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. 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. Fresh water base and salt water base plates were prepared with trimethylsilylacetic acid, fresh water base plates were prepared with 2- (trimethylsilyl)ethanol and R2A, LB and SWC plates were also used. Both TMSAA- organisms grew on R2A, LB and SWC plates and sodium acetate and sodium trimethylsilylacetate in liquid media. Neither TMSAA-organism grew on methanol, 2- (trimethylsilyl)ethanol, diethylsilane, triethylsilane or trimethylsilylacetic acid with cycloheximide. The Fueling Station organism exists partially in a hyphal state on plates and is purely hyphal on SWC plates (see Figure II above). However, not all colonies become hyphal and there is a possibility that these morphologies are different organisms so only non-hyphal colonies were used for further analysis. The Trunk River organism maintains a waxy white colony on all plates. Growth of TMSE-organisms was achieved on plates containing TMSE and methanol, but no growth was observed on TMSAA. This is unexpected because the most logical mechanism for microbial incorporation of this molecule is oxidization of the alcohol group to a carboxyl group to make TMSAA (see Chemistry section below). This taken together with growth on methanol suggest that perhaps these organisms are utilizing the methyl groups directly connected to silicon rather than metabolizing via the ethanol functional group. Growth curves were obtained for both TMSAA-organisms. The Fueling Station organism grows best in fresh water media (empirical data also observed on plates) with a doubling time of 100 minutes, while the Trunk River organism grows best on salt water media (empirical data also observed on plates) with a doubling time of 170 minutes (see Figure V).

Figure V. Growth curves for TMSAA organisms. Growth Curve - Fueling Station Growth Curve - Trunk River Mat

1 1 0 200 400 600 800 1000 1200 1150 1200 1250 1300 1350 1400 0.0041x OD600 y = 0.0003e 0.0069x y = 0.0012e 0.1 2 2 R = 0.9938 R = 0.9947 OD600

0.01 0.1 Time (min) Time (min)

DAPI staining was done to visualize the eukaryotic nucleus of the TMSAA- organism. Samples of thickly grown cultures (20 µL) were treated with DAPI mix (1 µL) and a wet mount was made directly without formaldehyde fixing. Interestingly, two very different types of DNA staining were observed. The Fueling Station organism shows a discrete nucleus off to the side of its large vacuole (see Figure VI right panels). The Trunk River organism shows a diffuse organization of DNA, possibly due to the multicellular nature of how this bug exists (see Figure VI left panels). Analysis of F420 showed no autofluorescence and does not explain this phenomenon.

Figure VI. Phase contrast/DAPI staining (left panels: 100x Trunk River Mat non- yeast organisms; right panels: 100x Fueling Station yeast-like organisms).

Chemistry: Trimethylsilylacetic acid (TMSAA) and 2-(trimethylsilyl)ethanol (TMSE) are related in structure, differentiated only by the oxidation state of carbon-1 (see Figure I above). TMSE is quite stable under physiological conditions however one can envision oxidation of carbon-1 by an organism to make TMSAA via an alcohol dehydrogenase- like pathway. TMSAA was chosen to provide organisms with a handle for reactivity. Although, carbon-silicon bonds are general quite stable, a clear mechanism for decomposition in aqueous media can be envisioned for this compound (see Figure VII). The oxophilic nature of silicon may be driving a spontaneous decomposition of TMSAA to trimethylsilanol and acetic acid. Such decomposition would produce a good substrate for growth leading to an artificial enrichment.

Figure VII. Potential spontaneous mechanism for the decomposition of TMSAA.

+ O H media Si H H O O O Si H H O

H O O HO Si H + OH tautomerization O

In order to address this issue HPLC and NMR analysis of TMSAA were attempted. HPLC was not useful since methods for resolving aliphatic acids require harsh conditions using an Aminex® ion exclusion column and a highly acidic buffer (5 µM sulfuric acid), conditions predicted to cause in situ TMSAA decomposition. HPLC under physiological conditions using a C-18 column, on the other hand, could not resolve the peaks sufficiently to provide meaningful data. NMR analysis was performed on a 400 MHz Bruker NMR at the WHOI Fye Laboratory of Marine Chemistry and Geochemistry with the help of Dr. Carl Johnson. Media solutions were diluted with deuterium oxide (1:1 mix) and a pulsed field gradient solvent suppression routine was used to collect 1H- proton NMR data without interference of a large broad water peak. Due to time constraints and lack of proper controls, enough data could not be acquired to answer all the questions regarding the stability of TMSAA, however abiotic decomposition could be confirmed. Figure VIII shows three 1H-proton spectra. The first chart (A) is a freshly prepared sample of TMSAA in SWB media showing the expected peaks at 0.06 ppm and 1.92 ppm. The middle chart (B) is the same media but incubated at 30 °C for 48 hours without microorganisms. Decomposition is clearly evident from the difference in integration between the two peaks of interest but also because of a small shift in the peaks from 0.06 ppm to 0.07 ppm and 1.92 ppm to 1.98 ppm, presumable a new silane compound and acetic acid. The third chart (C) is an inoculated version of the middle chart. Cells were grown for 48 hours then the media was spun down to pellet the cells and the supernatant was analyzed as above. TMSAA is clearly visible, no acetic acid is present (presumably consumed by the organisms) and there is a significant new silane peak representing the majority of silylated material in the media as in chart B, most likely trimethylsilanol.

Figure VIII. Decomposition analysis of TMSAA by 400 MHz NMR.

Sequencing and Phylogeny: In order to determine the phylogeny and a genetic characterization of these organisms 16s RNA and 18s RNA sequences were obtained. DNA was prepared from exponentially growing cultures and extracted using a bead beating kit (MO Bio Laboratories, Ultra CleanTM Soil DNA Kit; Catalog# 12800-100; Lot# SD5G18). DNA was observed by agarose gel and PCR amplification using both bacterial (16S-8F and 16S-1492R) and eukaryotic (Medlin A – 5’ CTG GTT GAT CCT GCC AG 3’and Medlin B – 5’ TGA TCC TTC TGC AGG TTC ACC TAC 3’) primers was accomplished using an annealing temperature of 48 °C instead of the usual 46 °C (see Figure IXa). The eukaryotic primers worked well even for directly picked colony PCR. Very little bacterial DNA was observed but further PCR amplification yield clearly visible bands that were submitted for sequencing (see Figure IXb), however usable sequences could not be obtained from these samples. BLAST of the eukaryotic sequences revealed two very different and distinct species. The Fueling Station organism was found to be 100% related to Issatchenkia occidentalis (in the tree Eukaryota > Fungi > Ascomycota > Saccharomycotina > Saccharomycetes > Saccharomycetales > Saccharomycetaceae), a common fruit fungus.1 The Trunk River Mat organism was found to be 99% related to Prototheca zopfii var. hydrocarbonea (in the tree Eukaryota > > > ). Interestingly, this organism is a non-photosynthetic algal which can grows on n-alkanes but optimally grows at pH 5 on acetate.2 It is also 3 reported to ferment glucose to make ethanol, CO2 and D-lactic acid.

Figure IX. Agarose gels after PCR amplifications.

Gel Lane Description (gels not on the same scale) Left gel (A) 1 Extracted DNA Fueling Station yeast-like organism (1:1 dilution) 2 Extracted DNA Fueling Station yeast-like organism (1:10 dilution) Eukaryotic 3 Extracted DNA Fueling Station yeast-like organism (1:1 dilution) Primers 4 Extracted DNA Trunk River Mat non-yeast organism (1:100 dilution) 5 Extracted DNA Trunk River Mat non-yeast organism (1:10 dilution) 6 Extracted DNA Trunk River Mat non-yeast organism (1:100 dilution) 7 Positive Control (known DNA) 8 Negative Control (no DNA) 9 Direct colony picking Fueling Station yeast-like organism 10 Direct colony picking Trunk River Mat non-yeast organism Right gel (B) 1 Extracted DNA Fueling Station yeast-like organism (1:1 dilution) 2 Extracted DNA Fueling Station yeast-like organism (1:10 dilution) Bacterial 3 Extracted DNA Fueling Station yeast-like organism (1:1 dilution) Primers 4 Extracted DNA Trunk River Mat non-yeast organism (1:100 dilution) 5 Extracted DNA Trunk River Mat non-yeast organism (1:10 dilution) 6 Extracted DNA Trunk River Mat non-yeast organism (1:100 dilution) 7 Positive Control (known DNA)

Conclusions: Enrichments using triethylsilane, diethylsilane, 2-(trimethylsilyl)ethanol and trimethylsilylacetic acid proved to be challenging growth substrates for microorganisms. Growth on trimethylsilylacetic acid lead to the isolation of two distinct organisms Issatchenkia occidentalis, a common variety fruit fungus isolated from various soil samples, and Prototheca zopfii var. hydrocarboneae, 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, presumably decomposing to acetic acid and trimethylsilanol. Direct NMR analysis of media proved to be a powerful tool in deconvoluting this problem where HPLC was inadequate. The exact kinetics of decay could not be determined due to time constraints, but most likely these organisms are good at growing on low levels of acetate and this concept is most likely the basis for the enrichment. A careful kinetic study of substrate decay and organism growth rate would answer this question conclusively. On the other hand, organisms growing on 2-(trimethylsilyl)ethanol may actually be silicon-carbon bond metabolizers. They were shown to grow on methanol but not trimethylsilylacetic acid, implicating that perhaps the methyl groups on the silicon are being manipulated rather than the ethanol group. Regarding future work, trimethylsilanol would also be an excellent substrate for this enrichment since it could be playing an inhibitory role in these experiments and the diffused nature of the DAPI staining in Prototheca zopfii could be an interesting phenomenon worth investigation.

References:

1) Abranches J, Starmer WT, Hagler AN. Microb Ecol. 2001 Aug; 42 (2):186-192. “Yeast-Yeast Interactions in Guava and Tomato Fruits”. 2) Uwe Roesler, Asia Möller, Andreas Hensel, Daniela Baumann and Uwe Truyen Int J Syst Evol Microbiol 2006, 56, 1419-1425. ”Diversity within the current algal species Prototheca zopfii: a proposal for two Prototheca zopfii genotypes and description of a novel species, Prototheca blaschkeae sp. nov.” 3) Ueno R, Urano N, Suzuki M, Kimura S. Arch Microbiol. 2002 Mar; 177 (3):244- 50. “Isolation, characterization, and fermentative pattern of a novel thermotolerant Prototheca zopfii var. hydrocarbonea strain producing ethanol and

CO2 from glucose at 40 degrees C.”