All right everybody. My name is Miller Tran. I'm the Associate Director of Research and Development for a small startup company here in San Diego called Triton Algae Innovations and today I'm going to talk to you guys all about the production of heterotrophic microalgae. And so, as I'm sure you all know by now, microalgae are a really diverse group of organisms. And typically when we think about algae, what we think about are organisms that perform photosynthesis. But, like I said these organisms are very versatile, so they're found everywhere, they're found in the soil, they're found in the water. And s, o what they've developed is an ability to grow in these different types of environments. And so, normally they would perform photosynthesis, but when they're in dark environments they have to find a way to survive. And so, a lot of these algae have developed a way to take organic carbon sources, and undergo a process called respiration to facilitate a process called heterotrophic growth. So before we get deep into the lecture, there's a couple of definitions that I want to make sure that everyone knows. So heterotrophic, what that basically means is an organism that can manufacture its own food instead of getting its food from photosynthetic energy, and it does this by utilizing organic substrates. A synonym is heterotroph; heterotrophs include all animals, protozoan, fungi, most bacteria, some aquatic and marine algae, and some cyanobacteria. Heterotrophs, just like organisms that are autotrophs, require nitrogen, phosphorus, trace elements, and oxygen for their growth and productivity. And then when we talk about heterotrophic growth, oftentimes we refer to it as aerobic fermentation. Now typically fermentation is a process without O2, but that is why we call it aerobic fermentation. So this is basically, growth of microalgae heterotrophically in a vessel using oxygen as a gas to perform respiration. So one of the key things that we need to understand in this lecture basically is, what is the difference between heterotrophic and phototrophic? And so, obviously for heterotrophic you need to have the ability to grow in the dark, for phototrophic you need to have the ability to grow in the light. Now when you combine the two, you're going to get a process that's called mixotrophic, where you're growing using an organic substrate and photosynthesis. But today we're just going to focus solely on heterotrophic growth. For heterotrophic growth you also require O2, whereas in phototrophic growth you require carbon dioxide. For heterotrophic growth, you require a reduced carbon source, and we'll go over a list of different reduced carbon sources. For phototrophic growth you don't require any reduced carbon source. One of the main advantages of doing heterortrophic growth is that you're able to achieve high densities, typically densities that go above 100 grams per liter, whereas for phototrophic algae typically your density is below 10 grams per liter. And for heterotrophic growth, because you're using a reduced organic carbon substrate, typically you're grown in complete containment, whereas for phototrophic growth you can be grown in containment or in open ponds, as we've seen in previous lectures. And so, what are the different organic carbon substrates that you can use? So you have your monosaccharides, which are individual sugar sub-units, so you have glucose, fructose, galactose. And then you have your disaccharides, which are like sucrose, lactose, and maltose, which basically are a combination of those monosaccharides. And then you also have your smaller organic carbon substrates, such as acetate, that the microalgae can use as a reduced carbon source to grow. And so, to understand how heterotrophic growth works, you have to understand the different ways that an organism can use these reduced carbon substrates. So on the left here, basically you see how an algae would typically use a substrate such as glucose. Because glucose is a larger molecule, to utilize these sugars basically what you have to have are sugar transporters, and so glucose will enter in through the cell, through a transporter it'll enter into the cytocell. Once inside the cytocell, you need a second transporter that allows the glucose to enter into the chloroplast. Now once in the chloroplast, that glucose will undergo a process called gluconeogenesis and it will form a substrate called GP3. That GP3 will ultimately be converted into acetyl-CoA and acetyl-CoA is one of the key substrates that allows respiration to occur. So acetyl-CoA will then go into the mitochondria, (which is shown there in the pink) and then once in the mitochondria, acetyl-CoA will be shuttled into this process called the TCA cycle, which generates ATP and reducing potential. And then from the TCA cycle, that energy is transferred to a process called oxidative phosphorylation, where oxygen is combined with the substrates coming from the TCA cycle to create a large amount of ATP. And that's what actually drives heterotrophic growth, allowing algae to achieve high densities. Now when you have a simpler compound such as acetate, the key differences between acetate and glucose, is that you don't require transporters. Acetate's so small that it's able to diffuse freely in and out of the cell. And so, once acetate enters the cell, it basically is converted from acetate to acetyl-CoA using an enzyme called acetyl-CoA synthetase. Now once it's changed to acetyl-CoA, there's two things that typically happen. That acetyl-CoA can go into the mitochondria where it undergoes that process, the TCA cycle and oxidative phosphorylation like we previously talked about, or it will be shuttled into this process called the glyoxylate pathway or the glyoxylate cycle. And during that cycle, that acetyl-CoA gets turned into a compound called succinate. That succinate then can also feed into the TCA cycle in the mitochondria, and then help to produce energy for the organism to undergo its metabolism and continue to grow. And so, there are a wide range of different algae that can be grown heterotrophically. And so, here's a list of about 20 different algae that are known to grow hypertrophically, and there are some references that you guys can go and look up and read a little bit more about. But typically you have your green algae like Chlorella and your Chlamydomonas reinhardtii, then you have your Dunaliellas and your Haematococcuses, and you have a bunch of different algae that will then be used to produce compounds like DHA and EPA. And so, these are just a list of the different algae that you can go and read a little bit more about, their processes differ depending on the organism and its makeup, but they're all very interesting to look at. At Triton we focus specifically on the green algae Chlamydomonas reinhardtii, which is probably one of the more robustly studied algae out there. I would say the two main algae that are typically grown in heterotrophic growth are Chlorella and [inaudible] which is the DHA producing algae. And so basically when you think about algae and the traits that you need to grow well heterotrophically there are a few that you really need to take into consideration. One, the obvious one is that algae has to have the ability to consume an organic substrate source. And in the absence of photosynthesis, you basically need to be able to consume that to have energy to grow. You need to have the ability to grow in light limited conditions. It's not to say that you can't grow heterotrophically with light, it's just that as the cells achieve this higher density, often times the light can't penetrate into the culture, so you have to have that ability to grow in light limited conditions. And you also, oftentimes when you're growing heterotrophically, you're growing in stainless steel tanks where you're devoid of light completely. And so without that ability to grow in light limited conditions typically those strains of algae aren't used in heterotrophically growth processes. And the last thing is your algae really needs to be able to be shear resistant and the reason for that is, is when you put your algae into stainless steel tanks one of the things that really drives the high density aspect of heterotrophic growth is the ability of the cells to take oxygen from the media that's being pumped into the media, sorry and take it into the cell and use it for respiration. Now, for that to happen basically you've propellers in the tank that will break up the gas molecules into smaller bubbles to increase the surface area of the gas allowing that gas to diffuse more freely into the cell. And when that happens the shear increases quite drastically in the tank with propellers going above a thousand RPM's per second. And so you can imagine the stress that that would cause. A lot of algae will just shear under that type of stress, and there are really a select few that can really tolerate that kind of force. And so, those are the pretty much the three key characteristics that we look for when we're trying to decide which strains of algae that we want to grow heterotrophically. So this is what the process looks like. So on the left here, basically, you have these 500 ml little tanks that we optimize our growth in. You can grow different types of algae in there. Here we have a chlamydomonas reinhardtii that is either yellow in the dark or green in the dark. Now, you can see that there are glass, so light in this case can get through, but because we're trying to optimize for a stainless steel condition typically what we'll do is we'll just cover them with a black shade to make sure that no light enters into the process that could really skew our results. From there, that culture can be transferred into a laboratory scale fermenter. Typically these are anywhere from one to even up to 200 liters. And basically in there what we'll do is we'll validate the processes that we've developed in the 500 ml culture. And so the processes that we typically will validate are pretty much your pH, the amount of oxygen that you're going to have entering into the tank, the temperature, the rotor speed. And so all of those little intricate details can be optimized in the 500 millimeter tanks. But before you go onto your pilot scale and your production systems you really have to validate them in the 20 liter tank. With a 20 liter validated tech transfer package, you can send that to any contract manufacturer with a fermentation facility and have them run that exact process and that process should really scale. And so in the next picture, the pilot scale systems typically from your 20 liter validation tank, you'll go into a larger either 200 to 3000 liter tank. This is an example here one of Triton's production partners and these are just a row of 3000 liter tanks that will have our algae growing in it, running the identical process that was developed in the 500 mill tank and validated in the 20 liter tank. And so one of the big advantages of growing algae heterotropically is that ability to scale and scale down your experiments, so that you can design the runs for the larger systems which is something that is really lacking in the photosynthetic algae growth community. The ability to scale down the process, and so again, because fermentation is such a well studied process not just for algae, but for yeast and other microorganisms, it's something that we've been able to learn from and to optimize our processes. And from there typically what you'll go into is a larger production tanks. This is where you actually get the economic benefit, the economies of scale. And so on the left and on the right here are basically examples of 100,000 liter tanks that can be used to grow our algae. And using the identical process again that was developed in the 500 milliliter tank without fine tuning too many of those little processes. And so for us that's hugely valuable because it doesn't require us to have a huge initial capital investment in these giant facilities because fermentation capacity around the States and around the world really is in excess right now. And so, it gives us the ability just to focus on the R&D and then pass the scale up problems, not the problems but past the scale up to our contract manufacturers. And then finally, from there you can go up to as large as 500,000 liters. This is a facility down in Brazil. It was a collaboration between TerraVia formerly Solazyme and Bunge and now they're actually growing strains of Chlorella in that facility up to 500,000 liters. And from there, I mean you can think about it from one liter all the way to 500,000 liters. The economy of scales are astronomical and you can probably produce algae in that scale below five dollars a kilogram which is essential for algae being widely accepted as a platform for different types of products. And so what types of products are we actually talking about here? And so I would say the first product that really put algae on the map was the Omega-3 fatty acids. Those are your DHA's and your EPA's. Now, the first company to do this was a company called Martek. They were subsequently bought by DSM for about a billion dollars. And basically what DSM has done is really corner the market for DHA production. And as you guys, I'm sure know when you go to the store where you go to Whole Foods, when you go to Vons everything out there has Omega-3 added to it. And typically that Omega-3 is not coming from fish. It's actually coming from a micro algae that's being grown by DSM and so you can see here on the left, infant formula. Almost all infant formula, high end infant formula at least now has DHA added into it. And that was a marketing campaign by DSM that worked out great. They've demonstrated that DHA is great for babies. And so now you typically find it in those high end baby formulas. Now, you also find it in your milk's, in your Minute Maid, in your butter and pretty much everywhere over the counter, you can find it. And so that really put algae on the map and demonstrated that growing algae heterotrophically could be a successful business. And then you have other things that are coming onto the market now. And so like I said, you have a lot of companies going into the Omega-3 fatty acid realm, but now you have companies like TerraVia who are producing a wide array of different products. And so you had them making skincare products, you had them making high end oils that were very, very similar to olive oil. In fact, slightly better characteristics than olive oil. When it came to cooking, you can make things like croissants with algae butter called Algawise that they were making. And more importantly they were making algae flour and high end algae protein products that could potentially one day replace our dependence on other non-renewable protein sources. And so really for us it's really exciting that the use of algae grown heterotropically and the different products that we can really get our algae into. And really I think we're just scratching the surface of what's possible. And I hope to come back maybe a year or two from now and talk to you guys about the products that Triton is developing. And so with that, I thank you guys for your attention and your time.
