[MUSIC] Hi, I'm Susan Golden. I'm a professor of molecular biology in the division of biological sciences at University of California at San Diego, and today I'm going to be telling you about cyanobacteria, one of the groups of organisms that are being researched and developed as potential sources of biofuels. Cyanobacteria organisms that in the past have been called blue-green algae, and very frequently, are still called blue-green algae, both with respect to algal biofuels and also in nutrition, as a nutritional supplement, as you see in this slide here. Cyanobacteria are one of the groups of organisms that we consider microalgae. When people are talking about biofuels, algal biofuels, they're usually talking about microalgae as opposed to macroalgae such as seaweeds and kelps. The microalgae include two very different groups of organisms. The unicellular algae of the plant kingdom, which are true algae. And the cyanobacteria, which in fact are not algae at all, but are bacteria. If you look at the relationship of organisms on the planet, what we know is that right now we can say that there are three major groups of organisms. The eucaryotes, which includes the plants. These are cells that have more complex structures and internal membrane bound organelles and, a group called the archae, which we won't discuss today. And then the domain, which are the bacteria and the cyanobacteria belong over here. So as you can see, the blue-green algae and the true algae are really very distantly related. However the reason that they've been lumped together is that they do something the same, which is very important, and that is that they carry out photosynthesis. And in fact, they carry out a very special kind of photosynthesis in which water is split to provide electrons to take carbon dioxide from the air and reduce it to a form that the cells can use. And the by-product of this splitting of water is the production of oxygen. Now, the reason that it was so confusing for so many years as to where the cyanobacteria belong in terms of their, relationship to other organisms and to the algae, is that there is a special relationship, and that is that plants and algae got their chloroplasts from cyanobacteria many millions of years ago. So, in fact, the photosynthetic process that we see in plants and and particularly as we're talking about today in algae came from their historic, ancient relationship to cyanobacteria. So cyanobacteria, as I mentioned, are prokaryotes. This means that they are bacterial in their cell structure. Like like other algae, like the eukaryotic algae, they carry out an oxygen-producing form of photosynthesis. There are organisms on the planet that carry out other kinds of photosynthesis, but we won't be, they're not considered algae, and we won't be discussing them today. Cyanobacteria are really very ancient. They've been on the Earth for at least 2.8 billion years, and possibly as long as 3.5, which is the longest we can push back, knowing that there were, that there was life on the planet. And, in fact, you can see here a picture of the original oldest-known fossils, which have been dated between 2.8 and 3.5 billion years ago. So cyanobacteria, very ancient. Producing oxygen, and in fact, they're the architects of our atmosphere. So the reason that we have an oxygen containing atmosphere now, is because of ancient cyanobacteria. And in fact, even now they're making somewhere around 30% of the oxygen that we're breathing. There are many that can also fix nitrogen from the air, which means that they can take nitrogen gas from our atmosphere, which is largely nitrogen, and convert that into a form that organisms can use. In other words, they can make their own fertilizer. They're an important primary producer, which means that they can take carbon dioxide out of the air and, and convert it to a form that can be used by other organisms, as well as by the cyanobacteria, and they also have a wide variety of metabolic capacities and they can live in many ecological environments. Now, cyanobacteria are very diverse, so we're really talking about a big group of organisms. And you often think about bacteria as being single-celled. And in fact, some Cyanobacteria are. However there're also many multicellular forms. Here you can see a filament of an Oscillatoria where each one of these little striations, each segment is in fact an individual cell. You can see Anabaena that looks like beads on a string. And these toughs of Tracadismey, Track, Trichodesmium. And also here is a spiral line a filament that would have individual cells there. So not only is there're a lot diversity in what the organisms look like, and what their cell structure is like. But also they have a lot of diversity in where they can live on the planet. So here you can see some growing on a rock in Antarctica. A hot springs map from Yellowstone, they are organisms that help to form the crust in deserts that keeps the keeps the soil from flying away and, also in the open ocean they're very abundant. Some live in symbiosis with plants, and some form mats that form very large three-dimensional structures called stromatilites, and in fact those fossils I showed you earlier were from ancient fossilized stromatolites. So they're really all over the planet everywhere from the poles to the equator and in between terrestrial and aquatic environments. Cyanobacteria have in the past been grown in mass culture, but usually not, not in the way we are thinking of using them now. So for example as a nutritional supplement Earthrise Farms for many years has been growing spirulina. To market as, in health food stores. And in China and in India, Mass culture of cyanobacteria has been used either as food, or a fertilizer in rice fields. And so here you can see blue green algae that have been grown up and packaged for use as a fertilizer in rice fields. Now in the modern idea of using cyanobacteria as a source of bio fuels we have to think of all kinds of ways to grow them as efficiently as possible, and with the lowest cost possible, and different companies and different research groups are approaching this in different ways. And some are using the open pond strategy. Others are looking into various kinds of bioreactors, including bioreactors that would be outside and would be able to use the sunlight to provide the light energy is needed. As I mentioned previously, some cyanobacteria can fix nitrogen from the air. Some also can produce hydrogen gas, so there has been research trying to look at cyanobacteria as a source of hydrogen, to use that as a fuel. Some can regulate their flotation, which could be useful for harvesting. Some grow as mats. And that could be a different way of being able to harvest cyanobacteria easily. Some make secondary products. And one thing this means is that there is already the chemistry, the underlying chemistry to be able to make various kinds of molecules. And if we can add additional enzymes that would change those molecules, we can exploit this possibility to make novel chemicals in that way. And, importantly for the ability to genetically manipulate them, some are naturally transformable. So it's very easy for some species to take up and incorporate DNA that we add from the outside. So how do we start developing cyanobacteria as sources of biofuels. One idea, is that you can extract naturally occurring molecules that the organisms are already making. They're taking CO2 from the air, they're converting that to sugars, these can be used to ferment, so that, other, other organisms can take that sugar and use it to make biofuels. And also, there are a lot of membranes, and a lot of lipids that are present in cyanobacteria and so these can be converted to biodiesel. And, also cyanoacteria makes some natural hydrocarbons. So the ability to enhance this natural production is another approach. And then one big idea is to is to metabolically engineer the strain, so that you can improve them. To make designer organisms that are making designer molecules. So instead of having, just extracting what's there and having to purify what you want, from among many other compounds. Get the organism to make and secrete some pure product such as perhaps jet fuel. And in fact, there has already been engineering of cyanobacteria to make a number of potentially useful products. So alkanes which are hydrocarbons that are useful as fuels here you can see isoprene. Here's an example of sugars being made and exported. And as I mentioned previously hydrogen gas. So many species have been engineered. But so far we're talking about rather low levels of products that are made, and using organisms that can't make, that can't be grown at scale yet to be able to make these products. So what do we need to be able to take cyanobacteria and really develop them as a source of fuels? We have to have really good microbial ecology, because you have to find good strains and better strains than we have now. You need to develop the genetics and molecular biology tools so that the strains can be improved. And also, to think of this as farming, to be able to improve the aquaculture so that the strains can be grown at a very large scale. And then there's also engineering that's involved to harvest, extract, and get the products out and refine them. So in terms of moving forward from where we are now, there are really two ways to go, and, and both of these need to be exploited. So we can think of a two-pronged approach in which model organisms that we can already work with well in the laboratory, can be used as a testing ground to learn how to modify pathways. But at the same time it's important to find candidate production strains, the ones that are going to grow well out in this mass large scale aqua culture and develop genetic tools to be able to work with those strains. And the idea is that if you can understand what pathways you want to develop and you can find the right strains and develop the genetic tools. Then we can engineer these production strains, modify their traits, bring in pathways from, from other organism and engineer the production strains to make the chemicals that we want them to make. And when I talk about model strains there are strains that have been used in various laboratories for many years, and so we have a lot of tools, genetic tools for working with them. And these include both unicellular and filamentous model strains. And the kinds of things that we refer to when we say genetic tools are the ability to transform them, which means, add DNA and get it taken up by the cells. Conjugation, which is another way of moving in DNA by mating with cells that are easier to transform. To have regulated promoters, so that we can turn genes on and off. And various libraries for which are collections of DNA used to knock specific genes out or to over express particular genes, and then also reporters. Reporters are genes whose activities easy to measure and these include for example florescent reporters and you can see an example of that here where a gene that codes for a green florescent protein has been engineered into antibena. And in this case it's been engineered to be expressed only in cells that are able to fix nitrogen, and in the other case here only in those cells that are not the ones that fix nitrogen. So, this is an example of a regulated promoter in which even the particular cell type can be can be controlled. And another organism that's been very important for for genetic engineering is Synechococcus elongatus, a unicellular cyanobacterium. It has approximately 2,700 genes and it's naturally transformable. This means that it's very easy to add DNA and have it taken up by the cells. And, it undergoes homologous recombination, which means that we can add in genes, and really get them plugged right into the chromosome of the cells, very easily. An example of engineering in this strain is that there are particular enzymes that you can add that will take the lipid molecules that are used. The cell uses to make membranes and it will nip off the fatty acid portion of that. And the fatty acid, then, is a precursor that can be used for, as a fuel. And what you can see here, here you see the cells that have not been engineered with such an enzyme and the one that looks hazy, it looks hazy because there's a layer of fatty acids that are laying across, floating across the top of the surface. So back to our idea of this two-pronged approach, I've given you some examples of using the model organisms to to go in and modify pathways like adding that enzyme to make them secrete fatty acids. But then the other part of that is to identify candidate production strains, and then develop genetic tools for them. And that way, we can get to the point of having really good, robust strains that can grow outdoors. So, what do I mean by a production strain? One that has the properties that you would want for being able to grow it large scale and to get them to produce these products that you want, whatever the desirable product is, very well. And there are a number of properties that are associated with being a good production strain. So how do you go about finding a production strain. Well, you have to start out just by going out and sampling some water, out in a situation that is similar to where you're going to have your pond. Under the right salinity that you want, under the temperature that you want, because those are going to be organisms that have evolved to be good for that situation. And then you have to isolate the strains, identify what they are, figure out what they're making metabolically and chemically. What, what does their lipid profile look like, for example. And then, once you've isolated a strain, very often, it won't grow at large scale. So the next step is to choose your strength that you want. Grow them up and test them outdoors and see if you can really grow them at scale. And we've been able to do this with a strain that we found in the Imperial Valley. And it would in fact grow up at large scale, it's a filamentous strain, it's really quite easy to harvest, it grows very robustly in these one acre ponds, and very importantly, we've been able to show that we can move, we can genetically engineer it. And very often strains, especially if you just go out looking for some new strain, the odds of being able to easily genetically engineer that are not very high, so this was quite lucky. And in fact this strain, which is called alectolimia, can be genetically engineered quite easily. And what you see here are un-engineered cells. The red here is just florescence from the photosynthetic machinery. And the green that you see down here is where that, a gene encoding that green florescent protein has been moved in. And so at this point, we are making real progress on being able to find production strains, potential production strains and then show that we can genetically engineer them and work with them the way we do our genetic models. And this puts us well on the way to being able to genetically engineer production strains.