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Remember building your clubhouse when you were a kid?
This is not too different. Getting started is pretty easy, follow our steps below...
You are going to have them. Think about these factors while choosing your growspace:
1. Cleanable surface
2. Where will spilled water go? Will it damage anything?
3. Can the cat, dog, of llama get at the cultures? (yes llamas, we have had llama related issues- its a thing)
4. Will you see it regularly? You will want to walk by it a few times a day to "give it a swirl" (algae like the attention)
5. Consider trays. In our lab we often use a cafeteria tray to keep projects organized, it makes them easy to move around together, and of course- contains the spill.
The key to all of this is "some high quality H2O!" (nod to the movie Waterboy). Our recommendation for sources of water are below:
1. Bottled spring water (UV or ozone sterilized)
2. Bottled drinking water (reverse osmosis municipal water with some salts added, but again UV ozone sterilized)
3. Reverse osmosis water.
4. Carbon filtered tap water (if your town uses chloramines, they will likely NOT be removed by activated carbon)
5. Wild water - Seawater or river water. You will need to filter to 1uM, then disinfect (see our discussion on disinfection) UV and ozone are probably the best means. Otherwise, boil the water, microwave works too.
Getting shocked really sucks. Our fearless founder, Matthew, had an "experience "with a Van de Graaff generator while working at Monterey Peninsula college. It gave him a healthy respect for electricity.
These are suggestions, we are not electricians. If you think you are in a dangerous spot, consult a professional.
1. Get a GFI- Ground Fault Interrupt. This gadget shuts the power off when a short is detected. Its good if you are concerned at all that you may spill water.
2. Get a power strip. You will likely need to power a light, heater, air pump. This can add up to ~100W when they are all on at the same time. Pick your supples appropriately.
3. Organize your power cords so that in the case of a spill, the water does not run to an unprotected area.
*Image- totally without permission. My absolute favorite youtube physicist is Canadian Mehdi Sadaghdar and his show ElectroBOOM is pee in you pants funny! Come to me for algae education, go to him to physics. Here is his link.
Most algae are photosynthetic (for the weird-os look up mixotrophic or heterotrophic algae).
We are in the process of manufacturing out own line of LEDs! But until we get that done, here are our suggestions in order of what we recommend and do not recommend:
1. An LED bulb- they work well, place cultures 1-10cm from lamp
2. A CFL bulb- They work well also, but put out a bit more heat than the LED, place 2-15cm from lamp.
3. LED or Fluorescent shop lights. We use both of these in our shop. You really don't need fancy grow lights (unless you want to show off to your friends).
4. An incandescent light: Ask your grandparents what an incandescent lamp is. For an incandescent lamp that is 60-100W, place the flasks 5-20cm away. They create a lot of heat and can overheat the culture and possibly melt your project or worse yet, start a fire.
5. Sunlight INSIDE of double or triple panes of glass. We do NOT recommend sunlight for most algae culture, it is too much light.
6. Direct sunlight. We do not recommend this unless the total volume of the culture is more than 40L, and the culture has a Secchi Disk Depth at least 150mm. Its too much light.
Blog post: How much light do I need?
Educational page: Lighting for algae cultures..
If there is snow outside, or you live in Seattle (sorry Seattle friends), you may need to think about a heat source.
Submersible heaters. These are good and can be purchased from us or from a pet shop. I like the models that you can adjust the temperature, gives a bit of freedom to do some experimentation. But I have had some of those fail (yes electrical shock) after 4-5 years of operation.
Your light source gives off heat. It is often enough to keep things up. You can test with a thermometer.
On top of a heat source... that is waterproof. Once, Matt suggested to a teacher to set her 500mL culture in her classroom on top of her computer because it gives off heat... he paused and then advised not to do that. However, atop a little refrigerator, or other heat source that is ~25C is often good. Use your best judgement.
Our favorite- Indirect heat by adding a cover.
Using a 10-gallon fish tank as an incubator works very well. A single LED light can increase the the temperature for a few flasks by 5-10C, if covered! Be fire safe!!!
Great! You are ready to get started.
Let the cultures breath
Unscrew the caps on the algae containers. During their travels, they have been locked into a plastic container, that does not allow gasses to exchange. This means that the water has a reduced amount of oxygen. Yes, oxygen! Algae cells respire just like animal cells (thanks mitochondria)!
Open the bottles by 1/2 turn, and set them somewhere that they will not be spilled.
Make your media
Media is the physical and chemical environment for your culture. ARS sells kits to make media. Each kit is pre-measured and weigh out for a specific volume of water.
1. Open your bottle of water (500ml for example).
2. Waste a splash of the water (~10mL), as the salts and nutrients will displace the container and may overfill.
3. Add the entire contents of the salt packet
4. Add the entire contents of the nutrients.
5. Shake the bottle to dissolve the salts.
6. Dance in a circle while shaking the bottle, we recommend singing "put the lime in the coconut", but Taylor Swift Shake it off works too.
How much media should I add at the start? The One-Fifth Rule
(aka: the four-parts-to-one-part rule)
We have to answer this question ten times a week. So here goes...
One part algae culture for four parts new media.
That means, If you buy 50mL of culture, you should add 200mL of new media! (one part to four parts)
Why- The anthropomorphic answer is "algae get lonely if they are too dilute".
The more scientifically accurate answer is more complicated, but two main reasons are below. The cells treat the water- the cells work together to bio-accumulate toxins from the water. Some of them will die, as they die, they will continue to absorb 'bad stuff' in your water, in a process call bio-absorption. The bio-absorption process is used in industry to pick up heavy metals from water, its pretty cool. Finally, the cells actually change- in the new environment of your lab, they may need respond by expressing different genes. This takes some time and energy. By introducing the change more slowly we try not to stress the cells to greatly.
Mixing the cultures:
Why mix at all?
First, you need to mix to re-suspend the culture. Cultures often sink when they are stressed (for example shipping across the country). Most cells are slightly negatively buoyant, meaning they sink. You will need to mix to get them back into the water column. Other cells, like spirulina, float when they are happy. Those floating mats of algae need to be mixed back into the water to keep the culture growing well.
The second reason to re-suspend is to break the boundary layer. Imagine you are a cell, who obtains nutrients near it cell membrane. After some time, the nutrients would be all gathered. The area around the cell where the nutrients are limited is called the boundary layer. With a large boundary layer the cell would need to either expend energy to get those materials, or move to another area where the materials were more plentiful (a reason for motility). By mixing the culture we break the boundary layer and give access the cell access to the nutrients.
Lastly, gas exchange! Algae make a lot of oxygen and need a lot of carbon dioxide. You need to get rid of the oxygen as it becomes toxic to the pigments above 300% saturation. I have seen ponds with over 500% oxygen, it smells absolutely happy! Carbon dioxide in the atmosphere is not very concentrated, so the ARS media has uses sodium carbonate and bicarbonate, so mixing to get carbon into our systems is not a priority. But if you are running a non-ARA media, consider adding CO2 gas.
How to mix:
Impellers- Fancy mixers used in photobioreacotrs
Air bubblers- inexpensive and good for cultures 2L and greater in volume. Smaller volumes and you end up splashing the media around increasing the rate of evaporation. Use an air stone to generate fine bubbles if you have filamentous algae.
Daily swirl- My favorite type of mixing. It gets you to interact with the culture every day.
What should I measure every day?
Secchi Stick Depth: This gives you a measure of the number of cells per volume in the culture. For many of our cultures, we have a chart of SDD to Cell count (see right).
Dry weight: This is filtering the algae onto a pad and physically weighing the dried mass of the algae per volume. Expressed as g/L.
Cell Count: this is a microscopic investigation where usually using a specialized slide called a hemocytometer. It is expressed as cells per volume or cells/mL.
Optical Density: If you have access to a spectrophotometer, you can quantify the biomass using a spetrophotometer. Keep in mind that the algae will absorb light across wavelengths differently. The two general wavelengths that are commonly used are 750nm (outside of the pigment absorbance range) and 540nM which is smack in the middle of the GREEN range that is most unused by photosynthesis.
pH: This is a measure of acidity of the culture. In practical terms pH is a measure of
the amount of CO2 in the system. Lower pH means more CO2 and higher pH means that photosynthesis has fixed a lot of it into biomass. In a production culture you will need to add CO2 daily.
Nitrate and or Phosphate: The famous N:P:K nutrients are absolutely important for algae culture. The 'K' or potassium is usually replete or in excess in most aquatic systems, so we generally do not measure it.
Our medias use sodium nitrate as the nitrogen source because it is easy to quantify using a test strip. Phosphate is also easy to quantify with a test strip or wet test.
In a production culture, you want to keep an eye on what is growing in your culture that aught not to be. Sacrificing a few drops of your culture is all you need to get an eye on what your cells look like... and who is an uninvited guest. I usually scan at the lowest power on my compound microscope with 40x magnification.
Bacteria are common in most cultures. Keep an eye out for grazers like rotifers. Amoeba are often difficult to get rid of. In most cases, you will want to waste the culture rather than try and get rid of the invaders.
How do i get my culture to gain volume and become more dense?
Increasing the volume of the culture.
Usually about 5-days after you start the culture, it will have passed through the lag phase of batch culture growth and entered logarithmic growth. You will visibly notice the culture getting more and more dense and the Secchi disk depth will decrease. When the density of the culture is less than ~30mm, you can consider "Scaling up" the culture, using the 10%-Rule.
To increase the total volume of the culture, add 10% more media to the culture each day. For example, if on day=5 you have 1000mL, you would add 100mL of new media. The next day=6, you would add 110mL of media. You should use your best judgement if you want to add more than 10% or less. Add more than 10% if your biomass becomes very dense with a SDD, less than 15mm. Conversely, if your SDD is increasing you are diluting the culture too much and you should consider less than 10% per day.
In every culture vessel, you are going to get some kind of biofouling. For our labs it takes about a month for the erlenmeyer flasks to become fouled. In production ponds, you should sweep the surfaces every single day.
To clean glassware:
Use dish soap and a brush. Be sure to get all of the biomass out of the bottles. If you have access to any acid, you should do an acid rinse inside and outside of the flasks. Rinse very well with water.