Silica sol-gel encapsulation of cyanobacteria: lessons for academic and applied research.

Cyanobacteria inhabit nearly every ecosystem on earth, play a vital role in nutrient cycling, and are useful as model organisms for fundamental research in photosynthesis and carbon and nitrogen fixation. In addition, they are important for several established biotechnologies for producing food additives, nutritional and pharmaceutical compounds, and pigments, as well as emerging biotechnologies for biofuels and other products. Encapsulation of living cyanobacteria into a porous silica gel matrix is a recent approach that may dramatically improve the efficiency of certain production processes by retaining the biomass within the reactor and modifying cellular metabolism in helpful ways. Although encapsulation has been explored empirically in the last two decades for a variety of cell types, many challenges remain to achieving optimal encapsulation of cyanobacteria in silica gel. Recent evidence with Synechocystis sp. PCC 6803, for example, suggests that several unknown or uncharacterized proteins are dramatically upregulated as a result of encapsulation. Also, additives commonly used to ease stresses of encapsulating living cells, such as glycerol, have detrimental impacts on photosynthesis in cyanobacteria. This mini-review is intended to address the current status of research on silica sol-gel encapsulation of cyanobacteria and research areas that may further the development of this approach for biotechnology applications.

Diffusion of dissolved ions from wet silica sol-gel monoliths: implications for biological encapsulation.

Divalent nickel (Ni(2+)), Cu(II)EDTA, methyl orange, and dichromate were used to investigate diffusion from hydrated silica sol-gel monoliths. The objective was to examine diffusion of compounds on a size regime relevant to supporting biological components encapsulated within silica gel prepared in a biologically compatible process space with no post-gelation treatments. With an initial sample set, gels prepared from tetraethoxysilane were explored in a factorial design with Ni(2+) as the tracer, varying water content during hydrolysis, acid catalyst present during hydrolysis, and the final concentration of silica. A second sample set explored diffusion of all four tracers in gels prepared with aqueous silica precursors and a variety of organically modified siloxanes. Excluding six outliers which displayed significant syneresis, the mean diffusion constant (D(gel)) across the entire process space of sample set 1 was 2.42×10(-10) m(2) s(-1); approximately 24% of the diffusion coefficient of Ni(2+) in unconfined aqueous solution. In sample set 2, the tracer size and not gel hydrophobicity was the primary determinant of changes in diffusion rates. A strong linear inverse correlation was found between tracer size and the magnitude of D(gel). Based on correlation with the tracers used in this investigation, the characteristic 1-h diffusion distance for carbonate species relevant to supporting active phototrophic organisms was approximately 1.5mm. These results support the notion that silica sol-gel formulations may be optimized for a given biological entity of interest with manageable impact to the diffusion of small ions and molecules.

Transcriptomic responses of Synechocystis sp. PCC 6803 encapsulated in silica gel.

Global gene expression of Synechocystis sp. PCC 6803 encapsulated in silica gel was examined by microarray analysis. Cultures were encapsulated in gels derived from aqueous precursors or from alkoxide precursors and incubated under constant light for 24 h prior to RNA extraction. Cultures suspended in liquid media were exposed to 500 mM salt stress and incubated under identical conditions for comparison purposes. The expression of 414 genes was significantly altered by encapsulation in aqueous-derived gels (fold change ≥1.5 and P value < 0.01), the expression of 1,143 genes was significantly altered by encapsulation in alkoxide-derived gels, and only 243 genes were common to both encapsulation chemistries. Additional qRT-PCR analyses of four selected genes, ggpS, cpcG2, slr5055, and sll5057, confirmed microarray results for those genes. These results illustrate that encapsulation stress is quite different than salt stress in terms of gene expression response. Furthermore, a number of hypothetical and unknown proteins associated with encapsulation and alcohol stress have been identified with implications for improving encapsulation protocols and rationally engineering microorganisms for direct biofuel production.

Evaluation of encapsulation stress and the effect of additives on viability and photosynthetic activity of Synechocystis sp. PCC 6803 encapsulated in silica gel.

Stresses imposed on the cyanobacterium Synechocystis sp. PCC 6803 by various compounds present during silica sol-gel encapsulation, including salt, ethanol (EtOH), polyethylene glycol (PEG), glycerol, and glycine betaine, were investigated. Viability of encapsulated cells and photosynthetic activity of cells stressed by immediate (2 min) and 24-h exposure to the five stress-inducing compounds were monitored by pulse amplitude modulated fluorometry. Cells of Synechocystis sp. PCC 6803 readily survive encapsulation in both alkoxide-derived gels and gels from aqueous precursors and can remain active at least 8 weeks with slight degradation in PSII efficiency. Post-encapsulation survival was improved in gels containing no additive when compared with gels containing PEG or glycerol. Glycerol was shown to have a detrimental effect on Synechocystis sp. PCC 6803, reducing ϕPSII and F(v)'/F(m)' by as much as 75%, possibly a result of disrupting excitation transfer between the phycobilisomes and photosystems. PEG was similarly deleterious, dramatically reducing the ability to carry out a state transition and adequately manage excitation energy distribution. EtOH stress also hindered state transitions, although less severely than PEG, and the cells were able to recover nearly all photosynthetic efficiency within 24 h after an initial drop. Betaine did not interfere with state transitions but did reduce quantum yield and photochemical quenching. Finally, Synechocystis sp. PCC 6803 was shown to recover from salt stress.