A tracer aided study on silicon chemistry in biological systems

Silicon (Si) is omnipresent in nature, and it is involved in important but diverse roles in a broad range of organisms, including diatoms, higher plants and humans. Some organisms, like the diatoms, need high amounts of silicon, and master silicon chemistry to a high extend using several enzymes. Other organisms which need silicon as an essential trace element apparently do not have the capability to handle silicon by any biochemical means and it was hypothesized that silicon chemistry as such plays a major role. The aim of the research described in this thesis was to gain more insight in the mechanisms behind the role of silicon in several organisms and to investigate to what extend silicon chemistry can play a role in biological processes. This study focused on the chemical or biological role of silicon in metal metabolism, on the risks that are connected to silicon polymerization, and on a possible application in biotechnology. For this a study was performed on Baker’s yeast Saccharomyces cerevisiae which often serves as a model organism for the eukaryotic cell (chapters 3 and 4), diatoms (chapter 5) and biofilms (bacterial communities attached to a surface, chapter 6). A silicon tracer was developed (chapter 2) to aid the studies described in this thesis. No-carrier added 31Si was produced by a 31P(n,p)31Si reaction by fast neutrons in the nuclear reactor of the Delft University of Technology. Several methods were investigated to remove the side product 32P. Anion exchange with Dowex resin gave the best results in total activity yield and specific activity, but precipitation with BaCO3 appeared to be the fastest and cheapest purification method, and sufficiently high yields were obtained as well. It was determined that the 31Si tracer was in the desired chemical form of silicic acid (Si(OH)4), and suitable to apply in biological systems. Since yeasts and biofilms do not possess any biochemical means to handle silicon, it is likely that any influence of silicon on these organisms has a chemical origin. This was investigated by studying the interaction of silicon and metals in these organisms. In chapter 3, the influence of silicon (as silicic acid Si(OH)4) on the growth rate and intracellular accumulation of a number of metals was investigated in Baker's yeast Saccharomyces cerevisiae, a model organism for the eukaryotic cell. It was found that the growth rate was not influenced by silicic acid up to concentrations of 10 mmol per liter growth medium and a slight growth inhibition was observed when silicate was present in an extremely high concentration of 100 mM. Intracellular metal concentrations were investigated in yeast cultures grown in normal culture medium without added silicate (-Si) or with the addition of 10 mmol/L silicate (+Si). Decreased amounts of Co, Mn and Fe were found within +Si grown yeast cultures as compared to -Si grown ones, while increased amounts of Mo and Mg were found. Zn and K were apparently unaffected by the presence of silicon. +Si enhanced yeast growth rate under low Zn2+ conditions, but decreased growth rate under low Mg2+ conditions. +Si did not alter the growth rates in high Zn2+ and Co2+ media. +Si doubled the uptake rate of Co2+, but did not influence that of Zn2+. It was proposed that these results could be explained by the formation of a polysilicate layer on the cell wall which changes the cell wall binding capacity for metal ions. The toxicity of silicic acid was compared to germanium (Ge, as GeO2), a member of the same group of elements as Si (group 14) and sometimes used in literature as a silicon analogue. Ge proved to be far more toxic to yeast than Si and no influence was found of Si on Ge toxicity. It was proposed that these results relate to differences in cellular uptake and that is not always possible to use Ge as a Si analogue. These results also indicated that a chemical mechanism, rather than a biological one, is important. This was further investigated by studying the influence of zinc and magnesium on Si-accumulation at several silicate concentrations in the medium by use of 31Si(OH)4 (chapter 4). Si-accumulation fitted well with Freundlich adsorption. Si-release followed depolymerization kinetics, indicating that silicate adsorbs to the surface of the cell rather than being transported over the cell membrane. Subsequently, adsorbed silicate interacts with metal ions and, therefore, alters the cell’s affinity for these ions. Since several metals are nutritional, these Si interactions can significantly change the growth and viability of organisms. In conclusion, the results show that chemistry is important in Si and metal accumulation in Baker’s yeast, and suggest that similar mechanisms should be studied in detail in other organisms to unravel essential roles of Si. The capability of silicon to adsorb on organic substances was also investigated in biofilms, bacterial communities linked together by extracellular polymeric substances (EPS) to study a possible application of silicon chemistry in a biological system. Biofilms have developed mechanisms to accumulate nutrients and organic substrates in their EPS matrix, probably to increase the substrate availability. It may be expected that the binding of ions by the EPS can result in interaction with silicon in the biofilm. Probably this interaction can be used for applications in civil engineering (e.g. biogrouting of soil). To study this the spatial distribution of silicate and phosphate binding in biofilms under different metal conditions was investigated (chapter 6). For this a new autoradiography method using 31Si (as silicic acid) accompanied by 32P (as phosphate) was developed. The equilibrium in silicon uptake was reached within minutes, so it was possible to quantify the 31Si signal, but for 32P this was not possible. Using this method it was shown that both silicon and phosphate bound heterogeneously to the biofilm. In addition, the metal concentrations in the growth medium affected the biofilm structure as well as the silicate and phosphate binding characteristics of the biofilm. In contrast to yeast and biofilms, diatoms master silicon chemistry to a high extend. Here it was investigated how diatoms cope with high amounts of silicon during valve formation as polymerization at/in vital structures should be avoided (chapter 5). Silicic acid uptake using 31Si(OH)4 was studied during valve formation in synchroneously dividing cells of the diatom Pleurosira laevis and other diatoms. Valve formation in diatoms requires bulk uptake and transport of silicic acid to the silica deposition vesicle (SDV). Two earlier proposed mechanisms for silicic acid uptake and transport were investigated: 1) uptake of silicon via silicon transporters (SITs) with subsequent intracellular transport, and 2) (macro)pinocytosis-mediated uptake. The SITs mechanism requires a controlled mechanism to stabilize the high amounts of reactive silicon species to prevent autopolymerization and simultaneously direct these species towards the SDV, whereas this problem does not play a role in the (macro)pinocytosis-mediated mechanism. Experimental data were correlated to systematically derived mathematical models for a compartmental analysis of the possible uptake/transport pathways, including those for both SITs- and (macro)pinocytosis-mediated uptake and transport. This study indicates that the experimental data on silicon uptake during valve formation match best with the model that describes (macro)pinocytosis-mediated uptake. This process not only explains observed surge uptake at high demands for silicon, but also suggests that another pathway exists in which SITs apparently are not involved. The study showed that the pinocytosis mechanism gave a good description of the uptake kinetics that were found in this study. This result offers a simple explanation for how the diatomic cell is able to fulfill its silicon needs without exposing the inner part of the cell to high silicic acid concentrations and the problems related to spontaneous polymerization. Further molecular and (bio)physical-chemical research is needed on diatom biosilicification. The results described in this thesis shed new light on the role of silicon chemistry in several bioprocesses. The influence of silicon on bioprocesses in yeast is probably from chemical origin, resulting from interactions with organic compounds and metals. These interactions, which also occur in biofilms, could also take place in higher organisms as well, and could probably explain at least part of the influence of silicon in biological processes in higher organisms. This may explain why despite many years of research biological binding sites or bioorganical compounds containing silicon have never been found (yet) except for some plants and for bulk consumers like the diatoms and some sponges. But when silicon chemistry itself is taken into account, enzymes and binding sites are probably not needed for silicon to do its job as an essential element. Further research is required on this subject to get clear-cut answers on this matter.Radiation, Radionuclides & ReactorsApplied Science