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+ Sulfite: HS- + 4H2O —> 8e- + SO4

2- + 9H+

Bicarbonates serve as electron acceptor for both methane and methanol while SO42- 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.        

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