PO S TE R A B S T R A C T S
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The CSIRO Collection of Living Microalgae:
Bioapplications

Vitrification cryopreservation approaches forreacalcitrant micro-algae species

Blackburn SI, 1,5Jameson I, 5Johnston, C, 1,4Nichols CM, 1,5Frampton
DMF, 2,5Mansour MP, 2,5Nichols PD, 2,4Robert S , 1,3,5Volkman JK
1,2,3,5

CSIRO Wealth from Oceans National Research Flagship
CSIRO Food Futures National Research Flagship
3
CSIRO Energy Transformed National Research Flagship
4
CSIRO Molecular Health Technologies
5
CSIRO Marine and Atmospheric Research
GPO Box 1538, Hobart, Tasmania, 7001, Australia
1
2

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1

Samantha L Gale, 2John F. Smith, 3Robin Tervit, 1Lesley Rhodes,
Serean L Adams

Cawthron Institute, 98 Halifax Street East, Nelson, New Zealand
33 Willowbank Place, Gerringong, NSW 2534, Australia
3
HR and HM Tervit Ltd, 231 Papamoa Beach Road, Papamoa,
New Zealand
12samantha.gale@cawthron.org.nz

Susan.Blackburn@csiro.au
The CSIRO Collection of Living Microalgae* is a living bank ofmicroalgae isolated from Australian waters from the tropics to Antarctica.
Wide microalgal biodiversity is reflected in a broad range of bioactive
compounds that have medical, human health, aquafeed, and energy
applications. In the CSIRO Wealth from Oceans National Research
Flagship, research on exopolysaccharides (EPS) from microorganisms
has revealed new adhesives, with microalgae from the Collection
being screened for their EPS production, along with development ofknown targets of EPS-producing bacteria from the extreme Antarctic
environment. Gene discovery in microalgae for biosynthesis of omega-3
long chain polyunsaturated fatty acids (LC-PUFA) that have an
impressive range of human health benefits is one focus of the CSIRO
Food Futures National Research Flagship. The goal is to introduce themicroalgal omega-3 pathway into genetically engineered crop plants,
thus ensuring a sustainable source of omega-3 LC-PUFA for the future.
Some microalgae are characterised by high oil content. The CSIRO
Energy Transformed National Research Flagship is investigating thepotential for producing biodiesel from algae. High biomass cultivation of
microalgae has the potential not only to produce oil and/or biomass forbiofuels, but also to mitigate CO2 and other greenhouse gases due totheir uptake during photosynthesis and growth.
*http://www.cmar.csiro.au/microalgae/

Cawthron maintains a nationally significant culture collection of
micro-algae for New Zealand as well as a number of aquaculture
species that are fed to shellfish larvae in hatcheries. Many of
the >150 strains in the collection are unique, with properties
not found in overseas isolates, and are being studied forcommercial and research applications. Cryopreservation has anumber of advantages: it is cheaper than ongoing maintenanceof cultures and safeguards strains from the risk of becoming
contaminated or lost due to genetic drift from serial subculture.
A standard cryopreservation method using 10-15% (v/v)
DMSO for cryoprotectant and controlled rate cooling has beensuccessfully applied to many of the algae species held but someremain recalcitrant, in particular the larger (>30µm) marine
dinoflagellates, for example, Procentrum lima. Vitrification is analternative cryopreservation method to controlled rate cooling andincorporates the pretreatment dehydration of cells or applicationof concentrated vitreous chemical cocktails so that ice crystal
formation is circumvented and a stable ‘glass’ is formed instead,when treated cells are directly immersed in liquid nitrogen. Criticalfactors such as dehydration and toxicity are controlled by tailoring
pre- and post- liquid nitrogen treatment parameters. Sensitiveequipment such as Differential Scanning Calorimeters assist in
vitrification protocol optimization by pinpointing thermodynamic
events, such as glass transitions (Tg), in vitrified cells. Initial trials
examining the dehydration and toxicity parameter thresholds of theselected marine micro-algae have begun and preliminary results
will be reported.
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Cyanobacteria isolation and growth at QHFSSMicrobial Culture Collection at the National
Institute for Environmental Studies (NIESCollection), playing a role as a core collection ofalgae in Japan

Lindsay Hunt, Toni Menjivar, Karen Reardon, Barbara Sendall
Queensland Health Forensic and Scientific Services, PO Box 594,
Archerfield QLD 4108, Australia

Fumie Kasai, 1Masanobu Kawachi, 2Mayumi Erata, 2Fumi Mori,
Kosei Yumoto, 2Mayumi Sato, 2Miwa Ishimoto and 1Yuuhiko
Tanabe1

Lindsay_Hunt@health.qld.gov.au
With increasing emphasis on Cyanobacteria and their impacts
on water quality, Queensland Health Forensic and Scientific
Services (QHFSS), officially started to develop, a Phycology
Culture Facility in early 2007, at the Coopers Plains laboratories,
in Brisbane. The purpose of the Facility was to supply high quality,
monoclonal phytoplankton material for research projects, (primarily
PCR based). Over 40 strains of cyanobacteria are currently
maintained at the Facility. An important aspect of the Facility has
been developing the capability of isolating environmental strains,using an isolation technique developed on site. Information
presented gives a brief outline of the isolation and growthtechniques employed at QHFSS to produce a cyanobacterial
culture from an environmental sample. Highlighted are important
aspects that need to be considered by scientists wishing toculture cyanobacteria, such as suitable sample choice, growth
media, isolation techniques and quality assurance. The isolation
techniques are relatively simple and can be further simplified asa small scale and informal project, utilising basic, inexpensive
equipment readily available in most laboratories or up-scaled, toa facility with dedicated resources and formal projects. The scaleand complexity of the culturing facility will depend on the desiredend use of the cultures, and the resources and facilities available
to a laboratory. This presentation may encourage other institutes
to attempt to develop their own small scale culture collections,
helping add to the collective knowledge of phytoplankton and theirbiodiversity.

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National Institute for Environmental Studies, Tsukuba, Japan
Global Environmental Forum, Tsukuba, Japan

kasaif@nies.go.jp

The NIES-Collection holds about 2,500 strains that consist ofvarious taxonomic groups including about 300 genera and 600
species. Among them, 2,096 strains are now available. We
distributed 600-700 strains per year in recent years. In 2002 the
NIES-Collection was selected to a core collection of algae in Japan
under the National BioResource Project funded by the Ministry of
Education, Culture, Sports, Science and Technology-Japan. Since
then, the NIES-Collection has increased its strain number andtaxonomic diversity in collaboration with University of Tsukuba andNational Science Museum. In addition, as a result of this project,
all algal strains maintained in the IAM Collection, which was theoldest algal culture collection in Japan, were transferred to the
NIES-Collection until the end of 2006FY when the IAM Collection
was closed. The NIES-Collection is characterized by four groupsof algal strains: 1) species relevant to environmental issues
such as water bloom and red tide, 2) experimental models, 3)taxonomically important species, and 4) endangered Charales andfreshwater red algae. For some of them, nuclear and organelle
genomes have been analysed. In addition to routine works in thecollection, we have intended to increase strain information such as
multilocus genotypic information using seven house keeping genes
in Microcystis aeruginosa, as well as SSU rDNA sequences inmorphologically simple cyanobacteria and eukaryotic microalgae.
The former is important to manage a large number of strains inthe same species and the latter is important to show phylogenetic
position of the strains in the culture collection.

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A culture collection of New Zealand alpine algae

University of Malaya Algae Culture Collections(UMACC): biotechnological and environmental
applications

1

Phil Novis and 2Gabriel Visnovsky

Manaaki Whenua – Landcare Research, P.O. Box 40, Lincoln
7640, New Zealand.
2
Chemical and Process Engineering, University of Canterbury,
Private Bag 4800, Christchurch, New Zealand1

novisp@landcareresearch.co.nz
The New Zealand alpine zone contains a wealth of microorganisms, largely unknown to science. We have establisheda culture collection of alpine algae collected mostly fromMt Philistine, Arthur’s Pass National Park. The collection
presently includes 15 chlorophyte species, 14 cyanobacteria, 2streptophytes, 2 diatoms, 1 xanthophycean, and 1 chrysophycean.
In addition to offering new data on algal diversity, the collection has
potential for investigations of the distributions of microbes and in
testing the extent of their dispersal, and in providing a resource formore applied research. We provide images of selected strains andtentatively assign relationships to isolates elsewhere in the world,
deduced using morphological characters and gene sequencing.

Siew-Moi Phang and 3Wan-Loy Chu
Institute of Biological Sciences, University of Malaya, 50603 Kuala
Lumpur, Malaysia
2Institute of Ocean and Earth Sciences (IOES), University of
Malaya, 50603 Kuala Lumpur, Malaysia
3International Medical University, Plaza Komanwel, Bukit Jalil
57000, Kuala Lumpur, Malaysia
phang@um.edu.my
1,2
1

The University of Malaya Algae Culture Collection (UMACC)
comprises more than 200 isolates from diverse habitats, ranging
from aero-terrestrial, freshwater, brackish to marine habitats. The
collection includes indigenous strains from Malaysia and the Polar
Regions. When the UMACC was initiated in 1987, the strains
were mainly used for the screening of high-value chemicals (e.g.
polyunsaturated fatty acids, carotenoids and phycobiliproteins)and bioremediation of agroindustrial wastewaters. Presently, thecultures of UMACC are used in the the screening for antiviral,
antineoplastic and antiinflammatory activity. Some of the algae
are being studied for removing colour from textile dyes using
suspension and immobilised cultures. The algae are also used forgenotoxicity testing of pollutants such as pesticides, heavy metals
and textile dyes. The collection of polar algae is very useful forour comparative studies on their response to global warming andUVR stress with tropical and temperate algae. With the increased
interest in biofuels, we are also exploring the potential of the
UMACC algae for biofuel production. In addition, the UMACC
serves as a valuable resource of tropical algae for DNA barcoding
initiatives.

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The Cawthron Institute Collection of Micro-algae(CICCM) supports cutting edge research

NIWA Blue-Green Algae Monitoring Service

Krystyna Ponikla, Lesley Rhodes, Andy Selwood and PaulMcNabbCawthron Institute, Private Bag 2, Nelson, New Zealandkrystyna.ponikla@cawthron.org.nz
The Cawthron Institute Culture Collection of Micro-algae is usedby researchers world-wide. Live cultures (many supported bycryopreservation) are supplied to Cawthron researchers working
on New Zealand government funded programmes and to approved
researchers at other research institutes and universities within
New Zealand. Over 10% of all cultures supplied are couriered
internationally. Cultures have been used for development ofstandards for new chemical analytical methods, developing andtesting molecular tools, the characterization of novel compounds
and determination of their toxicology. They have also been used
for the comparative study of ‘same’ species worldwide. Overthe last year a key research programme at Cawthron Institute,dependent on the collection, is the Seafood Safety programme(NZ FRST Contract CAWX0703). Study undertaken in thisprogramme includes research on novel toxin producers andtheir morphological and molecular identification. For example,
in February 2008, analyses were carried out (LC-MS, Cawthron
Biotoxin Laboratory) on fresh and frozen oysters collected from
Rangaunu Harbour, Northland. The frozen oysters were collecteda decade ago and at that time they had tested positive for an
unknown biotoxin by mouse bioassay. The recent study focuseson the toxin producer and 20 clonal micro-algal cultures have been
isolated from samples collected from Rangaunu Harbour. Other
projects include studies of toxins produced by subtropical microalgae to determine their potential impact on human health and on
New Zealand’s aquaculture industry in the future, particularly inthe light of climate change. The results of these studies are key
research outputs supported by the CICCM.

Karl Safi and Karen Thompson
National Institute of Water & Atmospheric Research (NIWA),
Hamilton, New Zealandk.safi@niwa.co.nz
k.thompson@niwa.co.nz
NIWA’s Algal Monitoring Service was started in Hamilton in 2003 to
help organisations monitor a range of water bodies to ensure thesafety of recreational and drinking waters. In December 2005, newdrinking water standards came into force in New Zealand. They
stipulate monitoring of drinking water sources deemed susceptible
to blue-green blooms and have set standards to trigger additional
monitoring and water treatment. New guidelines are now also
being considered for recreational waters. This poster offers an
overview of how Algal Services is able to help.

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Puwainaphycins J-M isolated from
Calothrix sp. TISTR 8906

PCR detection and quantification of key
microalgal species in oxidation ponds

Tomoharu Sano1, Hiroo Takagi1, Ayako Forchert1, Aparat
Mahakhant2, and Kunimitsu Kaya3
National Institute for Environmental Studies, 16-2 Onogawa,Ttsukuba, Ibaraki, 305-8506, Japan
2
TISTR culture Collection, Thailand Institute of Scientific andTechnological Research, 196 Phahonyothin, Chatuchak, Bangkok
10900, Thailand
3
Department of Environmental Sciences, Tohoku University, Aoba
01, Sendai, Miyagi 980-8579, Japan1

sanotomo@nies.go.jp
Up to the present time, a wide range of cyanobacteria (blue-green
algae) have been examined for biologically active compounds.
Prominent among these compounds are cyanotoxins such as the
microcystins, cyclic heptapeptides from freshwater cyanobacteria.
We have been screening cell-extracts of over 100 strains of
cyanobacteria for antifungal activity against plant pathogenic
fungi. The strain of cyanobacterium Calothrix sp., TISTR 8906,
showed strong activity against Colletotrichum truncatum andMacrophomina phaseolina. Previously, four major antifungal
compounds, puwainaphycins F-I, had been isolated from thecultured cells of TISTR 8906. Further investigation of the strain
TISTR 8906, four new compounds have been isolated fromthe same fraction of puwainaphycins F-I as minor components.
From the results of NMR experiments and amino acid analysis,
the structures of these compounds (puwainaphycins J-M) were
elucidated. The activities of these new compounds have been
investigating.

Sandra Visnovsky, Simon Bulman and Grant Smith
The New Zealand Institute for Crop and Food Research, Private
Bag 4704, Christchurch 8140, New Zealand
VisnovskyS@crop.cri.nz
With the aim of predicting pond health, we used moleculartechniques and microscopy to monitor algal populations in threeoxygenation ponds operating in Richmond, New Zealand. Over2 years, 18 microalgal species were isolated and ribosomal ITS
fragments amplified from their DNA. These DNA sequenceswere used to identify the algae by comparison to sequencesin GenBank. Ribosomal fragments were also amplified
and cloned from total DNA from pond samples. Species of
Chlorella, Scenedesmus, Chlamydomonas, Monoraphidium,
Ankistrodesmus, Kolliela, Dunalliela, Cyanobacteria, Phacus andEuglena were identified. The DNA sequences obtained were used
to design species-specific primers for a number of microalgae
including Chlorella, Scenedesmus and Euglena species that playa dominant role in pond operation. Species-specific primers werealso designed for Euglena acus UTEX 1304LB obtained from anoverseas algae collection. Species-specific primers were used forquantification of algae species in whole pond samples using SYBR
green quantitative PCR (qPCR). Euglena acus UTEX 1304LB wasused to calibrate our method for qPCR. We found that we could
consistently quantify this species when it was spiked into waterfrom real pond samples. Preliminary qPCR for Euglena acus,
Chlorella sp and Scenedesmus obliquus in pond samples gave
good amplification and reproducibility. Quantitative PCR basedon ribosomal sequences has the potential to accurately monitor
individual algal species within a complex mix of micro-organisms.

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