Karolina Koc-Michalska

Darren G Lilleker

Pawel Surowiec

Pawel Baranowski

Poland's 2011 online election campaign: new tools, new professionalism, new ways to win votes

This paper analyses the use of the online environment within the context of the Polish parliamentary election of 2011. Using traditional methods of content analysis we find that parties tend to adhere to a professionalised model of campaigning, adapting online tools to suit the objectives of the campaign. There also appears to be a recognition that their most likely visitors would be converts and so they attempt to mobilise supporters rather than convert browsers. New parties and candidates are more likely to target browsers; the latter offering a more personalised experience to online visitors. Importantly, when analysing the outcome of the contest we find that being online matters for candidates when controlling for all other variables. Equally the reach the candidate has, which may well influence their vote share, is dependent on offering a more personalised, representational image and having a frequently updated online presence that should encourage repeat visits. Cumulatively we suggest the future of online campaigning must not only focus on having a presence but using it in a way that appeals to a range of visitors, encouraging repeat visits, and that this strategy could have a positive impact on election outcomes.

Poland's 2011 online election campaign: new tools, new professionalism, new ways to win votes

Professionalization and elections in Poland

The professionalization of political communication is an evolutionary process [START_REF] Lilleker | Professionalisation: of what? since when? by whom?[END_REF], a process that sees political actors adapting to trends in communication in order to better engage and persuade the public and gather supporters and voters. One of the most interesting recent developments in communication has been the move towards social communication via the Internet.

The Internet is argued to affect every area of public communication, from commercial advertising and public relations to education [START_REF] Macnamara | The 21 st century Media (R)Evolution[END_REF]. Similarly the Internet is now embedded as a campaigning tool and digital interactive technologies have been adapted to the norms of political communication (Ward et al, 2008;[START_REF] Lilleker | Informing, engaging, mobilising or interacting: searching for a European model of web campaigning[END_REF]Lilleker & Jackson, 2011). Despite optimistic predictions that the Internet would reinvigorate democratic participation (Coleman & Blumler, 2009), technology is employed to meet campaign objectives and so party websites tend to offer limited interactivity (Kluver et al, 2007) and opportunities for co-production only under highly managed conditions [START_REF] Lilleker | Empowering the citizens? Political communication, coproduction and the harnessed crowd[END_REF].

Poland is interesting as a case study to extend analysis of the role the Internet plays in a campaign as the debates surrounding the impact of technology chime well with debates around the quality of democracy and civil society. Assessments of democratisation in Poland have centred around perspectives of civic life being 'half full' [START_REF] Roberts | The quality of democracy in Eastern Europe: public preferences and policy reforms[END_REF], or 'half empty' [START_REF] Mokrzycki | The Lost Dynamics. On the immaturity of Polish Democracy[END_REF][START_REF] Ost | Imagining and creating the enemy: trade unions in the new polish democracy[END_REF][START_REF] Pachulska | Polish democracy in transition[END_REF]: the former argues there is progress in democratic revival; the latter highlights the shortcomings of democracy in Poland. One deficit within the progress of democracy is that elections have become a 'promotional culture' spectacle [START_REF] Wernick | Promotional culture[END_REF][START_REF] Cwalina | Political Marketing: Theoretical and Strategic Foundations[END_REF] as opposed to a time of civic engagement. Audits of election campaigning (e.g. Instytut Spraw Publicznych, 2011) reveals that political parties in Poland engage in online political communication and, on a surface level, this 'communicative' development can be interpreted as an attempt to shift the focus of the political parties' communication in Poland towards interaction and engagement with, and mobilisation of, the Polish electorate [START_REF] Jakubowicz | Democracy and New Media, Keynote speech to the Polish Communication Association Conference, Political Communication in the era of new technologies[END_REF]. Internet usage by political parties and organisations has been conceptualised by Polish academics as a movement towards 'e-democracy' [START_REF] Grodzka | Społeczeństwo informacyjne[END_REF], yet this would entail more participatory and interactive behaviours being facilitated. Studies have shown that Polish parties lagged in this respect in the 2009 European parliamentary election (Lilleker et al, 2011), yet this was deemed to be a contest taken less seriously by campaigners [START_REF] Maier | Political Communication in European Parliamentary Elections Farnham[END_REF].

Poland is fertile for more focus to be given to the online dimension of the election campaign. [START_REF] Trammell | Rzeczpospolita blogów [Republic of Blog]: Examining Polish bloggers through content analysis[END_REF] report that since 1996, Internet penetration has been steadily increasing and reached a moderate average, compared to other European nations. Recent data (Eurobarometer Autumn 2011)revealed that the overall web penetration rate in Poland is 59% and is slightly below the EU mean (64%). Trends in social networking mirror developments in Western Europe and the US. Social Bakers (2011) find the number of Poles using Facebook grew by 79% over a six month period with over 2.4 million new users creating profiles. There are over 5.5 million Polish profiles on Facebook at the moment, which makes it the seventh largest country in Europe on Facebook1 . Alongside Facebook there are some 13.9 million Poles with profiles on the Polish social networking site Nasza Klasa.

Our paper focuses on the extent to which the Internet has a strategic role within the campaign through a content analysis of party and candidate websites at the 2011 general election. We explore how the Internet is used, who communication is aimed at and how it contributes to the election campaign communication and the contest's outcomes.

Professionalization and online campaigning

In the majority of advanced democracies, the Internet represents a fairly low cost way of reaching a significant amount of the population; therefore unsurprisingly it is now a feature of every election campaign. Early predictions that the nature of online communication, in particular that anyone can publish and interact with other users, could lead political communication away from an elite-centred broadcasting model were soon confounded. [START_REF] Margolis | Politics as Usual: the cyberspace revolution[END_REF] produced the first study of political communication online and found that this represented what they and subsequent scholars have referred to as politics as usual. Offline inequalities are directly reflected online, and smaller parties are least likely to have a website and use less sophisticated Web 2.0 tools [START_REF] Resnick | Politics on the internet: The normalization of cyberspace[END_REF][START_REF] Xenos | Politics as Usual or Politics Unusual? Position taking and dialogue on campaign web sites in the 2002 U.S. elections[END_REF]Kluver et al., 2007). Furthermore, the online dimension of a campaign tends to be designed to inform and engage browsers, mobilise supporters but offers as few opportunities for participation as the offline campaign. Interaction is allowed within private spaces, via email or online forms, but not the public interaction many engage in peer-to-peer within social networks. Scholars suggest that such uses of the Internet are inappropriate [START_REF] Kalnes | Norwegian parties and Web 2.0[END_REF]. The argument is that the online users likely to visit political campaign sites are sophisticated browsers and enjoy participating (sharing and creating). Static, non-interactive sites will be unappealing and of limited interest.

Low engagement on official party online presences does not mean that online conversations will not take place about the election, the general campaign or specific party campaigns. Due to the granularity of communication across online platforms, political communication exists within an ecosystem and the browsers experience of the campaign online will likely be co-created [START_REF] Chadwick | Recent Shifts in the Relationship Between the Internet and Democratic Engagement in Britain and the United States: Granularity, Informational Exuberance, and Political Learning[END_REF]. However, parties and candidates can choose to 'harness the power of the crowd' and allow their online supporters to add to and enhance the campaign [START_REF] Jenkins | Fans, Bloggers, and Gamers: Exploring Participatory Culture[END_REF] if they wish. The question is whether developing an interactive strategy signals a new form of professionalism in campaigning [START_REF] Howard | New Media Campaigns and the Managed Citizen[END_REF], one which conforms to the norms of online culture and communication, or whether the norms of the controlled professionalism associated with media management [START_REF] Gibson | Changing Campaign Communications: a partycentred theory of professional campaigning[END_REF] are transferred into online environments?

Literature on the professionalization of political communication argue that campaigns have become centralized strategically and have a sales oriented character, focused upon converting and persuading voters while also getting loyal supporters to the ballot box on election day. Campaign messages tend to be targeted, narrowcasted via mass media as well as direct channels of communication such as email, online forums and intranets [START_REF] Norris | Preaching to the Converted? : Pluralism, Participation and Party Websites[END_REF]. In addition, organizations adopted a more bifurcated strategy for their campaigning; while the central campaign command set out the core messages, communication is also the responsibility of local organizations, in particular the use of local email lists, intranets, forums [START_REF] Gibson | Changing Campaign Communications: a partycentred theory of professional campaigning[END_REF][START_REF] Katz | The ascendancy of the party in public office: party organizational change in twentieth century democracies[END_REF][START_REF] Norris | Preaching to the Converted? : Pluralism, Participation and Party Websites[END_REF]. Local organizations are also deemed responsible for using social networking and microblogging tools to reinforce and make locally relevant the national campaign messages. Professionalization has therefore necessitated a shift in organisational behaviour, one perhaps driven both by new communication technologies as well as broad social changes. Campaigns have clearly been adapted to a digital media landscape characterized by "abundance, ubiquity, reach and celerity" (Blumler & Kavanagh, 1999: 213). However, it is argued that even in 2011, it is the 24/7 mass media that remains dominant for campaigns, even within the US, yet we find new ways of characterizing campaigning that are designed specifically for the integration of the online environment.

Arguably, political communication and election strategy must, in the age of online social communication, ensure any single item of content is designed for multiple forms of consumption and disseminated in ways that can be collected by journalists, supporters or web browsers alike at multiple communication junctions [START_REF] Howard | New Media Campaigns and the Managed Citizen[END_REF][START_REF] Johnson | Campaigning for president 2008: strategy and tactics, New Voices and New Techniques[END_REF]. While there will be an informational component within communication, a range of interactive actions must be facilitated to reach the online political browser. Items created need to facilitate ease of sharing to permit messages going viral across the Internet [START_REF] Boynton | Going Viral -The Dynamics of Attention[END_REF] and can be commented on and adapted within the campaigns' ecosystem. As Howard (2006: 2) argues in describing the core tenets of hypermedia campaigning, parties and candidates must allow for and expect the "decomposition and recomposition of messages". These communicative processes permit co-ownership of communication across a wider agora and for reach of messages to be multiplied across networks.

In order to meet campaign objectives it is argued, the hypermedia campaign strategist must harness the online and offline information elite simultaneously and create a synergistic communicative process between nodes within the network. Online actions by political actors (a post to Twitter for example) feed into communication by online and offline communicators (journalists and bloggers) and these draw hits to other online features such as a campaign website which generate further sharing or interaction, which in turn can create broader offline and online attention, or resources in the shape of volunteers of donations. The hypermedia campaign is thus the response to the 21st campaign communication environment, it recognizes that to be successful one must both create a community as well as joining the communication ecosystem (see also [START_REF] Lilleker | The Internet in Campaigns and Elections[END_REF].

The 2008 campaign for the US presidency by Barack Obama perhaps exemplified the shift from post-modern [START_REF] Norris | Preaching to the Converted? : Pluralism, Participation and Party Websites[END_REF] to hypermedia campaign, post-modern campaigning focuses on targeted communication via all available media; the hypermedia campaign also receives data, in term of harvesting contact details but also feedback. Yet there appeared only superficial copying of the Obama model (Lilleker & Jackson, 2011;Lilleker et al, 2011), perhaps due to resource gaps but also due to caution around opening the campaign up to the online audience [START_REF] Stromer-Galley | Online interaction and why candidates avoid it[END_REF] Despite the arguments for adopting a full hypermedia strategy we hypothesise, based on previous studies of 'web campaigning' [START_REF] Xenos | Politics as Usual or Politics Unusual? Position taking and dialogue on campaign web sites in the 2002 U.S. elections[END_REF]Kluver et al, 2007;Ward et al, 2008;Lilleker & Jackson, 2011), and studies of election campaigning in Poland (Lilleker et al, 2011), that: H1: Polish political parties and candidates will evidence a hypermedia strategy but will focus on delivering information though engaging communicational tools and attempt to mobilise their supporters through their websites, but interactivity will be private or highly limited and controlled. H2: Within each category of communication style (informing, engagement, mobilisation, interaction) based on assessments of the opportunities potentiated for visitors, party and candidate websites will demonstrate widespread usage of a narrow range of features that focus on persuading and extending reach. Co-production, if permitted, will be highly controlled.

Within a professionalized, hypermedia age we would also expect parties to recognise who would be most likely to visit their sites and target those audiences specifically. [START_REF] Norris | Preaching to the Converted? : Pluralism, Participation and Party Websites[END_REF] finds that party websites tend to 'preach to the converted', so attracting loyal supporters, members and existing activists. Subsequent research [START_REF] Norris | Getting the message out: A two-step model of the role of the internet in campaign communication flows during the 2005 British General Election[END_REF] suggested the converted play the role of mediator, that campaigners could use the converted to share their communication and extend their reach within online networks. We suggest that there are a number of audiences that may be targeted during an election campaign and develop a coding scheme for detecting the extent to which sites are designed to be targeted or catch-all.

The first audience would be random browsers who may stumble across a site and would only stay if they are engaged by the content. High levels of eye-catching content and entertaining features at the front end of a website, as well as relevant and personally interesting content, will indicate browsers as a target audience [START_REF] Spink | Information-seeking and mediated searching. Part 1. Theoretical framework and research design[END_REF][START_REF] Marchionini | Exploratory search: from finding to understanding[END_REF]. Secondly, information seekers who would visit for professional reasons, in particular journalists seeking position statements, news feeds or similar simple ways for finding information or having it delivered directly (Panagopoulos, 2009: 7-8;[START_REF] Erickson | Campaign websites and hypermedia campaigning: lessons from the Ed Balls Labour leadership campaign[END_REF]. The third group we refer to as issue activists, individuals who want specific policy information and perhaps wish to interrogate party members on their position regarding a specific area of policy. In the case of candidates these may be local political activists and campaigners, at a national level outside lobby groups who do not have direct access to senior politicians or advisors but that parties or candidates may attempt to recruit [START_REF] Cober | Recruitment on the Net: How Do Organizational Web Site Characteristics Influence[END_REF] using specific forms of informative and interactive communication [START_REF] Stutzer | Political participation and procedural utility: An empirical study[END_REF].

The fourth group are supporters, the converted, these would be targeted with persuasion geared to bringing them closer to the campaign and would possibly be the main group for whom interactive mechanisms are designed (Norris, 2003: 42;[START_REF] Gerodimos | How to Mobilise Young People: Recommendations for NGOs and Civic Organisations[END_REF]. The fifth and final group are activists to whom most tools that aim at mobilisation are targeted and who may be especially active during the campaign [START_REF] Greer | Cyber-Campaigning Grows Up: A Comparative Content Analysis of Websites for US Senate and Gubernatorial Races, 1998-2000[END_REF]Lilleker & Jackson, 2011). Based on the literature on campaigning, however we hypothesise that: H3: Party and candidate websites will include features to appeal to each possible audience, however concentration will be within features most likely to supply information for journalists and campaign activists in order to extend the reach of the campaign.

Also consistent with professionalised campaigning is the concept of I-branding; the way that an organisation can communicate its brand personality through the use of communicational acts. We suggest that any online platform can have specific functions which relate to brand positioning and personality development [START_REF] Jackson | Interactivity and Branding, public political communication as a marketing tool[END_REF]. We argue that there would be three main strategies. The first is a pure sales strategy which is designed to target browsers and supporters through the use of engaging but persuasive devices [START_REF] Rohrschneider | Mobilising Versus Chasing: how do you parties target voters in election campaigns?[END_REF][START_REF] Neys | Political Internet games: Engaging an audience 2010 25[END_REF]. The second is one of personalisation [START_REF] Langer | A historical exploration of the personalisation of politics in the print media: The British Prime Ministers (1945-1999)[END_REF] which, for a party website, would focus on the leader or key figures within the party or movement, for a candidate it relates to self-presentation and perception management [START_REF] Goffman | Presentation of Self in Everyday Life[END_REF]Jackson & Lilleker, 2011). The third strategy is one of e-representation. This focuses on governance and representative issues and links to communication that targets specific supporter groups or issue activists but concentrates on demonstrating a record of delivery [START_REF] Coleman | Connecting Parliament to the public via the Internet Information[END_REF][START_REF] Small | Are We friends Yet? Online Relationship Marketing by Political parties[END_REF]. Given the literature on party campaigning and political marketing [START_REF] Small | Are We friends Yet? Online Relationship Marketing by Political parties[END_REF] and the candidate campaign strategies (Lilleker, 2007) we hypothesise: H4: Party websites will adhere to the key tenets of professionalization and concentrate on following a sales strategy within the online environment. H5: Candidate websites will be highly personalised and offer insights into the character and background of the individual representative while mirroring the overall sales campaign of their respective party.

The research largely contributes to understandings of how the Internet is used within the context of an election campaign, however we also seek to determine if an impact on voter choice can be determined. Focusing only on the websites of candidates, as there is sufficient number to perform regression analysis, we test whether there are any differentials in vote share from having a website (which we use as a dummy variable for online performance) when controlling for visibility in both online and offline traditional media. We therefore test whether candidates who are proactive online also gain coverage in the traditional media [START_REF] Negrine | The Rise of a Proactive Local Media Strategy in British Political Communication: clear continuities and evolutionary change 1966-2001[END_REF] and so gain higher visibility and a greater share of the votes. We also examine whether adherence to particular communication strategies, in particular those which support online community building, have any impact upon vote share. While this is best tested using real-time recording of voter interaction with citizens [START_REF] Baines | Measuring communication channel experiences and their influence on voting in the 2010 British General Election[END_REF], this was not possible and studies have tended to focus on push communication such as television news as opposed to comparing these to the pull communication online. Our approach is to build a general measure for visibility online and offline to test for any relationship with election outcomes. Our analysis is informed by a working hypothesis: H6: Candidates with a web presence, and who use the online environment to reach out to new voters and supporters, will gain greater support at the polls independent of the party they belong to or the visibility they gain in other media.

Analysing online political communication strategies

Our methodology draws on the longstanding and well-tested feature counting methods developed by [START_REF] Gibson | A Proposed Methodology for Measuring the Function and Effectiveness of Political Web-Sites[END_REF] with features added in order to accommodate the changing usages of the Internet. However, to understand the professionalization of online political campaigning it is necessary to move beyond simple categorisations in order to gain an understanding of the strategies which underpin website development.

Like previous studies [START_REF] Gibson | A Proposed Methodology for Measuring the Function and Effectiveness of Political Web-Sites[END_REF][START_REF] Foot | Web Campaigning[END_REF]Kluver et al, 2007) we divide features into providing information, being engaging, facilitating interaction and being aimed at mobilisation. However, when studying campaigning within a hypermedia era we need to go beyond these. We therefore focused on determining which of our features were most likely to be used to target specific types of website visitors; and which features best indicated adherence to our strategies of sales, personalisation and e-representation. We therefore focus on measuring strategic performance, building two new dimensions for the study of online election campaigning: targeting audiences and branding strategy.

Our data is developed from a content analysis of 172 candidate and 11 party websites.

For our analysis we chose candidates from all eleven Polish political parties who were on the top of the regional lists, a total of 331 candidates of which 172 had websites.

There are 41 electoral regions and 7 parties were present in all regions. We had to make a methodological choice which candidates should constitute a sample. Our sample design was driven by the assumption that those who topped their party lists would be the most active politicians and most likely to have an online presence due to their chances of being elected and their access to greater resources as well as being most representative of a highly professionalised communication strategy in Poland.

Content analysis was conducted one week before election day (1 st -7 th October), all websites were also archived2 . All the updates (number of entries, number of friends and followers were counted within two days of the elections). The content analysis identified the presence or absence of 89 features. The websites were coded by three coders, all coders passed inter-coder reliability tests (Cohen's Kappa (.72) and Krippendorff's Alpha (.72)), any irregularities were checked and corrected.

Categorising features as potentiating experiences, in particular engagement, is complex. Any new layer of analytical complexity raises epistemological issues.

Features in themselves are a priori in their ability to be communication events. The way that a feature is embedded by the creator determines how its use is intended; however actual usage, either as a perceptual or behavioural influence, is the responsibility of the individual visitor. Given our focus at the strategy or supply side, we propose that we can develop an understanding of what was intended based on study of the website as a series of communication events [START_REF] Jackson | Interactivity and Branding, public political communication as a marketing tool[END_REF]. Some researchers argue that the only way to discover the strategic intentions of the creators of political communication is through in-depth interviews (Vaccari, 2008a).

The danger here is that interview data can include a degree of post-hoc rationalisation based on outcomes, and strategists can play up or down their input and intentions based on failure or success [START_REF] Lilleker | Interviewing political elites: navigating a potential minefield[END_REF]. The true way for understanding the processes that underpin particular communication tactics is through observation, a highly time-consuming and complex procedure that depends upon gaining the trust of all those being observed. As [START_REF] Nielsen | Ground Wars: Personalised Communication in Political Campaigns[END_REF] argues gaining access is difficult, in particular to meetings where decisions are actually made. We argue that the website as an artefact for research is a static instantiation of strategy (Xenos & Foot 2000). In other words by understanding what features are utilised through the analysis of design of a party of candidate's online presence, and how features play specific roles within shaping users' perceptions and experiences, we can gain significant insights into the strategic role of the Internet within a campaign and how this could contribute to higher civic engagement within the context of an election.

The categorisation of features involved a series of discussions between the authors and other researchers involved in a range of projects (Lilleker & Jackson, 2011;Lilleker et al, 2010;Koc-Michalska & Lilleker forthcoming). We also conducted concept testing with web design specialists working within the Centre for Excellence in Media Practice at Bournemouth University. This delivered a categorisation strategy which permits us to understand how features can be read as indicators of specific strategies. The challenge is where features belong to multi categories, in particular when assigning these as targeting specific audience groups. However, through concept testing alongside data collection we determined that it was not a problem that not all features were discrete to specific categories and could apply to more than one targeting strategy.

In order to make direct comparisons between different parties we develop what we entitle an average online performance score (AOP), a commonplace tool in website analysis [START_REF] Vaccari | Surfing to the Elysee: The Internet in the[END_REF]. The AOP score was calculated by initially counting the number of features present for each category to create an overall mean, we then divided the mean score for each category by the maximum possible score. This technique allows us to compare performance within different categories of features (as each category contains a different number of features).

Measuring professionalization for online Polish election campaigns

The overall measures of performance (AOP) for parties and candidates (shown in Table 1) show two differing but compatible strategies at play within Internet usage during the 2011 Polish general election. Party sites offer a plethora of features and so a more sophisticated and aesthetically pleasing experience to visitors. Candidate sites are more basic, on average, with fewer features. Party sites also offer a richer range of experiences; there are a high number of opportunities for interaction and there is a clear focus on a strategy of mobilisation of supporters and activists. Information provision and engagement are balanced, suggesting that a significant amount of information was delivered using tools deemed as engaging, offering a rich, informative and persuasive experience with a range of opportunities to play a role in the campaign. Candidate websites tend to be informative; they also present information using engaging tools of communication and offer some opportunities for interaction. Candidates are less likely to engage with mobilisation strategies. The complementary strategies are likely to be reflected in who we detect parties and candidates expect to be visitors to their websites. Polish party websites predominantly provide information on their programme and values as well as offering a regularly updated news feed. Candidate websites have similar news feeds backed by information about their careers, education and backgrounds. Therefore, information appears to focus on positioning the party and candidate and aiming to persuade. The persuasive strategy also informs how information is delivered through more engaging forms such as video, with 10 out of the 11 parties and 95 candidates having a YouTube channel. Other popular engaging features are links to Facebook and Twitter, embeds from social networking, and features that allow sharing of content. In terms of mobilisation, eight parties sought donations and six of those had an online campaign that supporters could join.

Candidates mobilised their online visitors to communicate to them and for them only, relying on sharing via social networks. A similar difference can be found for the use of interactive features. All parties permitted contact through email or an online form, interactivity was then permitted through the use of social networks. Only two parties permitted visitors to make comments on their websites and so allowed debates to take place in public online within their own webspace. Candidates relied entirely on the free interactive spaces of Facebook used by 61%, YouTube by 55% and Twitter by 21%. The widespread use of free social networks by a total of 109 candidates and eight parties suggest that visibility is the prime driver, that both parties and candidates want to reach into the networks of as many online users as possible. Interactivity, for most candidates and parties in Poland, is an almost unavoidable by-product of having a presence in the spaces where people congregate.

The average online performances for targeting different audiences reinforces the notion of complementary but differing strategies and fits well to the overall strategic use of features by parties and candidates. As shown in Table 2, party websites do concentrate slightly more on issue activists and campaign activists, as well as general supporters, fitting with the focus on mobilisation. Issue activists tend to be served mostly through focused content provision; campaign activists through requests to act as well as spaces to go to receive material to share. Browsers are also well served, through the provision of front end sophistication such as videos and embedded materials from social networks, but mainly the targeting of browsers reflects the use of engaging ways for delivering persuasive information. Perhaps unsurprisingly a large number of features provide resources for professional information seekers.

Information can be requested, via email, or found onsite. Regularly updated rolling news, with RSS feeds, was used by the larger parties in an attempt to lead the news agenda.

Candidates appear to design sites that will appeal to their potential constituents who might browse the Internet seeking material to aid their voter choice. Alongside the personal details, many candidates offered similar features to parties such as embedded Facebook profiles or Twitter feeds, videos from YouTube or mainstream media and links out to their social network profiles. Targeting general browsers may not be the best use of resources but candidates seem to focus on this group while also providing information, largely about themselves as individuals, for journalists and those seeking general information. The overall strategies of parties and candidates (table 3) partially support our hypotheses and again reflect divergent but complementary strategies. As predicted parties focus mostly on using persuasive communication, however e-representation does not lag far behind. The balance between persuasion and e-representation reflects the shift towards the use of features that permit interaction which share space with engaging information provision. Intuitively one may expect there to be different strategies by parties within the coalition government and those in opposition.

Challengers tend to focus more on persuasion, often attacks on governing parties, while incumbents have to focus on their record. This was not the case in Poland. All parties offered a balance of persuasion and e-representation, but with a sales strategy predominating. Candidates offer a more personalised identity than parties, while balancing this with personalised information with a persuasive spin. Candidates foreground details about their careers and education, so positioning themselves as being credible potential representatives, as well as their backgrounds, family life and hobbies. The latter aspects reflect what is referred to as the demotic turn in society that has filtered into politics [START_REF] Turner | Ordinary People and the Media: The Demotic Turn[END_REF] where personal characteristics are used as part of a sales strategy. Ordinariness is used metaphorically to suggest being in-touch, of the people;

an elitist lifestyle, education or interests can be negatives [START_REF] Pels | Aesthetic Representation and Political Style[END_REF]. The personalisation is reinforced by aspects of a sales campaign that utilise the Internet to talk directly to voters. Prominent tools that permit a more direct, conversational style of communication were YouTube and the widespread use of social networking platforms.

The Polish Internet elections: making sense of web use and its impacts

As we predicted (H1), Polish political parties and candidates show evidence of a hypermedia strategy which focused on delivering information though engaging communicational tools matched with attempts to mobilise their supporters. However, due to the widespread use of social networking tools, interactivity is both private and controlled and public and controlled only through moderation of visitor's posts to their profiles. The use of social networking tools therefore lead to a shift from the topdown, politics as usual, communication style to one that potentiates a degree of participation. Similarly while the most commonly used features which deliver differing experiences (informing, engagement, mobilisation, interaction) are from a narrow range (H2), due to the use of social networking tools it is no longer possible to manage all possible forms of co-production. However, behaviours that are encouraged related more to extending reach through sharing rather than contributing to political debates.

In terms of targeting, party and candidate websites met expectations supplying features that appeal to the widest possible audience (H3), though, as hypothesised party strategists seemed to recognise the importance of mobilising supporters and activists. This strategy may reflect a more sophisticated approach to online campaigning, possibly one that has learned lessons from the Obama campaign. We suggest, therefore, that the Polish online campaign adhered to the central tenets of professionalization (H4) concentrating on a persuasive, sales strategy. The sales approach is matched on candidate websites (H5) which were highly personalised offering insights into the character and background of the individual representative.

The fact that over half of the profiles went beyond the traditional career curriculum vitae to include personal interests and lifestyle information as well as a range of pictures depicting them as both political actors and ordinary people suggest they believe that personality politics matters. This belief may be erroneous given they are elected with a party list system, however, candidates may well see a value in creating sophisticated web presences to demonstrate their credentials to both potential voters and party officials on whom they depend for their position on the list and promotion within the party [START_REF] Negrine | The Rise of a Proactive Local Media Strategy in British Political Communication: clear continuities and evolutionary change 1966-2001[END_REF].

Determining the impact of online campaign performance We attempt to explore the possible effect of different communication tools on two variables measuring the candidates success: vote share in elections (for all candidates in our sample, N=331)

and community reach (in terms of number of supporters) earned by candidates on different social network sites (friends and followers on Facebook and Twitter, for those who have online presence N=171). A set of explanatory variables is used: personal (in order to control for personal characteristics of each candidate (age, gender, years of incumbency)); party characteristics (to control for characteristics relating to the parties that candidates belong to (size, political ideology, party years of existence)) and campaign characteristics. Campaign variables control for spending of the party for each candidates (as the individual spend is not published in Poland this is a proxy created from the general amount of money spent by party divided by the number of candidates that the party proposed), frequency of coverage in traditional media (both online and offline editions) on the national and regional level in press, radio and television during the most intense period of campaigning, the final two weeks before the election day. Finally we use two different variables for online communication: simple online presence of the candidate (having/not having a website) as this represents a simple measure of interest in communicating online with potential supporters in elections; and online strategies (e-representation, personalisation and sales/persuasion strategy) as we suggest these meta-strategies will determine how browsers or supporters are targeted online and so determine the size of the online community gained.

Using regression (Table 4) we find, as would be expected, that incumbency (being a majority party in the parliament) and number of terms for incumbency, are key predictors of election outcomes. Given that these are candidates at the top of the party lists any other outcome would be counter-intuitive. None of the personal, demographic characteristics have predictive power. Candidates representing the parties with the longest histories (some created even before WWII) as well as those representing more right wing oriented parties have lower potential to gain votes. The proxy for party spending per candidate has no statistically significant impact.

Similarly there is no significant effect found for the level of coverage in the press or on radio. The only traditional media having any statistically significant impact on outcome is television, however there is a negative impact of coverage on national television but a positive effect for gaining coverage on regional television. This finding may appear counter-intuitive but is understandable in the Polish context; politicians who appear on main national television programmes are presented as either celebrities or cronies, the latter suggests involvement in corruption, rather than hard working representatives. While controlling for all the above variables (also in multilevel regression) we find that having an online presence is consistently a strong predictor of gaining a greater share of votes. Having an online presence, we therefore suggest, is a factor of gaining wider visibility based upon a proactive communication strategy and that the Internet may well play a key role in being visible, if only due to the remediation of online material [START_REF] Norris | Getting the message out: A two-step model of the role of the internet in campaign communication flows during the 2005 British General Election[END_REF] both by journalists and those within the online network of a candidate. .6639 Note: OLS regression. Dependent variable: % of vote share in 2011 elections (continuous) for 331 candidates (list leaders). Independent variables: gender (dummy, 1=women, 0=men); age (in years); Years of incumbency (number of terms in Parliament, continuous, 0 to 7); Web penetration rate in the region where candidate is present; Size of the party that candidates belong to (major vs. minor, fringe as reference); Political ideology of the party that candidate belongs to ((1=left to 11=right); Party years of existence (continuous). Campaign spending (money spent by party during campaign divided by number of candidate presented); presence in the traditional media (continuous, data from PRESS-SERVICE Monitoring Mediow); Online presence (dummy, 1 = have website, 0 = do not have website). We also find interesting relationships between the online strategies used, the stickiness (or frequency of updating) and the number of followers, friends or fans accumulated. Firstly, exploring whether a proactive online communication strategy had value in earning reach, we tested for correlations between the frequency of updates and the number of followers, fans or friends earned on their social media platforms. Using Spearman's rho correlation we find that the relationship between update frequency on Nasza Klasa and the number of followers is ranked at .670 (p<.000) for candidates and .857 (p<.000) for parties [for Facebook this is .831 (p<.000) for candidates and .573 (p<.000) for parties]. Usage of Twitter shows an even stronger relationship of .914 (p<.000) for candidates and .859 (p<.000) for parties. This suggests that a highly proactive communication can result in accumulating a following, one which may also have an electoral dividend. The Pearson's correlation between vote share in 2011 and community size was: for Facebook .407 (p<.000) and .376 (p<.000) for Twitter (however there was no statistically significant relationship for Nasza Klasa).

We also ran regressions with online strategies as possible factors influencing number of supporters. These data indicated the internal relationships between the online communication strategy and the reach of potential network enjoyed by a candidate.

Interestingly, alongside frequency of updates on social networks, we find a personalisation strategy has the highest dividend in terms of reach. We suggest that this is related to the fact that social networks are places for individuals to meet and that corporate style profiles do not engage the users of these platforms in the same way as one that is personalised. Similarly, where contact is permitted and conversations are allowed on policy issues or local politics, all of which fit with an erepresentation strategy, there is a relationship with the reach gained. If a sales campaign predominates, the candidate does not gain as large a following on social networks. Beyond the variables relating to strategy, only party size and the size of electoral (which seems obvious) show clear predictive power. Again, the party size variable would be expected to have power as it may govern resources, so determining levels of proactivity a candidate can pursue. participation that can only be moderated (Kalnes, 2011). The requirement for giving attention to these profiles, for both frequent updating and moderation, puts parties and candidates with fewer resources at a disadvantage. Resources enable greater proactivity in their communication; and using platforms popular among Internet users necessitates more interactive modes of communication. Whether parties and candidates will focus mostly on content creation, and relax attempts to moderate coproduction, is a strategic issue. The embedding of social networking into political campaigning may lead to more interactive forms of electioneering, it appears still a little early to tell. The imbalances in the use of the online environment we detect suggest resources lead to evidence of the same inequalities witnessed in many more developed democracies.

The use of the Internet in the Polish election of 2011 arguably allowed a range of ways in which Internet users could access information, much of which was persuasive, but also ask questions publicly and privately of parties and candidates.

Supporters, whether established activists or recent converts, were encouraged to play a role in the campaign; in particular the public display of support was encouraged.

Mobilisation tactics focused more on receiving donations than enlisting volunteers, but the clicktivist activities that were requested (Share or Like) are public and may lead to stronger levels of engagement. Though sporadic, the use of weblogs can also lead to much stronger forms of engagement with issues, rather than more simplistic content such as public appearances which candidates may promote through sharing pictures or links to news stories. Weblogs, or any spaces to comment on policy, were limited. This reflects a widespread caution among political parties to open up public discussion too far and face demands to alter policy due to coming under attack from an online flashmob [START_REF] Stromer-Galley | Online interaction and why candidates avoid it[END_REF]Lilleker et al, 2009). Thus, Poland evinces all the features of many democracies where parties and candidates are metaphorically 'dipping their toe' [START_REF] Jackson | Dipping their big toe into the blogosphere: the use of weblogs by the political parties in the 2005 General Election[END_REF] into online environments to gauge if there are benefits. The party tendency was to launch a sales campaign designed to appeal to activists and supporters, candidates follow similar strategies but aiming more at browsers. However, to win over voters it appears a more e-representational and personalised strategy, one which demonstrates a frequent presence online willing at least to keep their following informed not only about macro-policy issues but also the minutiae of the campaign. It seems social networks are best placed for such a strategy and so could be seen as positive for democracy, but wider usage for policy discussion with a greater number of Internet users is required prior to accolades and predictions of the birth or rebirth of a form of e-democracy. Being online may have a positive impact on voter choices between candidates but this may reflect that this is new, by the next contest the novelty may have worn off and it may not only be about being

*

  p<.10. **p<.05 ***p<.01

  The online campaign in Poland: indications why online mattersOur study of the use of the Internet during the 2011 Polish election indicates that Poland is catching up and election campaigning is becoming normalised and professionalised as with other Western democracies. Professionalism involves the adaptation of various platforms and tools to the norms of campaigning; however this results in a weakening in control over communication. Therefore adaptation is a twoway process. Parties and candidates have to some extent adapted to social uses of the Internet, migrating to social networking sites, microblogs and sharing sites, and by building blogtools into the architecture of their websites. These platforms also lead to adaptation, to the customs of communication on Facebook for example. To attract a following there is a requirement to relax control and create an open space for

Table 1 : AOP for Polish Political parties and candidates

 1

		Parties'	Candidates'
		AOP	AOP
	Information	.346	.326
	Engagement	.294	.280
	Mobilisation	.373	.157
	Interaction	.413	.264

Table 2 : AOP of audiences targeted by Political parties

 2

		Party	Candidates'
		AOP	AOP
	Browsers	.383	.450
	Information Seekers	.365	.324
	Issue Activists	.428	.259
	Supporters	.382	.297
	Campaign Activists	.407	.260

Table 3 : AOP for overall strategies employed

 3

		Parties'	Candidates'
		AOP	AOP
	Sales/Persuasion	.385	.340
	Personalisation	.214	.451
	E-representation	.381	.286

Table 4 : Regression analysis for election outcomes and personal and party factors and being present online

 4

	Vote share per candidate
	Coef.	Std err

Table 5 : Explaining how reach is earned within the online network

 5 Models are results of Poisson regression, robust. Dependent variable: community size (continuous, number of friends on Facebook + number of followers on Twitter + number of friends on Nasza Klasa). Independent variables: gender (dummy, 1=women, 0=men); age (in years); Years of incumbency (number of terms in Parliament, continuous, 0 to 7); Size of the party that candidates belong to (major and minor, fringe as reference); Political ideology of the party that candidate belongs to ((1=left to 11=right); Party years of existence (continuous); electorate size (number of voter to number of seats in the parliament per circumscription); Web penetration rate in the region where candidate is present. Online Strategies erepresentation, personalisation marketing (as in Table3); SN Updating (continuous)sum of number of entries in month before elections (7 th Sept. To 7 th October 2011) on Facebook, Nasza Klasa and Twitter. Citation in traditional media (23 rd September to 7 th October; press, radio and television; continuous, data from PRESS-SERVICE Monitoring Mediow)

		Model A	Model B
		Coef.	Std err	Coef.	Std err
	Online strategies
	E-representation	.170*	.098	.170**	.075
	Personalisation	.190**	.062	.121**	.048
	Sales	.007	.120	.031	.074
	SN updating	.004***	.001	.003**	.002
	Personal characteristics
	Age			-.006	.027
	Gender (men=0)			-.202	.337
	Years of incumbency			.060	.131
	Party characteristics
	Party size (major=1)			.708*	.375
	Political id			-.097*	.057
	Party years of existence			-.024	.032
	Other characteristic
	Regional web penetration rate			.033	.037
	Electoral size			.0001***	.000
	Citation in traditional media			.000	.000
	Pseudo R2			0.6503

Note: *p<.10. **p<.05 ***p<.01

The UK is first, with almost

million Facebook profiles, closely followed by Turkey with 26m and France with almost 21m. Data from www.SocialBakers.com

The data archives were downloaded to local computer at Sciences-Po, Paris. It was performed by TelePort Ultra provided by Tennyson Maxwell Information Systems, Inc.

online but interacting online. Poland evidences a professionalised political campaigning culture, one that is attempting to balance the potential and threat offered by the various tools offered by the Internet. The potential can be identified through reach to politically active groups, the threat arises when they want to start a public dialogue. These issues resonate across advanced democracies (Lilleker & Jackson, 2011), like their counterparts across the continent, political strategists in Poland are attempting to construct an understanding of what role the Internet can play in campaigning while academia dreams, perhaps, of the wider participatory potential.


domain: sdv info.info-au math spi

text:
Quentin Béchet

Ivan Feurgard

Benoit Guieysse
email: b.j.guieysse@massey.ac.nz

Filipa Lopes

The colorimetric assay of viability for algae (CAVA): a fast and accurate technique

Keywords: Algae, Chlorella vulgaris, Chlamydomonas reinhardtii, Erythrosine, Mortality, Viability

Determining the fraction of viable cells in algal cultures is critical to improve the understanding and control of algal microbiology, ecology, and biotechnology. Whereas current techniques for algal viability determination can be rather cumbersome, this paper describes a new assay that enables the rapid quantification of algal viability using only spectrophotometric measurements. This technique, henceforth named CAVA test, relies on the selective adsorption of erythrosine by non-viable cells and was validated on the algal species Chlamydomonas reinhardtii and Chlorella vulgaris. The results obtained by the CAVA test were in good agreement with the in situ measurement of oxygen production rates.

In addition, the CAVA test was shown to quantify the viability of algal samples regardless of the cause of death (heating, UV-irradiation or H 2 O 2 exposure). The CAVA technique has therefore the potential to offer fast and universal approach to measure the viability of algal samples.

Introduction

The ability to rapidly and accurately quantify the ratio of viable cells over total cells within microalgal cultures, henceforth defined as 'viability', is needed in various fields of algal research (e.g. ecology, [START_REF] Garvey | Applicability of the FDA assay to determine the viability of marine phytoplankton under different environmental conditions[END_REF]. For example, it is critical to determine if a certain stressor (e.g. toxicant, [START_REF] Gilbert | Rapid assessment of metabolic activity in marine microalgae: application in ecotoxicological tests and evaluation of water quality[END_REF]) causes cellular death rather than, or in addition to, activity inhibition to understand the mechanisms involved. The determination of viability is therefore critical to improve the design and control of algal biotechnology as algal cells found in full-scale cultivation systems are unlikely to be 100% viable due to, typically, viral-induced death [START_REF] Garza | The effect of cyanophages on the mortality of Synechococcus spp. and selection for UV resistant viral communities[END_REF], mechanical stress [START_REF] Brindley Alías | Influence of Power supply in the feasibility of Phaeodactylum tricornutum cultures[END_REF][START_REF] Gudin | Cell fragilitythe key problem of microalgae mass production in closed photobioreactors[END_REF], UV-irradiation (Häder et al. 1997) and rapid temperature changes [START_REF] Béchet | Mechanistic modelling of broth temperature in outdoor photobioreactors[END_REF].

Various experimental techniques enabling the quantification of algal cells viability have been described in the literature [START_REF] Markelova | A comparison of cittochemical methods for the rapid evaluation of microalgal viability[END_REF], Pouneva, 2000). The widely used 'plate cell count' consists of spreading a small volume of sample on a nutritive solid medium and counting the number of colonies formed after incubation (Allen and Smith 1966;[START_REF] Hwang | Survival of algal cultures after freezing by controlled and uncontrolled cooling[END_REF][START_REF] Madigan | Brock -Biology of microorganisms[END_REF]. The concentration of viable cells in the original sample can be determined by assuming that each visible colony originated from a single viable cell. Unfortunately, plate cell counts are often time-consuming: e.g. several days to weeks of incubation may be needed to detect colonies depending on the species studied.

Alternatively, pulse amplitude magnitudes (PAM) measurements can give information on the physiological state of algal samples that can be correlated to viability in certain cases (Drábková et al. 2007). Another type of viability assessment relies on the use of dyes that stain preferentially living cells [START_REF] Saga | Application of staining for determining viability of cultured algal cells[END_REF]. For example, Fluorescein di-Acetate (FDA) has been highly used in previous studies to assess microalgal viability [START_REF] Berglund | A rapid analytical technique for flow cytometryic analysis of cell vibility using Calcofluor White M2R[END_REF][START_REF] Clarke | Potential problems with fluorescein diacetate assays of cell viability when testing natural products for antimicrobial activity[END_REF][START_REF] Steinberg | Determining the viability of marine protists using a combination of vital, fluorescent stains[END_REF]. Once inside the cell, esterase from the living cells cleave the FDA molecule which results in the formation of fluorescein acetate that appears bright yellow-green under fluorescent light. Various dyes selectively stain dead cells only as they penetrate the damaged cytoplasmic membranes. Examples of dyes used in prior studies include visible dyes (methylene blue; Low et al. 1994, Evans blue;Saga et al. 1987, Direct brown;Venkata Mohan et al. 2008, etc.) and, more recently, fluorescent dyes such as SYTOX Green that becomes fluorescent once in contact with DNA (Breeuwer and Abe 2000). By counting the stained cells by microscopy, the viability of the sample can be estimated.

Unfortunately, while staining techniques are faster than the plate cell count, they usually involve microscope counting which is time-consuming. Cells stained by fluorescent dyes can also be counted by fluorocytometry but microbiology labs are not systematically equipped with fluorocytometers due to the high cost of this type of equipment. In order to avoid using costly equipment, Capasso et al. (2003) developed a new viability technique based on spectrophotometric measurements. The authors used 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium (referred to as 'MTS') as this dye is degraded by viable cells into a colored compound ('formazan'). The authors proved that the color development due to the formazan production was proportional to the number of viable cells. However, it is difficult to accurately estimate the fraction of viable cells with this technique because productivity may also vary with the physiological state of the viable algae (i.e. the test could underestimate true viability if viable cells under stress produce less formazan then unstressed viable cells).

This study describes the colorimetric assay of viability for algae (CAVA) which is based on the selective adsorption of erythrosine by dead algal cells and only involves spectrophotometric measurements. It has been experimentally shown that erythrosine was adsorbed by non-viable cells of 30 different algal strains [START_REF] Markelova | A comparison of cittochemical methods for the rapid evaluation of microalgal viability[END_REF]. Erythrosine was therefore selected as a 'staining dye' in this study. Chlamydomonas reinhardtii and Chlorella vulgaris were used as model strains representative of species broadly studied in algal biotechnology and with good potential for commercial applications [START_REF] James | Fatty acid profiling of Chlamydomonas reinhardtii under nitrogen deprivation[END_REF][START_REF] Mata | Microalgae for biodiesel production and other applications: a review[END_REF][START_REF] Spolaore | Commercial applications of microalgae[END_REF]).

Materials and methods

2.1. Algal strain and culture conditions Axenic C. reinhardtii (6145C; Nelson Cawthron Institute, New Zealand) was cultivated in TAP medium (Gorman and Levine, 1969) with ammonium replaced by nitrate at the same nitrogen molar concentration. Axenic C. vulgaris (isolated in New Zealand by Landcare Research, Lincoln, New Zealand, GenBank rbcL sequence: EF589154) was cultivated in sterilized BG-11 medium [START_REF] Andersen | Appendix A-Recipes for freshwater and seawater media[END_REF] enriched with nitrogen (0.25 g N-NO 3 -L -1 ) and pH-buffered with phosphate salts (KHPO 4 : 1.52 g L -1 ; K 2 HPO 4 : 3.1 g L -1 ). Following inoculation (from either solid or liquid axenic cultures) in aseptic 250 mL Erlenmeyer flasks, algal cultures were incubated under constant conditions of temperature (25°C), agitation

(orbital shaking at 160 rpm), atmospheric CO 2 partial pressure (0.5%), and illumination (14 W m -2 as photosynthetically active radiation at the culture surface). Cultures in the lightlimited growth phase (linear growth) were used for the viability tests.

Dry weight (DW) measurements

A known volume of algae was filtered onto a pre-weighted filter (Membrane filters, 0.45 µm, 47mm, MF-Millipore™, Merck Millipore, Billerica, MA, USA). The filter was then rinsed with reverse osmosis water in order to remove any salt from the filter and then dried at 105 o C

for one hour before being weighed. The DW concentration (expressed in the following in g DW L -1 ) was then calculated by dividing the difference in weight by the volume of sample filtered. The DW concentration was measured in duplicates.

Isotherm characterization

Algal cells were centrifuged and re-suspended in phosphate-buffered saline solution (PBS;

8.01 g L -1 NaCl; 0.20 g L -1 KCl; 1.60 g L -1 Na 2 HPO 4 , H 2 O; 0.27 g L -1 H 2 PO 4 ). PBS was used to maintain identical conditions of temperature and pH conditions in all the assays and did not affect cell viability (data not shown). The algae were then heated at 60°C for 25 min before being cooled down to 20 o C. The amount of erythrosine adsorbed on algae was found to stabilize after 25 min heating (see Online Resource S1). The solution of presumably killed cells was then diluted 2-fold and 4-fold to obtain a total of three algal solutions having different concentrations. Each of these solutions was exposed for 10 min to eight erythrosine concentrations (Erythrosin extra bluish, 1623-68-0 Sigma-Aldrich, USA) varying between 0.004 to 0.12 g L -1 . The amount of erythrosine adsorbed onto the biomass and the concentration of free erythrosine left in solution were then determined by spectrophotometry (see Section 2.4) and found to stabilize within 5 min of exposure (see Online Resource S1). In the subsequent experiments, 10 min exposure was therefore applied in order to determine sorption properties at equilibrium.

The Langmuir and Freundlich isotherms were tested in order to characterize the sorption of erythrosine by dead cells. Several microalgal concentrations were tested. The Langmuir isotherm is described as:

w w m C K C q q
(1)

where C w is the erythrosine concentration in the supernatant (g L -1 ), q is the specific amount of erythrosine adsorbed (g g DW -1 ), q m is the maximum specific amount of erythrosine adsorbed by dead cells (g g DW -1 ), and K (g L -1 ) is a saturation constant. The Freundlich isotherm is described as [START_REF] Gupta | Adsorption of a hazardous dye, erythrosine, over hen feathers[END_REF]:

n w f C K q (2)
where n, and K f (g 1-n -L n g DW -1 ) are constant parameters.

Viability assay

In order to perform the CAVA test, algae were first centrifuged and re-suspended in PBS (Figure 1). A first aliquot of re-suspended algal solution (0.6 mL) was poured in an Eppendorf tube and heated for 25 min at 60°C in order to completely kill the algae. This solution was then cooled down to 20 o C by soaking the tubes in a water bath and is henceforth referred to as the 'Killed sample'. A second 0.6 mL aliquot, henceforth referred to as the 'Sample', was transferred in another Eppendorf tube and kept at 20 o C. A third 0.6 mL aliquot of PBS was used as 'Blank'. Following the preparation of these three solutions, 0.4 mL of a 0.1 g L -1 erythrosine solution (in reverse osmosis water) at 20°C was added to each solution to reach an initial erythrosine concentration of 0.04 g L -1 . The three solutions were then maintained at 20°C for 10 min. The tubes containing the Sample and the Killed sample were finally centrifuged at 14,100 g for 5 min. The erythrosine concentrations in the supernatants of the three samples were measured by spectrophotometry at 526 nm (the maximum peak of light absorption by erythrosine solutions, determined by spectrophotometry). The optical densities were measured in duplicate for the Sample and the Killed Sample and in triplicate for the Blank (cuvettes: Thermo Fischer Scientific Cuvette 10x10x45mm polystyrene;

Spectrophotometer: Shimadzu UV-1800). Erythrosine concentration C w was found to be proportional to the optical density at 526 nm (OD 526 ) according to the equation:

w C OD 526 (3)
where the coefficient α was experimentally determined as 86.6 L g -1 (R 2 = 0.9996, N = 8).

Expression of the viability

The viability of an algal sample was determined from the optical densities of the supernatant the Killed sample (OD K ), the Sample (OD S ), and the Blank (OD B ). The following assumptions were made:

1. Only dead cells adsorb erythrosine;

2. The sorption of erythrosine onto dead algal cells can be described by a Langmuir isotherm;

3. The characteristic constants of erythrosine sorption (q m and K in Equation 1) are not affected by the mechanism causing cellular death;

4. Erythrosine losses by degradation and volatilization are negligible.

The validation of Assumptions 1 to 3 is discussed thereafter in the results and discussion section. Preliminary kinetic studies (Online Resource, S1) validated the Assumption 4.

Assuming there is no loss of erythrosine, a mass balance yields:

n w n a n Q Q Q , , (4)
where Q n (g) is the amount of erythrosine initially added in the tube, Q a,n (g) is the amount of erythrosine adsorbed by the dead algae, Q w,n (g) is the amount of free erythrosine remaining in the solution, and n stands for the solution analyzed (K -Killed sample, S -Sample and B-Blank). As the same amount of erythrosine is initially added in each solution, Q n can be determined as follows:

V C Q Q Q B w B S K , (5)
where C w,B (g L -1 ) is the concentration of free Erythrosine in the Blank and V is the volume of the solution contained in the tube (L). Assuming that only dead cells adsorb erythrosine, Equation 4 applied to the Sample and the Killed sample can be written as follows:

V C V X q Q Q n w n d n n w n a , , , , (6)
where q n is the specific amount of erythrosine adsorbed by algae (in g g DW -1 ), X d,n the dead cell concentration (in g DW L -1 ), V the volume of the solution (L), and C w,n the concentration of erythrosine remaining in the solution. In the case of the Killed Sample, the dead cell concentration equals the total cell concentration (X T , g DW L -1 ) and, assuming that erythrosine sorption is described by a Langmuir isotherm, Equation 6 becomes:

V C V X K C C q Q Q K w T H K w K w H m K w K a , , , , , , (7a)
When applied to the Sample, Equation 6 becomes:

V C V X K C C q Q Q S w d NH S w S w NH m S w S a , , , , , , (7b)
where q m,H (q m,NH ) and K H (K NH ) are the maximum specific amount of erythrosine adsorbed by the dead cells (g g DW -1 ) and the Langmuir's saturation constant (g L -1 ) for the heated (H) (and non-heated; NH) cells, and X d is the dead cell concentration in the sample (g DW L -1 ).

Combining Equations 3, 5, 7a, and 7b yield the two following equations:

K B K K H H m T OD OD OD OD K q X ' 1 , (8a) S B S S NH NH m d OD OD OD OD K q X ' 1 , (8b)
where K H ' and K NH ' are the products of α and K H and K NH , respectively. The viability ν is derived from the ratio of the concentration of dead cells, X d , over the total cell concentration

X T : K B S B K H S NH S K NH m H m T d OD OD OD OD OD K OD K OD OD q q X X ' ' 1 1 , , (9)
Assuming the values of q m and K are not affected by the mechanism causing cellular death (Assumption 3), the viability can be expressed as follows:

K B S B K S S K OD OD OD OD OD K OD K OD OD ' ' 1 (10)
where K' is the product of α and K H (or K NH assuming they are equal).

The viability of microalgae cultures exposed to different stresses and in different physiological states can be measured with this technique. In fact, comparing the quantity of erythrosine adsorbed by the Sample to the quantity of erythrosine adsorbed by the Killed Sample (Equation 10) enables accounting for the potential variations of adsorption properties of algal cells as long as Assumption 3 is verified.

Comparison with the measurements of oxygen production rates

Aliquots of killed and viable cells (from the same initial culture, the 'killed' cells being heated at 60 o C for 25 min and cooled down at 20 o C) were mixed at known volumetric ratios of 1:0; 3:1; 2:2; 1:3; and 0:1. The viability of the resulting five solutions was then assessed using the CAVA test and short-term in situ oxygen productivity (in mg O 2 L -1 s -1 ) was measured using the device presented by [START_REF] Béchet | Algal productivity modeling: a step toward accurate assessments of full-scale algal cultivation[END_REF]. This device included six cylindrical vessels The CAVA test was applied to monitor changes in cell viability in algal cultures exposed to UV-irradiation or hydrogen peroxide (H 2 O 2 ). For UV-irradiation, an algal sample was separated in solutions of around 150 mL placed in four different 1L-beakers. Two beakers were irradiated by a UV lamp (Philips TUV 30W/G30 T8) placed approximately 20 cm above the sample (UV-light intensity: 7.8 W m -2 ; ILT 1400 radiometer photometer equipped with a 255 nm filter) while the two other beakers were protected from UV light by an opaque cover.

The four beakers (two sets of two replicates) were placed in an orbital shaker (agitation at 150 rpm) to provide well-mixed conditions (no sedimentation was observed). Water evaporation was compensated for by weighing the beakers and adding reverse-osmosis water every day in 10). A statistical analysis on duplicate measurements revealed that the error on optical density was normally distributed (see Online Resource S2 for details). Monte-Carlo simulations were then used in order to determine how the error on experimental measurements impacted the accuracy of the viability estimations as described in Online Resource S2.

Results and Discussion

Characterization of the adsorption isotherm

Erythrosine absorption onto biological tissues has been shown to be characterized by both the Langmuir and the Freundlich isotherm for hen feathers [START_REF] Gupta | Adsorption of a hazardous dye, erythrosine, over hen feathers[END_REF]), de-oiled mustard [START_REF] Jain | Adsorptive removal of Erythrosine dye onto activated low cost deoiled mustard[END_REF], or de-oiled soya [START_REF] Mittal | Process development for the removal and recovery of hazardous dye erythrosine from wastewater by waste materials-Bottom Ash and De-Oiled Soya as adsorbents[END_REF]). However, and to the best of our knowledge, the adsorption of erythrosine by dead algal cells has never been characterized

and is the object of this first section.

According to Figure 2, the Freundlich isotherm could not represent erythrosine adsorption onto killed C. reinhardtii cells. On the other hand, both Langmuir and Freundlich isotherms could describe the experimental data collected for C. vulgaris (Figure 2). The Langmuir isotherm was shown to describe the sorption equilibrium over a large range of algal concentrations (0.24-0.96 g DW L -1 for C. reinhardtii and 0.43-1.7 g DW L -1 for C. vulgaris)

and erythrosine concentrations (0.01-0.12 g L -1 ). In addition, the parameters of the Langmuir isotherm, q m and K, (Equation 1) did not significantly change with the algal concentrations as

shown by the confidence intervals in Table 1. Langmuir's behavior was also observed when C. vulgaris cells were killed by exposure to hydrogen peroxide instead of heating (data not shown). High temperature is known to kill cells by protein denaturation and hydrogen peroxide is a strong oxidizer, affecting, for instance, enzymes in the Calvin Cycle (Drábková et al. 2007;Takeda et al. 1998). These results validate Assumption 2 (see Section 2.5) and the Langmuir model was subsequently used as the basis for calculating viability. In Sections 3.2 to 3.4, the half-saturation constants of the Langmuir isotherm (and the associated confidence interval) were taken equal to the values resulting from the fitting of the experimental points obtained for the three algal concentrations (values in bold in Table 1). The Langmuir isotherm suggests the binding of erythrosine as a monolayer. Differences in the cell wall biochemistry of the two algal species studied likely explain the difference of q m values found between these species (Table 1). Almost no erythrosine was adsorbed by non-killed cells, suggesting that the adsorption of erythrosine onto dead cells is likely associated with a loss of cell wall integrity causing binding onto otherwise unavailable intracellular material [START_REF] Markelova | A comparison of cittochemical methods for the rapid evaluation of microalgal viability[END_REF].

Comparison with oxygen productivity

In order to benchmark the CAVA test against other viability assays, oxygen productivity and colorimetric viability were measured in partly killed algal cultures. As can be seen in Figure 3, oxygen productivity was close to linearly correlated to the measured viability in algal samples containing different fractions of dead and viable cells, the uncertainty being mostly caused by experimental error. Because oxygen production by algae is the result of photosynthesis and cannot be performed by dead cells, the good agreement between the CAVA results and oxygen productivities indirectly indicates that living cells do not adsorb erythrosine, thus validating Assumption 1 (see Section 2.5). A slight limitation of the technique is the fact that the measured viability in the Killed sample was estimated to be close to 10-15% (Figure 3). This limitation is further discussed in Section 3.4. was not due to natural death as the viability of the algal sample not exposed to UV light remained constant over the duration of the experiment (Figure 4). In addition, the CAVA tests showed that the rate of cellular death seemed to increase, as expected, with the H 2 O 2 concentration for C. reinhardtii (Figure 5). The expected decrease in viability observed when cells are exposure to UV light or hydrogen peroxide suggests that the CAVA test is able to monitor the viability of algal samples killed by different techniques. This result is important as heating, H 2 O 2 and UV light cause death through different mechanisms: heating denatures proteins; H 2 O 2 is a strong oxidizer, and UV light damages DNA. In spite of these differences, the CAVA test yielded trends that were consistent with expected results.

Protocol optimization

Erythrosine concentration can be optimized to maximize the precision of the CAVA test. The accuracy of the measured viability depends on the amount of erythrosine adsorbed by algae.

This quantity is obtained by the difference between the amount of erythrosine initially introduced and the amount remaining the supernatant. As a result, the greater the amount of erythrosine adsorbed, the higher the accuracy. It is therefore recommended to use concentrated algal samples (e.g. > 1 g/L) to maximize the amount of erythrosine adsorbed by dead cells when using this assay. For example, in a hypothetical case of a 50%-viable sample of C. vulgaris, the confidence interval on the measured viability can decrease from ±20% to ±6% when the algal concentration increases from 0.5 to 2 g DW L -1 (see Online Resource S3

for details). Measuring the optical densities OD S and OD K in triplicates should reduce uncertainty further.

A limitation of the CAVA test is that the measured viability of cultures exposed to H 2 O 2 did not reach exactly 0% but stabilized at values close to 10% for C. reinhardtii and 15% for C. vulgaris (Figure 5). These plateau values were reproducible, as shown in Figure 5, and therefore unlikely caused by experimental error. However, this overestimation of viability was not observed when algae were killed by UV-exposure. Microalgae exposure to H 2 O 2 may change adsorption properties of the cells (i.e. values of K or q m ), affecting the viability calculation. As mathematically demonstrated in the Online Resource S4, the variability of K (due to physiological changes) does not significantly increase the uncertainty of the estimated viability. However, the value of the coefficient q m may differ between the Sample and the Killed sample due to different combinations of stressors involved (e.g. the combined action of Even if this study was limited to two algal species, erythrosine was shown to be adsorbed by more than 30 algal species including cyanobacteria by [START_REF] Markelova | A comparison of cittochemical methods for the rapid evaluation of microalgal viability[END_REF], suggesting that the CAVA test can potentially be applied to an important variety of algal species. In addition, the same protocol can theoretically be applied to different dyes such as Methylene blue [START_REF] Low | Binding of Basic Dyes by the Algae, Chara aspera[END_REF], Evans blue [START_REF] Saga | Application of staining for determining viability of cultured algal cells[END_REF], or Direct brown (Venkata Mohan et al.

2008

) which were shown to be adsorbed by dead algal cells. The technique is also relatively fast compared to other viability assays and is also practical as no microscopic observation is required. For example, the dual fluorescence assay proposed by Sato et al. ( 2004) and Shulze et al. ( 2011) using SYTOX Green and auto-fluorescence requires microscopy counting to distinguish living cells (appearing red) from dead cells (appearing green). Counting could be easily performed using a fluorocytometer but this type of equipment is expensive. In contrast, the CAVA test only requires a common and affordable spectrophotometer. To the best of our knowledge, the CAVA test is the first spectrophotometric viability assay that enables the direct quantification of the fractions of living and dead cells. The viability method presented in this study has therefore the potential to offer a fast and universal approach to measure the viability of algal samples. Among its possible applications, the CAVA test may be used to evaluate microalgal viability in outdoor cultivation systems where cells undergo stresses such as high temperature and UV-irradiation.

Conclusions

First, erythrosine adsorption onto dead C. vulgaris and C. reinhardtii cells could be described by the Langmuir isotherm. By applying the CAVA technique on partially killed samples containing both living and dead algal cells, the CAVA results were in good agreement with in-situ measurements of oxygen productivity. In addition, the CAVA test could quantify cells viability over time in algal cultures subjected to diverse mechanisms causing cellular death (heating, UV-irradiance, exposure to H 2 O 2 ) for the two algal species studied. Finally, the CAVA test can hypothetically be applied to any dye exclusively adsorbed by dead algal cells.

The technique has therefore the potential to offer a fast, practical and universal approach to measure the viability of algal samples. (X: algal concentration in g DW/L; units: q m : mg/g; K: mg/L; K f : 10 -3 g 1-n -L n /g n ; n: -). 95 % confidence intervals calculated using Monte-Carlo simulations are shown in parenthesis.

Values in bold characters result from the fitting of data from all algal concentrations.

Algae X Langmuir isotherm Freundlich isotherm q m K q m K K f n K f n C. reinhardtii 0.

Kinetic of erythrosine adsorption by killed cells

Killed samples of C. vulgaris were exposed to erythrosine (initial concentration 0.04 g L -1 ) for different periods of time. In parallel, a non-killed sample originating from the same algae sample was exposed to the same dose of erythrosine. The amount of erythrosine adsorbed by the samples was estimated by following the protocol described in the main manuscript. All the measurements of optical densities for the Sample and the Killed sample (OD S and OD K ) in the protocol of the CAVA test described in the main manuscript were made in duplicates, representing a total of 684 duplicates. The objective of this supplementary information is to explain how these duplicate measurements were used in a statistical analysis to estimate a confidence interval on the viability measurements.

Uncertainty on the optical density measurements

Figure S2-1 shows that the difference between two OD duplicates is not clearly correlated to the concentration of erythrosine in the cuvette (represented by the average OD). As a result, the difference between duplicates is likely to be due to factors other than the algal concentration such as pipetting errors or defaults of the cuvette (scratch, dust, etc.)

By assuming that the error on an OD measurement follows a normal distribution centered on 0 and characterized by a standard deviation σ, the difference between two duplicates should follow a normal distribution also centered on 0 but characterized by a standard deviation √2σ.

Consequently, the absolute value of this difference follows the half-normal distribution, represented by the following probability density function [START_REF] Bland | The half-normal distribution method for measurement error: two case studies[END_REF]: Figure S2-2 shows that the absolute value of the difference between OD duplicates indeed follows a half-normal distribution. As demonstrated by [START_REF] Bland | The half-normal distribution method for measurement error: two case studies[END_REF], the expectation of a variable X obeying a half-normal distribution is:

2 () EX (S2-2)
The standard deviation σ can therefore be estimated as follows:

2 X (S2-3)

Numerically, the standard deviation was found to be 5.77×10 -3 .

Calculation of the confidence interval on OD measurements

As OD measurements were made in duplicates, the OD values used in the formula of the viability (Equation 10 in the main manuscript) were the average ODs, expressed as:

1 av i i r OD OD N (S2-4)
where N r is the number of replicates of a measurement (e.g. 2 for duplicates) and OD i are the values of these replicates. As shown in the previous section, a sampled value of optical density OD i can be assumed to follow a normal distribution centered on the 'true' value of the OD (OD true ) and with a standard deviation σ. OD av can therefore be assumed to follow a normal distribution centered around OD true and characterized by a standard deviation σ/√N r .

The confidence interval on an OD measurement at 95% was therefore expressed as follows:

Calculation of the confidence interval on the isotherm parameters

The error on OD measurement during the characterization of the isotherm caused uncertainty on the fitted values of the isotherm parameters. Monte-Carlo simulations were performed in order to quantify the level of accuracy on the parameters K, q m , K f , and n.

Monte-Carlo simulations consisted on iterating the fitting of the isotherm parameters a large number of times (10,000 in this case). At each iteration, the values of the optical densities for each data point were randomly sampled around the measured value and within the confidence interval given by Equation S2-5. The confidence interval for each isotherm parameter was then calculated from the standard deviation of the 10,000 values generated during the Monte-Carlo simulations.

Calculation of the confidence interval on viability

According to Equation 10 in the main manuscript, there are two sources of uncertainty in the expression of the viability: the experimental error on the optical densities and the value of the parameter K'. The level of confidence on the optical densities was determined from Equation S2-5. The confidence interval on the parameter K' was taken equal to the confidence interval on K determined in section 2.4 multiplied by the constant α (see the main Manuscript for details).

For each viability measurement, Monte-Carlo simulations consisted on repeating the calculation of the viability by using randomly generated values for the different ODs and the parameter K' in Equation 10. The level of confidence on the measured viability was then calculated from the standard deviation of the multiple resulting viability values (here 10,000

values) obtained at each iteration.

S4: Impact of the uncertainty on K on the viability results

As shown by Equation 10 in the main manuscript, estimating the viability of an algal sample requires determining the value of the half-saturation constant K. In this study, this parameter was assumed to be constant despite the potential changes of algal adsorption properties due to physiological changes (e.g. change of cell size, cell wall composition, etc.). This section investigates how the variability of this parameter can influence the results of the CAVA test.

In a hypothetical case, let us consider a 50% viable C. vulgaris sample. Figure S4-1 shows the viability of the sample estimated with a coefficient K of 0.0378 g L -1 as shown in Table 1 if the true value of K varies from 0.0189 (50%) to 0.0756 (200%). As shown by Figure S4-1, such a large variability in the value of K does not significantly affect the estimated viability.

As a result, K can be assumed to be constant without significant loss of accuracy in viability. for different true K values (Hypothetical case: X = 1 g DW L -1 ). Plain line: Estimated viability; dashed-line: confidence interval at 95%.

(

  diameter: 49.2 mm; height: 40 mm) equipped with dissolved oxygen electrodes (Model DO50-GS, Hach) and placed above LED lamps (12V PHILIPS Endura LED 10W MR16 Dimmable 4000K). The sides and the top lid of the vessels were opaque and the bottom wall was made of a transparent polycarbonate plastic. A complete description of the device can be found in[START_REF] Béchet | Algal productivity modeling: a step toward accurate assessments of full-scale algal cultivation[END_REF]. The five algal solutions placed in the vessels of the device were exposed to the same incident light intensity (63±7 W m -2 as photosynthetically active radiation) and the temperature in each vessel was maintained at 22 o C. Dissolved oxygen concentration was recorded over time during the light and dark phases. The net oxygen productivity and the respiration rate were obtained by linear regression. The respiration rates in the dark and in the light phase were assumed to be equal. As a result, the gross rate of oxygen production in each vessel was obtained by adding the rate of respiration (in absolute values) to the rate of net oxygen production as algae respire in the light phase. 2.7. Application of the CAVA test in the case of death by UV-irradiation and H 2 O 2 exposure

3. 3 .

 3 Figures 4 and 5 show that the measured viability of cultures exposed to H 2 O 2 and UV light

H 2 O

 2 2 exposure and heating in the Killed sample versus H 2 O 2 exposure only for the Sample).Such difference could explain the 10-15% overestimation of viability seen in Fig.5. This explanation is consistent with the observations of[START_REF] Imase | Estimating the viability of Chlorella exposed to oxidative stresses based around photocatalysis[END_REF] who showed that H 2 O 2 may affect Chlorella sorokiniana by creating tiny holes in the cell membranes. This possible change of adsorption properties in the presence of H 2 O 2 challenges Assumption 3. In order to improve the method for viability determination when microalgae are subjected to hydrogen peroxide stress, further assays should be performed to characterize erythrosine adsorption on those cells.

FiguresFig. 1 Fig. 2

 12 Figures

Fig. 3 Fig. 4 Fig. 5

 345 Fig. 3 Comparison between the oxygen productivity and the measured viability of algal samples partly killed by heating (a: C. reinhardtii, 0.32 g DW L -1 ; b: C. vulgaris, 0.32 g DW L -1 ). The oxygen productivity was normalized to the oxygen productivity in the sample

Figure 1

 1 Figure 1 Click here to download high resolution image

Figure 2

 2 Figure 2Click here to download high resolution image

Figure 3

 3 Figure 3Click here to download high resolution image

Figure 4

 4 Figure 4Click here to download high resolution image

Figure 5

 5 Figure 5 Click here to download high resolution image

Figure S1- 1 :

 1 Figure S1-1: Evolution of the amount of erythrosine adsorbed by a C. vulgaris sample with the time of heating at 60 o C. The error bars represent the confidence interval at 95% calculated through Monte-Carlo simulations as discussed in S2.

Figure

  FigureS1-2 shows that the equilibrium between the solution and the algae was reached within less than 5 minutes exposure to erythrosine. In addition, as the amount of erythrosine adsorbed by the non-heated cells was constant over time, this indicates that erythrosine was not degraded through a chemical reaction or a biological mechanism, confirming then the Assumption 4 of the CAVA test presented in the main manuscript.

Figure S1- 2 :

 2 Figure S1-2: Evolution of the fraction of erythrosine adsorbed by C. vulgaris samples exposed to 70 o C for 25 minutes (a) and to 95 o C for 10 minutes (b). Red point line: killed sample; blue dashed-line: non-killed sample. The error bars represent the 95% confidence interval estimated using Monte-Carlo simulations as described in S2.

  Figure S2-1: Difference between two OD duplicates as a function of the average OD in the cuvette.

Figure S2- 2 :

 2 Figure S2-2: Distribution of the difference between OD duplicates and fitting (plain line) to the half-normal distribution described by Equations S2-1a and S2-1b (with σ = 5.77×10 -3 ).

Figure S4- 1 :

 1 Figure S4-1: Viability of a 50% viable C. vulgaris sample estimated with K = 0.0378 g L -1

  each beaker. The changes in viability in the four solutions were monitored daily. For H 2 O 2 exposure, H 2 O 2 (30% v/v; Code: H/1800/15, Fisher Scientific UK, Bishop Meadow Road, Loughborough) was added to algal cultures (volume ratio H 2 O 2 (30%): algal culture of 1:4 or 1:9, see Figure5for details) in a closed Duran bottle continuously mixed by magnetic stirring. Samples were withdrawn at regular intervals, centrifuged and re-suspended twice in PBS in order to avoid any degradation of erythrosine by remaining H 2 O 2 . CAVA tests were performed on the re-suspended solutions by following the protocol described in Section 2.4.

2.8. Monte-Carlo simulations for uncertainty estimation

Uncertainty on viability measurements was introduced by experimental error on the optical densities measurement (OD S , OD K and OD B in Equation

  Plain line: fitting with a Langmuir isotherm; dashed-line: fitting with a Freundlich isotherm. Values of fitted parameters are given in Table1. Error bars represent 95% confidence intervals calculated using Monte-Carlo simulations

	vulgaris in g DW L -1 : red circles: 1.7/0.96; blue diamonds: 0.85/0.48; black crosses:
	0.43/0.24).

Table 1 :

 1 Langmuir and Freundlich isotherms parameters for C. reinhardtii and C. vulgaris

Table 1

 1 Click here to download table: Table 1.docx

		96	84.6 (2.9)	21.4 (2.0)			418 (170)	0.623 (0.114)
		0.48	91.4 (5.0)	27.2 (4.0)	86.4 (4.1)	25.0 (3.1)	403 (231)	0.632 (0.128)	366 (121)	0.614 (0.090)
		0.24	86.3 (10.2)	29.0 (9.2)			387 (754)	0.626 (0.243)
		1.7	17.8 (2.2)	25.3 (7.1)			53.6 (16.2)	0.516 (0.087)
	C. vulgaris	0.85	25.1 (6.9)	44.9 (24.2)	22.4 (4.7)	37.8 (15.8)	64.8 (40.5)	0.540 (0.151)	58.6 (21.7)	0.529 (0.103)
		0.43	25.7 (76.5)	48.7 (364)			70.8 (681.4)	0.532 (0.292)

vulgaris: white: 6% / 1.3 g DW L -1 ; grey: 6% / 0.48 g DW L -1 . The error bars represent the 483 95% confidence interval calculated through Monte-Carlo simulations S3: Influence of the algal concentration on the uncertainty on viability