Microbial booster shots to increase energy yields from oil fields?

One of the major contributing factors leading to rapid exhaustion of fossil fuel resources is inefficient extraction methods. Currently oil extraction techniques only recover 40% of an available stock; in this ‘proof of concept’ study a researchers from the University of Oklahoma, have found that “oil residing in marginal reservoir samples [stranded oils] can be converted into methane by using a methanogenic microbial consortium as an inoculant.” This recovery of methane gas from underutilized hydrocarbon bearing resources via microbial biodegradation could provide a more efficient, cleaner burning, energy fuel.

The researchers exposed crushed residual oil bearing sandstone core material (from 200m depth with ≅0.013g oil per gram of core ≅ 30-40% oil core saturation) with an inoculant containing a methanogenic consortium of Archaea (already known for their methanogenic properties) enriched from gas-condensate-contaminated subsurface sediments along with a sterile fluid medium. Methane levels were monitored by gas chromatography (GC) of the incubation vessels headspace contents. DNA analysis was used to construct a phylogenic tree of the inoculation consortium.

Methane production varied between different core and inoculant combinations, but on average “0.15 to 0.40 µmol/day/g core (or 11 to 31 µmol/day/g oil), with yields of up to 3 mmol CH4/g residual oil” were observed (See Fig.1). Rates of methanogenesis were greatly reduced in incubations containing only formation oil without core material and production was totally absent in uninoculated incubations. These results shows that the microbial consortium is directly responsible for the methanogenesis and that an inoculation could potentially be used to recover methane from residual oil reservoirs. It should be noted that the core material play an, as yet, unidentified role in the process but is nonetheless vital for an effective methane yield. The DNA analysis run by this team identified many fermentative bacteria (e.g. Clostridia) but it is believed their role was as a syntrophic organism, exploiting byproducts of the methanogens themselves and were not directly involved in the breakdown of hydrocarbons. This information is useful as it allows the creation of more efficient inoculants in the future.

Methanogenic biodegradation of hydrocarbon sources already occurs naturally but the use here of an artificially magnified inoculation provides the bases for accelerating the process and making commercial use of methanogen consortiums. An application of an oil-to-methane recovery process in marginal reservoirs could significantly increase the energy yield from current global oil stocks and while in its infancy, the use of this technology could be of great interest to “help nations reduce reliance on foreign imports and increase the value of domestic reserve[s]”  and perhaps reduce the pressure to locate and exploit new oil reserves in sensitive areas. Unfortunately a large scale lack of global oil collected via traditional methods may be the impetus that finally pushes this recovery process to the forefront of energy production, and not environmental concerns.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2394919/

Gieg, L.M, Duncan, K.E, Suflita, J.M, (2008). Bioenergy Production via Microbial Conversion of Residual Oil to Natural Gas. Applied and Environmental Microbiology. 74 (10), pp.3022-3029

MGA Diversity and Function in the North Pacific Ocean

Marine Group A (MGA) bacteria were first discovered in 16S rRNA gene clone libraries produced from surface waters of the Atlantic and Pacific Oceans. However, almost nothing is known about MGA, which is currently a candidate phylum. The report expands on a previous study on the diversity of MGA in a range of O₂-deficient waters of the Northeast subarctic Pacific Ocean (NESAP), including an anoxic fjord, Saanich Inlet (SI). While MGA are ubiquitous in the dark ocean, they tend to be most dominant and diverse in interior regions of the ocean with distinct oxyclines, such as in oxygen minimum zones (OMZs) and permanent or seasonally stratified anoxic basins. Climate change is causing OMZs to expand and intensify, which could have large negative implications on ocean ecology and biogeochemistry, so this study aimed to contribute to a greater understanding of the metabolic diversity and ecosystem function of these bacteria to predict any systematic effects.

Phylogenetic analysis of MGA in the NESAP and SI

To compensate for the lack of reference genomes for MGA, 18 large-insert DNA fragments affiliated with certain MGA subgroups were identified to study MGA function and metabolism. 17 of these fragments containing MGA 16S rRNA genes were linked to 5 previously defined and 3 new MGA subgroups using metagenomic libraries.

The authors tested how the 16S rRNA-based patterns of MGA distribution contributed to the establishment of ecotypes along dissolved oxygen gradients, as a response to O₂  deficiency. 46 end-sequenced fosmid libraries from both NESAP and SI waters were screened for clones containing 16S rRNA sequences, and sequences for 14 fosmid inserts affiliated with MGA were exposed. Comparative analyses were also made against four publicly available MGA-associated large-insert fragments. This demonstrated a widespread difference in genomes, although patterns did not consistently reflect previous observations in clone libraries. There was a greater proportion of MGA distribution in the NESAP (open ocean) than in SI (coastal basin), where the highest number of end sequences were in dysoxic and suboxic samples, so it’s suggested that dominant MGA subgroups are adapted to O₂ deficiency in this area.  

Role of the metabolism of MGA in the marine sulphur cycle

Predicted protein-coding genes associated with oxygen deficiency adaptations and sulphur-based energy metabolism, such as polysulphide reductase (psrABC, or PSR), were discovered on multiple fosmids. The authors suggest that the sequences encoded on two particular fosmids from the NESAP and SI are associated with O₂-deficient environments. Two other fosmids were identified possessing proteins homologous to PSR, suggesting that specific MGA subgroups may have the capacity to generate energy via dissimilatory polysulphide reduction to hydrogen sulphide (H₂S) or dissimilatory H₂S oxidation.

So far, Wolinella succinogenes is the only microorganism in MGA to show evidence for the role of PSR in sulphur-based energy metabolism. The presence of PSR homologs on MGA-affiliated genome fragments suggests MGA may have a dominant role in the cryptic sulphur cycle of O₂-deficient marine systems.

Active sulphur cycles linked to sulphur-oxidising gamma and epsilonproteobacteria activities are found in oxygen-deficient marine systems, including OMZs and permanent or seasonally stratified anoxic basins. This paper gives initial indications of the scale of the metabolic processes of the MGA and their significance in the marine environment, based on the group’s diversity, but suggests a wider scope for further work. It would also be interesting to investigate how the role of MGA in the marine sulphur cycle affects other marine microbial communities, especially other anaerobic bacteria.

Wright, J.J., Mewis, K., Hanson, N.W., Konwar, K.M., Maas, K.R., and Hallam, S.J. (2013) Genomic properties of Marine Group A Bacteria indicate a role in the marine sulphur cycle. The ISME Journal, doi:10.1038/ismej.2013.152

Unicellular cyanobacterium symbiotic with single-celled eukaryotic alga.

In 2010, Candidatus Atelocyanobacterium thalassa (UCYN-A), a type of nitrogen-fixing cyanobacterium was found to have a degree of genomic streamlining, which is common in organisms involved in symbiosis. Unusually this cyanobacterium lacks Photosystem II (oxygen evolving component), RuBisCo (ability to fix CO₂) and the TCA cycle all-important components of photosynthesis. It was noted however, that UCYN-A has full nitrogen fixing pathways, as well as the genes for glycolysis, the implication being that it could provide valuable fixed nitrogen to a photosynthetic partner from which it would receive all of its fixed carbon. It is possible that UCYN-A gradually lost genetic information as a result of symbiosis.

To determine the potential symbiotic partner, samples were collected, sorted by flow cytometry and screened for the nitrogenase gene, nifH using quantitative PCR. This showed that the groups associated with the nitrogen fixing cyanobacterium were members of the photosynthetic picoeukaryotes (PPE) populations. Using 18S rRNA from the PPE and 16S rRNA from the associated sample it was found that PPE were exclusively prymnesiophytes and that UCYN-A was present in the microbial community. Within the prymnesiophytes the greatest relatedness was to Braarudosphaera bigelowii and Chrysochromulina parkeae. B. bigelowii is of particular interest as it has calcareous plates at certain stages in its life cycle. It is not certain whether UCYN-A has a calcareous stage during symbiosis, as it is not known at what stage the symbiosis occurs.

This symbiosis represents globally distributed and important (i.e. carbon fixation) groups of organisms, giving environmental importance to the symbiosis. This importance stems from the organisms involvement in a large portion of the vertical nitrogen flux in the oceans. It is already known that calcified prymnesiophytes are major contributors to the POM that is distributed throughout the water column. One of the questions raised by this apparent symbiosis is why this symbiosis has not ended with an endosymbiotic relationship, specifically the formation of a new nitrogen fixing plastid. The possible answers are numerous. Some of the ideas we discussed involved nitrogenase sensitivity to oxygen. This would mean that a nitrogen fixing plastid would have to be maintained in anoxic conditions within the cell. Another potential problem is the calcification state. Perhaps calcified plates would act as a barrier and prevent phagocytosis or perhaps the symbiosis is held intact by bonding to these plates and engulfing the UCYN-A would therefore not be possible.

Halogen In Situ Hybridization and Secondary Ion Mass Spectrometry (HISH-SIMS) were used to determine the flow of ¹⁴C and¹⁵N between the two partners. This showed that 95% of the nitrogen fixed by UCYN-A was transferred to the partner and 17% of the fixed carbon was transferred to UCYN-A. This technique also showed that there was an indentation on the algal surface where attachment occurred The indentation was smaller than would be expected, and some indentation could be seen at both poles of one alga which was on the verge of mitosis, implying that the symbiosis may occur throughout the life cycle of both the algal partner and the cyanobacterium.

This paper allows us to understand that there is a symbiosis occurring between some of the most important groups of microbes in the ocean. This opens up possibilities into what we may find in the future.

Thompson, A. W., Foster, R. A., Krupke, A., Carter, B. J., Musat, N., Vaulot, D., & Zehr, J. P. (2012). Unicellular cyanobacterium symbiotic with a single-celled eukaryotic alga. Science, 337(6101), 1546-1550

George & Ethan

Nitrogen-fixation and Transfer in Open-ocean DIatom-cyanobacteria Symbiosis

Driven by a building debate in the scientific world, regarding the discrepancy of N sources and sinks, Foster R.A et al decided to study the N₂-fixing events of diatoms; mainly the genera Hemiaulus, Climacodium and Chaetoceros, and their symbiotically related heterocystous, filamentous cyanobacteria; Richelia intracellularis, Crocosphaera watsonii and Calothrix rhizosoleniae.  These symbiotic diatoms have been observed internationally in blooms but N₂ and C fixation rates have never been monitored.  Previously, no advantage of the relationship had been identified but it was hypothesized that the cyanobacteria were providing their hosts with fixed N.

Long-term incubation of cultures from the Gulf of California and the sub-tropical North Pacific were performed and nanometer scale secondary ion mass spectrometer (NanoSIMS) was used to provide evidence that the fixed N₂ was being transferred to the symbiotic diatoms.

Diatoms are unable to obtain N from N₂ and so, they rely on dissolved inorganic nitrogen in the form of nitrate and ammonium, which exists at extremely low concentrations in the open ocean.  Within Hemiaulus, NanoSIMS was employed to show the quantifiable levels of the ¹⁵N label in each sample where, using epifluroescence, symbiotic diazotrophs were shown to be residing.  This verified that R. intracellularis was fixing N from N₂.  However, the diatoms’ chloroplasts were also enriched with N signifying that it was transferred from the symbiotic companion.

               The ¹⁵N label was most apparent in the cyanobacteria, C. watsonii  associated with Climacodium.  In Chaetoceros, the observed C. rhizosoleniae were also shown to be acquiring high-levels of N₂; this was especially abundant in the heterocyst and vegetative cells suggesting that the N was shifting from the C. rhizosoleniae, along the trichome and across the cell membrane of Chaetoceros.  This was also observed in the symbiotic relationship between C. watsonii and Climacodium.  These were previously unrecorded function.

It is interesting to note that in all cases studied, there was an equal or higher enrichment of N in the diatoms than in the vegetative cells.  Diatoms in oligotrophic conditions were thought to grow slowly due to the low concentrations of nutrients.  However, the team discovered that enrichment of the ¹⁵N label was saturated after 3hrs, faster than N-fixation was originally anticipatedin cyanobacteria. 

The transfer of the N was also surprising.  It was thought to be slower due to the placement of the cyanobacteria strains.  R. intracellularis is located between the frustule and the cell membrane, C. watsonii location is unknown and the C. rhizosoleniae is located extracellularly.  The rapid transfer of the N was due to the observed movement of N through the trichomes and the cell membrane (which was previously unobserved).

The growth rates of the diatoms and their symbionts were all found to be very similar.  But a difference in the growth rate of free-living C. rhizosoleniae and R. intracelllaris, and those cyanobacteria symbiotically existing with their hosts; the former having a much slower growth rate and a smaller terminal size.   The free-living cyanobacteria also showed slower N-fixation rates.

These relationships had always been assumed with little evidence, however, this paper clearly demonstrates the hypothesis that the cyanobacteria symbionts fully support the diatom’s need of N for cell growth and is significant enough for symbiotic diatoms to be included in N-fixation models.

In situ activity of a Prochlorococcus ecotype (eHL-11) from rRNA content and cell size

Prochlorococcus, a genus of unicellular cyanobacteria, is ubiquitous throughout the open ocean ecosystem. Many clades coexist, yet the genetic and physiological diversity found between clades allows them to occupy a range of different ecological niches thus resulting in different ecotypes, biogeographically structured along environmental gradients. Whilst community activity (comprising of different ecotypes) has previously been estimated, very little research has been conducted on individual ecotypes, though they are presumed to differ in their in situ activity.

The authors have used a novel molecular based approach, using cellular ribosomal RNA (rRNA), to measure specific in situ activity of two strains of eHL-11 clade.  By measuring side scatter (SSC), as an operational index of cell size, at different light levels specific growth rates was found. Using this data and plotting it against rRNA cell content (rRNA cell⁻¹) resulted in a linear correlation, a clade eHL-11 specific response. Though the correlation differed between strains, once data was normalised for cell size both strains showed the same slope thus showing a conserved relationship between rRNA cell⁻¹ SSC⁻¹ and growth rate (rRNA content and activity). From this the authors were able to assess the strain specific in situ activity and the response to environmental factors.

Diel variation
The patterns found are consistent with other Prochlorococcus strains in that many cell responses, both genetic and physiological, are tightly coupled with light intensity, showing a substantial diel cycle in order to optimise growth. Cell division was found to occur at night whilst both rRNA synthesis and biomass accumulation increased during the day owing to the observed increase in SSC and rRNA cell⁻¹ (similar in magnitude as driven by same mechanisms). The accumulation of biomass requires more ribosomes for protein synthesis, the energy for which is provided by photosynthesis, requiring light.

Effect of environmental variables
The authors sampled two geographically close stations, yet each with a distinct combination of environmental variables. At both sites: eHL-11 was the dominant clade of Prochlorococcus, the number of cells decreased rapidly once below the mixed layer (despite depth differences of the mixed layer), the cell size was small and constant within the mixed layer and growth rates were similar and ranged from 0.2-0.4 day⁻¹ within the upper 50m.

Though no subpopulation of Prochlorococcus were found throughout both of the water columns vertical trends in specific activity (rRNA cell⁻¹ SSC⁻¹) and inferred growth rates vary substantially between sites and do not follow the same trend as with cell concentrations. These differences could be attributed by many mechanisms including temperature, light and nutrient availability. It has been suggested by the authors that the variability in specific activity despite relatively constant cell counts may be due to the distribution of cells due to mixing, or abiotic pressures of viral lysis and grazing that specifically target fast growing or highly active cells.

I believe this approach has potential, particularly in it’s application on other clades as the rRNA sequences of bacteria are conservative and sufficiently diverse allowing for oligoprimers targeting specific clades. However there are certain limitations e.g. 1) the authors state that linear relationship between rRNA cell⁻¹ and specific growth rate is clade specific to eHL-11 in which case, would this impede the use of this method on other clades? 2) rRNA content can be affected by environmental variations and cellular physiological characteristics. The clade tested is relatively well understood yet many inferences and presumptions have been made from the observed in situ activity due to the variability in the responses to several environmental factors. For that, whilst useful for measuring in situ activity within this clade, I feel it’s difficult to deduce any rational behind the observed activity.

Lin Y., Gazsi K., Lance V. P., Larkin A. A., Chandler J. W., Zinser E. R. and Johnson Z. I. (2013) In situ activity of a dominant Prochlorococcus ecotype (eHL-11) from rRNA content and cell size. Environmental Microbiology, 15(10), 2736-2747

Marine Microbes: a Hidden Smorgasbord of Nanoparticles

   Modern nanotechnology is being held back by the absence of sustainable and green methods of synthesising nanoparticles; current methods are burdened with the financial and environmental expense of using toxic materials; plus they use 'top down processes' which demand large amounts of wastage and energy. The best candidate alternative is the manipulation of organisms using biotechnological 'bottom up processes'; they have been tentatively practised across the domains of life from microbes to plants. Microbes are suitable for nanoparticle synthesis because many are adapted to heavy metal exposure, which they extensively use metabolic processes such as absorption, precipitation and chelation which form nanoparticle  byproducts. Since marine microbes are diverse across a range of unique marine niches such as high salinity, extreme pressure, low nutrients and high heavy metal concentrations, their potential for yielding novel nanoparticles is promising; but interest in marine microbes is only gathering now and this resource has yet to be utilised.

   Most non-marine microbes secrete cell wall proteins to form extracellular complexes with metal ions, creating nanoparticle precipitates, which clump together into crystals and must be broken apart to yield nanoparticles for human use. Marine microbes have a radically different way of dealing with metal ions; before intracellular storage, they attach phosphate/carbonate/sulfide groups to metals and sequester them with peptides such as glutathione. The advantage of this for us is that because marine microbe nanoparticles are bound to peptides, they will not crystallise into bulky aggregations, increasing nanoparticle stability.

   This review reports demonstrations of marine microbe nanoparticle synthesis; two marine actinomycetes produced silver nanoparticles and a range of different particle properties were possible by altering temperature and silver nitrate concentrations. Six marine fungi were also able to synthesis similar nanoparticles; additionally, it was confirmed that each particle was surrounded by a stabilising peptide. Cubic silver nanoparticles across a range of sizes could be produced by a marine Pseudomonas isolate and marine microalgae have been shown to be capable of making silver nanoparticles which are antimicrobial towards human pathogens. Antimicrobial activity of silver nanoparticles from the marine yeast Candida sp. and cyanobacteria have also been reported to work against multi drug resistant pathogens. But its not all just silver; the marine yeast Rhodosporidium diobovatum was able to synthesis cubic lead nanoparticles.

   As this field currently stands, it seems our seas contain a vast library of nanoparticles with different potential uses in medicine, industry and biotechnology is awaiting discovery. The strong motives for tapping into this resource may be a major driving force in advancing marine microbiology, furthering our understanding in areas such as how to culture what is currently unculturable and the extent of marine microbial diversity.

Baker, S., Harini, B. P., Rakshith, D., & Satish, S. (2013). Marine microbes: Invisible nanofactories. Journal of Pharmacy Research.

A Cure for the Common Cold?

The common cold is an annoying, uncomfortable and inevitable 2 weeks of your year. Colds don’t only cause problems on a personal level, but the millions of lost work hours also cause an economic impact. They are caused by human rhinoviruses, the most known species of which are RV-A and RV-B.

Past research into combating these variant types have proved successful in lab cultures, targeting the protomer canyons, or “pocket” features. These are capsid grooves characteristic of Enteroviruses, and have residues conferring receptor recognition. Capsid-binding antivirals fit into these pockets and attach to the receptors, creating subdivided virus-drug complexes with pore like openings giving an entrance for the drug. Pleconaril for instance is reported to be 93% effective against these species.

 These complexes are roughly divided into two groups along the species divide, with differing canyon types.

However in 2006 a third species was discovered – RV-C. This species grows in under broader conditions than its counter parts, is less receptive to drugs, and worryingly found to cause half of cold infections in young children. This species avoided detection as it doesn’t propagate in typical cell culture systems. The 51 types recognised were discovered by direct sequencing from patient effluents!

Alternative attempts at culturing RV-C through fledgling studies using differentiated sinus and brachial epithelial cells at air-liquid interface (ALI) are promising. The only other study uses mucus membranes in primary human donor samples, which are few and far between! Also the variety of donors makes this technique variable and somewhat unreliable.

Both however don’t produce enough RV-C for biological research, so most information on the species comes from comparative studies with the wider known RV-A and RV-B species.

Holly Basta and co authors of this paper have used past genome alignments in combination with superimposed determined capsid structures of RV-A and RV-B types to create a high resolution 3-D model for The RV-C species. The model shows the vital Cα backbone present in viral components of RV-C, meaning the RV-C drug-binding “pocket” is superimposable on the RV-A and RV-B structures.

This structural model was termed C15, an isolate cloned into cDNA and tested for biological activity by mucus and ALI techniques.

The model showed C15 to have an altered surface structure to RV-A and RV-B. This accounted for it’s resistance to drugs engineered for RV-A and RV-B species, as it has different residues and receptors within­­­ the “pocket” structure.

Computer models found Pleconaril and some other drugs did fit into C15s’ “pocket”, but the diversity of residues in the viral “pocket” mean in practice these drugs are of no affect to viral growth. The authors commented they may at best find an alternate route “wiggling through tenaciously, despite the altered sequences”.

This will call for a RV-C specific drug in combination with the former RV-A and RV-B drugs, a path that will be eased significantly by the plethora of work already conducted studying RV-A and RV-B. Obviously much more research is needed in order to develop such a drug, but this model is significant in that it is a huge step in that direction, and can provide a basis for future tests and drug models.

So provided no more resistant rhinoviruses are lurking out of sight, you may find yourself picking up a packet of cold busting tablets in place of your paracetamol over the counter in the near future.  

- Basta, H. A., Ashraf, S., Sgro, J. Y., Bochkov, Y. A., Gern, J. E., & Palmenberg, A. C. (2014). Modeling of the human rhinovirus C capsid suggests possible causes for antiviral drug resistance. Virology, 448, 82-90.