Hi. My name is Frank Fields. I'm a research biologist here at the California Center for Algae Biotechnology at UC San Diego. Today I'm going to be talking about monitoring cultures of microalgae. So I'd like to first start with this graph which shows the typical growth of microalgae cultures. This shows the density of the culture over time. Cultures begin when we inoculate fresh medium at a low density and the cells may take some time to adjust to this new environment, so there is initial lag phase. This lag phase is followed by a period of rapid growth where cell division is at its highest and resources are being rapidly consumed. As these resources are consumed, something eventually becomes limiting to the culture stopping growth and divisions. This limitation sends the culture into what we call a stationary phase. And if the stationary phase persists long enough, cells will begin to die off or go into a resting stage waiting for the environment to get better. So understanding this basic growth curve is very important because we want to be asking ourselves, "What stage is my culture at as we're culturing algae?" So the first thing we can do to understand what stage it's at, is to quantify the biomass inside the culture. This is done by measuring the amount of dry weight that's inside of our culture. We can take a liquid sample, and pass it through a filter, and dry that sample, and weigh the mass of anything that was leftover. This slide here shows you a picture of what a filtration system looks like, as well as some filters on the bottom here. The green filters on top were harvested during growth phase, whereas the brown filters were harvested during the stationary phase. So we will see a change in color and density of the biomass depending on when we measure it during that growth phase. Now biomass quantification is the most direct measurement and usually the most important one. However, it does measure everything that's in that culture, including dead cells, inorganic matter, and contaminants. The next technique that we use is optical density. If you look at the picture of these three bags here, you'll see that the bags appear to have different densities to them. The way to measure optical density is to take a sample and shine a light through it in a machine called a spectrophotometer. The spectrophotometer measures the amount of light that was absorbed by the cells in your culture. As there are more cells in the culture, more light will be absorbed and you'll have a higher optical density. Optical density is very quick and easy way to measure your culture, and it tracks very well as you see on this graph here, with the dry weight from the previous slide. The next technique we use is to measure the cell number and size of the cells inside of our culture. These two pictures here show you the view of a specialized counting slide under a microscope. The cells on the left were measured during growth phase, whereas the cells on the right were measured during some sort of limitation. By knowing the amount of cells inside of each of those squares on that counting slide, we can calculate the number of cells per volume in our culture. Now measuring the size or the volume of the cells is also very important, because that will tell you if there's a change going on in the culture media and if the conditions are becoming unfavorable. This can also be correlated with a shift in composition inside of the cells. Now a lot of the times we're not growing the culture just for the biomass, we're growing it for a specific bioproduct, and we want to be monitoring that product as well during culture growth. This slide shows you a couple different techniques that we use to measure specific bioproducts. The top two images are showing you protein quantification. We use colorimetric assays to measure the amount of protein inside of the cells, and we can also use antibodies to measure one specific protein. The picture on the right in the slide shows you that on the fourth day, we had the highest amount of one specific protein and that would have been the best day to harvest that culture. The pictures on the bottom of the slide here, show you a fluorescent dye that binds to oil inside of the cells. This is very useful to track the oil now inside the cells as the culture is growing, and oil typically increases during limitation phases. So cultures can suddenly crash, and it's important to understand why, and keep an eye out for these limiting factors. There can be resource deficiencies, such as low nitrogen or phosphorus in the media, there can be unfavorable conditions, like a sudden change in temperature or lighting conditions, and there can be contamination or sickness. The picture on the slide shows you two ponds next to each other. The one on the left has crashed due to some limitation, whereas the one on the right remained healthy. So monitoring these potential limiting factors is also crucial to having a successful culture. Keeping an eye on the water chemistry and contamination level in any system is important. In open systems outdoors, it's imperative that you're monitoring weather on-site as well as weather forecasts so you can anticipate what to do next. So with all of these tools at your disposal, you should be able to assess the current state of your culture and decide if your culture is healthy enough to scale up to the next largest system, if you need to remedy the situation by changing the water chemistry, or adding something to decrease contamination, or if it would be best to harvest your culture as is and send the biomass for downstream processing, which we'll hear about in another lecture coming up. Thank you.
