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.

SiO

O

HSi

OH

HSiSi

HH

TMSAATMSETESDES

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.

(mM) Vol. (µL)

M.W. Density (g/mL)

B.P. (°C) Lot #

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

y = 0.0003e0.0069x

R2 = 0.9947

0.01

0.1

1

0 200 400 600 800 1000 1200

Time (min)

OD

600

Growth Curve - Trunk River Mat

y = 0.0012e0.0041x

R2 = 0.9938

0.1

1

1150 1200 1250 1300 1350 1400

Time (min)

OD

60

0

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.

SiO

O

H

OH

O

SiO

O

H

H+

OH

H

OHSi

O

OH

H

media

tautomerization+

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 > Viridiplantae >

Chlorophyta > Trebouxiophyceae). 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

reported to ferment glucose to make ethanol, CO2 and D-lactic acid. 3

Figure IX. Agarose gels after PCR amplifications.

Gel Lane Description (gels not on the same scale)

1 Extracted DNA Fueling Station yeast-like organism (1:1 dilution) 2 Extracted DNA Fueling Station yeast-like organism (1:10 dilution) 3 Extracted DNA Fueling Station yeast-like organism (1:1 dilution) 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

Left gel (A) Eukaryotic Primers

10 Direct colony picking Trunk River Mat non-yeast organism 1 Extracted DNA Fueling Station yeast-like organism (1:1 dilution) 2 Extracted DNA Fueling Station yeast-like organism (1:10 dilution) 3 Extracted DNA Fueling Station yeast-like organism (1:1 dilution) 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)

Right gel (B) Bacterial Primers

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.”