principles of bioprospecting for microalgae jerry brand utex...

Principles of Bioprospecting for Microalgae

Jerry Brand

UTEX – The Culture Collection of Algae

at The University of Texas at Austin

Workshop - Managing Seed Cultures of Microalgae Willie Wilson & Jerry Brand Algae Biomass Summit October 2, 2013

Fill a nonmetalic pan with non-chlorinated water.

Pour in a little water from a nearby lake or stream.

Add a little soluble complete plant fertilizer (approx. 1/10 of the concentration recommended for plants.

Set the pan in sunlight for several warm days.

A Simple Bioprospecting Strategy (Pan Bioprospecting)

Examples of Variations on Pan Bioprospecting

Fill a non-metalic pan with non-chlorinated water.

Pan A Pan B Pan C

Line bottom of pan with sand

add dilute fertilizer

Set in sun on cool days

Line bottom of pan with local clay

add dilute fertilizer

Set in sun on warm days

Add high-phosphate fertilizer

Set in sun on very warm days

Some Environmental Parameters that Can Be Varied When Pan Bioprospecting

Light intensity

Temperature

Source of inoculum (natural water, soil, a pure culture, etc.)

Fertilizer supplement (concentrated or dilute fertilizer, with or without soil, high or low nitrogen conc., nitrate or ammonium nitrogen, etc.)

Length of incubation time

If the same environmental parameters were used to encourage algal growth on two different occasions, would the results be expected to be the same?

Day 1

(nothing visible)

Day 6

(foamy brown water)

Fill a pan with non-chlorinated water.

Add a little fertilizer.

Pour in a little water from a nearby lake or stream.

Set in the sun for a period of time.

Day 5

(green water)

Timing is Everything

Pan Bioprospecting is actually an enrichment technique.

Place “enrichment“ medium in container

Inoculate with desired source

of algae

Incubate under selective (enrichment) set of conditions

Collect sample at just the right time

You are not likely to discover something valuable unless you:

know what you are looking for;

know where you are likely to find it;

know how to collect and maintain it.

Plan Ahead

Collect Smart

Prepare Unialgal Cultures

Bioprospecting Strategy

Enrich* and Screen**

*Design conditions that encourage faster growth of a microalga with desired qualities than other kinds of microorganisms.

**Devise a method for rapidly and selectively detecting the quality sought.

Plan Ahead

A. Know what you are looking for. B. Prepare a strategy and conditions for obtaining

and maintaining a desirable microalga.

What are you looking for?

Possibility 1: Seeking a particular kind of microalgae

e.g. Spirulina

Haematococcus

Nannochloropsis

Possibility 2: Seeking a particular quality?

e.g. Rapid growth rate

High neutral lipid (oil) content

Specific pigment composition

Plan Ahead

What are you looking for?

Possibility 1: Seeking a particular kind of microalgae

1. Learn where it normally grows

2. Learn how it normally grows

Plan Ahead

- rain water

- high organic matter, ammonium nitrogen (e.g. bird baths)

- rapid growth under moderate light, enriched medium

- change from green to red-orange color under bright light and/or nutrient-depleted medium

e.g. Haematococcus

What are you looking for?

Possibility 2: Seeking a particular quality?

Learn what conditions are likely to enrich for algae with that quality.

e.g. High red pigment content

Plan Ahead

- high light intensity (for carotenoids)

- enriched blue or blue-green light of low intensity (for phycoerythrin)

Be prepared to maintain and stabilize the microalga in culture after it has been collected.

Plan Ahead

How will the sample be taken from the field site to the site of its maintenance?

How will a desired microalga in the sample be identified?

How will the desired microalga be stabilized in culture?

- predators removed

- competitors removed

made unialgal

Collect Smart

Most collection sites will contain multiple kinds of microalgae at differing concentrations.

Many kinds of microalgae prosper only in a specific environment. A single collection site may contain multiple specific niches.

Collect Smart

Most kinds of microalgae flourish only in certain seasons.

strain density

Temp. (°C)

0 10 30 20 40 50

Consider a desired strain of microalgae growing in a natural body of water.

It thrives at a temperature of ~25 °C It tolerates temperatures between 19 and 30 °C.

strain density

Temp. (°C)

0 10 30 20 40 50

desirable collection temperature 22 °C (72 °C)

Desired strain

Strain B

Strain A

Time and Location of collection may be based on the temperature at the collection site at the time of collection.

strain density

Temp. (°C)

0 10 30 20 40 50

Simplified Typical Culture Distribution

What to do about this?

Desired strain Strain B

Strain A

Strain C

Collecting and Enriching For a Desired Strain

2. enrich for the desired strain

collect enriched sample at just the right time

Take an information sheet to the collection site and record detailed information about the site.

Collect Smart

Collection Site Data Sheet

Purpose of collecting

Collector ______________________________ Collection date _________________________

Time of Day ____________________________ GPS coordinates ______________________

Description of the location

Physical characteristics of the collection site

Chemical characteristics of the collection site

example

Example of Site Data Collected from a Recent Collection Trip

Sample identifier: Sample 1

Collection Date: 17 September 2013

Time of Collection: 14:45 local time

Collection site: Daya Bay, South China Sea

GPS Coordinates: N 22° 41’ 59’’ E 114° 32’ 54’’

Temperature: 30.7 °C

D.O.: 8.2 mg/L

Salinity 29.9 ppt

pH: 8.03

Physical description: Top 1-cm layer of mud core collected beneath 6 meters of water

Sample kept saturated with ambient water and stored in sealed plastic bag.

Some General Rules When Collecting and Transporting Field Samples That Contain Microalgae

- Collect and transport the field sample in an inert plastic container.

- Keep the field sample in its original substrate; for example: lake water from the collection site.

- Collect a sufficiently large volume of the field sample; generally at least 0.5 liters.

- Keep the sample in very dim light or darkness during transport.

- Keep the temperature of sample below its temperature at the time of the collection, but do not freeze!

- Avoid long transport times.

collect sample

Suppose we collect at 25 °C.

In addition to the desired alga, the collected sample is very likely to include:

- competitors,

- preditors,

- diseases.

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These must be removed from the sample before the desired alga is overwhelmed.

competitor

predator

disease

predator and competitor

desired alga

Plan Ahead

Collect Smart

Prepare Unialgal Culture

Bioprospecting Strategy

Enrich and Screen

Enrichment Techniques That Lead to Unialgal Cultures

A recommended single resource: various chapters in

Algal Culturing Techniques edited by R. A. Andersen Published by the Phycological Society of America

via Elsevier Press, 2005

A vast literature on various enrichment and purification techniques dates back over 100 years.

A Sequence of Enrichment Processes That Can Lead to Unialgal Cultures of Microalgae

1. Remove debris and biological contaminants that are easily separated from the field sample.

2. Examine distribution of organisms in the sample.

3. Use a rapid enrichment method such as cell-sorting technology so the desired alga is at a higher culture density than any undesirable contaminant.

2a. If necessary, use gradient of light, temp, nutrients, etc. to enrich for desired alga.

4. Use a dilution method to dilute out contaminants and retain the desired alga.

1. Remove debris and biological contaminants that are easily separated from the field sample.

Example: differential centrifugation

2. Examine distribution of organisms in the sample.

.

16˚C 40˚C

2a. If necessary, use gradient of light, temperature, nutrients, etc. to enrich for the desired alga.

mostly diatoms mostly green algae mostly cyanobacteria

3. Use a rapid enrichment method such as cell-sorting technology so the desired alga is at a higher culture density than any undesirable contaminant.

Image from: www.labome.com/ Image from pmc.ucsc.edu/

4. Use a dilution method to dilute out contaminants and retain the desired alga.

Summary

Field samples of algae are easy to obtain but acquisition of a desirable kind of algae or a desirable characteristic often requires carefully planning and implementing a strategy.

Field samples generally are a complex mixture of various kinds of microalgae and other organisms. Enrichment techniques must be employed soon after the sample is collected in order to remove harmful organisms and save the desired alga.

Cultures of microalgae are often not stable unless they are rendered unialgal.