Protocol for test tubes replication

The steps to duplicate algal strains at test tube level are described in the following paragraphs. All required equipment and consumables have to be .well cleaned and sterilised in advance.

Fig. 24.09 Pure strains kept under controlled conditions (photo STM Aquatrade)

Choose the test tubes that show the best algal populations at naked eye and check under the microscope a sample taken from each of them for contaminant organisms (use a new sterile pipette for each sampling). Then keep only the uncontaminated cultures and put them on a stand avoiding any stirring.

For each selected test tube:

1 prepare four sterilized test tubes with cap on a stand;

2 prepare at least 50 ml of sterilized seawater medium, enriched with half dose of the standard nutrients mix (see above);

3 heat necks and caps of all test tubes (new and old vessels) by means of a manually operated burner and let them cool;

4 fill each new test tube with 10 ml of seawater medium taken with a graduated pipette;

5 with a sterile 1 ml-pipette take 0.5 ml of mature culture from near the surface of the selected test tube;

6 Inoculate 0.1 ml of the old culture into each new test tube; be careful to make the drops fall freely into the culture medium without touching the tube walls; with one hand open and close the tube cap; with the other hand handle the pipette; do not mix or agitate the tubes;

7 place the used pipettes into the rinser cylinder;

8 after inoculation, heat the upper part of each new test tube thoroughly and cover with its cap, previously flamed; as an alternative, use sterilized hydrofobic cotton or flamed aluminium foil as stopper;

9 discard the old culture, if hot needed for other replicas, and clean the empty tube following the usual cleaning routine for glassware (annex 6);

10 write date and algal species on all new test tubes with a waterproof marker;

11 place the newly inoculated test tubes on a rack in a shelf reserved to pure strain cultures.

Protocol for purification of algal strains

Even under axenic conditions, pure strains can be contaminated by other algal species or micro-organisms: in such cases before up-scaling the cultures the microalgal populations have to be purified before being used as inoculum.

Two methods to purify contaminated algal cultures are of common use: successive dilutions of the original contaminated culture, and picking up of single cells from the original culture. Both techniques are also applied when new algal species are isolated from the wild.

Dilution method

A simple technique to purify a contaminated strain is to proceed with repeated sub-cultures obtained by progressive dilutions of the original sample. Dilution rates can be very high, depending on the number of the sub-cultures. In the procedure described below a dilution rate of 10-10 is reached. The same nutrient quality and environmental condition of the initial culture are used.

Example of dilution:

1. prepare 10 sterile test tubes with caps on a tube rack;

2. sterilize 500 m I of seawater in an Erlenmeyer flask, then fertilize it with half dose of usual mix of nutrients when cool;

3. using a sterile 10-ml pipette add 9 ml of enriched seawater to each tube, close loosely with flamed caps and number them 1 to 10;

4. put the test tube containing the strain to be diluted in the rack, remove the cap and flame its neck;

5. using a 1-ml sterile pipette take 1 ml and add it to the tube No. 1, then stir gently;

6. using a new sterile 1 ml-pipette repeat the previous step by taking 1-ml inoculum from tube No. 1 and inoculate it into tube No. 2;

7. repeat the same procedure with the remaining tubes, each time pipetting 1 ml from the previous tube (gently stirred) into the next one; flame necks and caps and let them coo);

8. keep under controlled environmental conditions.

When cell growth reappears, check samples of the tubes under the microscope and get rid of the tubes that are still contaminated, typically the initial ones, and keep only the purified cultures, usually in the more diluted tubes. Repeat the process using the last dilutions if necessary, and in any case at least every three months to always have a safe amount of purified cultures ready at hand.

Picking up method

Two systems which produce single cells are described below: the agar plate method and the capillary method:

Agar plate method

A solid medium can grant more stable conditions for the growth of the desired species. This technique needs some sterile equipment like Petri dish and platinum hooks.

Example:

1. prepare the solid medium by adding 1.5 g of agar powder to 100 ml of sterile fertilised seawater;

2. heat the medium using a Bunsen burner, stirring with a sterile glass rod to dissolve the agar, and pour it on 3 to 5 sterilised Petri dishes;

3. allow to cool and solidify;

4. using a sterilized 1 ml-pipette take 0.1 ml from the initial contaminated strain culture and drop it into each Petri dish;

5. spread the sample over the agar with a sterile platinum hook and incubate at desired environmental conditions placing the dish upside down, so that water drops will not form on the lid and then fall on the culture;

6. once algal colonies are observed, take a sample by means of a sterile platinum hook and check under the microscope

7. monospecific colonies should be kept in agar for successive replication or might be up-scaled by replication (i.e. a small portion of the colony together with some agar, using a sterile hook) on a 50-ml or 500-ml Erlenmeyer flask to continue in the liquid medium.

Capillary method:

This technique follows the dilution method, but the inoculum is obtained by selecting single cells of the desired species by means of a capillary pipette handled under a microscope.

The isolation or purification of cultured strains should be repeated as many times as required to produce contaminant-free cultures.

Upscaling culture conditions

As indicated above the upscaling of microalgae production starts from small containers (0.5 ml) and proceeds through various steps up to the mass production in PE bags (up to 450l). Each step involves an increase of the culture volume: when mature (i.e.: in log phase), the algal population of a smaller volume is sacrificed to replicate the same vessel and to inoculate larger vessels.

Small-medium size (0.5 to 10l) cultures of microalgae are usually kept in borosilicate-glass containers with large necks, such as Erlenmeyer flasks or other flat bottom round flasks (balloons) and carboys. The flasks are usually closed with a stopper made of sterilised cotton or of plastic which can stand .sterilization in autoclave.

Fig. 24.10 Petri dish with culture solid medium (photo STM Aquatrade)

All these cultures are provided with a proper medium to support algal growth (treated and fertilized seawater), good aeration supplemented with carbon dioxide (CO2) as an additional carbon source, and a strong light. In this way it is possible to reach quickly the log-phase growth. Aseptic conditions should be maintained to avoid contamination and culture crashes.

For practical purposes the main parameters for the cultures are usually the same for the different species of algae mentioned in the sections above:

Fig. 25.01-2 Algae flask replication (photo STM Aquatrade)

Temperature

For mass culturing purposes, the optimal temperature for the above mentioned algal species ranges between 20 and 24°C. Generally speaking, temperatures lower than 16°C and above 27°C will slow down growth rates, whereas those above 30°C are normally lethal. That makes room conditioning necessary as artificial lights and insufflated air from the aeration system can raise temperature dangerously.

Low temperatures are used for pure strains only, where growth should be kept as slow as possible. As low temperatures also affect bacterial growth, non-axenic cultures should be maintained at the lowest possible temperature consistent with a good growth, to prevent bacterial growth.

Salinity

In the selected species salinity does not represent a limiting factor within a range of 15 to 40 ppt.

Light

Light is the source of energy for photosynthesis and therefore in mass cultures algae are usually kept in continuous light. Fluorescent tubes are the commonest choice for providing light due to their low power consumption, low installation cost and limited heat production compared to bulb lamps. Spectral quality suitable for algal growth is provided by “Cool white” and “Daylight” models, often installed in equal numbers. The commonest tube size is 42” with waterproof contacts. See Part 4 Engineering for their installation in the hatchery’s algal unit.

Even if each algae species has its own preferred light intensity for best growth, for practical purposes intensity in mass cultures is kept in the range of 2 500-8 000 lux, while higher intensity is used on the small volumes shelves (5 000-8 000 lux) because of their higher cell density. Large volumes (bag cultures) can also be illuminated by natural daylight, entering from sufficiently large windows or from a continuous glass wall, provided that the cultures are .not directly exposed to external contamination.

Fig. 25.03 Lighted table used for mid size culture up scaling (photo STM Aquatrade)

Aeration

Aeration is used to maintain the culture in turbulent state, preventing settling of cells and exposing all cells to light. It also supplies carbon dioxide, which is fixed by the algae during photosynthesis, and provides essential pH stabilization.

Transparent PVC tubing, of 6 mm internal diameter, are commonly utilized to deliver air to the culture vessels. In small vessels this tubing is connected to glass pipes which fit the stopper and reach the bottom of the vessel, while in PE bags they are simply forced into a small hole near their bottom (the water pressure keep the holes sealed), whereas in tanks a weight keep them submersed.

Aeration should be moderate in the first two days of culture, then should be increased adjusting it according to culture growth. For this purpose screw clamps or cheaper plastic needle aquarium valves are required to adjust the air flow. If aeration produces foam, it is an early warning of culture troubles.

Carbon dioxide

As its normal content in air is low (approx. 0.03%), carbon dioxide is often supplied at 2% by volume to optimize culture growth. Commercial grade CO2 bottles are utilised, and the gas is injected into the main air pipe via a dispensing vessel in which gas bubbling reveals the gas flow. Since carbon dioxide is heavier than air, to prevent stratification the pipe makes some ups and downs after the point where it is injected.

Depending on the algal species, the CO2 supply, and the volume of inoculum, working cultures normally reach their log-phase in 5 to 7 days. At this point, cultures can be utilized either to start new cultures, to be fed to rotifers or to be used as “green water” in fish tanks..

The volume of the algal inoculum is usually 15 to 20% of the new volume. Smaller or larger inocula could be used to decrease or increase growth rate. In culture up-scaling, the new vessels have to be inoculated with a sufficiently high microalgae density in order to ensure a rapid growth and to limit the risk of contamination with different algae or other micro-organisms (protozoa, nematodes, fungi, etc.).

Scaling up protocol

The following section describes in detail the operation to replicate different volumes.

Inoculation of flasks from test tube

Follow the same procedure as previously described for pure strains. Each 0.1-1 flask will receive 50 ml of enriched medium and 0.5 ml of inoculum. At this stage, no aeration is required. When mature, each small flask will .inoculate a new 2-l flask.

Fig. 25.04 Alternative use of olives containers for .algae culture (photo Ittica Mediterranea)

Fig. 25.05 Very small PE bags of phytoplankton at .Ittica Ugento (photo STM Aquatrade)

Fig. 25.06 Medium and large size bags (photo STM .Aquatrade)

Inoculation of 2 and 6-l flasks from another 2 or 6-l flask:

1. prepare the necessary amount of new flasks filled with sterilised seawater, as well as all equipment required for the operation (pipettes, nutrients, cotton stoppers, aluminium foil, glass tubing, etc.);

2. select the mature culture that will be used as inoculum, checking a sample under the microscope for contaminants;

3. remove its cap and flame its neck; then close with a flamed aluminium foil stopper and let it cool;

4. in the meanwhile, add the fertilizing working solutions to each new flask, at a rate of 1 ml/l; using a new sterile pipette for each solution;

5. flame their necks and aluminium caps thoroughly;

6. when cool, remove the stopper and pour some algal culture of the old flask into the new vessels at a rate of about 10% of the receiving volume, avoiding to wet their neck with the inoculum, then gently shake flasks to mix the new culture;

7. flame thoroughly their necks, introduce the sterilized glass tubing for aeration and close tightly with sterilised cotton stopper (or any other type of sterile cap);

8. write date and algal species on the new flask;

9. place the flasks on the lighted shelf and connect to the air delivery system, adjusting its flow to a gentle bubbling;

10. after on hour, check all new vessels for a proper air bubbling.

Use only the upper layer of the old culture, leaving dead cells and debris in the flask used to inoculate the new ones. The size of the inoculum for small volumes is only 10% of the new volume because of the high cell density. Remember to get rid of every contaminated flask. The above mentioned procedure applies to the upscaling of .the other medium size vessels.

Fig. 25.07-08 PE bags inoculation with the help of an self-priming pump (photo STM Aquatrade)

Inoculation of a PE bag from a flask or from another bag

1. prepare the bag, either suspended to a rack or placing it inside a cylindrical frame made of wire;

2. fill the bag with sterilized water; leaving enough free space for the volume to be inoculated; wait a couple of hours to check for possible leaks; if found, seal them or replace the bag;

3. introduce nutrients and inoculum;

4. fit two PVC tubes for aeration to the bottom of the bag, connect them to an air line and adjust the air flow to a gentle bubbling;

5. add the nutrients using a graduated cylinder at the usual rate of 1 ml/l of each working solution;

6. select a mature culture that will be used as inoculum, either from a large flask or from another bag, and check a sample under the microscope for contaminants;

7. in case a flask is used as inoculum, remove its cap and flame its neck; then close with a flamed aluminium foil stopper and let it cool;

8. then, remove the stopper and pour the algal culture from the flask into the bag. The inoculum should be about 10% of the receiving volume;

9. if the inoculum comes from another bag, with a self-priming plastic pump transfer the inoculum from the old bag into the new one. The volume to be transferred should be about 15-20% of the receiving volume (a bigger inoculum is necessary to compensate for a less dense culture);

10. on each bag mark date, algal species, origin of the inoculum (bag or flask) and the bag serial number.

To reduce the risk of contamination, smaller bags are usually inoculated from flasks, whereas larger bags are inoculated from smaller bags.

Fig. 25.09-10-11 Large volume cultures (photo STM Aquatrade)

Monitoring algal populations

A regular check of microalgae cultures is essential to prevent crashes and to keep high quality standards. The main parameters to be monitored are: colour, density, pH and contaminant levels. As an example, a change in colour to opaque grey and a pH level lower than 7.5 may indicate a high degree of bacterial contamination. A lighter colour than normal may reveal insufficient nutrients or poor lighting. However, during the peak production season in the hatcheries a close monitoring of algal cultures can hardly be assured, due to the chronic lack of staff and time. Mass cultures are normally checked at naked eye by experienced staff and strict controls are usually restricted to pure strains and small vessels.

To partly by-pass this problem, the staff can do a good preliminary job outside the peak production season, when they are not so absorbed by production and there is some more time. Then test culture cycles can be closely monitored, the algal culture growth can be followed daily by counting the number of cells per ml with a haemocytometer (see below), and their average growth curves can be plotted against values obtained with a colorimeter. During more busy days, a colorimetric monitoring (comparing values against the test curves) gives a fast and reliable indication of algal cultures growth.

These test cultures are useful for a number of other reasons as it is possible:

• to determine the growth curve for each species of algae under local conditions;
• to devise criteria for quick identification of possible troubles (eg.; presence of foam, sedimentation pattern, changes in colour, etc.);
• to determine optimal utilization time, i.e. the age at which the algal population reaches the peak of the log-phase;
• to adjust environmental conditions to maximise production;
• to control possible contaminants and try countermeasures.

Amany Esmail

Manger GAFRD Web Site