The journal article I’ll be reviewing today is about enhancing bacterial quorum sensing – the way they talk to each other, to create microbial fuel cells that produce electricity and feed off nothing more than wastewater, cleaning it up in the process. Firstly, credit to Bonnie Bassler, who coined the phrase ‘tiny conspiracies’ (and most of the words in the title) to describe quorum sensing: she characterised it first, with beautiful bioluminescent bacteria – highly recommend watching this video if you want your mind blown.

OK so the paper is clever and interesting on a few levels: firstly the idea that microbes can be harnessed for energy production while simultaneously helping us to clear organic waste and other toxins from drinking water is something that is very attractive to energy companies that produce a lot of waste (albeit from ridiculous natural gas extraction methods like fracking). It is also interesting from a desalination point of view, where access to fresh water is restricted.

Way more importantly though, through experiments like these we learn more about bacterial communication itself. It shifts the focus away from the nasty bacteria that kill us and we try to fight off, to harnessing the trillions of species that live mutualistically on, in or around us and are indispensable for our continued existence on this planet.

Microbial fuel cells (MFCs)1

An MFC works like a normal battery, but the anode, the positive bit, is coated in bacteria. In the 1970s it was found that some bacteria can transfer electrons straight from the ‘cytochromes’ in their respiratory electron transport chain to this anode. In doing so, the bacteria oxidise (that is, steal electrons and oxygen from) organics pollutants that exist in wastewater generated from unconventional oil and gas production methods, to fuel their own survival and growth.

The bacteria provide the anode with a constant supply of electrons that can be connected to a cathode to produce a current. Because these anode surfaces things are tiny – 7um across for example, the future might be huge arrays to produce usable bioenergy.

The ability of the bacteria to coat the anode requires a ‘biofilm’ a protective layer of sugars and proteins produced by the whole population of bacteria simultaneously that provides them a niche – a place to thrive free of the terrors of a hostile outside environment. It is the formation of this film that is so crucial to the survival of bacteria in super saline, highly toxic places (such as in wasterwater), as well as to their effective function as electron donors on an electrode in an MFC.

So how do bacteria produce a biofilm?

Quorum sensing (for more, search the queen of QC: Bonnie Bassler). This is the name given to multiple methods of bacterial chit chat. Asocial, lonely bacteria howl a yearning “I’m here!” in the form of little chemical missives. Others shout back from the darkness. The population grows until a cacophonous threshold is reached – quorum – at which point all bacteria synchronise and do the same thing, like make a huge blanket of protective proteins and sugars – a biofilm. If this behaviour can be harnessed, and enhanced, then better biofilm utopias are made, providing MFCs with the necessary to protect themselves and produce more electricity.

Here, the experimenters (or rather, their recent ancestors) looked to the source – the hypersaline shale wastewater and runoff from fracking – to find their halophilic, quorum sensing bacterium: Halanaerobium praevelens2.

The genus means ‘salt organism that grows in the absence of air’ and praevelens is latin for ‘very strong, very powerful, prevalent’. These types of bacteria are descendents of the ones that some scientists believe first colonised the earth. We have sequenced its genome, so we know the broad strokes – the problem is, no-one knew the language.
Quorum sensing enhances biofilm formation and power production in a hypersaline microbial fuel cell3

chemicals

Figure 1: PQS (left) and Q (right) – skeletal structures of the quinolone quorum sensor molecules chosen

The first figure is more or less 3 unassailable in terms of criticism, it’s the chemical structures of two different chemical messages that tell these high salt loving bacteria to produce a biofilm. The experimenters basically made a lucky guess here. They went hunting, and found that similar bacteria, albeit ones that can’t survive in high pollutant, high salt conditions use quinolones like these to communicate. In the paper, there is almost no explanation as to what these are. They called it good though and asked, do they make a biofilm?

In the second figure of the paper, they add the “quinolones” to cells coating a polystyrene block in a dish. They then simply stain the sugary biofilm with a dye and measure its intensity in a fluorescence microscope. More biofilm = more dye, and they see an upregulation, which is increased with both chemicals. This sort of relative measurement works in this case, so it seems that just by adding the QS chemicals that the bacteria might produce themselves you can send the bacteria into overdrive and cause them to create a huge biofilm.


To confirm the effect of these two chemicals on increasing biofilm formation, a selection of ‘biofilm related genes’ were selected, and the amount of mRNA they produce was measured in the presence of PQS or Q.

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Figure 2: Researchers can choose a length of DNA (a gene) and measure the amount of this RNA produced from the gene, that relates to the amount of protein produced. The protein has a known function, which is inferred to be ‘increased’ or ‘decreased’ based on how much RNA the cell produces

The first gene shows an increase in expression (more mRNA), and codes for a protein that produces the lipopolysaccharides (fatty sugars) that are needed for biofilm production. There is a huge increase in this and the 2nd gene, coding for a ‘metal resistance’ protein – one that pumps heavy metals out of cells, which would otherwise kill them and are often present in these wastewaters. There is a big upregulation in expression levels of biofilm and metal resistance genes therefore, when bacteria are exposed to an excess of PQS and Q.

The researchers then claim that the next two genes CsrA, a quorum sensing enhancer, and ‘CapD’ – another biofilm manufacture gene are also upregulated. The increase is small, error bars large and there are no statistics – not even P values, so I would be inclined to ignore this result entirely.

The other criticism is that gene transcription does always relate to function. There are many examples in biology where gene transcription is increased massively, but the proteins themselves are regulated to a level where functionality of the system is not changed or is mitigated by compensatory alternative signalling pathways. They don’t address this anywhere in the paper.

So, a bit of a random selection of genes, and some odd conclusions based on small increases – but it does show that biofilm production is definitely increased, and the bacteria all switch on their metal resistance functions as well. Due to the greater effect on these genes of quinolone rather than PQS, for the rest of the paper the experimenters use quinolone (Q) only.

Right, now it gets interesting:

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Figure 3: Researchers set up a microbial fuel cell like the one above, then measured the voltage coming out in the presence of absence of pro quorum sensing ‘quinolone’

They simply set up a fuel cell, added the salt loving bacteria, then measured the voltage in the presence versus the absence of the pro-quorum sensing quinolonemolecule.

There was a ~150% increase in voltage with quinolone compared with no-quinolone. This level of electric energy production was maintained until the ‘fuel’ –  the saline water with organic solutes – ran out. At this point more fuel was added, and the bacteria then recovered their + 150% voltage output.

In a real system, at a powerplant, the fuel is simply wastewater, and would of course not be in short supply. It is also worth noting that these MFCs can be very small (7um across in some cases) and can be made into huge arrays. The sum of these voltages is supposed to produce a pretty sizable amount of electricity, and it is promising that adding a single pro-chatter molecule upregulates the system so consistently! Indeed, the quinolone was only added once – so when the bacteria decided to start their conversation and resultant multicellular biofilm production behaviour, they continued doing so at a high rate without a further push for 1750 hours! Very cool.

Finally, the experimenters do a ‘power density’ measurement, showing how much better this is when the bacteria are dosed with quinolone and have formed a huge biofilm. Convincing, if you ask me. Thousands and thousands of such anodes covering many cubic metres would produce useful amounts of power.


So it’s a cool paper. I agree with the researchers when they say they have utilised conserved pro quorum sensing molecules to enhance biofilm production, which enhances power output of these MFCs quite massively. I also agree that it is useful that as a side effect of the electricity production, these bacteria clean up wastewater by using organic toxins for energy.

My main criticism of the paper isn’t a traditional scientific one. Wastewater comes from fracking, a controversial solution to oil and gas shortages that produces a lot of this waste, requires a hell of a lot of water and harmful chemicals as input. These then seep into the groundwater, which can only be treated (as wastewater) when it is extracted for humans to drink. Only 30 to 50% of this stuff is recovered in this way, so there is a lot of it left in the groundwater that contaminates everything else around it.

It is logical therefore to treat wastewater with bacteria that produce electricity, but given the high environmental cost of fracking, it is also irresponsible to treat only the symptoms and not the cause of this problem. By treating the symptoms in this way, we tend to further ignore the cause (over reliance on natural gas and a lack of investment for credible alternatives), shifting the onus, and supporting short term thinking.

This criticism would not have been asked by reviewers (it is not a criticism of the experimental design) but the paper would have been improved by pointing out the other sources of wastewater, or the possible applications of such bacteria in producing electricity while also desalinating water where fresh water is hard to come by. This would have given a more holistic and future-looking lilt to the work.

To conclude, this is a simple paper that provides a simple way to directly enhance the (long term) conversation rate of halophilic (salt loving) non-canonical quorum sensing bacteria. It shows us a particular case where we can enhance their ability to produce electricity using organic waste, desalinating water in the process and making it potable. Quorum sensing is a pretty new area of study, spearheaded by an idol of mine (from afar) Prof Bonnie Bassler at Princeton. Combine this with halophiles, which are hard to culture in a lab, and you have to do some pretty clever science to get any answers. I was drawn to this paper because of this thoughtful approach (and the fact that I study mammalian T cells, which is very different!)

References

  1. Min, B., Cheng, S. & Logan, B. E. Electricity generation using membrane and salt bridge microbial fuel cells. Water Res. 39, 1675–1686 (2005).
  2. Ivanova, N. et al. Complete genome sequence of the extremely halophilic Halanaerobium praevalens type strain (GSL). Stand. Genomic Sci. 4, 312–21 (2011).
  3. Monzon, O., Yang, Y., Li, Q. & Alvarez, P. J. J. Quorum sensing autoinducers enhance biofilm formation and power production in a hypersaline microbial fuel cell. Biochem. Eng. J. 109, 222–227 (2016).

Written by: Michael Shannon