Pham Thi

Hong Hanh

Gautier Erwan

Saout Le

Ronan

Charles Amélie

Darné Olivier

Kim Jae

Etienne Redor

Pham Thi Hong

Hanh N°

Energy management systems and market value: Is there a link?

Keywords: G12, M14, Q56 Energy Management Systems, ISO 50001, Market value, Event study

This paper aims to advance in the knowledge of the economic impacts of ISO 50001 certification on firms' performance. The study hypothesizes that ISO 50001 is associated with improvements in market value of firms.

We employ, on one hand, event study methodology for a sample of 40 companies listed on different stock exchanges. On the other hand, we use market reaction to the announcement of ISO 50001 as a proxy for changes in firm performance. We reveal that market reaction to the adoption of ISO 50001 is negative but statistically insignificant. However, this result is not suggesting that getting ISO 50001 is a bad investment, but rather that inflated expectations of financial performance improvement due to the adoption of ISO 50001 has still been unfounded.

Introduction

In recent years, commitment to the natural environment has become an important variable, which is strongly affecting purchase decisions of customers. The new consumption tendency is fuelling private and institutional investment decisions towards socially and environmentally responsible investing. This has been also illustrated by the introduction of several international environmental management systems (EMSs) to the manufacturing industry. Among the popular EMSs introduced to the manufacturing industry, ISO 14001, which is developed by International Organization of Standardization (ISO), is known as a generic management system standard being relevant to any firm seeking to improve its environmental performance. By implementing ISO 14001 an organization would improve environmental performance, but does not have to specify its procedure as a requirement. Together with ISO 14001, in June 2011 ISO released an Energy Management System (EnMS), ISO 50001, which is also suitable for any organization -whatever its size, sector or geographical location. The main objective of ISO 50001, which is modeled after the ISO 9001 (Quality Management System) and the ISO 14001, is to improve energy-related performance and energy efficiency continuously and to identify energy reduction opportunities.

Until January 15 th 2013, ISO 50001 has been adopted by 1312 companies around the world. 2Following the development in EMSs, a number of empirical studies have tended to investigate whether environmental investments penalize or reward firm performance. Methodologically, these studies use either common micro-econometric approaches [START_REF] Ziegler | The Effect of Environmental and Social Performance on the Stock Performance of European Corporations[END_REF] or event study approach (e.g. Cañón-de-Francia and Garcés-Ayerbe, 2009; or [START_REF] Oberndorfer | Does the Stock Market Value the Inclusion in a Sustainability Stock Index? An Event Study for German Firms[END_REF], and measure firm's economic performance by the financial performance (notably profitability, cost efficiency and sales performance) or the market value. Despite the growing number of empirical studies, there are contradicting results regarding the relationship between firms' environmental management practice and their economic performance (e.g. [START_REF] Filbeck | The relationship between the environmental and financial performance of public utilities[END_REF][START_REF] Ziegler | The Effect of Environmental and Social Performance on the Stock Performance of European Corporations[END_REF][START_REF] Cañón-De-Francia | ISO 14001 Environmental Certification: A Sign Valued by the Market?[END_REF]. On the other hand, to the best of our knowledge, most recent quantitative researches have only focused on the benefits of adopting ISO 14001, but ignored the impacts of ISO 50001 certification that presents the latest international best practice in energy management. More importantly, despite being closely aligned to ISO 14001, ISO 50001 places more emphasis on the continual improvement of energy performance and is more appropriate than ISO 14001 in an organization where energy is a significant cost. In order to fill this research gap, we explore the shareholder value effects of energy performance by investigating the stock market reaction (abnormal returns) associated with the adoption of ISO 50001. In other word, we apply event study technique to resolve the question of whether ISO 50001 certification affects firms' market value.

4

The paper is structured as follows. Section 2 provides a literature review considering environmental management, certification and economic performance. This is followed by a description of our sample and research methodology. Section 4 presents and discusses the main findings. Concluding remarks are in the last section.

Environmental performance versus Economic performance

In the literature, either theoretical or empirical, the relationship between environmental and financial performance has highly attracted academic interest. From a theoretical perspective, [START_REF] Walley | It's not easy being green[END_REF] initially suggest that instances where environmental efforts can improve firm performance are rare. Likely, [START_REF] Jaffe | Environmental regulation and the competitiveness of U.S. manufacturing: what does the evidence tell us[END_REF] question the optimism of environmental requirements and assume that environmental practices and initiatives involve costs and have few financial benefits. In contrast, according to [START_REF] Hart | A natural-resource-based view of the firm[END_REF], firms' related efforts in order to improve environmental performance can create more valuable resources and be a source of competitive advantage. In other works, the Porter Hypothesis initialed by [START_REF] Porter | Green and competitive: ending the stalemate[END_REF] and subsequently considered by [START_REF] Xepapadeas | Environmental policy and competitiveness: the porter hypothesis and the composition of capital[END_REF] stresses the "freelunch" aspect in the possible relationship between firms' proactive environmental and financial.

These authors suggest that responding to new environmental advocates by applying innovations allows firms to improve their overall operations and then to decrease their production costs or to increase their productivity. In the same vein, [START_REF] Elkington | Towards the sustainable corporation: win-win-win business strategies for sustainable development[END_REF] shows that improving environmental performance could results in a rise in demand from "green" customers, who appreciate the ecological products. Moreover, for any firm, pointing out good environmental initiatives allows making new opportunities for acquiring a high ecological reputation and benefiting from premium pricing and increased sales [START_REF] Miles | Environmental marketing: a source of reputational, competitive, and financial advantage[END_REF]. On the other hand, firms may reduce costs and increase revenues through environmental management [START_REF] Ambec | Does it pay to be green? A systematic overview[END_REF]. Ambec and Lanoie also point out four opportunities to reduce costs (risk management and relations with external stakeholders; cost of material, energy, and services; capital cost; and labor cost) and three opportunities to increase revenues (better access to certain markets; differentiating products; and selling pollution-control technology). Indeed, showing circumstances that it pays to be green, makes opportunities for reducing costs and for increasing revenues. Despite the inconclusiveness in theoretical results, the studies finding evidence that good environment performance results in improved financial performance are predominant.

Regarding to empirical works, the results seem to be more inconclusive and even contradictory, highlighting such a complex relationship between environmental and financial performance [START_REF] Corbett | Extending the horizons: environmental excellence as key to improving operations[END_REF]. A large number of studies have replaced the traditional assumption about a trade-off relationship between environmental and financial performance by the novel hypothesis "It pays to be green". These studies confirm the existence of a positive effect of environmental performance on economic performance, which is measure by a set of indicators, such as returns on assets, sales and equity (e.g. [START_REF] Hart | Does it pay to be green? An empirical examination of the relationship between emission reduction and firm performance[END_REF][START_REF] Russo | A resource-based perspective on corporate environmental performance and profitability[END_REF] or by the intangible asset (e.g. [START_REF] Konar | Does the market value environmental performance[END_REF]. Other studies (e.g. Klassen and Mclaughlin 1996;[START_REF] Konar | Information as regulation: the effect of community right to know laws on toxic emissions[END_REF] find evidence that the positive impacts of green performance result from positive market reaction to firms' improved environmental responsibility. Unlike these listed studies, the relationship between environmental and financial performance is inconclusive or even negative in other empirical works. According to [START_REF] Telle | It pays to be green-a premature conclusion?[END_REF], the positive impact of environmental performance on economic performance, which is concluded in the studies using pooled regression, become statistically insignificant in the studies applying panel econometric techniques, in which the data heterogeneity is controlled. Lately, [START_REF] Ziegler | The Effect of Environmental and Social Performance on the Stock Performance of European Corporations[END_REF] consider a two-dimensional sustainability performance: the average sustainability performance of the industry in which a corporation operates; and the relative sustainability performance of a corporation within a given industry. They find that whereas the industry's average environmental performance positively affects the stock performance, the average social performance of the industry has a significantly negative influence. A negative relationship between environmental and stock performance of a firm is also previously concluded in [START_REF] Filbeck | The relationship between the environmental and financial performance of public utilities[END_REF].

Complementary to the studies exploring economic benefits of environmental performance, there are several researches looking for the relationship running from financial performance to environmental performance. For instance, [START_REF] Wagner | The relationship between the environmental and economic performance of firms: an empirical analysis of the European paper industry[END_REF] support the positive influence of financial performance on firms' environmental management. Accordingly, a good financial performance allows a firm to allocate more resources to prevention-oriented technologies and initiatives. Using Japanese data, [START_REF] Nakao | Relationship between environmental performance and financial performance: an empirical analysis of Japanese corporations[END_REF] also reveal that firms' financial performance has a positive impact on firms' environmental performance.

Together with a large number of researches studying the possible impact of environmental performance on economic performance, there are few empirical studies examining the relationship between EMSs captured by the adoption of ISO 14001, and financial performance of a firm. For instance, [START_REF] Watson | Impact of EMS implementation on financial performance[END_REF] tend to resolve the question of whether there is a difference in financial performance between firms that had introduced a certified EMS and firms that had not.

The authors find no significant difference between them across different economic sectors. In the same light, Cañón-de-Francia and Garcés-Ayerbe ( 2009) analyze whether ISO 14001 certification is interpreted by the capital market as a sign of environmental responsibility by using a sample of 80 large Spanish firms from 1996 to 2002. They find that adopting ISO 14001 has a negative effect on the market value of certain firms. This negative effect seems to be confirmed only in the case of less polluting and less internationalized firms. However, in the case of more polluting and more internationalized firms, there is no clear evidence supporting this negative relationship. Unlike the previous studies, [START_REF] Jacobs | An empirical investigation of environmental performance and the market value of the firm[END_REF] conclude that attainment of ISO 14001 certification results in statistically significant positive market reaction. Similarly, de Jong et al. ( 2014) asses the short-term and long-term impact of the ISO 14001 certification on financial performance by using a comprehensive dataset including a majority of ISO 14001 certifications in the United States. The authors provide clear evidence that the ISO 14001 certification process can help firms develop firmspecific capabilities, which will have a significant impact on the profitability of the certified firms. Differentiating from the above cited studies [START_REF] Inaki | ISO14001 certification and financial performance: selection-effect versus treatment-effect[END_REF] try to explore the possible bi-directional relationship between ISO 14001 certification and financial performance. Employing a multivariate panel data analysis, the authors find that firms with better average performance have a greater propensity to pursue accreditation but there is no evidence that improvements in performance follow certification.

Research hypothesis

Released 15 years after the introduction of ISO 14001, ISO 50001 has considered as the latest international best practice in energy management. Whereas ISO 14001 helps an organization to systematically identify and manage all environmental impacts in the broadest sense, ISO 50001 helps an organization to specify, develop and implement energy management system requirements in order to form an energy policy. Specifically, ISO 50001 allows an organization to identify its objectives, targets, and action plans related to significant energy use, which are intended to lead to reductions in greenhouse gas emissions, energy costs, and other related environmental impacts through systematic management of energy consumption. In fact, ISO 50001 can either be used in conjunction with ISO 14001, to help an organization to point out further opportunities for energy savings, or as a stand-alone management system standard for any organization in which energy use is a significant consideration from either an environmental or cost perspective. Despite several advantageous points (as displayed in Figure 1), ISO 50001 has not really attracted much attention of either firms or academic field. For instance, after 3 years of release, ISO 50001 has been adopted by only 1312 companies around the world, comparing to 14106 certifications of ISO 14001 from 1997 to 1999 (ISO). More importantly, at the time of writing, the impact of ISO 50001 adoption on financial performance investigated in any academic work.

Therefore, the aim of our paper is to fill this research gap by shedding light on whether better financial performance of a firm, which is measured by the market value improvement, is due to the beneficial effects of adopting ISO 50001. In other words, our main research hypothesis is that adopting ISO 50001 allows a firm to realize a cost-saving production model and then improves its business benefits.

<Insert Figure 1> 3. Methodology and Data

Event study

In this paper, we use event study methodology that allow us to evaluate the effects of ISO 50001 by examining the response of the stock price around the announcement of this event, which is displayed in as follows:

<Insert Figure 2> The main assumption of event study methodology is that market processes information about the event in an efficient and unbiased manner. According to event study, the event affecting a firm's valuation may be: i) within the firm's control; and ii) outside the firm's control. Adoption of ISO 50001 is considered as an event within the firm's control. Event study was initially applied for stock splits by [START_REF] Fama | The adjustment of stock prices to new information[END_REF]. [START_REF] Campbell | The econometrics of Financial Markets[END_REF] In this study, we estimate the model using data over a period of 120 days, which includes from day -139 to day -20 prior to the date of ISO 50001 certification. The estimation period ending 20 days before the ISO 50001 announcement allows us to exclude abnormal returns probably due to this announcement. Moreover, MacKinlay (1997) suggests that 120 trading days as commonly implemented in event studies for the estimation period. To translate calendar days into event days, we consider the announcement day of ISO 50001 as Day 0. On the other hand, we use (-1; +1) day event window, which is also recommended by [START_REF] Mackinlay | Event studies in economics and finance[END_REF] as most accurate since it allows for spillover effects in surrounding days and does not weaken the test's power. [START_REF] Mcwilliams | Event studies in management research: theoretical and empirical issues[END_REF] also show that using a long event window can complicate the control of confounding effects, and then reduce the power of statistical tests as well as result in false conclusions about the event's significance. Accordingly, a three-day window will be long enough to capture the significant effect of the event of interest.

In order to obtain estimated abnormal returns, we can use one of four models of normal returns suggested by [START_REF] Brown | Measuring Security Price Performance[END_REF] as follows:

Mean Adjusted Returns: The normal return for a security equals a constant Ki. In this model, the abnormal return for the security is: = with Rit is the period-t return of security i.

Market Adjusted Returns:

The normal return for a security at a given point in time equals the market return for that period. Accordingly, the abnormal return is: = with Rmt is the period-t return of the market portfolio security.

Market and Risk Adjusted Returns: Normal returns are assumed to be generated by a single index model, in which security returns are linearly related to market returns through stock betas. The Abnormal returns is determined as: = -with rft is the period-t risk-free rate and is the period-t stock beta, which is estimated over firm estimation periods.

Multiple Index Model Adjusted Returns: Normal returns for are assumed to be driven by multiple factors, such as market return, industry returns, firm size or other characteristics.

The estimation model of abnormal returns is: = ---⋯with Fim are different factors affecting the security i.

Following some recent empirical studies (e.g. Gupta and Golda, 2005;[START_REF] Dasgupta | Disclosure of environmental violations and stock market in the Republic of Korea[END_REF]or Cañón-de-Francia and Garcés-Ayerbe, 2009) we apply the market model to estimate abnormal return. This model supports a linear relationship between the return on a stock and the market return over a given time period [START_REF] Sharpe | Capital asset prices: A theory of market equilibrium under conditions of risk[END_REF]. Accordingly, the asset returns are given by: = + + (1)

with = 0 =
where t is the time index, i = 1,2,..., N stands for security, Rit and Rmt are the returns on stock i and the market portfolio respectively during period t, is the intercept of the relationship for stock i;

is the slope of the relationship for stock i with respect to the market return, and is the error term associated with stock i and the time index t.

As mentioned above, Equation ( 1) is generally estimated over a period of 120 days, which includes from day -139 to day -20 prior to the date of ISO 50001 certification. The event window is defined as the period from 1 day prior to the event to 1 day after the event. From Equation 1, the estimated values !and " of and respectively allow one to predict a normal return during the days covered by the event window. The prediction error or estimated abnormal return # $ for stock i on day t (the difference between the actual return and the predicted normal return), is then calculated as:

# $ = -% -& (2)
If we have more than one event in our sample, we can aggregate the abnormal returns across a number of events. Hence, the mean abnormal return for day t within the event window is given by:

'# $ = ( ∑ # $ ( * ( 3
)
where N is the number of announcements in the sample. On the other hand, if an event period consists of more than one day, we can estimate the cumulate abnormal returns over the period T1-T2 as follows:

+# $ , , , = ∑ # $ *. / *. 0 (4)
The next step is to test whether the abnormal returns are statistically different from zero. To do so, we use the traditional parametric t test developed by [START_REF] Brown | Using daily stock returns: the case of event studies[END_REF] and the nonparametric tests descripted by [START_REF] Corrado | A non parametric test for abnormal security price performance in event studies[END_REF].

First, the Brown-Warner test assumes that the mean abnormal returns are normally distributed independently and identically. Consequently the test statistic for any given day is given as:

1 = √3 45 6 77777 8 ~3 0,1 (5)
In Equation 5, the standard deviation of the residuals estimated from the estimation period is used as estimator of . With the assumption that the residuals in Equation 1or the abnormal performance are uncorrelated between the stocks = , the abnormal performance standard deviation is based on the standard deviation of each stock performance measure of the sample in the estimation period. Accordingly, while T indicates the length of the estimation period, the test statistics on day 0 is given as:

; < = = 0 > ∑ 45 ? > @0 0 > A ∑ 0 BC0 ∑ D45 6 E∑ FG 6 B B @0 H / B 6@0 > @0 (6)
where (T-1) degrees of freedom and Student's t distribution. In the Brown-Warner test, calculating the standard deviation of the residuals from the estimation period allows one to solve a probable cross-sectional dependence problem.

Second, the nonparametric test, the Corrado rank test, does not require the symmetrical cross sectional distribution of the excess returns and takes the excess returns' magnitude into consideration. In the Corrado test, all the time series observations of each stock are used. When the rank of the abnormal returns is shown by Kit= rank(ARit), the rank statistics at day 0 is as follows:

+ I=JK = ( ∑ L M ? E NO0 / < M P ( * (7)
where m is the number of total observations in the estimation period and event period. The standard deviation is as:

Q = A ∑ R ( ∑ D - S H ( * T . * (8)
Under the null hypothesis of no abnormal returns, the rank of the excess returns on day 0 turns into uniform distribution. [START_REF] Boehmer | Event-Study Methodology Under Conditions of Event-Induced Variance[END_REF] suggest that Corrado's rank statistics based on median is more resistant against the event-induced variance on day 0, and has a better performance than the Brown-Warner traditional test.

Data setting

As mentioned above, our empirical analysis uses the database provided by Reinhard Peglau -German Federal Environment Agency, which includes the date of adopting ISO 50001 for 1312 companies around the world until June 15, 2013. From this list, we can only select a sample of 40 companies, which are listed on the stock exchanges. The study will be carried out with 40 companies from several sectors and countries (Table 1).

The data on stock prices is collected from Bloomberg Database. On the other hand, in order to eliminate the possible unexpected effects of data heterogeneity, we introduce in our model two control variable. The first one is the standard error of market index controlling for the volatility of market portfolio. The second one is per capita financial consumption expenditure that allows us to control for the different consumption levels of each country having firms in our research list.

<Insert Table 1>

Empirical results

Table 2 presents the market reaction during the period of ISO 50001 announcement, including the day preceding the announcement (Day -1), the day of the announcement (Day 0), the day following the announcement (Day +1). The first column shows the estimated average cumulative abnormal returns +# $ derived from adopting ISO 50001 for each event window. It also provides the estimated mean abnormal returns '# $ for the day preceding/following the announcement (Day -1 / +1) as well as for the day when ISO 50001 is granted (Day 0). The Brown-Warner and

Corrado tests are presented in the second and third column. The last column shows the percentage of negative abnormal returns in each window.

As shown in Table 2, the mean abnormal returns for each event period [-1, +1], [0, +1], and [-1, 0] are all negative (-0.83%, -0.80%, and -0.37%, respectively). Similarly, during our reference event period [-1, +1], the abnormal returns for the Day +1, 0, and -1 are also negative but very small (-0.0007%, -0.0088%, and -0.0118%, respectively). Additionally, the percentage of negative abnormal returns in each window is very important (at least 69%). However, according to the tests of Brown-Warner and Corrado, the empirical results are not statistically significant. These results lead us to accept the null hypothesis of no abnormal returns due to the announcement of ISO 50001. In other words, the results indicate that the market reaction to ISO 50001 adoption is marginally negative but statistically insignificant.

<Insert Table 2> One possibility for the lack of a strong market reaction to the announcement of ISO 50001 could be due to the data heterogeneity in macroeconomic environment of each country, in firm size, in firm reputation and firm initial financial performance. For instance, the significant impacts of firm size have been concluded in several event studies (e.g. Klassen and McLaughlin, 1996;[START_REF] Hendricks | The effect of supply chain glitches on shareholder wealth[END_REF]. The fact is that a smaller firm has the greater impact of any event on its profit than a larger firm. Additionally, the announcement of any event is more important and remarkable for a small firm than a big firm which novel information is not rare. Following this consideration Jacobs et al. ( 2010) test for the dependence of market reaction on American firm size. They find that the dependence is in the theorized direction but statistically insignificant, implying that firm size does not influence the market reaction to any environmental initiative announcement. Consequently, in the literature, the dependence of market reaction on firm size is still inconclusive. For this reason, we will not divide the full sample into different subsample basing on the firm size. Indeed, dividing our data in different subsamples by firm size is not feasible due to the data unavailability and such a small number of considered firms.

On the other hand, it is plausible that market reaction could differ by country subsample. The market reaction may be positive, negative or insignificant, depending on macroeconomic conditions of each country. So that, by grouping all different firms in a single full sample, the average market reaction could be insignificantly different from zero. To overcome this issue, we should divide our full sample into different subsample basing on country characteristics.

However, a small full sample of 40 companies, once again, would not allow us to do so. For this reason, we can only redo event study analysis for a subsample of 14 German companies. The empirical results are presented in Table 3.

As showed in Table 3, the empirical results for German firm sample are, by and large, similar to those for the full sample in terms of sign and significance, but the magnitudes are different as would be expected. In other words, the abnormal returns are generally negative but statistically insignificant, and the percent negative abnormal returns are insignificantly different than the percent abnormal returns during the estimation period. The result suggests that the market does not react significantly to the ISO 50001 announcement also in the case of German companies.

Overall, our findings on the relationship between environmental performance and financial performance are different from those reported in the literature.

<Insert Table 3> The difference between the previous results and ours could be explained in two ways. First, while the environmental performance of a firm is captured by the adoption of ISO 14001 in previous empirical studies (e.g. [START_REF] Filbeck | The relationship between the environmental and financial performance of public utilities[END_REF][START_REF] Cañón-De-Francia | ISO 14001 Environmental Certification: A Sign Valued by the Market?[END_REF][START_REF] Jacobs | An empirical investigation of environmental performance and the market value of the firm[END_REF], this paper considers the adoption of ISO 50001 as an improvement in environmental or energy performance of a firm. Indeed, to our knowledge, we are the first to study the impact of ISO 50001 certification on firms' market value. While in the previous studies, in response to ISO 14001 adoption, the market may react positively (e.g. [START_REF] Jacobs | An empirical investigation of environmental performance and the market value of the firm[END_REF] or negatively (e.g. [START_REF] Filbeck | The relationship between the environmental and financial performance of public utilities[END_REF][START_REF] Cañón-De-Francia | ISO 14001 Environmental Certification: A Sign Valued by the Market?[END_REF], we find no evidence on the link between energy performance and firms' market value. This lack of consensus concerning the relationship between green performance and economic performance has also been justified in the literature. For instance, [START_REF] Telle | It pays to be green-a premature conclusion?[END_REF] suggests that the sign of this relationship strongly depends on the applied analytical method. In an earlier analysis, [START_REF] King | Does it really pay to be green? An empirical study of firm environmental and financial performance[END_REF] reveal that the link between lower pollution and higher financial valuation may be derived from firms' fixed characteristics. These authors also believe that the nature of the relationship between environmental and financial performance depends on the conditions in which environmental measures are implemented. On the other hand, [START_REF] Filbeck | The relationship between the environmental and financial performance of public utilities[END_REF] suggest that the impacts of environmental performance on financial performance are such complicate due to regulation requirements. Second, the lack of market reaction to the adoption of ISO 50001 concluded in our analysis may be due to the increasing frequency of such certifications over the time. Methodologically, one condition for applying event study is that the event (here, ISO 50001 announcement) must be novel information for the market. However, all 40 firms considered in our sample have ISO 14001 or other environmental awards and certifications before adopting ISO 50001. Thus, it is reasonable to consider that comparing to the announcement of preceding certifications, particularly the first environmental certification, the information on the adoption of ISO 50001 is less concerned, which in turn could not lead to any significant market reaction. Besides, while the first initiative for improving environmental performance (notably, the adoption of ISO 14001) may positively influence financial performance of a firm (e.g. [START_REF] Jacobs | An empirical investigation of environmental performance and the market value of the firm[END_REF], subsequent initiatives (notably, the adoption of ISO 50001) may result in costly environmental management and then a reduction in firm's benefit. This argument partially supports our evidence on a negative but insignificant relationship between market reaction and the adoption of ISO 50001.

Conclusion

Unlike a large number of studies focusing on the link between environmental and economic performance, the economic impact of environmental certifications has been underdeveloped in the literature. Furthermore, the existing works have only attempted to advance in the knowledge of the economic effects of ISO 14001. To the best of our knowledge, we are the first to empirically verify the economic impacts of ISO 50001 on the firm market value.

Based on an event study, the empirical results lead us to reject the hypothesis that ISO 50001 certification generates positive abnormal returns in firms' market value. Typically, we find that the market reaction to ISO 50001 is negative but statistically insignificant. The lack of significant

Data collected by Reinhard Peglau -German Federal Environment Agency

Acknowledgements

The author gratefully acknowledges financial support from the Chair Finance of the University of Nantes Research Foundation.

The author also thanks Reinhard Peglau (German Federal Environment Agency) and Amine Boutabba (Université d'Evry Val d'Essonne for providing ISO 50001 certification data.

The author gratefully acknowledges financial support from the Chair Finance of the University of Nantes Research Foundation.

impact of ISO 50001 on firms' market value could be due to two main reasons. The first one is that the link between environmental (captured by ISO 50001) and economic performance is quite complicate as mentioned in [START_REF] Guenster | The economic value of corporate ecoefficiency[END_REF], the financial performance related to environmental performance is not evidence in itself. Thus, we need more time and a larger data sample to completely verify such a complicate relationship. The second one is that compared to other environmental certifications, in particular ISO 14001, ISO 50001 is lately released. This late release, on one hand, has not really attracted firms' registration. That is why, after three year of implement, there are only 1312 companies adopting ISO 50001. On the other hand, for investors, the announcement of ISO 50001 is much less interesting than that of the first environmental certification -ISO 14001. The market, therefore, may react weakly or not at all to the adoption of ISO 50001.

Although we are the first to provide an empirical analysis about the relationship between ISO 50001 and firm's financial performance, there are some limitations in our work. The first one is to consider the market reaction to the announcement of ISO 50001 as a proxy for changes in financial performance, which could be measured by other alternative indicators. Second, although event study is a well-known technique to examine the possible economic impacts of any event in financial economics literature, this method is only applied for the traded firms. Third, due to data availability, our sample including 40 companies around the world is not large enough to further develop the empirical study. Given the relevance of these limitations, our future research should continue to investigate the economic consequences of environmental or energy management system certification by employing other econometric methods, using other financial performance indicator, or developing our empirical analysis also for non-traded companies.

To conclude, the lack or the link between energy and financial performance is not suggesting that adopting ISO 50001 is a bad investment, but rather that the expected role of ISO 50001 in improving firms' financial performance has been unfounded. In this regard, "How does it pay to be green?" seems to be a more important question than "Does it pay to be green?". In other words, our empirical finding suggests a novel question of how a firm pays to be green in order to improve its business benefits.


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Grafting odorant binding proteins on diamond bio-MEMS

Keywords:

Odorant binding proteins (OBPs) are small soluble proteins found in olfactory systems that are capable of binding several types of odorant molecules. Beside, cantilevers based on polycrystalline diamond surfaces are very promising as chemical transducers. Here two methods were investigated for chemically grafting porcine OBPs onto polycrystalline diamond surfaces for the development of biosensors. The first approach results in random orientation of the immobilized proteins over the surface. The second approach based on complexing a histidine-tag located on the protein with nickel allows control of the proteins orientation. These grafting methods were confirmed using electrochemical impedance spectroscopy, fluorescence imaging and X-ray photoelectron spectroscopy analysis. The sensing performances of these OBP modified transducers were assessed. The second grafting method led to typically 20 % more sensitive sensors, as a result of better access of ligands to the proteins active sites and also perhaps a better yield of protein immobilization. This new grafting method appears as highly promising for further investigation of the ligand binding properties of OBPs in general and for the development of non-specific biosensors arrays for artificial olfaction applications.

Introduction

The demand for volatile organic compounds (VOC) detection and identification has increased over the years due to the needs for environmental monitoring, homeland security or medical applications, to name only a few. Amongst the sensing technologies available for VOC detection, biosensors mimicking the way that noses operate to discriminate odorant compounds are highly promising (Sankaran et al., 2012). Odorant binding proteins (OBPs) are important proteins present in many vertebrates and insect olfactory systems. They are small proteins, involved in the perireceptor events associated with olfaction (Dal Monte et al., 1993;Spinelli et al., 1998;Pelosi et al., 2001;Briand et al., 2002). They were initially discovered by Pelosi and co-workers in the nasal olfactory mucosa of bovines and other mammals as a soluble protein able of binding 2-isobutyl-3-methoxypyrazine (IBMP), an odorant molecule with an extremely low detection threshold (Pelosi et al., 1982). Vertebrate OBPs are about 17-20 kDa; they belong to the superfamily of lipocalins and are ai l orga ized i a β-barrel structure, with a calyx-shaped a it , ade up of eight a tiparallel β-sheets with a short segment of αhelix at the C-terminus (Flower, 1999). The ligand binding site is located i the ore of the β-barrel, as clearly demonstrated by the use of a selenium-containing ligand, 2-amino-4-butyl-5propylselenazole (Napolitano et al., 1992). Their ability to bind diverse odorants, together with their high conformational stability are attractive characteristics for the design of label free biosensors in particular for the detection of small organic molecules (Garibotti et al., 1997;Vincent et al., 2000;Persaud et al., 1996;Pelosi et al., 2013). Label-free biosensors are generally based on optical, electrochemical or gravimetric transduction methods. Transducers based on microcantilever technology are attractive for ligand binding proteins based biosensors that do not involve electrochemical processes, because they can offer high sensitivity, they can be miniaturized and are suitable for parallelization into arrays. Microcantilevers have recently opened new perspectives for instance for the detection of different toxic metal ions, volatile organics, explosives, etc. (Baller et al., 2000 ;Lavrik et al., 2004 ;Goeders et al., 2008;Boisen et al., 2011;Hwang et al., 2011 ;Spitzer et al., 2012). We report on the use of poly-crystalline diamond micro-cantilevers combined with OBP receptors for the detection of small volatile organic molecules. Diamond is highly desirable for the design of microcantilevers because it exhibits e eptio al e ha i al properties, su h as a er high You g's modulus in the order of 1000 GPa (Bongrain et al., 2009) as well as an excellent hardness and robustness. Moreover, its carbon nature offers wide opportunities for stable grafting of wide a range of bio-receptors via covalent C-C binding (Härtl et al., 2004;Agnès et al., 2010). The extreme sensitivity of diamond microcantilevers to surface stress variations upon chemical or biochemical interactions occurring on their sensitive surface was demonstrated recently (Bongrain et al., 2011). In this case, the high You g's odulus of dia o d allo ed the a tile ers to e operated i the resonant regime in the liquid phase while retaining a reasonably high quality factor (Q-factor).

In the present study, the immobilization of OBPs on diamond surfaces is investigated. Thus, we explore immobilization of a wild-type OBP (wtOBPpig) as well as a mutant OBP (m6hisOBPpig), both from pig, on diamond microcantilevers using two different original grafting strategies: (i) hexanoic acid (ha) covalently attached to hydrogen-terminated diamond surfaces followed by 1-ethyl-3-[3dimethylaminopropyl]-carbodiimidehydrochloride/sulfo-N-hydroxysuccinimide (EDC/NHS) coupling with OBP and (ii) a method based on diamond modified with nitrolotriacetic acid nickel complex (NTA-Ni 2+ ) able to bind specifically to histidine-tagged proteins. Immobilization of OBPs was validated using electrical impedance spectroscopy (EIS), fluorescence methods and X-ray photoelectron spectroscopy (XPS). The first immobilization strategy results in random orientation of the proteins over the sensor surface whereas the latter approach enables orientation of the proteins due to the controlled position of the histidine tag attached to the N-terminal of the proteins. The sensing performances of the two resulting sensors are compared for IBMP, a reference compound commonly used for the characterization of OBPs affinity constants, as well as for 2,4-dinitrotoluene (2,4-DNT), used as a surrogate for the explosive trinitrotoluene (TNT).

Material and methods

Boron Doped Diamond Electrodes (BDD) fabrication

BDD films with a boron concentration typically of 2 × 10 21 at • cm -3 were synthesized by microwave plasma enhanced chemical vapor deposition (MPECVD) over a p-Si (100) substrate using a SEKI Technotron Corp. AX6500X reactor with a gas mixture of methane, hydrogen and trimethylboron at typically 800 °C with a pressure of 25 mbar and a microwave power of 2600 W. After growth the electrodes are naturally hydrogen terminated. Some of the electrodes used for control experiment were oxidized under ultraviolet (UV) light/ozone exposure using an excimer UV lamp (Heraeus) generating intensive radiation at 172 nm.

Diamond Microcantilever fabrication

Diamond microcantilevers were fabricated following a process previously described (Bongrain et al., 2009). In brief, silicon moulds prepared by deep reactive ion etching over 4 inches wafers were seeded selectively with diamond nanoparticles using standard clean room patterning approaches. Then, the cantilevers were grown by MPECVD inside the moulds and finally release by dry etching. Cantilever dimensions were 210 µm X 140 µm. Before any grafting procedure the diamond cantilever surfaces were re-hydrogenated at 700°C in hydrogen plasma. The cantilevers geometry led to a resonance frequency of 145000 +/-9900 Hz and a Q-factor in the order of 600 in air and in the order of 100 in liquid. An SEM image of a typical cantilever used in this study is shown in Fig. S1 in supplementary file.

OBPs preparation

OBPs preparation is reported in detail in supplementary data.

First method (1) of OBPs immobilization

All chemicals used for OBP grafting were purchased from Sigma-Aldrich, France. The first method of OBPs immobilization (Fig. 1(a)) consisted of grafting in a first step a hexanoic acid radical covalently onto hydrogen terminated diamond surface using a process described elsewhere in the case of aminated biotin and ferrocene (Agnès et al., 2010;Bongrain et al., 2011). This covalent immobilization of aliphatic primary amines on hydrogen terminated diamond has been explained by Ruffinato (2010) and co-workers as being a nucleophile substitution involving the formation of carbanion at the diamond surface in the presence of OH -ions in solution. The reaction of the protonated amine with the labile carbanion results in the release of ammonia and C-C covalent grafting of the aliphatic compound over the diamond surface (Ruffinato, 2012). Thus aminocaproic acid (aca) solution at a concentration of 1mM was prepared by dissolving the adequate amount of aca in a 0.2 M phosphate buffer. The hexanoic acid radical was grafted over the diamond surface by dipping the hydrogen terminated diamond substrates (electrode or cantilever) in the aca solution for 10 min followed by thorough rinsing in deionised (DI) water. Next the immobilized carboxylic acid was activated with EDC/NHS (30 mM/ 60 mM) treatment for 2 hours [START_REF] Fischer | Surface Plasmon Resonance[END_REF]. This protocol enabled subsequent binding to the wtOBPpig protein. Here the substrate was exposed to wtOBPpig solution (0.95 mg/mL in 50 mM NaHCO 3 at pH=8.00) for two hours. Finally the substrates were rinsed thoroughly with DI water and stored in phosphate buffered saline (PBS) solution.

Second method (2) of OBPs immobilization

A second original approach (Fig. 1(b)) was investigated in an attempt to control the orientation of the proteins immobilized on the surface. Here the method consisted of immobilizing a nitriloacetic acid group from 1 mM solution of Nα,Nα-Bis-(carboxymethyl)-L-lysine hydrate ≥ 97. % (NTA) onto the diamond surface using the same process used for immobilization of aca. Subsequently, the diamond surface was exposed to 1 mM Nickel(II) chloride hexahydrate (99.99%) solution for 2 h at room temperature. The resulting NTA•Ni 2+ complex was formed thus enabling the OBP immobilization. NTA has the ability to bind to Nickel (II), which complex has a high affinity with histidine groups (Johnson et al., 2004;Blankespoor et al., 2005;Brellier et al., 2009). Hence m6hisOBPpig were finally immobilized by exposing the Ni-NTA terminated diamond surfaces to m6hisOBPpig solution (1.0 mg/mL in 20 mM PBS, pH=8.00) for 2 hours. Again the substrates were rinsed thoroughly with DI water and stored in PBS solution.

Fig. 1. Summary of OBP immobilization on hydrogenated diamond surfaces using grafting method ( 1), (a) and using grafting method (2), (b).

EIS measurements

In order to characterize the surface coverage of OBP onto the diamond surface, EIS measurements were performed. The electrochemical characterizations were carried out in a 3 electrodes setup where BDD is the working electrode, and platinum wires both the counter and pseudo-reference electrodes, respectively. Ultrapure DI water (Millipore Direct Q3) was used to make all the solutions. EIS was carried out in equimolar (1 mM) potassium ferricyanide(III)/ potassium hexacyanoferrate(II) trihydrate (Acros Organics) dissolved in 0.5 M potassium chloride solution (Acros Organics). The specific transfer resistances (Rt) were experimentally determined before and after each immobilization step by EIS over a frequency range of 50 kHz-10 mHz with logarithmic point spacing and potential amplitude of 0.01 V root-mean-square (rms) while the BDD electrode was maintained at open circuit potential. EIS spectra were recorded after each grafting step for both immobilization procedures, starting from the hydrogen terminated diamond electrode surface to OBPs grafted surface. Nyquist plots were also recorded with oxidized diamond electrodes, used as negative control since in the experimental conditions used the amine terminated primary linker (aca or NTA) can only be immobilized on hydrogen terminated surfaces (Agnès et al., 2010). Hence OBPs can theoretically be immobilized only on such surfaces that contain the immobilized linker.

Fluorescence imaging

Further confirmation of protein immobilization was obtained by fluorescence imaging. For this purpose, two identical samples were prepared as follows. The hydrogen-terminated surface was patterned by conventional photolithography using a process similar to that described elsewhere (Härtl et al., 2004). Since attachment of hexanoic acid or NTA radicals via the amino-group is only possible on hydrogen-terminated surfaces, the purpose of this experiment was to demonstrate that the proteins are immobilized indeed only where the diamond surface was originally hydrogenated and therefore where a linker molecule has been attached. It verifies also that no non-specific adsorption of the proteins occurs over the control (oxidized) areas. Thus, a photosensitive resin was deposited onto hydrogen-terminated diamond substrate a d patter ed × spots) by photolithography. The samples was then oxidized in the unprotected areas under oxygen plasma (2 mbar oxygen, 50W, 30 s) in a reactive ion etching equipment. The resin was then removed and the grafting procedure applied on the whole sample. After the last step of OBPs grafting, a first sample was immersed in a fluorescein solution then rinsed vigorously with DI water. Fluorescein has the ability to bind the OBP thus offering the possibility to visualize under UV excitation the presence of OBPs on the diamond surface. The same experiment was done two months later with the second sample in order to verify whether the OBP was still present and active over the surface.

Fluorescence imaging was also used to verify the presence of OBPs on cantilevers after the immobilization step as well as after several exposures to the target chemical. Fluorescence microscopy images were recorded at exc = a d em = 570 nm.

XPS surface analysis

XPS analyses were used in this study to characterize the NTA-Ni grafting on hydrogenated diamond surface. The methods and experimental conditions used for XPS surface analysis are reported in detail in Supplementary data.

Cantilever resonance frequency measurements

The dynamic response (resonance frequency shift) of the biosensors was achieved by Laser Doppler interferometry using a Polytec setup consisting of an interferometer (OFV534) and a vibrometer controller (OFV5000), coupled to a network analyzer. The operation principle of this equipment has been described elsewhere (Bongrain et al., 2011). This setup can detect vertical oscillations in the picometer range, while cantilevers excited by the piezoelectric cell exhibit amplitudes between tens to one hundred nanometers at their resonance frequency. The micro-cantilevers were mounted in a liquid cell with a constant volume (200 µL) underneath which an external piezo-electric vibrator was placed to allow structures actuation through acoustic waves. A stabilization step was necessary when the cantilever environment changed from air to liquid. This stabilization time is due to temperature stabilization in the liquid cell, adsorption of dissolved salts from the buffer solution on the cantilever surface (Godin et al., 2010) and surface stress stabilization at the cantilever-liquid interface. Exposure to the different test solutions was carried out as follows. 50µL of the blank solution was pipetted out from the cell and replaced by the same volume of either IBMP solution or 2,4-DNT solution so that the cantilevers remained immerged throughout all experiments. Gentle stirring was carried out following analyte addition to ensure homogeneous concentration during measurements. This did not affect the measurements since the kinetics involved through binding with OBPs are typically in the order of several minutes. The resonance frequency shift measurements were recorded with a sampling time of 20 s. proceeds by a inner-sphere transfer mechanism and the electrode kinetics is highly sensitive to the electrode surface conditions. Thus the presence of immobilized organic species over the electrode surface causes a significant decrease in the electron transfer rate and therefore an increase in Rt. In contrast, when the same experiment is performed using an oxidized surface, virtually no change in Rt resistance is observed after the different grafting steps. This suggests that hexanoic acid groups and subsequently OBPs are indeed grafted only on the hydrogen terminated diamond surface as expected but not on the oxidized surface.

Results and discussion

Fig. 2 (c) su arizes the Rt alues e tra ted fro the Ra dle's odel for oth ele trodes and for both grafting method. In the case of the hydrogen terminated electrode, Rt is increased from typically 78 Ω to 896 Ω fro the la k ele trode to the OBP grafted ele trode using method (1), respectively.

The results were very similar and immobilization was also successful only on the hydrogen terminated electrode with the grafting method (2), based on NTA grafting followed by the NTA-Ni 2+ complex formation which enables the m6hisOBPpig immobilization via histidine group. Here the Rt values increased from typically 72 Ω to 1630 Ω fro the are diamond electrode to the OBP grafted electrode, respectively. The significantly higher Rt value obtained for the OBP grafted electrode using method (2) tends to indicate that this latter method is more efficient and more OBPs are present over the electrode surface.

After immobilization of m6hisOBPpig on the electrode surface using method (2), the surface was exposed to imidazole (200 mM in PBS at pH=7.4). Imidazole has the ability to binds competitively NTA against Ni 2+ , which has for effect to release OBPs off the surface. This experiment was carried out in order to verify that OBPs were indeed linked to nickel rather than being non-specifically adsorbed onto the surface. Fig. S2 (see Supplementary data) shows that after exposure to imidazole solution, the transfer resistance Rt indeed is very significantly reduced from typically 1677 Ω to 394 Ω, most presumably as a result of OBPs detachment. This tends to give further evidence that OBPs are indeed attached to the diamond surface via complexation of nickel. Fig. S3 (see Supplementary data) reports the assessment of OBP immobilization kinetic by recording the evolution of Rt upon exposure of NTA-Ni modified electrode to m6hisOBPpig solution at a concentration of 1.0 mg/mL. The resulting curve is showing that a plateau is achieved after typically 200 minutes (T 90% = 140 min).

Fluorescence imaging

OBP immobilization was also evidenced by fluorescence imaging using the protocol described in section 2.4. Fig. 3 shows a resulting fluorescence microscopic image of wtOBPpig immobilized on hydrogen-terminated diamond patterns. This experiment confirmed the presence of OBPs only on the region where the diamond was originally hydrogenated. The absence of any fluorescence signal on the oxidized area confirms also that no significant non-specific OBP adsorption occurs outside the pattern. The same experiment was repeated with the second grafting method and led to the same result. No significant difference in fluorescence was observed when the process was repeated using a second sample that was stored in air for two month after grafting the OBPs.

Sensor performances

The detection of IBMP and 2,4-DNT with diamond microelectromechanical systems (MEMS) sensors grafted with m6hisOBPpig was investigated using both methods ( 1) and ( 2) as described previously.

At start, the resonance frequency of cantilevers was measured in air and then in an aqueous volume of 200µL at pH=8. As expected the resonance frequency strongly decreases from typically 145 kHz to approx. 35 kHz when dipped into the buffer solution. This is due to damping resulting from the increase of viscosity of the surrounding liquid medium (Vancura et al., 2008). Then, a volume of 50 µL of this solution was replaced by the same volume of either IBMP or 2,4-DNT solutions at various concentrations to cover the concentration range from 60 µM to 1.32 mM for IBMP and 2,4-DNT, after dilution. After each subsequent analyte addition, a significant increase of the resonance frequency was observed. The new frequency values were recorded when a plateau was reached. Each value is given for a set of five different cantilevers in order to assess the reproducibility of the entire process. Each data point is the mean value recorded for the five cantilevers. The highest frequency shift recorded was in the order of 300 Hz in the case of IBMP exposure for a concentration of 1.32 mM. The stability of the OBPs on the surface of the cantilever was also explored. Experiments were performed with the same cantilever over 2 month periods with no significant change in sensitivity after 2 month storage in air at room temperature. RSD for figure 5 are reported in detail in supplementary data in Table S1. Calibration curves for both biosensors in response to the target analytes is shown in Fig. 5. In all cases, the sensor response is linear with the analyte concentrations. Table 1 summarizes for each sensor the values of sensitivities, with the time constants at different concentrations of odorant concentrations extracted by fitting a first-order exponential regression on the response curves. The highest sensitivities and response times are achieved for IBMP. This is expected since the affinity constant of porcine OBP for this compound is known to be very high (Pelosi et al ., 1982;Pevsner et al., 1985;Baldaccini et al., 1986;Pevsner et al., 1990;Burova etal., 1999). Nevertheless, the sensitivity to 2,4-DNT is also comparatively high and confirms the fairly high sensitivity of porcine OBP to aromatic compounds as reported by Vincent et al., 2000;Ramoni et al., 2007 andWei et al., 2008. Furthermore, the sensitivity of the NTA-Ni-m6hisOBPpig sensor to both odorant molecules is significantly higher than the sensitivity of the aca-m6hisOBPpig sensor, here typically 20 % higher for both IBMP and 2,4-DNT.

The sensors modified by NTA-Ni complex linker was found to be generally more sensitive to odorants and more reproducible than the sensors modified with aca-m6HisOBPpig. This could be explained by the better access of ligand molecules to the active sites of the proteins and also perhaps a better yield of immobilization of the proteins when using the NTA-NI linker. In both cases the sensitivity toward IBMP and 2,4-DNT was fairly close. This is expected since porcine OBP is relatively nonspecific and has the ability to bind a broad range of molecules. The response of the cantilevers is mainly due to surface stress variations arising from slight conformal changes of the proteins upon binding. Even though those conformal changes are known to be small, a reasonable sensitivity was observed and a linear relationship was found between concentration of ligand and frequency variations. This result suggests that orientating the protein over the surface has a significant effect on the overall se siti it . "i e the a ti e site of the age ole ule is fa i g the outside e iro e t ore molecules are likely to bind the receptors. A better yield of the grafting reaction resulting in more OBPs being immobilized over the surface as suggested by a larger increase of Rt upon grafting with method (2) (Fig. 2 (c)) may also contribute to the better sensitivity. Nevertheless, for both IBMP and 2,4-DNT, the time constants for the sensors grafted with method (2) are significantly higher than those measured with method (1), therefore confirming that the active sites of the proteins are probably more easily accessible in this case. Interestingly also, one can notice in table 1 as well as on the error bars in Fig. 5 (a) and (b) that the calculated standard variations are significantly smaller for method (2), indicating that method (2) is more reproducible. In fact, one could easily interpret that when OBPs are randomly oriented over the surface; the number of active sites available varies randomly from sensor to sensor, resulting in the significantly higher standard variations. On the other hand, when all sites are equally oriented, they are more likely to offer similar number of binding sites, thus leading to more reproducible sensors responses, provided that the density of active sites is constant from one sensor to another.

Ta le . Co pariso et ee se siti ities s a d τ for IBMP a d , -DNT detection using the two OBP grafting methods with SD and RSD.

In the gas phase, adsorption of molecular species over the transducer surface contributes to increase the cantilever effective mass, which generally results in a decrease of the cantilever resonance frequency. However in liquid media the cantilever mass sensitivity is significantly reduced due to viscous damping and fluid load effects (Chen et al., 1995;Hwang et al., 2006). In the latter case, the resonance frequency of the cantilever may be influenced by hydrodynamic interactions and/or by surface stress variations. Since the binding of the odorant molecules in our case does not involve much charge transfer from or to the OBP, the overall surface charge of the transducer is not likely to be significantly affected by the presence of adsorbed analytes. Moreover, the cantilever response being positive, one can assume that the response is mainly arising from surface stress variations over the cantilever and not from mass loading. A positive response could also be attributed to a loss of mass due to detachment of the OBPs upon conformal changes. It is believed this is not happening because the response was fully reversible after many response cycles. Moreover fluorescence imaging performed on cantilevers after several measurements confirmed the presence of OBPs over the surface of the cantilevers. The evolution of the resonance frequency in liquids following mass and bending stiffness variations may be described by equation ( 1) where m is the effective mass of the cantilever, E is the Young's odulus of the aterial o posi g the a tile er (here 980 GPa), w, t and l are the width, thickness and length of the cantilever, k and m are the stiffness and effective mass variations, and u is the mass per unit length applied by the fluid. u can be extracted from the ratio of the measured cantilever resonance frequency in air and in the liquid according to equation ( 2), where the contribution of the quality factors is neglected (Bongrain et al., 2011).

√ √ (1) √ (2)
It has been shown that the binding of an odorant molecule can induce local conformational changes in OBP cavities. Such ligand-binding induced conformational changes and folded structure changes generally modify the protein spectral properties and have been studied by UV or Fourier transform infrared spectroscopy. Those conformal changes depend on the type of OBP and the shape of the odorant molecule, as well as on the conformation of the ligand in the OBP cavity (Nespoulous et al., 2004). According to the literature, the porcine OBP lipocalin structure is less affected by the presence of ligands than rat OBPs for instance (Burova et al., 1999). Indeed, very few specific interactions were established inside the porcine OBP cavity between the odorant molecule and the amino acid sidechains, which did not induce large conformational changes (Hajjar et al., 2006). The crystallographic structures of porcine OBPs complexed with odorant molecules revealed that different odorant molecules can interact in the cavity, with alternative conformations, thus revealing the absence of specific interactions and the role of porcine OBPs as non-specific carriers, able to recognize efficiently a large number of molecules belonging to different chemical classes (Vincent et al., 2000, Vincent et al., 2004). This explains probably the relatively small difference in sensitivity between DNT and IBMP. The maximum stiffness variation upon the largest change in resonance frequency obtained during exposure of NTA-NI-m6hisOBPpig modified cantilever to 1.32 mM IBMP calculated using eq (1) was found to be 1.7 N.m -1 . Since the effective stiffness coefficient of this cantilever is fairly high, approximately k = 100 according to equation k=Ewt 3 /4l 3 , this corresponds to a stiffness variation of 1.7 %. This is relatively weak when compared for instance to the sensitivity of diamond cantilevers grafted with carboxylic acid groups which exhibit a k value as high as 6% upon deprotonation of the acid. This supports the assumption of low conformal changes of the OBP upon ligand-binding and hence the low effect on surface stress. The corresponding sensitivity to IBMP over stiffness variations was found to be 0.163 Hz.mN -1 .m for NTA-Ni-m6hisOBPpig and 0.161 Hz.mN -1 .m for aca-m6HisOBPpig cantilevers, respectively. This is almost one order of magnitude below the sensitivity of similar diamond cantilevers to 32base-pair DNA hybridization of probe DNA immobilized over their surface (Bongrain et al, 2010). Indeed in the latter case the sensitivity is expected to be higher since steric effects are more significant due to the large increase in density of DNA strains and the overall charge over the surface. Nevertheless the sensitivity of the diamond cantilevers is sufficient to allow monitoring the ligand binding to OBP in the µM concentration range.

Conclusions

We reported on the grafting of porcine OBP onto diamond microcantilevers using two different chemical approaches enabling either random or oriented immobilization of the proteins over the surface. Surface immobilization of the proteins was validated by impedance spectroscopy, fluorescence imaging and XPS analysis in the case of the oriented method using NTA-Ni complex linker. This grafting technique provided sensors with better sensitivity for the detection of IBMP as well as 2,4-DNT, a simulant for explosive TNT, in the µM concentration range. This is highly promising for further investigating the ligand binding properties of OBPs in general and for the development of reliable non selective biosensors that may be used in vapor sensing applications such as for the detection of explosives. The low specificity of this biosensor will be a certain inconvenience to overcome.

  Fig.2 (a)shows typical Nyquist plots recorded on a bare hydrogen terminated electrode as well as on the same electrode following immobilization of aca and subsequently wtOBPpig by EDC/NHS coupling. The same plot is presented in Fig.2 (b) for the same immobilization attempt carried out onto an oxidized BDD electrode. The impedance measurements in this case can be analyzed by fitting the impedance spectra to the Randles equivalent circuit(Wang et al., 2004). Here the intercept of the observed semi-circle with the Z RE axis at high frequency gives the value of the solution resistance (Rs), whereas extrapolation of the semi-circle to low frequencies yields an intercept corresponding to Rs + Rt, where Rt is the charge-transfer resistance. It can be seen clearly that in the case of the hydrogen-terminated electrode, Rt increases slightly from the blank hydrogen terminated surface (black curve) to the aca grafted sample (red curve). Then a further increase, much larger this time, is observed with the same electrode upon OBP immobilization. Electron transfer for Fe(CN) 6 -3/-4

Fig. 3 .

 3 Fig. 3. Fluores e e i ros op pi ture o ser ed at e = a d e = 570 nm of diamond patterned by oxidation and functionalized with OBP immobilization after the immersion in a fluorescein solution.

Fig. 4 .

 4 Fig. 4. High resolution Ni 2p core level XPS spectrum of NTA-Ni modified hydrogenated diamond surface.

Fig. 5 .

 5 Fig. 5. Concentration response profile for NTA-Ni-m6hisOBPpig and aca-m6hisOBPpig based biosensors (a) upon exposure to different concentrations of IBMP, inset: kinetics response curves upon a specific concentration of IBMP; (b) upon exposure to different concentrations of 2,4-DNT. Errors bars: +/-Standard deviation (SD) calculated from 5 measurements carried out on 5 cantilevers.

Acknowledgements

Financial support from the European Commission Research Innovation -7 th Framework Programme (FP7-SECURITY project n° 285203) is gratefully acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found in the online version.

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Sylvain Jay

Gilles Rabatel

Nathalie Gorretta

IN-FIELD CROP ROW STEREO-RECONSTRUCTION FOR PLANT PHENOTYPING

Keywords: In-field phenotyping, 3D modeling, Structure from motion, Plant/soil discrimination, Plant height retrieval, Leaf area retrieval

This article presents a methodology for crop row structure characterization developed for phenotyping purpose. It is based on 3D modeling performed using Structure from Motion. The model provides an accurate crop row reconstruction and serves as a basis to retrieve plant height and leaf area. To do so, a robust discrimination method using both color and height information is proposed to separate vegetation from soil. The point cloud is scaled and the plant surface is approximated by a triangular mesh. The efficiency of our method was assessed with two data sets collected in outdoor conditions. Height estimation was extremely accurate since both average error and error of reference measurement had the same order of magnitude. Strong correlations and low errors were also obtained for leaf area estimation. The proposed method thus provides an interesting tool for phenotyping.

Introduction

In a context of a greener and more competitive agriculture, developing efficient phenotyping methods has become a topic of major interest for plant breeding. Numerous parameters have to be retrieved, especially those characterizing the plant structure, such as height or leaf area. 3D reconstruction performed by stereovision-based methods has proven to be an interesting way to retrieve these structural parameters. In controlled indoor conditions, many solutions have been proposed [START_REF] Bellasio | Computer reconstruction of plant growth and chlorophyll fluorescence emission in three spatial dimensions[END_REF][START_REF] Thiagó Santos | Image-based 3d digitizing for plant architecture analysis and phenotyping[END_REF][START_REF] He | Growth prediction of a transplant population using artificial neural networks combined with image analysis[END_REF][START_REF] Chéné | On the use of depth camera for 3D phenotyping of entire plants[END_REF], but unfortunately, most of them are not adapted to phenotyping (low throughput, dedicated to a single variety, ineffective under natural light). In outdoor conditions, the reconstruction process and estimation results can be substantially affected by various phenomena such as wind or heterogeneous background and lighting. As a matter of fact, only few authors conducted field studies [START_REF] Kise | Development of a stereovision sensing system for 3D crop row structure mapping and tractor guidance[END_REF][START_REF] Leemans | A method for plant leaf area measurement by using stereo vision[END_REF].

To deal with such conditions, [START_REF] Lati | Estimating plant growth parameters using an energy minimization-based stereovision model[END_REF] have recently proposed a novel approach for the estimation of height, leaf cover area and biomass volume. Their method can even handle occluded areas to some extent (i.e. overlapped leaves). Such 3D-based measurements often necessitate to discriminate plant from background, since only plant-related information is used to retrieve structural parameters. Most usual discrimination methods involve thresholding a vegetation index map obtained from RGB bands [START_REF] Meyer | Verification of color vegetation indices for automated crop imaging applications[END_REF]. On the other hand, [START_REF] Lati | Estimating plant growth parameters using an energy minimization-based stereovision model[END_REF] used a hue-invariant transformation that is robust under natural lighting. However, like every other color-based discrimination method, this approach fails if the contrast between plants and soil is low, which makes their 3D-based measurement method ineffective in such cases. This study proposes a 3D modeling based methodology to characterize crop rows in outdoor conditions (i.e. to retrieve structure, plant height and leaf area). Special attention is given to the methodology robustness against heterogeneous lighting and low vegetation/soil contrast. Moreover, other phenotyping-related needs (non-destructive, automatic, fast) are considered. We use Structure from Motion (SfM) to obtain the crop row 3D model, whereas most authors usually used classical stereovision. Structural parameters are then retrieved from this model. This paper is organized as follows. Section 2 presents the proposed approach. The data sets as well as some details about the implementation of the method are described in Section 3. Section 4 shows the results of 3D reconstruction, discrimination and growth parameter retrieval. Lastly, Section 5 draws some conclusions.

Proposed approach

The framework of the proposed method is presented in Fig. 1. A first part is related to the 3D reconstruction performed using SfM. In a second part, various post-processing steps are applied to the 3D model in order to retrieve the growth parameters of interest.

3D reconstruction

Stereovision allows to retrieve object 3D structure from 2D images acquired from different view angles. Using SfM, these view angles are obtained by moving a single camera round the object of interest. This motion is either unconstrained or constrained by a translation stage. In this latter case, the camera is translated at a given speed and acquires an image at every ∆x, where ∆x is the baseline. Therefore, every point of the scene is seen in several images that correspond to several view angles. Knowing the position of a given point in several images and successive camera positions, the point position can be estimated by ray intersection. An advantage of using SfM compared with classical stereovision is that it does not need any prior camera calibration. Indeed, the high number of images and use of a unique camera allow to simultaneously estimate camera properties and 3D model. The three usual main steps of SfM are described in the following paragraphs. The first step consists in retrieving and matching similar feature points that are seen in two overlapping images using the scale-invariant feature transform (SIFT) [START_REF] Lowe | Distinctive images features from scale-invariant keypoints[END_REF]. This method has been designed to provide keypoint descriptors that are invariant to scale, rotation, translation and exposure. Examples of such extracted keypoints obtained for a couple of images are shown in Fig. 2a. In the second step, these keypoints are used as inputs of an iterative algorithm that simultaneously estimates their 3D position and the camera internal calibration, position and orientation for every image. At each iteration, an image is added and a bundle adjustment is performed in order to limit error accumulation [START_REF] Hartley | Multiple View Geometry in Computer Vision[END_REF]. The computed sparse point cloud (corresponding to keypoint positions) and the camera successive positions are presented in Fig. 2b. Lastly, in the third step, a dense point cloud is produced from estimated orientations and positions of the camera. For this purpose, a matching process using cross-correlation is implemented. For a given pair of overlapping images, a pixel in the first image is associated with the pixel that is located on the epipolar line in the second image and that maximizes the cross-correlation criterion. This operation is repeated for every couple of images so that the estimated position of considered point is less sensitive to noise. An example of crop row reconstruction is shown in Fig. 2c.

Post-processing

Once the crop row structure has been modeled, various additional processing steps have to be implemented in order to retrieve growth parameters from the 3D model. First, the point cloud has to be given an appropriate scale that allows to retrieve height and area values in SI units (resp. cm and cm 2 ). Then, an important step is to discriminate soil-related pixels from plant-related pixels, since only the latter is used to estimate height and leaf area. In order to increase the class separability obtained when using only color information, we propose to take into account spatial information as well, and use together the height H and Excess Green Index (ExG) [START_REF] Woebbecke | Color indices for weed identification under various soil, residue, and lighting conditions[END_REF]. Indeed, the higher the point, the more likely it belongs to the vegetation class. To do so, after having estimated the crop row structure, we perform discrimination using a clustering algorithm with two classes (vegetation and background). Plant height is finally retrieved by computing the difference between the plant highest height and the mean ground height. Concerning leaf area, an additional post-processing step is needed in order to reconstruct the plant surface. A triangular mesh is produced from the point cloud and the leaf area is then approximated by summing every polygon area.

Materials ans methods

Data

We assessed the efficiency of our method with two data sets collected in outdoor conditions. Different sensors, acquisition means, lighting conditions and crop row structures were considered. The first data set comprised images acquired with a digital single-lens reflex (DSLR) camera (Canon 500D). Four plant species or varieties were imaged in order to deal with different plant structures: sunflowers (Helianthus annuus), Savoy cabbages (Brassica oleracea var. sabauda), cauliflowers (Brassica oleracea var. botrytis) and Brussels sprouts (Brassica oleracea var. gemmifera). The camera was looking towards nadir and images were acquired approximately every 10 cm by manually moving the camera at about 1 m above the ground level (depending on plant height). The second data set was collected in sugar beet (Beta Vulgaris) fields in Vimy (northern France) within the framework of AKER project 1 . Two sugar beet varieties subjected to four different nitrogen applications were imaged. To do so, a digital CCD camera was mounted on the translation stage of the Becam phenotyping platform 2 (Benet and Humbert, 2009) (see Fig. 3). The camera was looking towards nadir at 1.20 m above the ground level and acquired images every 4 cm along the crop row so that every point in the scene was imaged from a dozen view angles. For both data sets, a tape measure was disposed into the scene before acquiring images. This allows to scale the 3D model and obtain measurements in centimeters and square centimeters.

Reference height measurement was performed manually by measuring the distance between the mean ground level and the plant highest point. To perform reference leaf area measurement, limbs were harvested, disposed on blank sheets and flattened with a pane of glass before scanning. Leaf-related pixels were then extracted by image processing. Finally, leaf area in cm 2 was obtained from leaf area in pixels using pixel size calibration. In the second data set, only one mean measurement of leaf area (carried out on four to six plants) was available for each row. Height and leaf area ranges for the two data sets are reported in Tab. 1.

1 AKER is a project funded by the French National Research Agency and partly devoted to sugar beet phenotyping 2 The Becam phenotyping platform was developed by Irstea for plant counting and belongs to the French Sugar Beet Technical Institute (ITB).

(a) RGB images.

(b) Estimated 3D model.

Implementation

Regarding 3D reconstruction, SfM was implemented with MicMac, a digital surface model freeware developed by IGN (2007). Section 2.1 only describes an overview of the main steps performed by MicMac. For more information, the reader can refer to the associated documentation (IGN, 2007).

Regarding post-processing steps, scaling was done thanks to a reference object disposed in the scene (see the yellow tape measure in Fig. 2). Discrimination was performed using H and ExG with the k-means (KM) clustering algorithm [START_REF] Lloyd | Least square quantization in pcm[END_REF] implemented with two classes. Lastly, plant surface was approximated using the ball-pivoting algorithm [START_REF] Bernardini | The ballpivoting algorithm for surface reconstruction[END_REF] implemented in Meshlab.

In order to assess height and leaf area estimation results, we used the coefficient of determination (R 2 ), root mean square error (RMSE) and mean absolute error (MAE). In particular, RMSE and MAE have been defined by [START_REF] Willmott | Advantages of the mean absolute error (MAE) over the root mean square error (RMSE) in assessing average model performance[END_REF].

Results and discussion

3D reconstruction

An example of reconstruction built from the first data set is presented in Fig. 4 (Savoy cabbages). Sugar beet row reconstruction obtained from the second data set is shown in Fig. 2c. In every case, SfM gave an accurate crop row 3D model. Moreover, this method was robust against acquisition means and sensor quality, which were different within the two data sets. In the first data set, the translation motion was performed manually, moving the camera horizontally above the ground level. As a result, the camera altitude, view angle and baseline between two successive positions differed from an image to another. However, this does not affect the reconstruction process, which makes the method flexible and easy to implement (no need for a specific imaging setup). In the second data set, good reconstruction results were obtained even though the sensor quality was lower.

Reconstruction was performed with a 3.60 GHz processor and 4 GB of RAM.

Overall processing time was comprised between 30 seconds and 60 seconds per image, depending on the crop row structure complexity and the number of images.

Discrimination vegetation/soil

The interest of using height for discrimination is illustrated in Fig. 5. In the RGB image shown in Fig. 5a, both sunflower and background have similar ExG values. Moreover, the background is heterogeneous so the ExG range is wide (between 0 and 0.8). In this case, color is not a relevant parameter to discriminate both classes since there is only one mode on the green histogram in Fig. 5b. On the other hand, at least two distinct modes are seen on the red histogram showing the height distribution. In fact, there are one main cluster for the background and one or more clusters for the plant. This scatter plot shows that height information can remove ambiguity occurring when using color only.

Plant height estimation

Fig. 6 shows the results of height estimation performed on the first data set. Estimated values are plotted against actual values for every studied species. We obtained a strong linear correlation and low average errors between estimated and actual values (R 2 = 0.99, RMSE = 1.1 cm, MAE = 0.85 cm). RMSE and measurement error (due to rough ground, moving leaves, etc) had the same order of magnitude, which demonstrates that crop row 3D models give extremely accurate estimates of plant height. Moreover, Fig. 6 shows that the proposed method obtained similar R 2 , RMSE and MAE for every species, thus proving that the method can handle various crop structures.

Leaf area estimation

For the first data set, in Fig. 7, estimated leaf areas are plotted against actual leaf areas for every species. We also obtained a strong linear correlation and low average errors between estimated and actual values (R 2 = 0.94, RMSE = 85 cm 2 , MAE = 59 cm 2 ). It shows the potential of our method for leaf area estimation in outdoor conditions. Estimation results were good for every single species.

Relative MAE were 12% for sunflower and Brussels sprout, 16% for cauliflower, and 21% for Savoy cabbage. These figures prove that performance remained stable for the considered plant structures. For the second data set, leaf area estimation results are shown in Fig. 8. For each row, the actual mean value is compared with the mean value estimated with the proposed approach. Sample means and standard deviations (shown with error bars) were computed from four to six plants.

The estimated mean leaf areas were close to the actual mean leaf areas. Indeed, RMSE and MAE computed with these mean values were 66 cm 2 and 45 cm 2 respectively (i.e. 13% and 9% resp.). The actual mean leaf areas were in the intervals specified by sample standard deviations except for row 6. In this latter case, results were probably less accurate because of many overlapped leaves that were not handled by the proposed method.

Conclusions

This study presents a methodology to retrieve crop row 3D structure, plant height and leaf area. It has been designed in order to fulfill phenotyping-related needs to some extent (non-destructive, automatic, robust, fast). The crop row 3D structure is retrieved by SfM using images acquired by a single moving camera. Various post-processing methods are then implemented to estimate plant height and leaf area. In particular, we propose a plant/soil discrimination method that is robust in outdoor conditions. This method provided good results for various crop structures, lighting conditions, sensors and image acquisition means.

Fig. 1 :

 1 Fig. 1: Framework of the proposed method.

  (a) Keypoint detection and matching for a pair of images. (b) Sparse 3D point cloud (camera positions and orientations in green). (c) Dense 3D point cloud.

Fig. 2 :

 2 Fig. 2: 3D reconstruction steps.

Fig. 3 :

 3 Fig. 3: Becam phenotyping platform.

Fig. 4 :

 4 Fig. 4: Savoy cabbage row reconstruction performed with 38 images.

  Distribution of ExG and H.

Fig. 5 :

 5 Fig. 5: Example of low-contrasted scene.

Fig. 6 :

 6 Fig. 6: Height estimation results obtained with the first data set.

Fig. 8 :

 8 Fig. 8: Leaf area estimation results obtained with the second data set. The errors bars are the sample standards deviations computed from four to six plants.

Table 1 :

 1 Height (H) and leaf area (LA) ranges for the two data sets (min-max).

				Data set 1		Data set 2
		Sunflower Savoy cabbage Cauliflower Brussels sprout Sugar beet
	H (cm)	15-65	10-17	19.5-22.5	11.5-17.5	×
	LA (cm 2 ) 27-1201	99-331	360-538	80-573	378-622

RHEA-2014, Madrid, Spain, 21-23/05/2014

Acknowledgments

This study was funded by the French National Research Agency, within the programme "Investissements d'avenir" with the reference ANR-11-BTBR-0007 (AKER project). We are grateful to the French Sugar Beet Technical Institute (ITB), which made the Becam platform available to us and carried out leaf area measurements within AKER project.


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 1993, 11, pp.395-418. hal-01012319
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