Team:Aix-Marseille/Interviews
On March 26th 2020 we were fortunate, to have a skype interview with Sylvain BALLU, he is in charge of the green tides monitoring program at CEVA company. CEVA stands for “Centre d’Étude et de Valorisation des Algues” that could translate into “algae study and valorisation research center”. CEVA was founded in 1982 as an association. Today it is a semi-public company which is interested in the reasons for the phenomenon and at the same time is looking for treatments and ways of valorizing these algae (in cosmetics for example).
They see valorization as plan B for companies, but their primary objective is to make these algae disappear. Indeed, their presence has a strong economic impact, collecting them is very expensive but it costs even more to compost them.
Mr Ballu's core business is monitoring the phenomenon, they organize air patrols, go to the field, analyze the surface area of deposits every month, ...
They have been monitoring this phenomenon since 2002 and therefore have gathered tons of data and gained a very accurate vision of the phenomenon.
They are able to correlate the environmental conditions with the variation in quantity and quality of the green algae. He also explained that if the winter was mild, a good quantity of nutrients would be in the water in April, which is favorable to the proliferation.
Mr. Ballu explained that the flow of nitrate affects the blooms of algae.
This flow is caused by agricultural practices but also by the climate.
In Brittany, the natural parameters are unfavorable to water quality, the climate is mild and the soils are relatively warm, so the mineralization of organic matter in the soil continues even in winter. When we have organisms in the soil that live and proliferate continuously we fragment the organic matter by making nitrate. Nitrate is a natural element that is normally very scarce, that's why historically we have fallow periods to let the soil rest. On top of that, it rains a lot in Brittany, so there is sometimes 3 times more water in the soil than it can evapotranspire. It is estimated that 130mm of water is saturated and we have about 3 times this amount during the winter, it washes out the soil profile and all the nitrate already present after the previous crop continues to mineralize. The nitrate will be carried beyond the refining zone by the excess rainfall. It is therefore harder in Brittany than elsewhere not to have a nitrate leakage.
And agricultural practices don't help. Indeed, there is a lot of agriculture, 60 to 80% of the land is occupied by agriculture in Brittany. Moreover, it’s an intensive agriculture, pork but also mostly cattle in some places. The fact that the systems are productive even in small family farms does not help. It is therefore necessary to find farming systems and practices that are difficult enough to implement without condemning the farms economically. Nitrate levels in watercourses have already been greatly reduced, but this is not enough.
Several actions have been put in place, they have taught farmers to fertilize their land a little less (with fertilization grids, putting the right dose at the right time). There was also the implementation of CIPAN (intermediate nitrate trap culture): after the harvest, they will plant a specific crop that is only there to capture the nitrate present in the soil at the end of the harvest + the mineralization of the fall (as the soil continues to mineralize, if a crop is brought in, the nitrate instead of being mobile in the soil will be immobilized in the form of a plant that will not be leachable, this organic matter is then returned to the soil and will be used for the next crop. However, he explained to us that we should change the system and not the practice, we want to move towards good practices but some are difficult to implement.
He told us that historically the green algae had predators, during the proliferation, "grazers" were put in place (top down control), it is a control by predation (periwinkles, sea hares...). But now the green algae only grow on rocks, they grow in bays that are sandy and agitated by the swell and these grazers can not live in these environments.
He then listed the possible or envisaged solutions.
There is first of all that of collecting to avoid that the quantity increases from one year to another. It works, they did it in China by asking students and the army to collect (10 000 people) but here we do not have these means and it is not something that the iGEM team can act on.
One of the other axes is to optimize denitrification in wetlands, it can be by making sure that wetlands are not drained. There is work at the SAGE level (water development and management scheme) to make sure that farmers put the wetlands back into wetlands even though sometimes they have been drained. He also cited a solution put in place by Professor Genin of the University of Nancy. He uses mixed CR2,CR3 compounds and uses the oxido-reduction properties of iron to boost denitrification in wetlands. The problem is that treating large volumes of water (2 cubic meters per second) is complicated in reverse osmosis but it also has a problem of river physiology. We can de-nitrify 2 square meters per day but it is not enough. Moreover we cannot remove all the oxygen from the watercourses for the ecosystem (fish for example). Then, one of the solutions that he finds interesting for us, would be to improve denitrification upstream of agriculture, by denitrifying the agricultural effluents before putting them in the plots. But that's a fairly high cost and it would only solve part of the problem. Then he talked to us about the valorization of algae. He started with methanization and co-methanization. Currently they know how to do it but the yield is not very good. He listed several negative points and this technique. Then we discussed the bioethanol track, which also has its drawbacks. In both cases, one of the major problems is that there is not the same amount of algae all year round on the beach, almost systematically forcing us to have an ulva culture. And in the case of bioethanol it has to be in a very low nitrogen environment because with little nitrogen we will have more starch with less protein.
This interview was very interesting, it allowed us to have a global vision, in order to make more targeted researches.
Sylvain Ballu then made an appointment with us by phone when we had more specific questions, but referred us to one of his colleagues, Renan Pierre, who is better able to answer.
Indeed, after this interview it seemed to us more appropriate to start on the valorization of green algae. Because green algae are present because of several factors, even if we managed to reduce one of them it does not treat the problem. Moreover there are several factors on which we cannot act, it is administrative and political measures which must be taken.
Renan Pierre gave us a telephone interview following our call with Sylvain Ballu.
He explained to us that the technique of methanation and co-methanation involves several difficulties. One of the reasons is the carbon-nitrogen ratio which is not very adapted for an optimal methanation, but also the mineral load of the ulves which is 30/40%, in terms of the methanation process, the reactor is clogged with a lot of inert matter. He explained to us that with algae there is a problem of inhibition by other molecules (such as polyphenol) and difficulties with resistant polysaccharides that limit the conversion in relation to the organic matter we have. And since there are no microorganisms capable of degrading the dead polysaccharides there are losses. The problem also arises for ethanol.
The problem for our team if we choose this project is the methanizer which is a rather complex system to stabilize, which needs a continuous homogeneous input flow and which is expensive, we don't have it so it would be quite complicated to do the laboratory part.
In the case where we do not have the right carbon-nitrogen ratio we could go on a share of algae in a terrestrial plant flow (e.g. 10% algae). Trying to optimize conditions to be able to integrate 10 or 15% of algae in the methanizer without disturbing it too much. Use existing systems.
The biggest problems for us are the management of hydrogen sulfide which is very toxic, the problem of corrosion of the equipment and the equipment itself.
For bioethanol there is always a mineral problem. Also there is not so much source of glucose in the algae so we end up with a rather diluted hydrolysate. It is therefore complicated to reach high concentrations of ethanol. One of the limits with ulva or other algae is that you can ferment glucose, you can ferment mannitol and eventually galactose on brown algae but a lot of sugars in the algae are not fermented to ethanol by classical organisms. This is perhaps where there are interesting things to do at the biological level, for example on the ulva where the main sugar is rhamnose, could we manage to ferment ethanol with rhamnose? On brown algae which are also used in bioethanol projects, there is some work to succeed in converting alginates to ferment them, but it is not yet very advanced. Where there would be a challenge: to significantly improve bioethanol production would be to succeed in fermenting marine monosaccharides.
Profitability is not very good. Some companies are working on ethanol from micro-algae that would be able to produce ethanol themselves.
He explains to us that the best solution for the supply would be to make a culture in a basin and to use the collected algae in addition, since the cost of collection is high, we might as well use them. But the advantage of pond culture as a supplement is that we can overproduce starch by depriving the algae of nitrogen.
To have more informations about biométhanization we had contacted Amr Chamaa from LIGER to have more information to do biomethanization.
In his company they tried to producing biomethan from algae but the performance achieved was not profitable, so now they use others raw material. they obtained with green algae 35 m^3/tons of wet material, this low efficiency has to be improved.
LIGER uses the trash that was made by Olmix after the production for animal feeds or for compost. This compost is used by farmers in fields to bring nutrients to cultures, but its composition is different from that of fertilizers. To do methanation they did not identify a specific strain, they had used a culture media with several families of bacteria that come together to make methanation. He talked of the production of biomethan from farms products that also use families of bacteria. From his point of view a strain responsible for methanation was not identified yet, and this is why a culture media with families of bacteria were used.
His company did not do the identification of the strain involved in methanization because the PCR and data analysis that were needed for all the bacteria had a huge cost. He thinks that an efficient method to produce biomethan from algae will be extremely useful nowadays.
On april 7th, we contacted Philippe Potin because he is the coordinator of the project GENIALG and IDEALG, which is a PhD in marine biology at the CNRS. He is specialized in algal degradation enzymes (polysaccharides) and in the biological properties of these algal polysaccharides especially for their revalorization.
There has already been synthetic biology work with algae grown in Chile, a company of the University of Berkeley, a start-up supported by many companies including oil groups (in the 2010’s) to produce bioethanol. They worked on E.coli and S.cerevisiae by playing on alginate algae. They made a publication in Science to show the feasibility of the project. They added a gene (in operon) to degrade the alginate that came from Vibrio. The problem is that the frame was not operating on the total biomass of the algae, it was only operating on alginate which represents 30 to 40% of the biomass. There are also other compounds that can intervene with the degradation process and be toxic such as polyphenols.
There is also in Marseille, the AFMB laboratory working on the metabolization of sugars: the cazymes.
The interest would be to find enzymes that degrade biomass. Indeed, bacteria capable of degrading the wall (polysaccharides) of ulva are sought. They have published in Nature an article on the synthesis of pathways allowing to have products capable of degrading green algae.
There is also a company called Olmix working on the degradation potential of algae to improve the yield of algal products. They use 2 to 3 thousand tons of fresh algae. They have the potential to work more but there are problems with access to biomass. Notably technical and environmental: the harvest must be done without disturbing the ecosystems (fish nurseries, marine nature parks), so this poses problems at the regulatory level.
According to him, synthetic biology cannot solve the problem of green tides but at least limit their impacts by making economic use of them to limit their weight on communities. And looking for a solution to the problem of nitrate contamination in the soil seems difficult according to him.
We must also take into account the issues of recovery and safety. Because of the H2S released by algae on the beaches, which can be dangerous, one driver died.
In order to limit these risks it would be necessary to work on the sulfur bacteria, for example by mutating its sulfate-reducing activity. Indeed, because in a few hours they are already active and the objective to recover them is that this is done in less than 24 hours.
We must take into account this important problem which conditions the approaches that we can have on these algae.
There can be volume problems because large quantities are needed and present all year round to be able to create biomaterials. And in France we don't have such a huge volume in the end (150 000 tons of algae in Brittany because 50,000 tons are counted, but to have a good estimate we must multiply this figure by 3).
There are also logistical problems because yes there is a lot of work on optimizing the biomass but it is difficult to transport the algae because 80 to 90% of their composition is water…
There are a lot of questions to ask and working on ulves is a good option.
The application of synthetic biology to marine biomass is an important issue. Because the cost price is not expensive so the question is relevant. Moreover, it would provide new molecules or substitute products.
Do you think that bioethanol is a good option for revalorization?
Bioethanol is not realistic in terms of biomass, marine biomass represents nothing vs. terrestrial biomass. There are 32 million tons of fresh algae worldwide. It's tempting because we all have what it takes to do it, but we must aim for other approaches, for example products with high added value, because it's not on the volume that we will be able to act. We need to see if there are synthetic biology approaches that can combine the two and especially to see if we can improve bio-refining techniques.
There are recent works that can be interesting, in particular the question of whether to stay on a known chassis? Why not develop other chassis that can be efficient for other applications? How can we associate these organizations with engineering?
Today there are people who are trying to tackle the biomass of algae with extracts from molluscs, abalones. They are interested in the co-products of abalone culture to degrade the algae biomass. It is from aerobic degradation of the biomass in aerobic for safety issues, that a better production yield can be achieved. Because algae as such are not usable.
The most important thing is that our chassis can secrete enzymes that degrade algae. We won't be able to degrade all the biomass but we will have degraded compounds (simple sugars) in greater quantities. As long as there is no molecule that interferes with them.
On March 26th, we had contacted Ann Sophie Burlot, RetD project manager for Olmix, to better understand how and why algae are revalued.
She told us that the green algae harvest takes place between May and September. Her company conducts research on algae production in April. Algae growth is linked to the presence of nitrates and phosphorus in the water. It is difficult to say if the amount of algae has increased, but there are still a lot of algae that washes up on the coast and we cannot succeed in harvesting them all. They are not very far from the coast, they are 1 or 2 hours away. They do not press the seaweed at the harvest site. They do not prevent the sulfate-reducing activity of the bacteria, but some are being done today to improve the stability of the algae.
The authorities want to stop the growth of the algae. Moreover, an anti-algae plan is already in place since 2017 and applied by the farmers which consists on the one hand in reducing the fertilizers used and on the other hand in harvesting the algae on the beaches for their revalorization (on this last point they are in collaboration with the authorities). They plan to produce seaweed in Asia and import it to France, which represents a challenge for his company. She stated that the algae are not rich in nitrates, they are consumed for the production of proteins. Algae are not used to produce biofuel because the techniques we know today are not profitable. She knows a company that uses algae to produce biomethane for vehicles in Brittany, it is called LIGR and they work with her. The waste after the production of bioethanol must be composed of proteins, minerals, pigments. They could make animal feed from proteins, there is an alga called Spirulina which is consumed in CHAD by humans thanks to its 60% protein content. It is not possible to produce bioethanol and biomethane, we must take the example of LGR.
On April 3rd, Erwann Loret, a CNRS researcher, answered our mail. We had asked him in what way the algae found in the Mediterranean Sea can be similar to those found in Brittany, he told us that Ulva Lactuca is a green algae responsible for the green algae in Brittany. It can have several phenotypes but an English publication from 20 years ago showed that this algae was genetically similar and that there was a single species. The Breton ulva grows very well in the Mediterranean.
We also asked him about the limits to the production of methane and bioethanol. He told us that, as he says in his review, the problem of extracting bioethanol from Ulves is salt water. For him it will not be as competitive as bioethanol from sugar cane.
For methanation, he told us that methane is a gas that produces a greenhouse effect much greater than CO2. It is also an explosive, which is a problem for wheat grain silos for example.
Thanks to all our bibliographic research we have discovered that some bacteria like Formosa agariphilia have different genes close to each other which are responsible for Ulve's polysaccharides (Ulvan) degradation. These sets of genes are named Polysaccharide Utilization Loci (PUL). To have a better understanding about them we decided to contact a specialist : the bioinformatician Nicolas TERRAPON. He works at the Laboratory of Architecture and Function of Biological Macromolecules AFMB. They gave us an interview on April 16th.
We asked him about his experience, he answered that he arrived 6 or 7 years ago in his current lab. He was working on the phylum of bacteroidetes, which have the particularity of having PULs, a set of dedicated operons, grouped in the same place, which cut polysaccharides, which is not at all systematic in bacteria. At the beginning, they studied the PULs of Bacteroidetes in the microbiota, which consume a lot of sugar. The process begins with the secretion of one or 2 endoactive extracellular enzymes that cut the polysaccharides in the middle of the chain and release oligosaccharides. Then SusC and SusD, junk proteins will intervene. SusC is the transporter that internalizes oligosaccharides into the cell to cope with competition for nutrients. SusD, it serves as a platform. These molecules are very conserved in PULs. They can be accompanied by SusE/F which bind to the oligosaccharides but which are not preserved. Finally, endo and exo active cazymes (which attacks the ends of polysaccharides) will degrade the sugars until they obtain assimilable sugars. However, the enzyme content can be very different depending on the sugar caught.
And, then we asked him to tell us more about PULdb and CAZy, he replied that in their team they do literature monitoring. Either the enzyme resembles those of an existing family and they bring it into that family. Either it comes from a new family and they therefore create a new one (For example PL37 and PL40 from Formosa).
For example, next to the domains of PL28, there were domains which following blasts and determination of their HML profiles were identified as a new family of Ulvan lyase. Ulvan lyases are very fashionable nowadays.
Then about the PUL 14 he tells thanks to CAZy, we can see that PUL 14 generally contains CAZymes PL24/25/28/40/37. PL24 can be found in Bacteroidetes and Gammaprotéobacteria too, PL28 only in Bacteroidetes. Pl37 is little found outside of Bacteroidetes. In addition, it allows you to have references.
PULdb makes it possible to locate a PUL on the genome of an organism from its number. This is not 100% correct but it does give you an idea. To do this we start from SusCD. However, not all marine bacteria have SusCD. But when 3 cazymes are present in a group of 4 enzymes, we speak of Cluster of Cazymes. That’s why it’s best to tick it off when searching CAZy. So if we go to CAZy and type PL24 by checking the cazymes cluster, we see all the species with PL24 and therefore all the PULs and clusters containing it.
And they wanted to know what are the different enzymes contained in PUL and can they be used separately. He said in addition to SusC/D, we have different cazymes. GH (glycoside hydrolase) needs one molecule of water to cut 1 oligosaccharide while a PL does not. PLs can produce compounds like unsaturated esters that only GH can cut (GH105, 88). These GH are found in the same PUL. We can also often find sulfatases, for example with PL40 and 38, because the sugars of algae are often very sulfated. It is not enough to put the enzyme cocktail in a test tube, because not all proteins can work at the same time. They have a very organized sequential mechanism with very specific enzymes. But the great advantage of bacteroidetes is that all the enzymes sought are found in the same place and are not scattered throughout the genome. In addition, if 2 enzymes are very often next to each other they surely have very related roles.
Finally we asked if we could transform bacteria with an exogenous whole PUL. He doesn't think so because a PUL is too big. But there is a lot of redundancy there due to duplication events. It is therefore necessary to identify the enzymes essential for degradation in order to reduce the size of the fragment to be transferred.
PUL are mainly self-regulated (the regulator is nowhere else in the genome). The regulators are often HCT, GntR and AraC (typical HCH domain very widespread). They are regulated by their monosaccharides. If these monosaccharides are transformed into something else by molecular biology, there may be concerns in their regulation.
There are other issues:
- SusC and SusD don’t work in E.coli. In addition, monosaccharides can be eaten by E.coli.
- For Bacteroidetes, there is not too much of a model for production.
- Some species prefer certain sugars. So two species with the same PUL can have different efficiencies because not the same preferences.
Any species with the following Cazymes can be of interest: PL37/40/38/105 GH3/106/78 (exo) and a sulphate. These enzymes should be sufficient.
In formosa there is too much sulfatase for example. It is also necessary to test which enzymes of the different species are the most effective. The order of the enzymes other than SusC and D is not important.
Cazymes have also been found in Gammaproteobacteria such as Pseudoalteromonas species CV and LOR but which are less specific for ulvans. However, they contain more exogenous degradation proteins. It has enzymes, but it may not have all of the necessary enzymes present in the genome in that same place.
PL40 and possibly PL38 can also be found in alphaproteobacteria and PL37 in Firmicutes. But we are not sure they have the same role (by analogy).
You can take enzymes from different bacteria to create a composite PUL. It is also possible to mix the exogenous PL 40 and 27 from Pseudoalteromonas and the GH from Formosa, for example.
It is also important to look at the market price (BACdives) of these bacteria and the ease of cultivation.
Then, on May 28th, we contacted, Frederic Besson research officer at the BIAM institute for the CEA of Cadarache. The CEA is a research and technological development center for energy in Europe.
It works on the production of biofuels from lipids.
He told us that unlike macroalgae which are essentially used for their polysaccharides and other polymers, the microalgae are used for their capacity to accumulate reserve lipids (~50% of dry weight in certains conditions). These lipids can serve to produce biodiesel (diester), with the same way that we do diester with the palm or rape oil. It’s not really profitable because the culture of microalgae on a large scale is still expensive and we need to collect microalgae (>99% volume of culture is in the water), then extract the oil from cells and transform it into diester. All of these are expensive and energy guzzlers.
They are essentially doing fundamental research on lipid metabolism from a model microalgae (Chlamydomonas). They are lipids specialists and they try to understand the oil synthesis pathway for a much more long term increase in the oil content of cells without decreasing the speed of cell division (oil only accumulates in microalgae when they are put in nitrogen deficiency, which stops the growth of biomass). And also to try to make the microalgae capable of transforming its lipids in hydrocarbons.
So they don’t work at all on the deconstruction of macroalgae polysaccharides and unfortunately, he can’t help us on this aspect.
Thanks to all these interviews, we were able to make very precise bibliographical researches. The opinion of professionals is essential, they know their field perfectly and can advise us directly.