Alkyl Silanes as a Source of Carbon for Microbial Growth Eduardo J. Martinez Microbial Diversity Course Woods Hole, MA Tuesday, August 1, 2006 Abstract: Alkyl silanes have not been described as substrates for microbial growth and no precedent exist for naturally occurring carbon-silicon bonds. Enrichments on minimal media using alkyl silanes as the sole carbon and energy source may be a novel method for the isolation of new microorganisms or the discovery of new enzymatic pathways. Triethylsilane, diethylsilane, 2-(trimethylsilyl)ethanol and trimethylsilylacetic acid were used in enrichments under aerobic shake tube conditions. No growth was observed on the unfunctionalized silanes but 2-(trimethylsilyl)ethanol and trimethylsilylacetic acid were successful substrates. Organisms growing on 2-(trimethylsilyl)ethanol took several weeks to grow up so pure cultures could not be obtained, but they are clearly bacteria (about 2 µm in diameter) and can also utilize methanol as a carbon source. Trimethylsilylacetic acid in media at pH 5 selects for eukaryotes. Two very different species were isolated. Issatchenkia occidentalis is a common variety fruit fungus isolated from various soil samples. Prototheca zopfii var. hydrocarbonea is a non- photosynthetic algal which can grow on n-alkanes and exhibits very interesting cell fission morphology. Unfortunately, trimethylsilylacetic acid was shown to be unstable under the aqueous incubation conditions, decomposing to acetic acid and trimethylsilanol. However, decay kinetics could not be measured to compare with growth rates and determine if this decomposition was significant. Background: New carbon sources are useful in the discovery of novel microbial life and metabolism. Alkyl silanes have not been described as substrates for microbial growth and furthermore no literature could be found on the natural occurrence of carbon-silicon bonds. Without selective pressure on microbial communities requiring metabolism of this bond, it is difficult to envision how an enrichment based on alkyl silanes would proceed. The processing of silicon-carbon or silicon-hydrogen bonds would likely require either unknown biochemistry or interesting utilization of existing enzymes. Alkyl silanes are completely unnatural in origin and used as synthetic reagents in pharmaceuticals or bonding agents in latex manufacturing. Power Chemical Corporation in China produces 30,000 metric tons of silanes per year, a limited production compared to other industrial processes, however this signals a potential need for organisms that could be used in bioremediation of these compounds if they begin to concentrate in the environment. There are a variety of commercially available alkyl silanes to choose from as potential substrates for microbial growth. Alkyl silanes were chosen based on cost, steric hindrance, water solubility, and functional group activation (see Figure I). Triethylsilane (TES) is one of the most common silanes in organic synthesis. It is a reducing agent used as a surrogate for hydrogen gas and ultimately produces triethylsilanol as a by-product. The silicon-hydrogen bond in triethylsilane is relatively activated (ΔH°Si-H = 72 kcal/mol compared to ΔH°C-H = 100 kcal/mol) but the silicon- carbon bonds are stericly hindered and relatively stable (ΔH°Si-C = ~80 kcal/mol compared to ΔH°C-C = 81 kcal/mol). Diethylsilane (DES) is similar to triethylsilane, the silicon-hydrogen bond is slightly more stable but less stericly hindered. 2- (Trimethylsilyl)-ethanol (TMSE) and trimethylsilylacetic acid (TMSAA) have a hydroxyl and carboxylic acid functional group respectively, providing a handle for reactivity and water solubility. Additionally, the silicon-carbon bonds in these molecules are more accessible to enzymatic manipulation since they are in the form of methyl instead of ethyl groups. Generally, silicon is considered highly oxophilic providing a substantial energy sink going from silicon-carbon to silicon-oxygen bonds (ΔH°Si-O = 103 kcal/mol compared to ΔH°C-O = 83 kcal/mol) in all of these compounds. Figure I. Structure of alkyl silanes. H H O Si Si Si H O Si H H O DES TES TMSE TMSAA Table I: Energies of carbon and silicon bonds with heteroatoms.* Bond Bond Dissociation Energy (kcal/mol) Bond Bond Dissociation Energy (kcal/mol) C-C 81 Si-Si 45 C-O 83 Si-O 103 C-H 100 Si-H 72 C-Si ~80 * Lowry, T. H.; Richardson, K. S. Mechanism and Theory in Organic Chemistry, 3rd ed, Harper and Row Publishers, New York 1987. Media: Media was prepared from modular components, vitamins and the silanes were added after autoclave sterilization (see Table II). The sterile media was dispensed (5 mL) into sterile 18mm test tubes and covered with Morton caps. The alkyl silanes (purchased from Aldrich) were added without filter sterilization as a neat aliquot into each test tube according to Table III. Addition of TMSAA caused the solution to become pH 4.5 in FWBS and pH 4.5 in SWBS. Neutralization with sodium hydroxide afforded pH 6.7 in FWBS and pH 6.5 in SWBS (normally pH 6.9 and pH 6.8 respectively). Plates were made with washed (3 x D.I. water) Bacto Agar (15%) using FWBS - 0.2% v/v Trimethylsilylacetic acid (12 mM), FWBS - 0.2% v/v 2-(Trimethylsilyl)ethanol (14 mM) and SWBS - 0.2% v/v Trimethylsilylacetic acid (12 mM). Table II. Media Recipe. 500 mL Fresh Water Media (FWBS) Amount 500 mL Salt Water Media (SWBS) Amount 100 x FWB 5 mL SWB <500 mL 0.5 M NH4Cl 5 mL 0.5 M NH4Cl 5 mL 150 mM Potassium PO4 5 mL 150 mM Potassium PO4 5 mL 1 M Na2SO4 0.5 mL 1 M Na2SO4 0.5 mL 1 M MOPS Buffer, pH 7.2 2.5 mL 1 M MOPS Buffer, pH 7.2 2.5 mL Trace elements 0.5 mL Trace elements 0.5 mL Milli-Q water <500 mL Post Autoclaving: Post Autoclaving: Vitamin solution 0.05 mL Vitamin solution 0.05 mL Vitamin B12 solution 0.05 mL Vitamin B12 solution 0.05 mL Table III. Alkyl silanes. Alkyl silane Abbrev. Conc. Vol. M.W. Density B.P. (°C) Lot # (mM) (µL) (g/mL) Diethylsilane DES 20.1 13.0 88.22 0.681 56 00315KD Triethylsilane TES 20.0 16.0 116.28 0.728 107-108 12401TD 2-(Trimethylsilyl)ethanol TMSE 19.6 14.0 118.25 0.829 174-175 04309CE (Trimethylsilyl)acetic acid TMSAA 19.9 16.0 132.24 0.820 39 (mp) 18322HC Sampling and Inoculation: Samples were collected from a variety of locations: sea water sediment, beach sand, marsh sand, wilderness soil, xenobiotic contaminated soil, fresh water ponds and mud (see Table IV). Each sample was inoculated into the appropriate media (salt water base or fresh water base) and shaken at 30 °C. Table IV. Sampling Sites (taken on the afternoon of 2006.07.14). Site Description Garbage Beach Sand Surface sand resting under decaying seaweed. Trunk River Mat Surface mat above anaerobic layer on the edge of Trunk River. Eel Pond Sediment Surface sediment below high water mark on the edge near Loeb. Cedar Swamp Water Sediment on the water line. Forest Soil Moist cool shaded area near mushroom off the bike path. Fueling Station Dark soil sample from the Falmouth Coal Co. under the fueling station. School Street Marsh Surface sediment on the water line. Sewage Plant Falmouth Sewage Water Treatment Facility Enrichment: The enrichments were monitored by visual inspection once daily looking for turbidity. After 48 hours 20 µL were transferred to a fresh tube of media and incubated with shaking at 30 °C to separate from carbon source in the inocula. Table V summarize the results after 2.5 weeks of incubation, colors represent different cell morphologies and presumably distinct organisms. Notice that different organisms were derived from TMSAA depending on the pH of the solution. Table V. Enrichment Summary. Site Media DES TES TMSE TMSAA TMSA-Na Garbage Beach Sand SWBS - - - - na Trunk River Mat SWBS - - - + + Eel Pond Sediment SWBS - - - - na Cedar Swamp Water FWBS - - + - na Forest Soil FWBS - - + - na Fueling Station FWBS - - + + + School Street Marsh FWBS - - - + na Sewage Plant FWBS - - - + na (-) = No growth after 3 weeks; (na) = Not attempted. Colors represent different cell morphologies. Microorganism Characterization: Four days after inoculation the first organism grew up using trimethylsilylacetic acid media from inocula coming from the Fueling Station, School Street Marsh and Sewage Water sites. All three enrichments produced the same organism, namely a budding yeast-like microbe. Notable features include a very large central vacuole; two or three phase bright structures connected to this vacuole and 1-2% of the time a small fast moving object inside the vacuole (see Figure II). Six days after inoculation a second morphology emerged also from the trimethylsilylacetic acid media but from Trunk River Mat inoculum. This microbe also appears eukaryotic in nature but is clearly not yeast- like. Various structures are surrounded by a large clear sheath, disruption of this outer layer by applying pressure to the cover slip causes the vacuole-like object to spill out along with very small motile material (see Figure III). Fourteen days after inoculation, growth was observed using 2-(trimethylsilyl)ethanol media from inocula coming from Cedar Swamp, Forest Soil, and Fueling Station sites. These are clearly bacterial in origin and extremely small (2 µm in diameter). Two morphologies seem to exist, namely short rods and spherical cocci, however it is difficult to determine microscopically if this observation is genetic or phenotypic in nature (see Figure IV). Figure II. Yeast-like organisms (left panel: 160x DIC wet mount; right panel: 16x agar plate; bottom: 4x dissecting scope agar plate). Figure III. Non-yeast organisms (left panel: 160x DIC wet mount; right: 16x agar plate; bottom: 160x DIC wet mount). Figure IV. Bacterial organisms (160x DIC wet mount). After several passages in liquid culture the microbes were plated out to obtain pure colonies.