer [26]. A common two step sol–gel process employs metal alkoxide precursors, such as tetraethoxysilane (TEOS), Si(OC₂H₅)₄, which is hydrolyzed in the presence of an acid catalyst and alcohol solvent. Subsequent condensation and hydrolysis reactions then reach an equilibrium, forming a sol, or colloidal suspension, of metal oxide clusters. With subsequent basic catalysis, condensation reactions accelerate and the clusters aggregate into a randomly cross-linked, polymeric gel. With aging, the gel becomes increasingly dense and cross-linked as solvent is expelled from the pore volume. Finally, a heat treatment can be used to remove the solvent and fully condense the gel into a microstructure suited for the application. The literature on sol–gel processing in the context of fabricating optoelectrical devices is expansive.

Recently, this process was borrowed from materials science to demonstrate encapsulation of biological components, first with viable yeast cells [7]. Silica sol–gel was selected for its biocompatibility, and S. cerevisiae for its tolerance to ethanol, which is evolved from the hydrolysis of TEOS to silicic acid, Si(OH)₄. Immobilization of biological components was not new, but using a purely inorganic solid matrix was a novel approach. Unlike most other commonly used organic encapsulation matrices, which can swell and are vulnerable to biological degradation, silica sol–gel creates an inert matrix that restricts cell growth. As a result, cells may enter an altered steady-state metabolism that yields increased production of secondary metabolites. The rigidity also prevents cell outgrowth, maintains the mechanical integrity of the matrix, and prevents contamination by other organisms. This allows for applications in biosensors and bioreactors where fluid streams with analytes of interest or fresh substrate can pass over porous matrices containing immobilized biological components. Several recent reviews are available [10], [27], [28], [29], [30].

Despite the variety of biological components that have been successfully encapsulated in silica sol–gel, the use of this technique for biosolar H₂ production by viable phototrophs has not been investigated previously. The matrix is transparent and therefore suitable for phototrophs and photoactive proteins, as was demonstrated with photoactive proteins in 1992 by Ellerby et al. [8]. More recently, sol–gel encapsulation has been used for the development of a biosensor based on immobilized algal cells [31], [32] and an alternate method using colloidal precursors has also been used to encapsulate multiple strains of Synechococcus [20]. Our evaluations indicate that approximately 12% of incident light is absorbed by the gels described in this report. We found this effect to be consistent across all gel compositions and to remain stable (after 24 h) for the duration of the study (data not shown), illustrating that most of the incident radiation can be available for photosynthesis. Therefore, this approach may be well suited for immobilizing Synechocystis sp. PCC 6803 and potentially for improving H₂ production. Cyanobacteria use most of the energy collected during photosynthesis for the production of biomass. It has been suggested that cells may direct no more than 15% of their available energy toward H₂ production [33]. However, if growth is restricted, that percentage could potentially increase. In a different format, Laurinavichene et al. demonstrated that cells of Chlamydomonas reinhardtii immobilized on glass fibers produced approximately 2.5 times more H₂ than cultures under otherwise identical conditions in liquid suspension [34].