Cryopreservation and coservation Day 2005

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CryoLetters 26 (4), 231-238 (2005)

@ CryoLetters, clo Roya! Veterinary College, London NWl OTU, UK

CRYOPRESERVATI ON AND CONSERVATI ON OF MICROALGAE:THE DEVELOPMENT OF A PAN-EUROPEANSCIENTIFICAND

BIOTECHNOLOGICALRESOURCE (THE COBRA PROJECT)1* 2 . .J. 2 2 2 3

J.G. Day , E.E. Benson , K. Hardmg , B. Knowles , M. ldowu , D. Brenmer, L. Santos , F.Santos3, T. Fried14, M. Lorenz4, A. Lukesova5, J. Elster6, J. Lukavskl, M. Herdman7, R.

Rippka7 and T. HaU8

ICCAP, Scottish Association for Marine Science, Dunbeg, Argyll, PA37 1QA, UK.2University of Abertay Dundee, Bell St., Dundee, DDl 1HG, UK. 3Dept. Botfu1ica,Univ.Coimbra, 3000 Coimbra, Portugal. 4SAG, Albrecht-von-Haller-Inst. Pflanzenwissenschaften,Univ. Gottingen, Untere Karsptile 2, 37073 Gottingen, Germany. 5lnst. Soil Biology CAS, NaSadkach 7, 370 05 Ceske Budejovice, Czech Republic. 6Inst.BotanyCAS, Dukelska 135, CZ379 83 Trebon, Czech Republic. 7Inst. Pasteur, 28 Rue de Dr Roux, 75724 Paris, France.8AQUAARTlS, 19 rue de la Dutee, 44806 St. Herblain-cedex, France. [email protected]

AbstractMicroalgae are one of the most biologically important elements of worldwide ecology andcould be the source of diverse new products and medicines. COBRA (The COnservation of avital european scientific and ~iotechnological Resource: microAlgae and cyanobacteria) isthe acronym for a European Union, RTD Infrastructures project (Contract No. QLRl-CT-2001-01645). This project is in the process of developing a European Biological ResourceCentre based on existing algal culture collections. The COBRA projects central aim is toapply cryopreservation methodologies to microalgae and cyanobacteria, organisms that, todate, have proved difficult to conserve using cryogenic methods. In addition, molecular andbiochemical stabiliiy tests have been developed to ensure that the equivalent strains ofmicroorganisms supplied by the culture collections give high quality and consistentperformance. Fundamental and applied knowledge of stress physiology form an essentialcomponent of the project and this is being employed to assist the optimisation of methods forpreserving a wide range of algal diversity. COBRA's "Resource Centre" utilises InformationTechnologies (IT) and Knowledge Management practices to assist project coordination,management and information dissemination and facilitate the generation of new knowledgepertaining to algal conservation. This review of the COBRA project will give a summary ofcurrent methodologies for cryopreservation of microalgae and procedures adopted within theCOBRA project to enhance preservation techniques for this diverse group of organisms.

Key words: AIgae, algal biotechnology, BRC, cryoinjury, cyopreservation, culture collection

This paper was originaIlypresented~at a joint meeting of the National Institute for BiologicalStandardsand Control and the Societyfor Low TemperatureBiologyentitIed"Current Trends inCryobiologyand Lyophilisationheld4-5lhSeptember2003.

INTRODUCTlONMicroalgae are a highly diverse group of microorganisms comprising eukaryotic

photoautotrophic protists and prokaryotic cyanobacteria (sometimes called blue-green algae).They are virtually ubiquitous in euphotic niches, and as primary producers, they contributehalf of global photosynthetic activity (1). Furthermore, they form the basis of the food chain

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~for >70% of the world's biomass (18). Microalga1 cultures are a va1uable environmental,ecological, taxonomic and biotechnological resource, however, their commercial exploitationis, relatively speaking, limited. The European exploitation of algae and their products,remains (as compared to otber globa1 economic regions) limited and the industry is in itsinfancy. This is despite the fact tbat a number of nutriceutica1 algal products sold into tbehealth-food market have been commercialIy successful. The best known is I1SuperBlueGreen@ Algae", produced by the American company CelI Tech, which had a gross marketvalue of over $100 million in 1999 (11). Other product areas include aquaculture feed,"green" fertilisers, pigments, vitamins, antioxidants and anti-microbial agents. Algae havesignificant potential as sources of novel bioactive compounds and one of the partners in thisproject (AQUAARTIS), who have interests in neutraceuticals, pharmaceuticals and"functional foods", is amongst tbe world-Ieaders in a1galbiotechnology. At present. the vastmajority of alga1cultures are maintained by routine serial sub-culture, a technique that cannotguarantee culture stability or security. Genetic and phenotypic stability is of critica1importance to all users of biological resources and, in particular, to the commercialisation ofmicroalgae as all biotechnological products and processes require stable starter-cultures. Theauthors have had personal experience of significant changes in performance/ growthcharacteristics alga1 strains maintained by serial subculture (Table 1) and loss of strainsproducing potentially commercial metabolites (T HalI, unpublished data) when they weremaintained by this technique. Tbe exact events which cause the loss of these features are mostoften not apparent and one of tbe aims of the project is to \lse molecular techniques to monitorrecovered cultures for these changes.

Table 1. Examples of characteristics that may be lost on maintainingalgae by routine serialsubculture

Unstable characteristic Alga(e)

*Spine production*Change in optimum growth rateKetocarotenoid production

"Irreversible shrinkage of diatom frustuleAlkaloid neurotoxin production*Phycobiliprotein productionGas vacuole production

Micractinium pusi/lumTetraselmis suecicaChlamydomonas nivalisAI!freshwater diatomsAnabaena fIosaquaeMany cyanobacteriaMany cyanobacteria

*Characteristic stable in cryopreserved samples; i.e. pre and post-cryopreservation strains arephenotypically indistinguishable.

Cryopreservation is the method of choice to preserve biodiversity as derived germplasmor celI lines, and this circumvents the need for repeated sub-culturing and mitigates againstthe destabilising changes associated with repeated and long-term culture in the active growingstate (3,10). This approach has been observed to ensure the stability of a variety of charactersthat may be unstable on employing a conventional maintenance regime (Table I). However,many strains are difficult to maintain in vitro and are reca1citrant to the availablecryopreservation methods. Where this results in very low levels of viability in cryopreservedsamples post-thaw, this raises the concem that one could potentially select a freeze-tolerantsubpopulation that was not fulIy representative of the original strain. This concem hasstimulated some algal researchers working in culture colIections to suggest that there shouldbe a minimum acceptable post-thaw viability level and these have ranged from 10-60%(9,16). Suboptimal protocols may also influence the "quality" of the preserved material andwork undertaken in the COBRA project has demonstrated up to a 3000x increase in the

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/incidence of gas-vacuolated-deficient mutants in Microcystis aeruginosa PCC 7806 onemploying5% (w/v) polyvinylpyrrolidone(PVP) as a cryoprotectantin a standard2 stepprotocol(7).

The key problems relating to development of preservation methods for algae include therigid, sometimes irnpermeable, cell wall and the presence of complex cell structures such asvacuoles, flagella insertion points etc. These can make penetration of cryoprotectants (CPAs),effective dehydrationlcryodehydration and successful freezing without lysis difficult toachieve. Current methodologies for cryoprotection utilize CPAs such as dimethyl sulphoxide(DMSO), methanol and glycerol in rapid and slow rates of cooling (Table 2). Thus theCOBRA project has at its core the objective to apply in vitro conservation, cryopreservationmethods to algae and cyanobacteria (see www.cobra.ac.uk). Furthermore, it was recognisedby the EU as a mechanism that could facilitate, and help underpin, the development ofEuropean biotechnology industries.

Table 2. Cryopreservation methods employed for microalgae & cyanobacteria

Method ReferencesSuccess

1 DMSO (5% v/v), or no cryoprotectant;One step ~LN2

2 DMSO (5% v/v), or methanol (10% v/v);Twostepcooling15minat -30°C ~LN2

3 DMSO (5% v/v), or methanol (10% v/v);

Two step cooling 1°C min-1 to - 40°C,then -40°C for 15 min ~LN2

Microcystis aeruginosa,Ch/orella spp

5,8,9

Chlorococcales (250 strains)/other small unicellular algae

9, 15, 16

Eukaryotic Algae (>365 strains) 2,9

THE COBRA PROJECT

Outline structure oj theprojectThe COBRA partners bring a wide range of expertise to the project including: Biological

Resource Centre (BRC) management, cryobiology, algal stress physiology, ecology,molecular biology, electron microscopy and algal biotechnology. In addition, they includerepresentatives of a range of different sectors including: Culture Collections, Biotechnologylndustries and Academia (Table 3). The Project is organised into five interconnected WorkPackages (WPs), some of the on-going activities in these WPs are outlined in more detailbelow.

Table 3. The COBRA consortium

Organisation Acronx.m

Natural Environment Research CouncilUniversity of Abertay DundeeInstituto do Mar., Univ CoimbraUniversit~UG6ttingenInstitute of Soil Biology CASInstitute of Botany CASInstitut Pasteur, ParisAQUAARTIS

NERC/CCAPUADIMARUni-GOEISBlBIP

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Status Country

Gov UKHES UKHES PHES DGov CzGov CzPNP FSME F

~Technology transJerand establishment oj 'cryobanks'(WP 1)

Technology transfer has been a core element ofthe COBRA project which was initiatedwith a Technology Transfer Workshop (18-22 Jan 2002), attended by 44 scientists of whom36 work directly on the project and this number included principal investigators, technicalstaff, post-doctoral researchers and post-graduate students. The focus on technology transferhas continued as a vital element and has been developed via the websitehttp://www.cobra.ac.uk and email discussion groups. Reciprocallab visits between membersof the consortium, as well as consortium meetings/ focus group meetings at conferences andproject meetings have further facilitated technology transfer and "trouble shooting".Experience within the project has confirmed the value of this focus on training and hasprovided some useful training tools that can be adopted for other areas of cryopreservationresearch.

As part of COBRA new cryostorage equipment and facilities have been set up in fourdifferent centres (NERC, Uni-GOE, lMAR and lB) providing national specialist preservationresource centres in each respective country. In addition, the storage facilities at IP wereexpanded. These COBRA facilities have enabled establishment of a rolling program of thecryopreservation of cultures currently maintained in vitro, in five of the specialist Europeancentres (NERC, IMAR, Uni-GOE, lB and IP). Furthermore, these developments incryobanking resource centres have enabled the transfer of key commercial strains fromAQUAARTIS to NERC (CCAP) where they have now been successfully cryopreserved.Excellent progress has been made by the partners and >1500 additional strains have beencryopreserved to date (Table 4) as a direct result ofthe effective training on standard methodsthat has been established in the COBRA project. The most effective method appears to have

been: pre-treatment with DMSO (5% v/v), followed by a two step protocol (1°C min-l to

-40°C, then -40°C for 15 min before plunging in liquid nitrogen. The 1°C min-I cooling ratewas achieved using either a conventional controlled rate cooler, or a "Mr Frosty" passivefreezer. However, large numbers of strains continue to be maintained by routine subcultureand this represents a significant risk to the long term conservation of these organisms.

Total 1693 3109 -50% holdingslNumber of additional strains cryopreserved using conventional and novel approaches.21naddition, a collection of 400 cryopreserved cyanobacterial cultures has been lodged at the CCAPand this will be accessed into the Collection in due course.

Characterization oj the injury events during cryopreservation and the ejjects on cells:assessing current and novelprotocols (WP2)

Whilst empirical application of existing methods can produce some successes, it is vitalfor the preservation of those microalgae which remain recalcitrant to preservation toinvestigate the various elements of cryopreservation methods and thus identify the critical

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Table 4. Cryopreserved algae maintained by the COBRA partners: progress in the first twoyears

Partner No.1 Cel! type Total no. % of algal holdings, cryo. cryopreserved

CCAp2 110 Microalgae & cyanobacteria 711 >60%IMAR 326 Microalgae 326 <40%Uni-GOE 398 Microalgae & cyanobacteria 398 <40%lB 526 Microalgae & cyanobacteria 526 >60%ISB 274 Microalgae & cyanobacteria 274 >60%IP 59 Cyanobacteria 874 >60%

/aspects that need to be optimised. This area of activity has employed investigative physical,microscopic and biochemical tools to elucidate those stages at which critical damage occurs inreca1citrantorganisms and uses this information to optimise protocols for each group of algae.In addition, fundamental knowledge of algal stress physiology has been used to developnew/improved methods for recalcitrant organisms.

Much of the focus has been on the Euglenoids, a group particularly difficult tocryopreserve. Detailed profiling of key cryopreservation parameters has been undertaken ashas the optimization of osmotic, evaporative and chemical desiccation treatments to ensurethe formation of stabl~\ vitrified glasses on exposure to cryogenic temperatures characterisedusing differentia1scanning calorimetry (DSC). As a result, an optimised, generic Euglenoidcryopreservation protocol has now been formulated for Euglena strains, held at threeEuropean centres (CCAP, UAD and Uni-GOE). This was achieved by comparing novelvitrification methodologies (specifically encapsulation/dehydration) with, and without, two-step freezing protocols (passive cooling in the vapour phase followed by plunging in LN2)with and without methanol cryoprotection. This work has stimulated the publication of anoverview ofthis approach (13) and further research-orientated papers are in preparation.

One centre (lB) has successfully applied simple silica gel desiccation pre-treatment(without alginate encapsulation) to Chlorella vulgaris, Nostoc sp., Anabaena flos-aquae,

, Scenedesmus obliquus, and S. acuminatus. Out of 30 strains of soil algae cryopreserved byencapsulation/dehydration by ISB, using osmotic dehydration with sorbitol (0.5 M) andsucrose (0.5-0.75M), 26 species recovered, 13 of them more successfully in comparison withconventionall or 2 step protocols (4). .

Knowledge of the physical properties of storage-recalcitrant algae and cyanobacteria isbeing collated for vitrification protocol deveJopmentusing DSC which studied the water-statebehaviour of alginate bead-encapsulated/dehydrated algae. These properties will be comparedwith those of organisms that reside in habitats that confer adaptive advantages to freezing anddesiccation stress is still in progress. Fundamental knowledge has already been used at UADto improve cryoprotection strategies by manipulating osmotica, changing the metal cationcontent of media to minimise damaging free radical mediated Fenton chemistry and throughthe application of the iron chelating agent desferrioxamine which impairs the production ofhydroxyl radicals that may be raised on recovery of cryopreserved cells. At present, UAD, inassociation with Uni-GOE and CCAP are developing a theoretical model to examine theinteraction between cryoinjury, photooxidation and photosynthesis. This will be tested at theexperimentallevel-using free radical and antioxidant markers.

A visual digital imaging technique (NAJA-lmage Analysis linked to a Graphic GIMPprogramme developed by ISB) is being used to monitor re-growth and morphology ofencapsulated/dehydrated soil algae. This has also been applied to measure changes incWorophyll content in cryopreserved algae. At ISB, cryomicroscopy studies together withtluorescence kinetic measurements revealed that cryoinjury of most cryopreservationrecalcitrant species appeared during cooling to -40°C or even -20°C (4). In parallel, UAD isexamining photoxidative injury in EugJenoids and exploring the biochemical basis ofstrategies to reduce tree radical stress.

Development and assessment oj genotypic and phenotypic post-preservation stability tests(WP3)

It is important to carryout pre- and post-preservation characterisation using phenotypic(morphology; growth rate, growth conditions, metabolite formation) and genotypic (InternalTranscribed Spacer (ITS)-1,2 rDNA sequencing and Amplified Fragment LengthPolymorphism (AFLP)) methods. These indicators are being used to determine the success ofpreservation. For a total ot 37 strains from 5 groups ot microalgae (including cyanobacteria)AFLP patterns have been determined prior to cryopreservation, immediately after thawing

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and after two weeks of recovery under standard culture conditions. To date, no differences inthe AFLP banding pattem before and after cryopreservation have been detected (12). Thiswork is ongoing and is to be published in its entirety in due course. Highly iteratedpalindrome - 1 (HIP1) ext. profiles (6) for the investigation of a large portion of the genomehave been established by IP for cyanobacteria1 strains. This approach allowed inter-specificdifferences to be determined within most of the genera examined and intra-specificdifferences to be identified within Microcystis aeruginosa (6). HIPl ext. profiles have theadvantage over AFLP in that they are independent of bacterial contaminations which mayinfluence AFLP banding pattems.

A number of phenotypic testing procedures to evaluate post-storage performance havebeen developed or assessed during the project. A miniaturized growth measurement (A750nmmeasurement in 96 well microtiter plates) has proved rapid and well suited for axenic as wellas xenie strains of motile, planktonic and filamentous species from different algal groups.Good results were even obtained for species that appear not to be suitable for cell or colonycounting methods. The measurement of photosynthetic capacity through changes influorescence induction (Kautsky effect; measured before freezing and after a 12h recoveryphase in the dark after thawing) has been applied to more than 200 strains covering thefollowing groups: Chlorophyceae, Bacillariophyceae Zygnematophyceae and Euglenophyta(14). It was observed that the photosynthetic capacity of many Zygnematophyceae andEuglenophyta was temporarily lost after thawing, but recovered in those strains that showedgood post-thaw growth.

A digital image analysis method for celllcolony counting developed by IMAR has beenapplied to strains from their culture collection that grow on agar plates (17) and a method forestimating viability based on oxygen production during photosynthesis has been implementedfor strains unable to grow on agar plates. The measurement of photosynthetic activity(chlorophyll fluorescence measurements) of algae at the level of single cells using afluorescence kinetic camera mounted on cryomicroscope (developed by partner ISB anddescribed .above) has been investigated. This approach may also be employed to assessviability of algae in alginate beads and those grown on solidified medium in Petri dishes.

Evaluation oj preservation and genetic stability testprotocols (WP4)The development of new and optimized cryopreservation protocols in specialist research

labs during the first phase ofthe COBRA project (see sections above) was an important step.However, the robustness and repeatability ofthese methods in a broad range oflaboratories isan essential process to confirm or validate their general utility. This validation process wasundertaken by engaging laboratories of varied experience both within and outside thecollaborative COBRA framework (Table 5) and reported (with the aid of WP5 below) to thewider culture collection community outcomes ofthe assessment and validation.

In addítíon, at the end of the project a workshop will be held and this will be open tostakeholders and the user community. The process of validation is not simply a process ofdistríbuting protocols and awaiting results from participants. The participants must own theprocess and be involved in establishing a protocol for the validation process inc1udinghowthe results will be collated, evaluated and published.

The role oj lnJormation Technology to disseminate cryopreservation data (WP 5)It has been an important aim of this project to provide an overarching IT infrastructure

ensuríng that knowledge, expertíse and research outputs are effectively dísseminatedthroughout the project consortium during the project. This has Web-site construction anddevelopment, software to enable knowledge sharing betwcen partner databases (seewww.cobra.ac.uk). Standardised database packages and guidance materials have been providedfor most partners and a prototype centralised guery portal created and tested by partners. In

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addition, Knowledge Engineering Research related to alga1 conservation has been initiated,developed and implemented. Most partners use a common strncture, whieh has simpHfieddata interrogation and research into database query optimisation and mediation has beenundertaken. However, with a general acceptance of a standardised database strneture, the needfor extensive ontology-based intelligent query faci1ities has been avoided. Instead,establishment of experimenta1"cases", and the applieation of Case-Based Reasoning (CBR),to aid the eonservation researeh is eurrently under investigation. It is planned to attaeh anexperimental CBR faci1ityas an artificial intelligence capabi1ityfor uti1isationof the avai1ablereseareh data. It is a1soplanned that the exercise of establishing cases wiU aet as a catalyst toenable reflection on the qua1ity of the data being reeorded and on the strncture of theunderlying databases.

Table 5.protocols

Laboratories undertaking validation of cryopreservation and stability/viability

Validatinglab

Cryopreservation protocols Viabilityl stability protocols

AFLP

ISB

lBIP

ExternalvalidatorExternalvalidator* alternatively,epifluorescence microscopy (cyanobacteria)** commerciallyavailable passive freezing device for use in -70°C freezers

NERCIMAR

Uni-Goe

2 step "Mr Encapsulationl Vital Re-growth KautskyControlled Frosty"** dehydration staining* (CFU) effect

Rate Freezing+ + + + + ++ + +

+

+

+

+

+ + + + +

+

+

+ +

+

+

+

+ +

+ +

+ + + +

+ ++ + + +

+ + + + + +

THEFUTUREIt is planned to maintain the momentum of the COBRA project by building on the soHd

foundations established in the first years of the project. The main objectives being to achievethe Milestones and Deliverables planned (see, www.cobra.ac.uk) and in particular thecryopreservation of major sections of the participating collections, the validation ofpreservation and stabi1ity/viabi1itytesting protocols, the development of novel IT knowledgesharing system(s) and the continuation of fundamental research. In the longer term, andfollowing project completion, the COBRA partnership envisages that the outputs andactivities of the consortium will benefit the wider European conservation community. Thiswill be manifest by the eontinued virtual coordination of a critical mass of expertise andknowledge in cryogenic storage methodologies that will assist conservationists andbiotechnologists. The fundamental knowledge acquired pertaining to molecular stability andcryoinjury will also have generic interest for and relevance to other cryobiological disciplines(e.g. medical, higher plant, aquaculture and animal biodiversity eonservation). Finally, the ITcomponent (WEB-based communications and Knowledge Management and Sharing) of theCOBRA project has the potential to help develop generic "Virtual Infrastructures" as well ascohesive management strategies (see, www.cobra.ac.uk). These may be applied as "models"

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to assist large-scale and dispersed European partnerships and the expanding EuropeanResearch Arena. In addition to products and processes directly derived from microalgae andcyanobacteria, they offer potential expression hosts for biotechnology products. Thedevelopments within the COBRA project are overcoming some of the critical technologyhurdles that currently obstruct the provision of good quality cryopreserved stocks of cells thatwill be vital to support the use of microalgae in biotechnology. This includes the ability tostore samples in support of patent applications.Acknowledgements: The authors thank the EV (Contract No. QLRI-CT-2001-01645) forfinancial assistance and all those involved in the COBRA project including: Jane Pearson &Christine Butterwick (NERC); Claire Johnstone & Isobel Pimbley (VAD); Nuno Laranjeiro,Hugo Osorio & Raquel Amaral (IMAR); Julia Mueller, Hella Timmermann, Dr Dierk Mende& Dr Dominik Hepperle (SAG); Marie Sabacka, Marek Stibal. Katerina Machova & DrVladislav Cepek (IB); Pavel Hrouzek, Tomas Hauer, Jan Kastovsky & Martin Lukes (ISB);Dr Katia Comte (IP).

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2. Beaty MR & Parker BC (1992) VA.J. SeL 43,403-10.3. Benson EE (1999) Plant Conservation Biotechnology, London: Taylor & Francis.4. Benson EE (2003) in COBRA Year 2 Report (ed) Day JG. FPV Contract no. QLRI-CT-

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81hInternational Conference on Applied Algology, Monteeatini Therme -Italy 16 Sept-1 Oet 1999.

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14. Lorenz M, Miiller J, Mende D & Fried1 T (2003) in Abstracts oj The Third EuropeanPhyeological Congress, Belfast, 1reland,21-26 July 2003.

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Bristol: Biopress Ltd.Acepted for publication 22/6/05

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