Introduction: Simple Algae Home CO2 Scrubber - Part II Algae Cultures and Breeding

In Part I we built a simple home CO2 scrubber using algae. I'm writing this instructable to demonstrate how to culture and cultivate algae using more or less laboratory style procedures but substituting some readily available items for more traditional laboratory glassware.

I encourage you to experiment and understand how to successfully cultivate algae. The techniques and process outlined here will work for most algae strains.

Please bear with me, this is a work in progress. I normally publish open source software where the policy is "Publish early and often". The content is constantly being revised and I'm open to collaboration on many elements.

For instance the crude incubator could easily be supplemented with a sound design using plastic sheets with hinged access panels and portholes with oversized rubber gloves mounted in them. And so on.

In Part I we used a readily available algae source to create a healthy algae culture which gets fed with air, sunlight and liquid plant food to provide CO2 scrubbing on a small scale.

The original sample probably had a wide variety of micro-organisms, many of which are probably bubbling harmlessly away in the soup. But we're not here to create bio-soup, we're here to make algae.

It will be very nice for you if you have a microscope but you do not need one. It should be possible to obtain a single species algae culture and breed it up to production reactor populations without one. If you happen to have one, as I do not, then you can whole hog nuts and breed a pure culture from a single cell.

In any event if you want to get serious about algae you'll need to learn to culture and cultivate algae. These skills can be applied to special purpose algae species obtained from the appropriate channels. There is an appendix with more detail on obtaining single species specialized algae cultures from universities and research institutions. Expect to pay about $75 for a culture which, with sound breeding practices, is sufficient to create pure feedstock. Put some of that in a bioreactor full of pure water, nutrients and you're done.

For the rest of us we're going to isolate some algae cultures from our birdbath sample in a Petri dish (or substitute such as I use) with Agar (or a substitute).

From that we're going to identify some single culture strains (one batch of green stuff). We're going to multiply those strains in some small bottles to understand the process of scaling up a culture.

At the end we're going to create our production breeder reactor which will consist of one culture reactor which is used to maintain a constant population and two breeder reactors used to create feedstock for production reactors (in the case of CO2 scrubbers) or for feeder reactors which are moderate sized (3-5 gallon) reactors for producing feedstock for above ground pool based open pond or vertical growth closed reactor systems.

Okay with all said and done let's take a look at what we're going to need...

Step 1: Bill of Materials

1 case Ball brand Mason jars - If you're going to breed algae you're going to have a variety of strains to nurture at various stages. Just do yourself a favor and get a case of the quart size. There is also a very handy half gallon size which is suitable for larger cultures.

A lab journal for recording strains, experiments and results.

Several .5L clear plastic water bottles, labels removed and run through the dishwasher.

A couple of 6+ inch pieces of reasonably stiff wire, picture hanger wire isn't stiff enough and coat hanger wire is the worst case because it's hard to bend but will do.

Calcium Carbonate - Chalk or 500 mg Calcium dietary supplement made from calcium carbonate

Some soil to use for preparing algae media. Should be good rich soil from a variety of sources.

There is nothing special about the particular soil used However, several considerations are probably important, including the following:

1. The soil should be a loam, with a mixture of particle sizes (sand, silt, clay).
2. It should contain a moderate amount (15 - 20%) of very-well-decomposed organic matter.
3. It must not contain pesticides, especially herbicides.
4. It should be soil that has been aged (preferably for 6 months or more) under moist conditions and not, for example, fresh potting soil, soil that contains fresh manure, or soil to which a commercial fertilizer was recently applied.
5. A slightly acidic soil derived from granite or other igneous rock is preferable to soil obtained from calcareous soils.
6. Particulate matter in the soil such as gravel, Perlite, or vermiculite are not necessarily damaging but can be of considerable nuisance when wishing to quantitate the amount of soil used in the medium or when handling algae that are physically associated with the soil. Particulate organic matter, such as compost that is only partially degraded, should be avoided "altogether.

Aluminum cake pan for heating and foil to line it. 2 quart pot with cover for boiling.

6 Petri dishes (see picture) or a reasonable substitute. Most scientific supply stores will offer prepared dishes with Agar. If these are available use them.
Almost any shallow, wide mouthed container that can be easily covered with Saran Wrap(TM) and sealed with a rubber band will do
I will be using baby food jars with the labels removed.

Glassware will need to be cleaned thoroughly or sterlized between uses.

At minimum place them opening down in the dishwasher and when removing them after the cycle completes keep them upside down until the cap is screwed on. Keep the cap inverted until just before closing the bottle. This will minimize accidental contamination.

A couple of gallons of fresh water will do nicely. Its probably worthwhile to pick up one of the 3 gallon bottles of fresh water available at the supermarket. Especially if you're thinking about any sort of small volume production you will need several of these for use as feeder reactors.

Materials to build the culture incubator. This will be used to breed algae at various stages as we reduce the population samples, then incubate the targets and attempt to get identifiable algae strains. It is not strictly speaking necessary to use an incubator for the hobbyist. For anyone who is serious about cultivating algae and plans on obtaining cultures of more exotic algae from scientific and academic supply houses an incubator is probably appropriate.

20 gallon aquarium (does not need to be watetight)
Heavyweight clear plastic
The side from a zipper plastic blanket bag my wife had
Plastic garbage bag would be okay. White is best
Strong Tape
Small lamp that can use up to 75-watt bulb
Bulb size will vary based on desired temperature
On/Off electrical timer set for 18/6 on/off cycle
Thermometer (preferably in a clear plastic case)
If anyone knows of an inexpensive temperature controlled thermostat I'd be pleased to hear it.

Step 2: Prepare the Soilwater Medium

Soilwater is an essential component for cultivating algae. While the liquid plant food provides most of the essential nutrient algae also require other trace minerals which are extracted from the soil. In this step we will create a standard soil-water medium is used to supplement the plant nutrients.

First we will prepare a standardized green house soil based on a recipe from the University of Texas at Austin.

This is an adaptation of E.G. Pringsheim's biphasic soil-water medium. Variations of this medium are suitable for isolation and for growing algae to secure "normal" growth forms. Soilwater is not a well-defined medium, yet not all soil is suitable for culturing a broad range of algae.

UTEX uses some greenhouse soil they got from Indiana University. For a long shelf life it must be kept dry and away from light. UTEX keeps it in sealed 5-gallon plastic containers, keep a few quarts around in Mason jars for future use.

Prior to its use in soil-water media, treat soil in batches by placing it in a heat-resistant pan lined with aluminum foil, fill the soil to a so depth of 1/4 inch, and bake at 300 degrees (150C) for 2 hours. After it cools, cover the pan with aluminum foil and store in darkness at room temperature. Avoid excessive moisture during storage.

Now its time to prepare the medium. Add 5 tsps of green house soil for each liter of water and 5mg of calcium carbonate. Bring to a low boil and simmer for 15 minutes. Cover, allow to cool and refrigerate. Repeat daily for 3 days. Allow to refrigerate overnight and bring to room temperature before use.

Use a large pot, make a couple of gallons. Parcel it up into the Mason jars and save what you don't use.

This is the water medium we will use to culture our algae.

Step 3: Preparing Agar and Culture Plates

Prepared agar plates are inexpensive and readily available from scientific supply stores.

If, for whatever reason you don't care to use prepared plates you can make agar plates from tablet, powdered, or bottled agar by following a few simple instructions.

Agar can be obtained from health food stores and high end supermarkets with health food departments. The clerks will be able to help you if you ask if they carry 'the dietary supplement agar and calcium supplements with calcium carbonate because a friend recommended them" instead "Hey, you guys got any stuff for breeding algae?"

Keep the Petri dishes or other culturing containers clean (sterile) and closed until just before use. Lift the lid as little as possible when filling the containers and cover immediately to prevent contamination.

Preparing Tablet or Powdered Agar:

Dissolve 1 bar, 10 tablets or 7 grams of powdered agar in 1/2 quart of the soilwater we prepared in the last step. Warm the agar in a hot water bath by placing it in a glass bottle. Heat water on the stove until just about boiling, remove from heat and place the agar bottle in the water. The microwave is another good way. Simply heat up a bowl of water and place the bottle with agar in the center.

As it warms the agar will begin to melt, warm the agar until it becomes liquid. Remove it from the heat and let it cool a bit. Then add 7 drops of Schultz Liquid Plant food and mix well to distribute the nutrient evenly throughout the agar.

Pour enough of the liquid into each container to cover the bottom by about 1/8 inch and place on a counter to cool. The agar will set up like stiff gelatin at room temperature.

If the agar doesn't set up properly try again with more agar. Extra agar may be stored and re-liquified in a hot water bath. Keep the samples covered and be careful to avoid contamination.

Storage: Stack agar plates upside down in the refrigerator. Do Not Freeze! The purpose of placing the plates upside down is to prevent condensation from dripping down onto the agar surface which could then facilitate movement of organisms between colonies.

For those who want to more rigidly controlled independent work the formulation for LB (Luria Bertani general purpose nutrient agar) is: 9.1 g/L tryptone, 4.6 g/L yeast extract, 4.6 g/L NaCl, and 13.7 g/L agar. 500 ml of agar will pour ~ 25 large Petri dishes (100 mm diameter) or 50 small Petri dishes (60 mm diameter).

Next we're going to build an incubator for culturing algae. It's a warm, safe place for the algae to grow in controlled conditions as the population is bred up.

Step 4: Building the Culture Incubator

This is used to keep the cultures in a warm, well lit environment free from contamination. The plastic "door" drops down to provide a shield and care should be taken. Okay, the world will not end if your algae culture is tainted or if the aseptic conditions are compromised. But what the heck if you're going to go through all this wash your hands and keep things covered as much as possible.

20 gallon aquarium (does not need to be watetight)
Heavyweight clear plastic
Strong Tape
Small lamp that can use up to 75-watt bulb
Thermometer (preferably in a clear plastic case)

Turn the aquarium so the opening faces the front instead of the top
Cut the plastic slightly wider than the opening and about 2 inches longer than the height of the opening
Tape the plastic to the top of the aquarium so that the plastic falls over the opening. This is the door
Place the lamp in the aquarium, letting the cord hang out the front
Place the thermometer in the aquarium so that you can read it without opening the "door"
Experiment with different sized bulbs until you find one that keeps the temperature around 70 degrees ( for algae breeding, for stock culture maintenance the algae is maintained at a lower temperature)

Okay, that' it. In the next step we're going to place some culture samples on the agar plates we created in the last step and place them in the incubator. After about 2-3 an algae culture should begin to be visible on the agar.

Step 5: Preparing to Create the Culture

The basic methods of algal culture have changed little over the years and the various steps in the process leading to production-scale cultures are shown in the picture.

Now we need the wire in the bill of materials. Take a piece of wire about 6" or long enough to reach to the bottom of the culture plates you are using. For Petri dishes this is obviously quite short but if you're using mayonnaise jars you'll need something long.

Using a pair of needle nose pliers make a loop in the end of the wire. Bend this at a 90 degree angle and wrap some tape around the other end to form a handle. Using pliers hold the round tip in a flame (match or candle) until the tip glows red to sterilize it. Let it cool down in the incubator.

We're going to use this tool to spread tiny samples of algae on the agar plates and allowed to grow. After a few days we'll identify some uniform populations and we'll use this tool again to gather samples from those populations and put them in the soilwater media to breed an identifiable algae strain.

Now we're going to get our algae sample. If you're using the reactor we built in Part I then you'll want to extract a small portion of that for easy handling in the incubator. Remove the bubbler assembly from the reactor. Note: It's easiest if you hold the top and turn the bottle underneath it. You'll want to have a pitcher or tall glass handy to put it in.

Now we're going to fill the Mason jar at least half way. To reduce contamination risk open the lid of the Mason jar only enough to allow easy pouring. Put the lid back on the Mason jar and place it in the incubator.

Top of the culture reactor with soilwater media and replace the bubbler. This method insures that there is a steady supply of culture media available.

Place as many Petri dishes in the incubator as you want to culture, probably 4-6 is enough. Do not remove the lids from the culture plates until you use them.

Now we have to record this strain and its heritage. Where did it come from and when?

I number my strains using a location name and a date in YYYYMMDD format for easy comparison. A '-S' suffix identifies samples and cultures from that sample are identified with '-CXX' extensions.

So a sample from the Delta Mendota canal from Jan 1, 2008 would be DM-20080101-S and the cultures from that would be identified with -CXX extension indicating which culture. Record the culture ID and other information in the lab journal.

Create a label (masking tape and pen will do) and label each of the Petri with it's culture identifier. This isn't necessarily the order in which the plates are filled but it is the sample ID the culture(s) which result will have.

Now we're ready to create our first culture. Go wash your hands thoroughly up to the elbows. Let me clarify what this means. A surgeon will scrub each surface of the hand including the sides and tips of fingers at least 10 times. That would be overkill.

Step 6: Sampling the Algae Medium

Okay now we're going to move completely inside the incubator where we have our sample tool, the Mason jar of algae media and our Petri dishes. Turn off the light first (just in case you were warming things up).

Remove the lid from the Mason jar and using they eyedropper dip into the algae medium and get a sample. Put the lid back on the mason jar and open up Petri dish #1. Put one drop of algae medium onto the plate and use the sample wand to make a Z shape most of the way across the agar but not all the way to the edge.

Close the Petri dish and repeat this process for each sample you take. When you are done remove the sample wand and the Mason jar from the incubator. Hopefully you've placed the incubator in a location where light is readily available (indoors near a window?) and the light bulb supplements the heat. Use a lower wattage for steady supply since the heat will build up in the incubator over time. This is okay algae like it hot.

After about 5 or 6 days the cells will have grown enough to form colonies. These colonies on this first streak plate often tend to grow together, so it is difficult to pick off colonies with only one species. You may have to scrape some colonies off the first plate and re-streak them on a second plate in order to get good separation.

Okay now we're going to breed the individual colonies to produce cultures of the individual species.

Step 7: Breeding the Culture

In order to get visible growth as quickly as possible we will stage the individual colonies in a series of ever increasing reactors until we get a production batch. The first of these we'll call a multiplier reactor. Use a small flask ( 150 ml or so ), a baby food jar or a mason jar with a small amount of prepared nutrient medium (soilwater + plant food ).

Prepare as many jars as you have clearly separated individual colonies to sample. With multiple tries you will become adept at streaking the plates in ways which result in more colonies (and require less 'seed' stock).

Label the jars to match the sample designation and add a serial culture ID. Note in the log book anything particularly interesting, size, color what have you that might differentiate this culture from others.

Once you can clearly identify individual colonies scrape them off and place them in the multiplier reactors, cover the reactors and leave in the incubator. After a few days you should have visible algae growth.

If you have a microscope you should be able to track initial population growth. The chart shows algae growth rates through the exponential portion of the growth curve. After that algae shadowing becomes to come into effect.

With a microscope it is possible to start with a known population ( in theory one cell to provide a uni-strain culture) and breed up.

These reactors have a finite amount of nutrient, and when that is exhausted, the growth will stop and eventually they die. Experience will teach how long these types of cultures last and help to identify peak color conditions.

After that to continue the culture you must "sub-culture" by transferring this culture into a culture reactor containing fresh growth medium. I typically transfer from the breeder reactor to a .5 ml water bottle containing fresh growth medium. Open the lid a quarter to a half turn (you want it water tight but not air tight to vent). It is possible to use a bubbler apparatus on these but I find if I turn them upside down and shake them a couple of times a day they do fine.

After a week or two the breeder reactor will reach max density and should be transferred to a culture reactor such as one the we built in part I.

The pump we use in Part I will easily support multiple reactor vessels. These should be connected in parallel Single and multiple 'Tee' adapters are available at PetSmart and other fine aquarium supply stores as well as the specialty hardware section of your local ACE or Lowe's.

Simply split the output from the pump and distribute it to as many culture reactors as will bubble air through.

Monitor the color of the algae and experiment with the amount of nutrient solution required to maintain growth rates.

At this point you should have several well identified strains with which to experiment with variations in light, nutrient and so on and so forth.

Step 8: Maintenance of Stock and Starter Cultures

Stock cultures, otherwise known as master cultures, of the preferred species are the basic foundation of culture. They are normally supplied as monospecific (uni-algal) cultures from reputable culture collections maintained by national institutions or research laboratories. Since they are valuable, they are normally kept in specialized maintenance media, on nutrient enriched agar plates or slopes, under closely controlled conditions of temperature and illumination. A special area or room off the algal culture room is usually allocated to this purpose.

Stock cultures are used only to provide lines of starter cultures (also known as inocula) when required. Every effort should be made to minimize the risk of contaminating the stock and starter cultures with competing microorganisms. The sterile procedures described below should be followed to ensure that contamination does not occur.

Stock cultures are normally kept in small, transparent, sterilizable containers. For example, 500 ml glass, flat-bottomed boiling or conical flasks fitted with a cotton wool plug at the neck, suitable for containing 250 ml of sterile medium, are ideal. Stock cultures are also often maintained in agar medium with suitable nutrients in Petri dishes or on slopes in test tubes.

Stock cultures are best kept in a cooled incubator at 4 to 12�C (50F) (according to preference), illuminated by low wattage (8W) fluorescent lamps or LED bulbs that provide at least 450 lux.

Alternatively they can be kept in cool conditions close to a north-facing window (out of direct sunlight), or in a cool room illuminated by fluorescent lamps. The objective is not to allow rapid growth, but to maintain the cultures in good condition. The cultures are not aerated, nor is carbon dioxide introduced.

It is necessary to sub-culture stock cultures at monthly intervals to maintain them in a vigorous and healthy state. Simply put 1-1.5 oz stock culture into another reactor containing nutrient medium.

The original stock culture can be kept for a few weeks in the event that the new stock culture fails to grow. The stock culture transfer procedure is best performed in a cabinet that has been sterilized by ultra-violet light to further reduce the risk of contamination.

Step 9: For More Information...

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