Looking for phototrophs that use methane and methanol as electron donor

Amin Omairi-Nasser The university of Chicago

Microbial Diversity - 2014 Abstract

Phototrophs are the organisms that capture photon and use their energy to acquire energy in form of ATP. They also use the energy from light to carry out various cellular metabolic processes. This process will also require and electron donor and an electron acceptor. While H2O is the electron donor in oxygenic phototrophs (cyanobacteria) other compounds (methanol, sulfite etc…) are used by anoxygenic phototrophs (purple and green bacteria). Methane is one of the compounds that have a high redox potential, however no phototrophs that uses methane were yet found. In this study I enriched for phototrophs that uses methane and methanol as electron donor. I designed a media, collected samples from fresh water and seawater and enriched cultures at different wavelength. Enriching Introduction

The ability to fix carbon from light energy is distributed through a diverse selection of bacterial groups, and involves a similarly wide variety of chemistries (Overmann and Garcia- Pichel, 2006). Different organisms have evolved to use different wavelengths of light to transfer electrons from a variety of electron donors ultimately to carbon dioxide (Hohmann-Marriott and Blankenship, 2011). Of those capable of photosynthesis, only one group (cyanobacteria) of related organisms uses water as an electron donor, resulting in the production of molecular oxygen. The advent of this process almost certainly marked the shift in the Earth from an anoxic to oxic atmosphere. The consequences of this event on the trajectory of life's evolution are incomparable. While oxygenic photosynthesis is the most studied and known process it only occurs in one of the 5 groups that preform photosynthesis. Anoxygenic photosynthesis occurs in 4 groups of bacteria: Phototrophic green bacteria, phototrophic purple bacteria, Heliobacteria and Acidobacteria. It is likely that some form of anoxygenic photosynthesis was a precursor to the complex machinery necessary for oxygenic photosynthesis (Trost et al., 1992). Because of this, the modern anaerobic phototrophs, belonging exclusively to the bacterial kingdom, represent model systems to study photosynthesis in its simplest forms. H2S is a wide used electron donor by many organisms due to its high potential energy. Both Purple bacteria and green bacteria have groups that use H2S along with other sulfur reduced compounds (as thiosulfate); they are known as purple sulfur bacteria and green sulfur bacteria. However, many bacteria are able to use other compounds as electron donors such as organic, fatty and amino acids, alcohols (Methanol, Ethanol etc…) and aromatic compounds (Overmann, 2001).

Methane is known to have a relatively good redox potential and considered as a good electron donor. Phototrophs that use methane as electron donor were never been identified before even though many attempts have been made. In this study I tried to enrich for phototrophs that use methane or methanol as electron donors. Both methane and methanol appear to be suitable electron donor for all known reaction centers as their redox potential is higher than all, cyanobacteria, purple and green bacteria reaction center. Samples from Eel pond and Cedar swap representing seawater and fresh water, respectively were used. I was able to get purple bacteria and green bacteria enrichments in both cases however I wasn’t able to complete the characterization of the obtained strains.

Figure 1. Comparison of electron transport pathways in oxygenic and anoxygenic organisms (from Blankenship, 1992). Abbreviations: Cyt bc1, cytochrome bc complex; P840, reaction center bacteriochlorophyll; P680 and P700, the reaction center chlorophyll of photosystem II and photosystem I, respectively; Pheo, pheophytin; QA, and QB bound plastoquinones; PQH2, reduced plastoquinone; Cyt bL and Cyt bH, different forms of b-type cytochromes; FeS, iron-sulfur centers; Cyt f, cytochome f; PC, plastocyanin; A0, chlorophyll; A1, phylloquinone; FX, FA and FB, iron sulfur centers; Fd, ferredoxin; FNR, ferredoxin/NADP+ oxidoreductase; NADPH, nicotinomide adenine dinucleotide phosphate (reduced form). Some organisms derive their energy from electron donating inorganic molecules such as hydrogen gas or sulfur compounds and are not dependent on current or past photosynthesis for their survival.

MATERIALS AND METHODS

Enrichment Samples from Cedar swamp (fresh water) and Eel pond (seawater), Woods Hole, MA were collected in a 50mL Falcon tubes. Samples were collected from the top of the sediments were it’s possible to have methane and light. The medium contained the following components: Artificial seawater base or Fresh water, 10 mM NH4Cl, 1 mM KH2PO4, 250 µM NaSO4, 20 mM MOPS buffer (PH 6.5 and PH 7.2 for samples collected from Cedar swap and Eel pond, respectively) trace elements, Multivitamin solution, 5 mM NaHCO3. Enrichments were performed by adding different electron donors and by incubating the cultures at different wavelengths of light (White light, 860 nm and 660 nm). Most of the enrichments were supplemented with DCMU. Cultures and media were prepared in the anaerobic chamber when necessary. Electron donors were used as follow:

Electron donor Notes Methane Saturation of the head of the bottle by pumping methane gas for 30 sec Methanol By adding to the media methanol (different concentration; 1% = 246 mM) Na2S 100 µM to 8 mM were added when needed

Absorption spectra Whole cell absorption spectra were measured directly from bottles or on the plates using the SR-1900 Series Spectroradiometer (Spectral Evolution Inc.) A white lamp was used as a light source and the detector was placed, either on the other side of the bottles or on the top of a colony or a drop (depending what’s measured).

Free reaction energies calculation Thermodync was used for the calculation of free reaction energies for actual conditions of activity and temperature (Damgaard and Hanselmann, 1991)

SEM Hitachi Analytical TableTop SEM, TM3030 was used for collecting images and for elemental analysis. Samples were washed with water 3 times then placed on a Millipore membrane.

Results

Calculation of free reaction energies using Thermodyn

I decided to enrich bacteria using Methane, Methanol and H2S as electron donor. Bacteria using the two later are known to exist while no phototrophs using methane have been identified. In order to check if methane as well as methanol and H2S are has a favorable potential energy to give electrons to electron transport chain component, their electron potential was calculated. Under anoxic conditions were only methane, methanol or sulfite will be present we assumed that their reaction as electron donors will be as following:

- - + Methane: CH4 + 3H2O —> 8e + HCO3 + 9H - - + Methanol: CH3OH + 2H2O —> 6e + HCO3 + 7H - - 2- + Sulfite: HS + 4H2O —> 8e + SO4 + 9H

2- Bicarbonates serve as electron acceptor for both methane and methanol while SO4 serves as electron acceptor for sulfite. Both compounds are present in the media. Note that the E of the reaction as indicated in figure 2 is calculated in a way were our molecule of interest are the substrate which is not the way of presenting it in general (Slonczewski, 2010). The sign of the potential energy should be inversed to make a good comparison in figure 1.

Figure 1A show that Methane, Methanol and H2S have a redox potential energy of -0.295 mV, -0.364 mV and -0.233 mV, respectively. This potential is much higher than water (0.8 mV). Therefore they could play the role of effective electron donor. Figure 1B show the redox potential energy of the different component of electron transport chain in different bacteria.

Figure 2. Curves showing the energy potential of Methane (blue), Methanol (purple), sulfite (red) and water (green) relative to the abundance of electrons.

Collection sites

Samples were collected from Cedar swamp (Fresh water) and Eel pond (sea water). Cedar swamp is known to have methane-producing bacteria while Eel pond have various hydrocarbon products due to the presence of boats (oil spilling etc…). I assumed that these locations might contain phototrophs that use methane and methanol as electron donor. Samples were collected from the surface were I supposed that light will penetrate and methane or methanol could reach.

Figure 3. Pictures representing sampling sites. A. Cedar pond (source of fresh water samples). B Eel pond (source of sea water samples.

Enriching for phototrophs using methane as electron donor

Enriching for these phototrophs was attempted several times during the course. In Attempt 1, I started cultures for both FW and SW samples and they were incubated under white light. My idea was to enrich for any organism that could use Methane as electron donor.

Figure 4. Attempt 1 for enriching Methane using phototrophs. A. The bottle containing FW samples (Cedar swamp) showed positive growth while (B) no growth was recorded in bottles from SW (Eel pond).

Figure 4. shows that I obtained green algae (Chlamydomonas) and diatoms from the first enrichments (FW samples). I suppose that in the first inoculation we would be adding many other components to the medium. These components might favor the growth of easy growing organism. Based on the first attempt I decided to repeat the inoculation (Attempt II). DCMU was added to all bottles to inhibit the growth of cyanobacteria (and chloroplast containing organisms). I also incubated the samples under three different light regimes; White light, 660 nm (favors green bacteria growth) and 850 nm (favors purple bacteria growth). After 4-5 days, only bottles grown at 850 nm showed some growth. FW samples turned green while SW samples turned purple. Samples were passed to new bottles and after two days whole cell absorption spectra were measured for both enrichments (figure 5). Whole cell absorption spectra show the presence of Bchla in the SW culture (purple culture). This suggests the presence of purple bacteria in the culture. The culture looked heterogeneous on the light microscope suggesting the presence of mixed culture. This was confirmed when I plated the culture on a SW plate and incubated it with methane. In order to identify the bacteria that are present in the culture and most importantly the purple bacteria that might be using the methane as electron donor, PCR was performed to amplify the 16S DNA using primers 8F and 1492R. Amplicons were cloned in pGEM-T vector (Promega) and transformed in E. coli. Plasmids were then prepared from 13 different clones and submitted for sequencing. Sequences showed the presence of 7 different bacterial families with high hits (>98%) for Desulfovibrionaceae (genus Lawsonia) and Chromatiaceae (genus Thiorhodovibrio). Three sequences corresponded to Archaea but with low similarity. Some Thiorhodovibrio have been previously described as purple sulfur bacteria (Overmann et al., 1992) but never been shown to use methane. The similarity with a known Thiorhodovibrio is between 51% and 87%. This suggests that the culture we have is new Thiorhodovibrio that might be able to use methane as an electron donor.

Whole cell absorption spectra were also analyzed for the FW samples (green culture). It’s important to mention that while some growth was detected after 4-5 days (like for the SW bottle), the culture turned clearly green after around 10 days. Whole cell absorption spectra show a peak at 751 nm, which correspond to Bchlc. This suggests that the culture contains green-sulfur or non sulfur bacteria. The growth on 860 nm could be explained by the fact that Bchlc can absorb at this wavelength but much less efficiently. Note that since the culture grew slower I didn’t have time to prepare DNA a send for sequencing like I did with the SW culture.

In order to confirm if methane is the electron donor and the carbon source that is used by the Both FW and SW cultures, I started new cultures with following combinations: • + Methane/ +Na2S • +Methane/-Na2S • -Methane/+Na2S (Methane was replaced by N2/CO2)

SW samples were grown at 860 nm. However, since SW culture looks to contain a green bacteria with Bchlc that prefer to absorb light a 660 nm; duplicates were prepared for the SW cultures and were grown at both 860 nm and 660 nm. These combination would give an idea weather methane or sulfite is used as electron donor or at least if methane is important for the growth of the culture.

Figure 5. Pigment analyses for samples grown at 850 nm. A and C. Pictures of bottles containing cultures that grew from SW and FW, respectively. B. Whole cell absorption spectra for the purple culture that grew from SW inoculum. Spectrum shows two peaks at 852 nm and 801 nm that correspond to Bchla. D. Whole cell absorption spectra for the GREEN culture that grew from the FW inoculum. Spectrum shows one peak at 751 nm that corresponds to Bchlc.

Methanol using phototrophs

As mentioned before, methanol was also a good candidate to be tested, as electron donor since it’s potential energy was favorable. Inoculation from SW and FW sediments was performed in duplicates in corresponding media that contains 0.1% methanol as sole electron donor. Bottles were incubated at both 660 nm and 850 nm. Under 850 nm, only the FW bottle incubated turned purple. The growth was relatively fast and bottle started turning purple on the second day. Under the 660 nm light, only the SW bottle turned green. The growth however was much slower than the one observed at 850 nm. The culture in the bottle turned slightly green after 12 days of incubation.

Figure 6. Inoculation of FW and SW samples in Methanol containing media. Duplicates of each inoculum were made and incubated at two different light wavelength (660 nm and 850 nm)

Characterization of the Methanol growing purple bacteria

To further analyze the purple bacteria that were growing at 860 nm whole cell absorption spectra were measured (figure 6). The spectra show the presence of Bchla, which correlates with the assumption that the bacteria are purple bacteria.

Figure 7. Whole cell absorption spectra for the purple culture that grew from the FW inoculum. Spectrum shows two peaks at 861 nm and 800 nm that correspond to Bchla.

In order to verify if the purple bacteria was using the methanol as the sole electron donor and/or carbon source, I decided to test their growth in the presence and absence of methanol. Figure 8 shows that no growth occurred when there was no methanol supplied in the media. At the same time growth was also affected by the concentration of methanol. Cultures growing with 0.1% methanol grow better than cultures with 1% methanol in their media. These observations confirm that the enriched purple bacteria requires methanol for growth. To further analyze the effect of Methanol on the culture cell growth was monitored during several days under different methanol concentration (figure 8B). Growth curves shows that the optimum concentration of methanol is 0.1% and that 1% of Methanol is also relatively well tolerated by the organism. It’s also worth mentioning that a slight growth was observed in the 0% methanol media. This might due to the presence of other purple bacteria in the media that doesn’t require methanol. The used media for this experiment was supplemented with 50 µM of sulfite which could be used by these microorganism and explain their slow growth due to the almost absence of the source of an electron donor.

Figure 8. Methanol is required for the growth of the analyzed purple bacteria. A. Growth of the purple bacteria I obtained in the absence (0%) or the presence (0.1% and 1%) of methanol. B. Curve growth of the purple bacteria using three different methanol concentrations.

The culture was plated on FW plates supplemented with methanol. Purple and white colonies were obtained and sequenced. Sequences show that the purple bacterium belongs to Rhodocyclaceae family (which is a purple bacteria) and that it’s 74% similar to Zoogloea. Previous studies had shown the presence of purple bacteria that can grow on methanol like Rhodopseudomonas acidophila that grow on a less % than what I’ve used (Quayle and Pfennig, 1975) We also measured the whole cell absorption spectrum of the green methanol using bacteria (figure 9). The spectrum shows a peak at 704 nm when measured in the bottle and 721 nm when measured from a drop on a filter. The pigment probably corresponds to Bchle. However the culture should have been further analyzed. The pigments should have been extracted and analyzed and the culture should have been tested under different methanol concentration. But since the culture was growing slowly there was no time to accomplish these analyses.

Figure 9. Whole cell absorption spectra for the green culture that grew from the SW inoculum. Spectrum shows two peaks at 704 nm (when measured in bottle) and 721 nm (when measured from a drop on a filter). The pigment probably corresponds to Bchle.

High H2S concentration resistant purple bacteria This was a side project that started during first week’s enrichments. While our group was enriching for purple bacteria we tried to enrich purple bacteria growing on 5 mM sulfite. It’s believed that green sulfur bacteria can tolerate up to 5 mM of sodium sulfite (similar to sulfite) while most of purple sulfur bacteria prefer to grow on a 1 mM sulfite. When I started my project I characterized further this culture. The culture was grown on three different concentrations of sodium sulfite (figure 10). Cultures growing on 1 mM look more homogeneous while once I increased the concentration the culture tend to grown on the bottom of the bottle and form a hard shelf. I tried to measure growth curve but it was a bit of a challenge. It was clear that the culture was growing but the curve didn’t correspond the growth because it was difficult to keep the same amount of cells every day.

Figure 10. Purple bacteria culture grown with increasing concentration if Sodium sulfite.

In order to check whether the bacteria are incorporation the sulfur or they are just resistant I analyzed them under the microscope using phase contrast. Figure 11 shows that with more sulfite in the medium cells tend to accumulate a diffracting component in their cytoplasm. This component is probably sulfur. In Figure11.C the white dots correspond to cells have diffracting components. I verified that there were cells because they were moving like cells when I was looking at them.

Figure 11. Light microcopy micrographs representing purple sulfur bacteria grown at different Na2S concentration.

In order to check if the diffracting components correspond to sulfur or to another element, I analyzed the same sample presented in figure 11.C using SEM coupled with EDS (Figure 12). SEM shows the presence of granules that looks gray (Figure 12.A) when analyzed with EDS, the granules looks to be sulfur granules (Figure 12 B, C and D). This confirms that these purple bacteria are metabolizing sulfite and integrate sulfur in their cytoplasm. It’s worth mentioning that not all the cells were integrating sulfur. This could be explained by either I didn’t have a pure culture or the amount of sulfur wasn’t saturated. I tried 10mM of sulfite and the cells were growing. I also plated them to have pure colonies and restart the test but there was no time to continue the analyses.

Figure 12. SEM and EDS analyses for the purple sulfur bacteria grown on 8 mM sulfite. A. SEM micrographs of the same samples observed in figure 11.C. B. EDS of (A). C. Elemental analyses of one of the granules in (A), analyses show the presence of sulfur in these granules. D. Elemental analyses (B). S, sulfur; C, Carbon.

Conclusion The enrichment I made using methane or methanol as electron donor were promising. Both purple bacteria and green bacteria were obtained based on pigment contents and sequencing. However the time in which the experiments is done is short especially considering the growth rate of the obtained cultures.

Acknowledgments I would like to thank Kurt Hanselmnann, Arpita Bose and Jared Leadbetter for the scientific discussion. I would like to thank the entire faculty for all the various discussions and for giving me the opportunity to attend the course. I also would like to thank my P.I Prof. Robert Haselkorn, the “Planetary Biology Internship Scholarships” and the “Simons MD Scholarship” for my financial aid support for the course.

Bibliography

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