Guillaume Levrier

Tracking and Mapping the Use of CRISPR-Cas Systems for Research in China 1 Tracking and Mapping the Use of CRISPR-Cas Systems for Research in China

Scientists have floated the idea of a ''Sputnik 2.0'' technological race between the People's Republic of China (PRC) and the United States of America in CRISPR-based genome editing research. This quantitative analysis of articles published between 2010 and 2020 shows that research centers based in the PRC have succeeded in making CRISPR-based genome editing a standard tool. A corpus of 18,863 academic documents containing the acronym CRISPR in their abstract shows that although PRC-based research institutions were slower to start publishing on CRISPR, they have now outpaced the publication rate of institutions located in the European Union (EU). While U.S.-based institutions have kept their leading position in basic research, PRC-based research has become momen-tous in agriculture-related fields. This corpus hence illustrates how deeply the international landscape of life sciences research has shifted since the Human Genome Project, mostly to the PRC's advantage.

Introduction

Genome editing, a set of techniques derived from a capacity to trigger an organism's DNA repair mechanisms following an endonuclease-induced disruption at a targeted site, has become mainstream 1 in research contexts between 2010 and 2020. Much of this success as a tool for life sciences academic research has been attributed to the technological developments following the discovery of Cas proteins' potential as tracking and cleaving mechanisms. Some commentators have argued over the existence of a new "Sputnik 2.0" race 2 between the US and China. According to this view, different regions of the world would be competing to achieve what could be called a "nuclease supremacy". While this analogy might appear to be far-fetched, the People's Republic of China (PRC) policy interest in life sciences has indeed skyrocketed over the past twenty years. A first stone was set with the ninth five-year plan (1996-2001), which mentioned the importance of biotechnology. The thirteenth five-year plan (2016-2020) explicitly lists genome editing techniques (基因 编辑技术) as part of the "advanced biotechnologies" (发展先进高效生物技术). The same document argues that developing such technologies are to make the PRC an international leader in this field (提升我国生物 技术前沿领域原创水平,抢占国际生物技术竞争制高 点) (see Appendix 1). Recent publications [START_REF] Gao | Genome engineering for crop improvement and future agricul-ture[END_REF] show that PRC-based institutions have made considerable progress in crop improvement, notably using baseediting.

This study seeks to determine to what extent research institutions in the PRC have taken advantage of CRISPR-based genome editing as a platform for research. Using the open-source software PANDORAE (see Methods section), all the publications available on Elsevier's Scopus service containing the acronym CRISPR in their abstract for the years 2010 to 2020 were retrieved and analyzed. These requests yielded 18 863 results, which make up the core corpus used for this research.

Material and Methods

The corpus was retrieved and visualized using PANDORAE's automated request feature (FLUX) to Elsevier's Scopus service. This software is free and open source, which makes data interpretation and reproducibility easier compared to commercial solutions. Its features ad-hoc data cleaning (through the CHAEROS processes) and visualization (through TYPES) algorithms [all these processes and PANDORAE's complete code are available online on GitHub], making the use of other solutions such as Derwent Data Analyzer, Clarivate analytics, Bibexcel, InCites or CiteSpace redundant. However, PANDORAE normalizes the data it retrieves to the CSL-JSON (Citation-Style Library -JavaScript Object Notation) format, and can hence yield explorable visualizations based on these different data sources or sorting algorithms.

An important caveat, directly linked to the nature of the database used, must be acknowledged: this dataset is relevant in terms of orders of magnitude only.

The metadata made available by Scopus is rich but necessarily imperfect. Some affiliations might be improperly listed, others might be missing. It does not provide users with a reliable way to distinguish reviews from research articles. Identical documents might be listed several times under different names, or as different versions of the same work. Chinese authors' names are latinized and abbreviated by the service, which makes authorship attribution difficult to manage. Whenever possible, PANDORAE's processes use DOIs to sort data, and tries to use the longest available name form. However, one should not overestimate the precision of this data set. Although it is relevant to explore developments of thousands of articles on the scope of decade, as is done in this article, more data cleaning work would be needed to support more precise assertions. It should also be noted that PRC and CN (CN is the ISO 3166-1 alpha-2 code for China) are used interchangeably in this article, both designating China. CN being a two-letter abbreviation, it is used in the code alongside the other two area abbreviations (EU and US), which can prove practical for substring selection purposes.

To respect application programming interface (API) rate limits, a total of 11 requests were made, following the syntax hereunder. The last two characters were changed according to the year requested. In this URL syntax, the %20 corresponds to a blank space, which can be misleading in this formula. Once parsed, the end of the sequence ''%2020XX'' becomes ''_20XX,'' where _ represents a blank space. The requests were made on January 22, 2021. Requests made at a different date will yield different results, as articles are corrected, retracted, or retrospectively added.

ABS(CRISPR)%20AND%20PUBYEAR%20%3D%2020XX

Each request was geocoded using PANDORAE's matching algorithm. Each encoded affiliation city was attributed a longitude and latitude when available. Each resulting data set was then converted to the CSL-JSON format and sent to a Zotero library instance for hand curation.

To provide more insights on the use of CRISPR among disciplines and subfields, the ISSN of the publication in which each retrieved document was published was then stored in a dedicated data map. Each discrete ISSN was then requested through Scopus' ISSN API service, which yielded its profile. These journals profiles contain (for this specific corpus) 0 to 8 different keywords qualifying their editorial line. For this dataset, this request yielded a total of 212 subfields sorted in 27 categories (see Appendix 2 for the full list).

The eleven resulting collections were then exported from this library and rebuilt using a Go language script written for that purpose. The affilRebuild.go (see supplements) script iterates over all the documents contained in all the CSL-JSON files available in the same directory. Each affiliation listed in a document is then added to a map whose keys are four potential targets: EU, US, CN and Other. The Go map is hence declared as a typed variable (such as map[string][]map[string]interface{}). The first string is the area key, and the value is a slice of Go-parsed JSON objects, in our case articles retrieved from Scopus and encoded in CSL-JSON by PANDORAE. If the country belongs to pre-Brexit EU28, it will be added to the EU category. The script then outputs a series of CSL-JSON documents by country (or, in the case of EU country, an "EU" file), in which are listed all the documents which have at least one affiliation located to that country.

Following this script's logic, a document listing several affiliations will be duplicated as many times in the sorting map. The datasets produced by this script hence do not weigh documents, but instead weigh affiliations. A document with no affiliation listed will be dismissed. To provide more insights on this weighting, the same script also produces a document profile (Fig. 1). This profile is a map of the listed affiliation structure of each article. Two main features appear: most of the articles retrieved list only one affiliation; and most of the articles which list two or more affiliations list two affiliations in the same block (EU, US, CN/PRC or Other).

This dataset provides a global overview of how CRISPR has been written in article abstracts in different countries over the past decade. By coupling this data to Scopus' "cited by" data (as of January 2021) for each article and its the journal keyword qualifications, a much deeper perspective can be produced. The fieldRebuild.go script hence merges the US, EU and CN datasets with each article's citation count and their journal's keyword to generate a series of graphs per field (see Appendix 2).

The datasets where then re-imported into PANDORAE for visualization through its TYPES processes. The geotype (Figs 45) represents articles which list at least one affiliation in PRC and maps them by city. On these figures, a city's radius is a logarithmic function of the number of articles which list an affiliation within its walls. This log function is necessary to ensure a very wide range of cities, some hosting numerous large institutions listed as affiliations by thousands of articles in this field, others hosting a single laboratory relevant to that same field, remain visible on a single visualization. Red edges between cities represent at least one instance in which institutions from these cities have collaborated on an article. The chronotype (Fig 6 ,7) styles a circular timeline based on the weighted dataset: each dot listed is one instance of an article per affiliation. Red edges connect instances with same authors. To make up for the weak quality of the date encoding in the original dataset, PANDORAE substitutes any missing month and day by the 15th of January. The beginning-of-year cluster is hence a data quality artefact, though such a cluster might exist (in much smaller proportions). The fieldotype (Fig 10) is a series of stacked area charts displaying the number of papers published in the field (dark color) per area each year and the cumulative citation count of articles published in that area that year (light colors) as of early 2021. An important methodological point must be highlighted here: if an article was published in a journal qualified with X keywords (here, X being comprised between 0 and 8), it will figure in each of the X relevant graphs (one for each subfield identified by that keyword). This can be particularly impactful for highly cited papers published in high factor journals with multiple keywords (see Fig 8). However, the keyword distribution (Fig 4 ) shows that its profile is similar for the three areas. Three features must however be acknowledged: • the percentage of EU publications in journals with no keywords is higher than the other two areas, which means that the EU publications will be underrepresented in Fig 10 andAppendix 2. • the percentage of US publications with only one keyword is higher than the other two areas, which means that it is less represented in cumulative terms (on the totality of Appendix 2).

• The "Global" category is an average of US, EU and CN, and does not take into account the "Other" area.

Results

The retrieved corpus illustrates the rise of CRISPR in academic abstracts between 2010 and 2020 (Fig. 2). The rise in publications validates the idea that a "CRISPR decade" did occur in life sciences research between these dates. Through the affiliation listings, and the profile weighting detailed in the "Methods" section, one can also observe that US-based research institutions have collectively published more articles than any other region in the world (Fig. 3). They also make up most the hundred most cited articles in the corpus (Fig 5 ). A second look shows that the PRC has caught-up with, and arguably surpassed, the EU in terms of number of publications. While intra-regional collaboration weighting artifacts logically deepen the gap between regions, it appears unlikely that they would do so to such an extent that it would narrow a gap of 362 items (which would still amount to more than 300 items using average weightings).

The affiliations' geocoding highlights the importance of international and national cooperation. Cooperation between research institutions in the US and in the PRC seem to be much deeper than cooperation between the ones based in the EU with their Chinese counterparts (Fig. 6). Another observation is that intra-PRC collaboration appears to be mostly to the benefit of Beijing-based institutions ( Fig 7). A more quantitative approach gives additional insights (Fig. 8). Intra-PRC collaborations are strongly carried by the collaborations listing the Chinese Academy of FIG. 2. Number of registered keywords distribution per area. This graph shows for example that >35% of the articles written by authors with an EU affiliation are published in journals with no Scopus-registered keyword. Global is EU, CN, US average, excluding other countries.

Science as co-affiliation). This is in the same order of magnitude as the US average (12 articles). The EU average is significantly higher (24 articles), which can be explained by the listing of several large national research institutions (such as France's Centre National de la Research Scientifique). International collaborations account for a very limited number of total collaboration (see blue curve in Fig. 8 and right part of the table). Out of the 30 international institutions that collaborate most with PRC-based research centers, 20 are based in the US. A chronological perspective (Fig. 9) on the dataset shows that teams that have started using CRISPR in all three areas have kept using it over the years. Zooming in on two periods (Fig. 10) confirms that collaborations between PRC and US teams through time seem to be more dynamic than with EU institutions. A more in-depth analysis (Fig 11) of this chronological data shows that affiliations in the PRC have not only caught up in "mainstream" CRISPR disciplines, such as Molecular Biology, Biochemistry, Cell Biology or Biotechnology, but have also become prominent in more specific fields, with a strong focus on Agricultural sciences. Areas such as Plant science, Agronomy and Crop Science, Animal science and Zoology, Insect science and others feature leading PRC academic institutions in terms of both publications and citations. This is consistent with previous geographical studies of genome editing intellectual property [START_REF] Martin-Laffon | Worldwide CRISPR patent landscape shows strong geographical biases[END_REF] . The main difference between authors in the PRC, the US and the UE seems to be on high-impact articles. While their publication profiles are similar for the average article (Fig. 12 a.,b.,c.), the higher-impact articles published by US-affiliated authors (Fig. 12 d.) are singularly more impactful than the higher-impact articles of either EU-based or PRC-based authors, though EU-based authors appear to still have a slight edge on that measure. This is confirmed by ranking the most 'productive' affiliations of each area (Fig. 13). The US would be in a similar position as the EU and the PRC if not for the affiliations based in the Boston area and the San Francisco Bay area, which on their own account for five times the 20 first most productive institutions of either the PRC or EU area.

These observations can also be confirmed through a different perspective, that of ranking journals in which these articles have been published by number of papers published and number of citations (Fig. 14 a &b). Unsurprisingly, high impact-factor journals have mostly published highly cited articles written by authors linked by a US-based affiliation (Fig 14 b). However, PRC-based authors do publish comparatively more in journals such as Frontiers in Plant Science and Plant Biotechnology Journal, which is consistent with the keyword-based analysis presented in Fig 11.

These quantitative perspectives on the PRC CRISPR research landscape can hence be summarized as follows:

• PRC research institutions were slower to start publishing articles using CRISPR than US or EU based institutions.

• The publication rate of PRC institutions has kept accelerating between 2015 and 2020. It remains below the rate of US institutions but is now higher than the rate of EU institutions.

• The average article appears to have the same impact in the three compared areas. However, US institutions based in either the Boston or San Francisco areas have a major edge on very high impact articles.

• Publication in the PRC have a level of atomicity comparable to the US's. The average PRC institution published 15 articles relevant to this corpus over the past decade. When collaborating with other institutions, they mostly collaborated with other PRC-based institutions, and appear to have remained loyal to these institutions.

• PRC-based institutions do have an edge in specific fields. This is especially salient for Agriculture-related sciences. : tabular representation of the same data, showing the number of articles published by authors listing this affiliation (Num.), the cumulative number of citations of these articles (Cit.), the number of articles divided by the cumulative citations (N./C.), the city in which the affiliation is based in according to Scopus and the affiliation's name.

Discussion

The PRC was the only "developing country" to participate to the International Human Genome Project (HGP). Its contribution amounted to 30 centimorgans, or 1% of the total length. This was considered an impressive feat [START_REF] Wang | The international Human Genome Project (HGP) and China's contribution[END_REF] for the country at the time. The data presented in this article shows that the scale of PRC's contribution to life sciences in the decade leading to 2020 has radically changed. It has now replaced the EU as the main challenger to the US, and possibly its main partner, in this area. Does this meteoric rise give substance to the idea that a race for "nuclease supremacy" would currently be under way? Is genome editing considered to be a technological platform so transformative that it could rank as a strategic asset? Do policymakers in the US and in China believe CRISPR to be relevant to the future of sovereign power?

The dynamics of CRISPR's adoption as a lever for more epistemic progress do show that different countries have invested discretely in its early promises. For some part, these differences can be explained by structural factors. The US hosts world-leading universities in the life sciences and has managed to keep them competing for the top position in genome editing research. At the other end of the spectrum, EU countries have largely failed to challenge the US's leadership and to ramp up its funding for research programs. Brexit seems poised to weaken scientific research on both parting sides, which will degrade Europe's position further. This sharply contrasts with the PRC's position, as PRC-based institutions are becoming ever more present in CRISPR-powered research. The PRC's rise in these research fields is not uniform, which shows it was able to capitalize on its preexisting structures to build a comparative advantage in some fields. US-based institutions have built up on their early discoveries to take the lead in terms of number of publications and citations in the life sciences, making it hard for PRC-based labs to challenge their positions. But this advance relies a lot on the past glory of setting the first landmark articles for a new method: publications since 2018 show a much more levelled landscape. In the meantime, some PRC institutions have focused their efforts on Agriculture-related fields, with great success in Plant sciences and Agronomy and Crop sciences. In doing so, they have kept their edge in these fields and made it sharper with CRISPR. This progress did not happen deus ex machina. The 2000-2010 decade did host the rise of leading PRCbased institutions specialized in DNA sequencing [START_REF] Wu | DNA sequencing leads to genomics progress in China[END_REF] . The results of this scientific and industrial investment continue to bear fruit today [START_REF] Dun | Chinese DNA sequencing company raises a record $1 billion[END_REF] . But if the PRC-based laboratories were so prompt in taking full advantage of what genome editing newly allowed, it is also due to the very nature of CRISPR-Cas techniques. The "slower, cheaper, more efficient" slogan is slowly fading into irrelevance, as other engineerable nucleases are less and less resorted to outside of therapeutic contexts. Laboratories now use CRISPR in the same way they would have used PCR in the previous decades [START_REF] Rabinow | Making PCR: A Story of Biotechnology[END_REF] . Would not that be the relevant time to invest in searching the next biotechnological revolution? We must enhance our embryos so that students can all be brilliant, become engineers and highly skilled people _ _ _ China is starting to do it! Non médecin et banal citoyen, j'ai fait séquencer mon génome par une compagnie -hélas non-française-puisque ce n'est pas encore possible en France. Donc, ces données sont stockées quelque part aux États-Unis où en Chine ou ailleurs.

As a non-MD and average citizen, I had my genome sequenced by a company -Alas a non-French one-since it isn't possible yet in France. So, these data are stashed somewhere in the US or in China or elsewhere.

Les États-Unis et la Chine ont pris une avance considérable dans ce domaine très important et il est temps pour l'Europe de se mettre au diapason.

The US and China have taken a considerable advance in this very important domain, and it is time for Europe to catch up.

Les enjeux actuels:sociaux; économiques et politiques.. The frontier between fixing and augmenting is blurry. […] In addition, even if these technologies are strongly framed in France, they will never be elsewhere, in the US or in China. Who can prevent someone to go there and have their genome modified? Le problème c'est que personne ne sait ce que veut dire capacités "normales". C'est subjectif. Ce qui est normal pour quelqu'un ne l'est pas pour un autre. D'autant plus que si on encadre ces technologies en France, elles seront autorisées ailleurs, aux USA ou en Chine. N'importe qui pourra s'y rendre et s'augmenter, puis revenir en France. Aucune loi ne pourra les empêcher.

The problem is that no one wants "normal" capacities. It's subjective. What is normal for someone isn't for someone else. Plus, if we limit these technologies in France, they will be authorized elsewhere, in the US or in China. Anyone will be able to go there and augment themselves, and then come back to France. No law will be able to prevent that. Mais, toute loi française pour interdire les augmentations ne s'appliqueront qu'en France. Quid des autres pays ? Qui pourra empêcher le citoyen lambda français d'aller aux USA ou en Chine, modifier son génome, et revenir en France ? But, all French laws to prevent augmentations will only be applied in France. What about other countries? Who will prevent the average French citizen to go to the US or to China, modify their genome, and come back to France?

This question, as illustrated by the diversity of reactions 9 to a recent international report, is proving to be a political minefield. Controversies around governmental responses to the covid-19 pandemic might also have a durable impact on the future of molecular biology. This very specific context might explain why the recently published fourteenth 5-year plan only mentions "genetics" once, as part of a longer list containing other cutting-edge technology such as AI, quantum technologies or hydrogen energy [在类脑 智能、量子信息、基因技术、未来网络、深海空天开 发、氢能与储能等前沿科技和产业变革领域,组织实 施未来产业孵化与加速计划,谋划布局一批未来产业].

Outside of China, the idea of a new Sputnik race lives on. The fact that the DARPA has funded genomeediting related projects 10 can be interpreted as indicating that some policymakers in the US consider the country to be in a scientific competition deserving State intervention. But a race with whom? Science Magazine reported in 2020 11 that out of the 189 scientists investigated by the NIH for failure to "disclose financial ties to foreign governments", 93% were targeted for alleged ties with the PRC. Fifty-four scientists have resigned or been fired. Such uncontrolled technological transfers to the PRC are apparently considered to be a threat by US authorities.

The PRC's new status has also already been, to some extent, as acknowledged by citizens interested in these issues. The prospective uses of CRISPR-Cas techniques were listed as one of the topics for debate during the French government's public consultation over the revamping of its bioethics framework, which happened in the first half of 2018. The presented array of comments (Table 1) highlights how the US and the PRC have become, in the view of the French citizens who chose to take part in this exercise, equivalent in terms of both perceived technical capacity and perceived lack of appropriate regulatory frameworks. The underlying idea, that the two areas are developing these techniques at breakneck speed to reap its (hypothetical) benefits, indicates that this race also exists in the minds of those who consider themselves to be spectators to it.

Conclusion

This quantitative study finds that the PRC-based institutions appear to be both the main rivals and best partners of US-based institutions in genome editing research, and that PRC research is particularly salient in Agriculture-related fields. The swiftness of the PRC's research focused progress and the reaction of US authorities both substantiate the idea of a "technological race" going on over genome editing between the two countries.

These findings call for a qualitative comparison of the policies implemented in the UE, the US and China to promote genome editing research. Such research could elucidate what "genome editing" is to policymakers, and how government policies have been discussed, designed and used to fuel this rapid progress.

FIG. 1 .

 1 FIG. 1. Document affiliations profiles, showing the 20 most prevalent profiles. The rectangle on the bar chart indicate a broken scale. A document profile is a specific mix of listed affiliations, using four categories of affiliations (CN, EU, US, and Other). The most prevalent profile in the data set is 1 U.S. institution listed. The ninth most prevalent is 1 CN institution listed and 1 U.S. institution listed. Here, CN is equivalent to PRC. CN; PRC, People's Republic of China.

FIG. 3 .

 3 FIG. 3. Top 100 articles of the corpus by citations. Affiliations are attributed as one or more per area. FIG. 4. PANDORAE geotype showing documents of the corpus listing at least one PRC-based affiliation. Edges link cities hosting collaborating institutions.

FIG. 11 .

 11 FIG. 11.(Continued).

FIG. 12 .

 12 FIG. 12. Contour density by marching squares of journals for articles published by US-based (a), EU-based (b), and PRC-based (c) authors. X-axis figures citations, Y-axis the number of articles. The chosen extents aim to display the repartition of average articles for each area. (d) Shows the performance of top articles for each area, listing the number of articles published in this journal (Num.), the cumulative number of citations of these articles (Cit.), the number of articles divided by the cumulative citations (N./C.), and the publication's name.

FIG. 14 .

 14 FIG. 14. Stacked bar chart ranking publications that published (a) the most articles from the corpus (b) articles with most cumulative citations in the corpus. Bars are stacked by area of author affiliation.

  mondiaux aussi: que fera la loi Française face àç la question du clonage (voir Chine et USA) The current stakes: social, economic, political… Global too: what will the French law do with regards to cloning (see China and USA) La frontière entre réparer et augmenter est floue. […] De plus, en admettant que ces technologies soient fortement encadrées en France, elles ne le seront pas ailleurs, aux USA ou en Chine. Qui peut empêcher une personne s'y rendre et modifier son génome ?

Table 1 .

 1 Anonymous written contributions to the « Etats Généraux de la bioéthique » online platform set up by the French government (first half of 2018)

	FR

Acknowledgments

Many thanks to the reviewers for their perspectives which helped me build a much deeper article than I had initially envisioned. Many thanks also to Sciences Po's METAT team for their advice and technical help in the early days of PANDORAE and to Lou Safra, Thierry Vedel, and Jean Chiche for their insights on early versions of this work.

Funding Information

This research is funded by the Agence Nationale de la Recherche, grant ComIngGen -18-CE38-0007-01.

PANDORAE and the scripts used for this paper are

open, free and available on GitHub.


I-Micropollutants problematic  Definition  Factors influencing the fate of micropollutants II-Examples of research work on ISDND and anaerobic digester  Identification of micropollutants to be taken into account in ISD impact studies  Diffusion of micropollutants through the sealing barriers of storage facilities  Biogeochemical cycles of metals in ISD bioreactors  Biodegradation of organic micropollutants during methanization

How to define micro-pollutants?

The term "micropollutants" means organic or mineral substances whose toxic, persistent and bioaccumulative properties may have a negative effect on the environment and/or organisms even at low concentration.

They are present in many products that we consume daily (drugs, cosmetics, phytosanitary products, insecticides, etc.), at home or in industry.

Progress in laboratory analysis is increasingly highlighting their presence in the aquatic environment at extremely low concentrations, in the order of one nanogram per litre or microgram per litre (hence the term micropollutants). Some of these substances are liable to have potentially chronic direct or indirect effects on ecosystems (e.g. the feminisation of fish due to endoctrine-effect substances in the aquatic environment), and even on human health.

What are the micro-pollutants families?

Lead, cadmium, mercury, arsenic, antimony, radon, uranium This law allows to determine the solubility of a volatile substance in a liquid solvent with which this gas is in contact.

The more Ki is great the more the substance is volatile The Henry's law constant depends on the temperature:

Important property to know the mode of exposure to pollutants

Polarity: Octanol/water partition coefficient Log Kow, is a measure of the differential solubility of chemical compounds in two solvents (octanol / water partition coefficient).

Log Kow = Log(C oct /C eau ).

This value makes it possible to apprehend the hydrophilic or hydrophobic nature of a molecule Indeed, if the Log Kow is positive and very high, it expresses the fact that the molecule considered is much more soluble in octanol than in water, which reflects its lipophilic or hydrophobic character, and vice versa.

Relationship between polarity and bioaccumulation

Nature of analyzed leachates

Metals biogeochemical cycles in MSW landfill



  MetalsPolycyclic aromatic hydrocarbons are organic compounds containing only carbon and hydrogen-that are composed of multiple aromatic rings PAHs are uncharged, non-polar molecules found in coal and in oil deposits. They are also produced by the incomplete combustion of organic matter (for example, in engines and incinerators or when biomass burns in forest fires). Some carcinogenic PAHs are genotoxic and induce mutations that initiate cancer; others are not genotoxic and instead affect cancer promotion or progression An adduct formed between a DNA strand and an epoxide derived from a benzo[a]pyrene molecule (center); such adducts may interfere with normal DNA replication. compounds containing the bisphenyl structure with different numbers (one to ten) and arrangements of chlorine atom attached PCBs were widely used as dielectric and coolant fluid, in transformers, capacitors PCBs are persistents in environnent (non biodegradable, hydrophobic…) PCBs are carcinogenic Dredging of PCBs contaminated sediments PolyBrominated Diphenyl Ethers (PBDE) Polybrominated diphenyl ethers or PBDEs, are organobromine compounds that are used as flame retardant. The family of PBDEs consists of 209 possible substances, which are called congeners. PBDEs have been used in a wide array of productsshare the environmental long life and bioaccumulation properties with PCBs PBDEs can cause cancer in people Volatility/solubility : Henry's Law Constants  Polarity: Octanol/water partition coefficient (Log KOW)  Biodegradability : Half life time VOLATILITY/SOLUBILITY : HENRY'S LAW This law establishes a relation between the partial pressure Pi of a pure gaseous body and its molar fraction X L in a solvent : Pi = X L . Ki Ki is the Henry's law constant



  Time required for half of a quantity or concentration of a pollutant to disappear from the biotope or a contaminated organism photooxidation, thermooxidation ...) leading to the more or less complete disappearance of a molecule.DegradationBiological degradation carried out by bacteria, fungi and obtaining metabolites of lower molecular weight.BiodegradationPrimary biodegradation = partial attack of molecules; may result in more persitant, more bioavailable, and sometimes more toxic metabolites than the initial molecule Ultimate biodegradation: complete degradation of the molecule; leads to CO 2 , CH 4 , water, mineral elements. IMPORTANCE OF THE SPECIATION Speciation consists, beyond the measurement of the total concentration of an element in a sample, to separate, identify and measure individually all the chemical forms of this element Biogeochemical cycle of mercury Course Outline I-Micropollutants problematic  Definition  Factors influencing the fate of micropollutants II-Examples of research work on ISDND and anaerobic digester  Identification of micropollutants to be taken into account in ISD impact studies  Diffusion of micropollutants through the sealing barriers of storage facilities  Biogeochemical cycles of metals in ISD bioreactors  Biodegradation of organic micropollutants during methanization 29 MSW Landfill leachates impacts   Recalcitrant micropollutants to leachate treatments Contamination of surface water Transfers of micropollutants through the sealing barriers Contamination of subsoils and groundwater Transfers of micropollutants through the sealing barriers Two basic modes of transfer in the sealing barriers : advective transfers (defects in the geomembrane) The organic compounds were analyzed both in the dissolved and particulate phases Compounds analyzed: Analysis campaign : main results  Presence in large quantity of bisphenol A (antioxidant) and phthalates (plasticizer) which are plastic additives with reprotoxic properties  Presence also of some VOC (mono-aromatic, chlorinated solvent ...) with carcinogenic properties Presence of PAHs, PCBs and PBDEs mainly on the particulate phase because these compounds are not very soluble  Pharmaceuticals and hormones have not been found

of the toxicity of the organic fraction

		EDA approach to ISDND leachates
	Toxicity test
	Targeted mechanisms	Methods	Detected molecules	References
	Génotoxicity		SOS Chromotest	Genotoxics et pro genotoxics: HAP, nitrosamin , pesticides…	Quillardet et Hofnung, 1985
	Dioxin receptor (AhR)	EROD activity	Dioxins et dioxin-likes, HAPs, PCBs	Laville et al., 2004
	Estrogen receptor (ER)	Luciferase activity	Steroïds naturals et synthetics, Alkylphenols, Bisphenol A…	Pillon et al., 2005
	Raw leachate  Toxicity tests results	Aerated leachate	Treated leachate
					Activités Activity
	Samples	Site		Characteristics Génotoxicité Dioxin-like Oestrogénique Genotoxicity Estrogenicity
	Site1-LB	Site 1	Lixiviat brut provenant de plusieurs casiers d'âges différents. Site 1 -Lix Brut + ++ Raw	+++
	Lixiviat Site1-LA Site1-LT Site2-LB Site2-LA	Site 1 Site 1 Site 2 Site 2	Lixiviat prélevé dans le bassin d'aération recevant le lixiviat site1-LB. Site 1 -Lix Aéré -+ Aerated + Perméat prélevé en sortie de traitement (évapo-concentration suivie d'une osmose inverse) du site1. Site 2 -Lix Aéré -+ ++ Aerated Lixiviat prélevé dans le bassin d'aération recevant le lixiviat site2-LB. Site 2 -Lix Brut -++ ++ Raw Lixiviat brut prélevé au niveau de la station de relevage du site 2. Site 1 -Lix traité ---Treated
			Site 2 -Lix traité Treated	-	-	-

Site2-LT

Site 2 Effluent de sortie de traitement (réacteur membranaire suivi d'une filtration sur charbon actif) du site 2.

Evaluation

Source de l'oestrogénicité du lixiviat étudié Estrogenicity origin of the landfill leachate

EVALUATION OF BIODAVAILABILITY


Clarisse Hamadache

Adrien Laviron

Mathieu Ehrhart

Corentin Hiver

Jürgen Kiener

Anne Meyer

Jean Peyré

Vincent Tatischeff

Vincent Tatische↵

Development of a Compton telescope calorimeter module for MeV-range gamma-ray astronomy

Keywords: gamma-ray imaging, gamma-ray polarimetry, artificial neural network, position sensitive detector, MAPMT, SiPM

A Compton telescope for imaging and polarimetry

The instrumental developments in gamma astronomy in the MeV energy range are a cornerstone for various scientific topics. By considering the sensitivity of gamma astronomy observatories as a function of energy, shown in figure 1, there is a lack of sensitivity around 1 MeV. In this range Compton e↵ect is dominant, and a strong background is present. However, various scientific topics can be explored in this energy range, such as nuclear processes, compact objects, cosmic rays, and gamma-ray bursts. We can use several tools to study these phenomena, such as the temporal aspect of the emission, spectrometry, imaging, and polarimetry. The sensitivity of a future space observatory, dedicated to the MeV energy range, should be increased by one to two orders of magnitude compared to past observatories. For this purpose, we propose a novel Compton telescope composed of a silicon strip detector tracker and a position-sensitive calorimeter. The Figure 1: Sensitivity of di↵erent observatories in X -ray energy range (see [START_REF] De Angelis | The e-ASTROGAM mission. Exploring the extreme Universe with gamma rays in the MeV -GeV range[END_REF]) calorimeter components are scintillators coupled with pixelated photodetectors either MultiAnodePhotomultiplier Tubes (MAPMT) or silicon PM (SiPM).

For several years we have been developing a Compton telescope which is the best concept for the science we expect. In this context, we are involved in 3 projects, currently in progress. We work in the frame-work of an ASTROGAM-like [START_REF] De Angelis | The e-ASTROGAM mission. Exploring the extreme Universe with gamma rays in the MeV -GeV range[END_REF] large space observatory for gamma astronomy which is a candidate for a future ESA mission. In fields other than astrophysics, we will soon achieve the ComptonCAM project. Comp-tonCAM is a portable gamma-camera for the nuclear industry, built on the same principle as the one for astronomy. It is dedicated to imaging the environment, planned to be used in nuclear facility removal [START_REF] Tatische↵ | ComptonCAM[END_REF]. A third project, COMCUBE, relies on a constellation of nanosatellites in low earth orbit. COMCUBE is a dedicated observatory for gamma-ray burst detection and polarimetry [START_REF] Laviron | COMCUBE: A constellation of CubeSats to measure the GRB prompt emission polarization[END_REF].

Instrumental requirements

A Compton telescope allows the energy and direction determination of an incoming gamma-photon by reconstructing a Compton interaction thanks to at least two layers of detectors. In our case, the first thin layer (the tracker or scatterer) favors the Compton e↵ect and gives the position r 1 of a first energy deposit E 1 . The second one, thicker (the calorimeter or the absorber), absorbs the di↵used gamma-ray and gives the position r 2 of the second energy deposit E 2 . We determine the incoming direction angle ✓ as

✓ = arccos " 1 m e c 2 1 E 2 1 E 1 + E 2 !# (1)
where m e is the electron mass and c the velocity of light. We then locate the origin of the incident gammarays as a circle in the sky from the interaction position of the gamma-rays in the detector and the energy deposits (see figure 2 left). By observing several gamma rays from one source, we localize it at the circles' intersection and image it (see figure 2

right).

A Compton telescope is also intrinsically sensitive to the linear polarization of gamma-rays. We consider the Klein-Nishina di↵erential cross-section for linear polarization of gamma-ray

d KN d⌦ = 1 2 r 2 e E 0 E ! 2 " E 0 E + E E 0 2 sin 2 ✓ cos 2 ⌘ 0 # (2)
It is a function of the electron radius r e , the energies of the incident and di↵used photons E and E 0 , and ✓ the di↵usion angle of the di↵used gamma-ray regarding the direction of the incident photon. It is also a function of ⌘ 0 , the angle between the direction of the incoming photon's electric field and the plan of the Compton scattering. We define it as the polarisation angle ⌘ 0 with respect to a reference axis (see figure 3 top). By reconstructing the ⌘ angle, we can determine the ⌘ 0 angle as a minimum of the distribution. The source polarization fraction ⇧ is then obtained from the modulation amplitude µ 0 compared to the ideal modulation amplitude µ 100 for a 100% polarized gamma-ray source; the ⌘ angle distribution of an unpolarized gamma-ray source (0% polarized) being uniform (see figure 3 bottom).

The imaging and polarimetry quality of a Compton telescope depends on the precise determination of the energy deposit and the gamma interaction localization in the tracker and the calorimeter. The tracker is made of double-sided strip detectors, so the interaction localization does not present di culties. However, it is a primary issue in the calorimeter, which is a thick (more than 1 cm) monolithic scintillating crystal.

Calorimeter Module

Operation principle and machine learning tools developed for performance characterization

The type of calorimeter we study is composed of a scintillating crystal of 5 ⇥ 5 ⇥ 1 to 2 cm 3 , mainly cerium bromide (CeBr 3 ) crystal. It is optically coupled with a Hamamatsu photodetector, MAPMT H12700A, or SiPM array S14161-6050HS-04, both with 64 channels of 5.2 ⇥ 5.2 cm 2 , providing a pixelated photodetector of about 6, 25 ⇥ 6, 25 mm 2 pixel size. The reading electronics is a dedicated system called ROSMAP for MAPMT and ROSSPAD for SiPM, both provided by IDEAS company (Norway). Figure 4 shows an exploded view of the module (MAPMT version). The principle of the position reconstruction relies on the analysis by machine learning algorithms of the scintillation light emitted. A gamma-ray penetrates the crystal and interacts at the (x,y,z) coordinates according to the defined crystal axis (figure 5, top). A part of the scintillation light emitted from the interaction point migrates toward the photodetector and is collected by its 64 pixels. We call "event" the distribution of light on the 64 pixels, represented both in 3D and 2D on the bottom of figure 5. By analyzing the shape of the distribution light with machine learning algorithms, we reconstruct the 3D interaction position. We use a supervised learning process; the algorithms we use for localization are artificial neural networks (ANN) as multi-layer perceptrons. It means that we provide a set of events (each composed of 64 values in input corresponding to the 64 pixels) with known positions of interaction (1-z coordinates -or 2 -(x,y) coordinates -values as output) for the first phase of training. We developed the algorithms from the Python Keras framework [START_REF] Chollet | [END_REF] with Theano [START_REF] Theano | Theano: A Python framework for fast computation of mathematical expressions[END_REF] backend. After training, the resulting algorithm can reconstruct unknown positions from light distributions. The neural network architecture and parameters have been optimized by extensive data analyzing work for several gamma-ray energies and irradiation positions on the surface of the crystal (x,y position reconstruction) and also on the side of the crystal (z position reconstruction). An experimental setup called a scanning bench that provides training data is presented in figure 6; the calorimeter module is fixed on arms that move horizontally and vertically in front of a gamma-ray beam provided by a collimated radioactive source. The first stage of data analysis categorizes events into three groups:

• a so-called one-peak gamma interaction (an example is shown in the bottom of the figure 5);

• two-peaks gamma interaction (see figure 7 top);

• and the so-called scattered event (see figure 7 bottom).

The two last categories are not well reconstructed by neural network algorithms. A morphological cut based on a test of maximum pixel clusterization excludes those events; they represent less than 10% of the total number of events at 662 keV.

Once the calorimeter module was deeply studied, we obtained a spatial resolution based on the di↵erence between the reconstruction and irradiation positions. The

= v u t 1 N p N p X i=1  ⇣ x p x rec i ⌘ 2 + ⇣ y p y rec i ⌘ 2 , (3)
gives the spatial resolution calculated for the N p collected at the irradiation position p at coordinates (x p , y p ) and reconstructed at the positions (x rec i , y rec i ). We then determine the average 2D spatial resolution over the detection area xy =< xy >. For z coordinates, similar indicators, z and z , have been used. For a MAPMT calorimeter module, at 662 keV, the morphological selection keeps & 90 % of the events, and the spatial resolution achieves xy ' 3.8 mm, z ' 2 mm.

Concerning the spectrometry performance of the MAPMT calorimeter module, a raw spectrum relying on the addition of the charge collected by the 64 channels provides an energy resolution of about 8.3% at 662 keV. We then considered the dependence of the energy resolution as a function of the 2D position (x,y) of the interaction of the gamma-ray. Indeed, a larger part of the scintillation light is lost by absorption or di↵usion when the gamma interaction occurs close to the crystal's border, and therefore collected charge is lower, which leads to a degradation of the energy resolution over the 64 channels. We considered the center of the crystal as the reference position. By applying a correction factor relative to the shift in the 137 Cs spectral line position between the central position and the others, we obtained an energy resolution of 4.7%.

All these studies and results performed to characterize the spatial and energy resolution of the calorimeter module in the MAPMT version are published in [START_REF] Laviron | Optimization of CeBr 3 position-sensitive calorimeter module[END_REF].

SiPM calorimeter module

The MAPMTs of the calorimeter module primarily used can be replaced by a Silicon detector (SiPM). In the case of SiPM, there is no need for high voltage and, the gain of thickness reduces the volume and the mass of the instrument, which is always a significant benefit to obtain for space applications or a hand-held camera. The calorimeter module in the SiPM version is built in the same way as before; it is a cerium bromide scintillating crystal of 5 ⇥ 5 ⇥ 1 cm 3 optically coupled with a Hamamatsu SiPM array of 64 pixels. The read-out electronics for SiPM is a dedicated system called ROSS-PAD provided by IDEAS company (Norway). Figure 8 shows the size comparison of the two calorimeter module versions, MAPMTs vs. SiPM. To characterize the spectral and spatial performance of the module, we used the methods described in section 2.1. The spectral response depends on the position; there is a loss of scintillating light near the borders by absorption or di↵usion. The e↵ect is more significant at higher energy, as shown in the light map response over the SiPM surface array in figure 9.

Figure 10 shows clearly the shift in the 137 Cs spectral line position between the central irradiation position and two other ones closer to the borders. One can easily see the degradation in the spectral resolution by simply summing the photodetector channels. However, after applying the correction factor as a function of the event interaction position, we obtain an energy resolution of 5% at 662 keV.

We performed the first 2D (x,y) spatial resolution study. We collected data sets for ANN training with a collimated 137 Cs source. We observed the three event categories shown in figure 7 in the same proportion as in section 2.1. After the reconstruction with the ANN, we plot the resulting xy map of 2D spatial resolution at 662 keV before and after the morphological selection (see figure 11).

In figure 12, we can see the surface of the crystal (x,y coordinates); we have several irradiation positions with a gamma-ray beam, and around each irradiation position, the corresponding 3D position reconstruction of events (events are selected in energy and morphology). We observe, as expected, a degradation of the reconstruction position near the borders of the crystal. The SiPM photodetector in the calorimeter module gives similar performances to those obtained with a MAPMT. It is an encouraging result for the di↵erent Compton telescopes or cameras we want to build. However, there is still work in progress to improve results and test this type of photodetector deeply to be used in astrophysical and/or high radiation environments. In the near future, we will continue working on:

• in-depth study of the electronics parameters

• spatial resolution at a larger energy range and in z-coordinate

• thermal study in a climatic chamber

Preliminary SiPM calorimeter for space application

For a future space observatory instrument, we need to reduce as much as possible the volume of the di↵erent components of the material. For this purpose, we are testing new electronics from Weeroc company (France) with a lower thickness than the ROSSPAD electronics described in section 2.2 (see figure 13). The Weeroc electronic module is based on the Citiroc ASICS, and several required implementations for the Compton imaging data treatment have been done (timestamped data, regular reset time). For the COMCUBE project, the backend electronics board based on an AL-TERA FPGA will be removed and replaced by a Xilinx board providing the backend electronics for all the detctors of the gamma-ray instrument. A preliminary spectrum of the collimated 137 Cs source is shown in figure 14; we measured an energy resolution R = 5.1%.

Compton telescope prototype: first gamma image and first gamma polarization measurement

The COMCUBE collaboration is currently building a 1U CubeSat size scale model of the Compton telescope to test the performance of the proposed instrument (1U CubeSat size being 10 ⇥ 10 ⇥ 10 cm 3 ). The main subsystems include two double-sided silicon strip detectors (DSSDs), a side calorimeter module to enhance the effective area of the telescope, a main calorimeter including four scintillating crystals, a power supply board, and a coincidence and data acquisition electronics. Important tasks for the realization of this scale model are:

• the dimensioning of the data flow and onboard storage;

• the development of the data processing software;

• the realization of the mechanical system;

• the management of the system operation and telemetry.

Before building the 1U scale model of the instrument, we did some experiments in the laboratory to test the imaging and polarimetry principle with both detector types (tracker and calorimeter) of the Compton Telescope. Figure 15 shows an image of the COMCUBE detector test bench under development. The assembly of the first DSSD with a calorimeter module allowed us to test the Compton imaging technique by making the first images of a gamma source placed at 1 m from the telescope prototype. The measured spectral resolution (6.0% FWHM at 662 keV) and angular resolution (about 7.5 FWHM at 662 keV) are in good agreement with the Compton telescope performances predicted by the numerical simulation.

A polarization measurement using a scattered gamma-ray beam is shown in Figure 16. The device, designed by numerical simulation, generates partially polarized photons of about 356 keV (theoretical polarization fraction ⇧ ' 60%). The measured polarigram (bottom) agrees with the theoretical prediction. Thus, the measurements of the signal modulation µ 100 ⇧ = 0.252 ± 0.023 and of the polarization angle ⌘ 0 = 109 ±3 are consistent with the simulation results: µ 100 ⇧ = 0.210±0.018 and ⌘ 0 = 106 ±2 . The obtained modulation factor, µ 100 ' 0.4, is in good agreement with the prediction for a quasi-monochromatic source of energy 356 keV observed at ✓ = 20 . Thus, these first results of a laboratory gamma-ray polarization measurement are very promising.

Conclusion and future works

After testing the 1U model in the laboratory, the collaboration wishes to carry out an onboard stratospheric balloon experiment to gain operational experience with the instrument in the near-space environment. In particular, we wish to test the detection of an astrophysical source -the Crab nebula and pulsar -in a background of radiation and cosmic particles representative of that in low Earth orbit. We will also study the radiation hardness of the detectors and other payload components and their operation in an environment with a temperature range similar to that found in low earth orbit.

Figure 2 :

 2 Figure 2: Compton telescope imaging principle. An incoming gamma-ray interacts by Compton e↵ect in the first detector D 1 , and the di↵used photon is absorbed in the second detector D 2 . The origin of the incident gamma-ray is determined as a circle in the sky from the interaction positions of the gamma-rays in the detectors and the energy deposits. Several incoming gamma-rays from the same point source give one circle's intersection that allows source imaging.

Figure 3 :

 3 Figure3: Polarization principle measurement. Top -Compton interaction: incident photon on electron e , and 0 di↵used photon. ⌘ 0 is the angle between the direction of the incoming photon's electric field Ẽ and the plan of the Compton scattering. We define it as the polarisation angle ⌘ 0 with respect to a reference axis. Bottom -Polarigram: the signature of a polarization measurement. By reconstructing the ⌘ angle, we can determine the ⌘ 0 angle as a minimum of the distribution. The source polarization level ⇧ is obtained from the amplitude µ 0 = (P max P min )/(P max + P min ) compared to the ideal modulation factor for a 100% polarized gamma-ray source, µ 100 .

Figure 4 :Figure 5 :

 45 Figure 4: Picture (exploded view) of the calorimeter module. From left to right, the scintillating crystal, the MAPMT, and the read-out electronics.

Figure 6 :

 6 Figure 6: Scanning bench

Figure 7 :

 7 Figure 7: Two typical light distribution morphologies observed in the training data set. Top: two-peaks gamma interaction; bottom: socalled scattered event.

Figure 8 :

 8 Figure 8: Complete calorimeter module version for size comparison. From left to right: picture of a MAPMT; picture of a calorimeter module, from top to bottom: electronics, MAPMT, crystal (side view); picture of a calorimeter module: the same as on the left but with a SiPM instead of a MAPMT; picture of a SiPM array.

Figure 9 :

 9 Figure 9: Relative detector spectral response through its surface measured with a collimated source and determined by the gaussian fitted energy to the spectral line for di↵erent irradiation positions spaced with a 3 mm pitch. The reference energy is the maximum of the fitted energies. Top: measurement for the 59 keV line of 241 Am; bottom: measurement for the 662 keV line of 137 Cs.

Figure 10 :

 10 Figure 10: Spectra of the 662 keV line of a 137 Cs source at di↵erent irradiation positions, (0, 0) being the center of the crystal, (14, 14) an intermediary position, and (22, 22) a position close to a corner

Figure 13 :

 13 Figure 13: Left: picture of the SiPM arrays plugged with Weeroc electronics; right: picture of both SiPM calorimeter module left with the Weeroc electronics and right with the ROSSPAD electronics (for size comparison)

Figure 14 :

 14 Figure 14: Preliminary spectrum with Weeroc electronics for SiPM calorimeter version. Spectral resolution is R = 5.1% at 662 keV with collimated 137 Cs source

Figure 15 :

 15 Figure 15: Top -Captioned photograph of the COMCUBE 1U prototype being assembled. The DSSD and its readout electronics are framed in red, the calorimeter module in green, the coincidence electronics in yellow and the power supply board in cyan. The telescope is placed in a climatic chamber whose temperature is regulated at 20 C. Bottom left and right -Compton images obtained with a 137 Cs source, (E = 662keV) placed at 1 m from the telescope and at two angular positions: (✓, ) = (0 ,0 ) (left) and (20 ,0 ) (right).

Table 1 :

 1 2D spatial resolution xy at 662 keV for both MAPMT and SiPM calorimeter versions.

Acknowledgments

This work is supported by the European Union's Horizon 2020 Program under the AHEAD2020 project (grant agreement n. 871158), and by Andra under the "Investing in the Future Program" of the French Government.


Cu

Stables = Les autres 16 O, 17 O, 18 O, 14

Pb 87 Rb  87 Sr Pas de transformation en fonction du temps -

Réaction en deux étapes:

  Pétrole transporté par le pétrolier Erika (S1) 1 échantillon d'une nappe de pétrole (S2) 16 résidus pétroliers (S3-S18) 10 échantillons de plumes d'oiseaux (S19-S28)ECHANTILLONS COLLECTES APRES LA MAREE NOIRE DE L-518 kJ.mol -1 de nitrate(Thauer et al., 1977) kJ.mol -1 de nitrate(Thauer et al., 1977) Remarque : le N 2 O est un intermédiaire à la fois de la dénitrification et de la nitrammonification(Welsh et al., 2001) Les autres paramètres, soit n'ont pas d'effet significatif sur la production de N 2 (positif ou négatif), soit sont liés au H 2 S ou à l'acétate 32En accord avec résultats de Brunet etGarcia-Gil (1996) Injection de H 2 S sous forme gazeuse dès l'injection de nitrateLes réacteurs c et d  concentration H 2 S >> 35La présence de H 2 S dissous à une concentration supérieure à 0,5 mmol.l -1 a bien empêché le nitrate d'être réduit en N 2Les réacteurs c et d  pas de dénitrification 37Démonstration formelle de la réduction du nitrate en ammonium La nitrammonification remplace la dénitrification dans les réacteurs c et d 38

-Étude expérimentale en microcosmes (flacons à plasma) d -Voies de réduction du nitrate (3)

-Étude expérimentale en microcosmes (flacons à plasma) d -Voies de réduction du nitrate (5)

-Étude expérimentale en microcosmes (flacons à plasma) f -Reproduction du phénomène… (2)

Application


A IDENTIFICATION

Elaboration de stratégies opératoires des méthaniseurs par approches méta-omiques

Les digesteurs anaérobies sont considérés comme insuffisamment maîtrisés car le microbiome, qui est l'acteur clé du processus, reste encore largement méconnu. De nos jours, les méthodologies omiques permettent d'effectuer des diagnostics fonctionnels du microbiome utiles pour élaborer des stratégies de gestion.

Conception de nouvelles stratégies opératoires pour lever les verrous de la méthanisation et étendre ses champs d'application par des approches méta-omiques

La digestion anaérobie (DA) est un processus de dégradation de la matière organique qui produit à terme un biogaz riche en méthane pouvant être converti en énergie. La méthanisation est encore considérée comme insuffisamment maîtrisée du fait de l'insuffisance de gestion du moteur microbien des réacteurs anaérobies car ce dernier reste encore largement méconnu. De nos jours, les méthodologies d'écologie microbienne omique permettent d'effectuer des diagnostics fonctionnels du microbiome. Les objectifs industriels sont : i. Proposition de stratégies de gestion microbienne des digesteurs anaérobies ; ii. Définition d'indicateurs microbiens de performance optimale et d'indicateurs d'alerte précoce des défaillances du processus de digestion anaérobie ; Les objectifs scientifiques sont : i. Développement d'une approche méta-omique permettant de faire un diagnostic du microbiome ; ii. Développement de méthodes biostatistiques d'exploration de données permettant la fusion de données méta-omiques et de données opérationnelles; iii. Évaluation de l'importance des propriétés du moteur microbien pour maintenir la stabilité fonctionnelle et la robustesse des bioprocédés.

Méthodologies omiques d'écologie microbienne appliquées à des perturbations clés de la digestion anaérobie pouvant mener à une défaillance du procédé.

Afin d'atteindre les objectifs du projet, la stratégie a consisté non seulement à étudier le fonctionnement de la communauté microbienne en régime stable, mais également sa réaction aux perturbations opérationnelles. Dans le projet, nous avons appliqué des méthodologies omiques d'écologie microbienne (métataxonomie, métagénomique et métabolomique) à différentes perturbations clés connues pour entraîner une défaillance du processus de digestion anaérobie des boues d'épuration. Au cours de ce projet, trois types différents de perturbations de la digestion anaérobie des boues d'épuration (concentration en ammoniac (tâche 1), variation de la composition du co-substrat (tâche 2), modification de la température (tâche 3)) ont été étudiés. Cette approche innovante repose sur l'utilisation de méthodologies omiques pour obtenir des informations sur les microorganismes présents, les fonctions exprimées et les métabolites produits, ainsi que sur l'étude des variations de ces indicateurs en fonction des conditions opératoires. L'analyse de ces données par des approches biostatistiques innovantes a permis d'extraire les corrélations pertinentes entre les différents types de données.

Résultats majeurs du projet

Des approches innovantes utilisant des méthodologies omiques d'écologie microbienne et des méthodes statistiques pour l'analyse du microbiome ont été proposées pour aller plus loin dans la description du microbiome. Par exemple, des phylotypes clés spécifiquement sensibles à des inhibitions ont été identifiés. Certains pourraient être utilisés comme bio-indicateurs pour contrôler le fonctionnement des digesteurs. Certaines stratégies de gestion microbienne des digesteurs anaérobies ont également été proposées (utilisation de matériaux réactifs pour contrer l'inhibition due à l'ammoniaque, utilisation de la température comme levier pour améliorer le bilan énergétique des digesteurs…).

Perspectives

Certaines stratégies de gestion microbienne des digesteurs anaérobies pourraient être mises en oeuvre sur site (utilisation de matériaux réactifs pour lever des inhibitions, de la température comme levier pour améliorer le bilan énergétique). Une autre perspective serait de développer des outils de diagnostic rapide pour évaluer le bon fonctionnement des digesteurs et prévenir les défaillances. Les développements méthodologiques récents permettent désormais des approches permettant de capturer les particularités biologiques des systèmes de DA et de développer des outils de diagnostic rapides.

Production scientifique

Le projet a été valorisé sous la forme de 9 publications (7 publiées, 2 soumises) dans des journaux internationaux à comité de lecture (Waste Management, Chemosphere, Journal of Proteome Research, Bioresource Technology, Chemometrics and Intelligent Laboratory Systems). Les résultats ont fait l'objet de 10 présentations dans des conférences internationales et 3 dans des conférences nationales.

Illustration

Légende : Concept du projet digestomic

Informations factuelles

Le projet DIGESTOMIC est un projet de recherche industrielle coordonné par INRAE. Il associe aussi AgroParisTech et SUEZ. Le projet a commencé en octobre 2016 et a duré 58 mois. Il a bénéficié d'une aide ANR de 446 565 € pour un coût global de l'ordre de 1 700 000 €

B.2 RESUME CONSOLIDE PUBLIC EN ANGLAIS

Design of anaerobic digestion new operational strategies using meta-omic approaches

Anaerobic digesters are considered as not being sufficiently controllable because the microbiome, which is the key player of the process, still remains largely unknown. Nowadays omic methodologies allow to perform functional microbiome diagnostics which can be useful to develop microbial-based management strategies.

Design of new operational strategies to overcome anaerobic digestion technical barriers and extend its fields of application using meta-omic approaches

Anaerobic digestion (AD) is a process of degradation of the organic matter which ultimately produces biogas rich in methane that can be converted to electrical and thermal energy. Methanization is still considered as not being sufficiently controllable due to the limitation of microbial-based management of anaerobic reactors. Indeed, the microbiome, which is the key player of the AD process, still remains largely unknown. Nowadays omic microbial ecology methodologies allow to perform functional microbiome diagnostics. The industrial goals of the Digestomic project are: (i) Proposal of microbial management strategies of anaerobic digesters, (ii) Definition of microbial indicators of optimal performance and early warning indicators of anaerobic digestion process failures. The scientific goals of the Digestomic project are: (i) Development of a meta-omic approach in order to make a microbiome diagnostic, (ii) Development of biostatistics data mining methods for the fusion of meta-omic data and process parameters, (iii) Assessment of the importance of the properties of the microbiome to maintain the functional stability and robustness of the process.

Omic microbial ecology methodologies applied to different known key anaerobic digestion perturbations that could lead to some process failures

To achieve the project goals, the strategy was not only to study the microbial community during steady state functioning but also its reaction to operational and environmental disturbances. We applied the omic microbial ecology methodologies (metataxonomics, metagenomics, metabolomics) to different known key perturbations that could lead to some process failures during sewage sludge anaerobic digestion. Three different types of anaerobic digestion perturbations have been studied (i.e., ammonia concentration (task 1), co-substrate composition variation (task 2) and temperature modification (task 3)). This innovative approach of the project is based on the use of omics methodologies to obtain information on the microorganisms present, the functions expressed and the metabolites produced, and also on the study of the variations of these indicators according to the operating conditions. The analysis of these data by innovative biostatistics approaches has allowed the extraction of the relevant correlations between the various types of data.

Major results of the project

Innovative approaches using omic microbial ecology methodologies and statistical frameworks for the analysis of the microbiome were proposed to go further in the description of the microbiome. For example, key phylotypes specifically sensitive to inhibition were identified. Some of them could be used as bio-indicators to control digester functioning. Some microbial management strategies of anaerobic digesters have also been proposed (use of reactive materials to counteract ammonia inhibition, using temperature as a level to improve plant energy balance…)

Perspectives

Based on the results obtained, some microbial management strategies of anaerobic digesters could be investigated and implemented on-site, such as using reactive material to overcome ammonia inhibition or using temperature as a lever to improve plant energy balance. One other important perspective would be to develop biological diagnostic tools to rapidly assess the correct functioning of the digesters and prevent strong inhibitions or failures. Methodological developments now allow more feasible and faster approaches capable of capturing the biological peculiarities of the AD systems and to develop fast diagnostic tools.

Scientific production

The project was valorized in the form of 9 publications (7 published, 2 submitted) in international peer-reviewed journals (Waste Management, Chemosphere, Journal of Proteome Research, Bioresource Technology, Chemometrics and Intelligent Laboratory Systems). The results were presented in 10 international conferences and 3 national conferences.

C MEMOIRE SCIENTIFIQUE

Mémoire scientifique confidentiel : non

C.1 RESUME DU MEMOIRE

Anaerobic digestion (AD) is a process of degradation of the organic matter which ultimately produces biogas rich in methane that can be converted to electrical and thermal energy. Methanization is still considered as not being sufficiently controllable due to the limitation of microbial-based management of anaerobic reactors. Indeed, the microbiome, which is the key player of the AD process, still remains largely unknown. Nowadays omic microbial ecology methodologies allow to perform functional microbiome diagnostics.Innovative approaches using omic microbial ecology methodologies and statistical frameworks for the analysis of the microbiome were proposed to go further in the description of the microbiome. For example, key phylotypes specifically sensitive to inhibition were identified. Some of them could be used as bio-indicators to control digester functioning. Some microbial management strategies of anaerobic digesters have also been proposed (use of reactive materials to counteract ammonia inhibition, using temperature as a level to improve plant energy balance…)

C.2 ENJEUX ET PROBLEMATIQUE, ETAT DE L'ART

Anaerobic digestion (AD) is a process of degradation of the organic matter which ultimately produces biogas rich in methane that can be converted to electrical and thermal energy. In a context of environmental protection and research for increasing energy efficiency, methanization arouses a renewed interest because it allows converting waste into both an energy resource and an organic amendment that can be used for manure spreading on soil. The number of organic matter deposits is continuously increasing, making anaerobic digestion an important potential source of energy. Over the last years, numerous studies were carried out to optimize the management of reactors and maximize their efficiency. However experience feedback shows that digesters remain poorly instrumented and that their operation mainly relies on the know-how of the developer. Furthermore, the dynamics of the populations and industries can cause sudden and unpredictable changes of quantity and composition of the waste to be treated. Until recently, methanization was considered as not being sufficiently controllable to confront these changes and to present the required flexibility. Such a situation was mainly due to the limitation of microbial-based management of anaerobic reactors. Indeed, the microbiome, which is the key player of the AD process, still remains largely unknown. Nowadays omic microbial ecology methodologies allow to perform functional microbiome diagnostics [START_REF] Vanwonterghem | Linking microbial community structure, interactions and function in anaerobic digesters using new molecular techniques[END_REF] which could help (i) to build real microbial management strategies of anaerobic digestion and (ii) to set microbial indicators of optimal performance and warning indicators of process failures [START_REF] Koch | Microbiomes in bioenergy production: From analysis to management[END_REF]. During the project other research teams have confirmed the interest of omic approaches for AD diagnostic purposes [START_REF] Venkiteshwaran | Correlating methane production to microbiota in anaerobic digesters fed synthetic wastewater[END_REF][START_REF] References De Vrieze | The active microbial community more accurately reflects the anaerobic digestion process: 16S rRNA (gene) sequencing as a predictive tool[END_REF]. The industrial goals of the Digestomic project are: (i) Proposal of microbial management strategies of anaerobic digesters, (ii) Definition of microbial indicators of optimal performance and early warning indicators of anaerobic digestion process failures. The scientific goals of the Digestomic project are: (i) Development of a meta-omic approach in order to make a microbiome diagnostic, (ii) Development of biostatistics data mining methods for the fusion of meta-omic data and process parameters, (iii) Assessment of the importance of the properties of the microbiome to maintain the functional stability and robustness of the process.

C.3 APPROCHE SCIENTIFIQUE ET TECHNIQUE

To achieve the project goals, the strategy was not only to study the microbial community steady state functioning during but also its reaction to operational and environmental disturbances. In the project we applied the omic microbial ecology methodologies to different known key perturbations that could lead to some process failures during sewage sludge anaerobic digestion. During this project, three different types of sewage sludge anaerobic digestion perturbations were studied (i.e., ammonia concentration (task 1), co-substrate composition variation (task 2), temperature modification (task 3)). This innovative approach is based on the use of omics methodologies to obtain information on the microorganisms present, the functions expressed and the metabolites produced, and also on the study of the variations of these indicators according to the operating conditions. The analysis of these data by innovative biostatistics approaches has allowed the extraction of the relevant correlations between the various types of data. The experimental design, omic methodologies and statistical analysis used were as follows :

Experimental design

For task 1 on the effect on ammonia during AD, we decided to study the microbiome dynamics alongside the ammonia accumulation speed. We then compared the effect of different speeds of ammonia addition in semi-continuous bioreactors. Six semi-continuous stirred tank reactors (Bioprocess Control) with a working volume of 5 L (7 L total) were used.

For task 2, batch digesters were used to study the influence of different mixtures of substrates and to determine the most appropriate ratio. Batch experiments were carried out in 1L glass bottles under mesophilic conditions, in the dark and without agitation. A second experiment in semi-continuous bioreactors was performed to study the effect of the modification of the feedstock composition during AD.

It is to note that for task 3 on the evaluation of temperature as an operational lever, we did a full-scale experiment on a 190,000 people equivalent French WasteWater Treatment Plan (WWTP). Two 3,000 m 3 digesters handling the same sludge were operated in parallel, under the exact same operating conditions. During a first period of 4 months, both digesters were operated under mesophilic conditions (35°C) and monitored to verify that they behaved in the same way. Then Digester A was kept running in the mesophilic mode during the entire experiment (control reactor : 35°C) while the temperature of digester B was abruptly decreased from 35°C to 30°C, and after 2 Hydraulic Retention Time (HRT) of stable operation , it was abruptly decreased from 30°C to 25°C (experimental reactor). After 3 HRT at 25°C, the temperature of digester B was restored to 35°C until the end of the experiment.

Analytical methodologies

-Metataxonomics (16S metabarcoding) V4-V5 region of the 16S rRNA gene was amplified by PCR. Sequencing was performed on Ion Torrent Personal Genome Machine using Ion 316 Chip V2 (Life Technologies) and Ion PGM Hi-Q View Sequencing Kit (Life Technologies) according to the manufacturer's instructions. Sequencing data was processed by the Torrent Suite Software.

-Metagenomics Libraries were prepared with the TruSeq NGS Library Prep Kit from Westburg (WB9024). The sequencing of the genomic DNA was done using the Illumina NextSeq 500.

-Metabolomics As the objective during this specific work was to study the metabolome as a whole, untargeted methodologies were used. Liquid-chromatography mass spectrometry (LTQ-Orbitrap) and Fourier-transform ion cyclotron resonance (FTICR) with direct infusion were used to analysis the supernatant, that is the dissolved extracellular metabolites.

Statistical analysis

Different statistical approaches were employed to integrate and analyze the data. Classical methods such as PCA (Principal Component Analysis), ICA (Independent Component Analysis) were used to extract relevant information from the different data sets. More refined approaches, such as ComDim (Common Components and Specific Weights Analysis), or specifically designed correlation approaches were employed to draw links between different types of omics. Finally we also developed specific frameworks for the time course analysis of data.

C.4 RESULTATS OBTENUS

Task 1: Microbial management strategies to prevent ammonia inhibition during AD

The influence of ammonia accumulation on the microbial dynamics was evaluated. In semicontinuous reactors ammonia was added at different speeds. The results showed that the faster the speed of accumulation was, the more the digester performances were impacted. A longitudinal analysis was performed by carrying out a temporal sampling. This analysis allowed the assessment of the adaptation capacity of the microbes when subjected to different conditions of ammonia addition. Moreover, microorganisms specific to the ammonia accumulation were identified (Lachnospiraceae, Dysgonomonadaceae and Ruminococcaceae) and could be used as potential bio-indicators to forecast digester inhibition. In order to propose a strategy to prevent ammonia inhibition during AD, we decided to study the effect of Zeolite which is a mineral support able to counteract ammonia inhibition, although the mechanisms behind this mitigation ability remain unclear. In order to unravel them, different physical treatments were applied to the zeolite before it was added into digesters containing ammonia. In general, in absence of ammonia the zeolite did not influence the microbial composition and the digester performances, while a significant effect was observed in presence of ammonia. A discriminant analysis coupled to a sparse method allowed to highlight the impact of the zeolite on the microbial syntrophy needed for the propionate degradation. Additional data from the literature were included in order to evaluate the genericity of the results. The results showed that the addition of zeolite in presence of ammonia led to the modification of the microbial community, especially of microbes involved in a syntrophy for the degradation of volatile organic acids. This resulted in the decrease of the inhibition.

Task 2 Microbial management strategies to overcome dysfunction during co-digestion

The influence of the feedstock composition on the AD microbiome was studied. Digesters were fed with different mixtures of substrates (grass, fish, sludge). The results showed that the substrate composition played a significant role in the microbial diversity and specificity which could explain the different digester performances observed between the batch reactors. An integrative analytical framework was specifically used to link the microbial activity to the substrates degradation. It allowed the identification of potential degraders of specific molecules. The capacity of the microorganisms to adapt to a modification of the feedstock composition was also evaluated in semi-continuous reactors.

Task 3: Evaluation of temperature as an operational lever Low temperature anaerobic digestion (30°C and 25°C) was successfully performed at full-scale for several months. During the low temperature operation, the reactor remained stable but the specific biogas production decreased by 20%. Despite this significant decrease in biogas production, the energy balance is still positive, leading to an increase in the amount of biogas available for valorization. If some changes in the microbial structure have been observed during temperature decrease, after the mesophilic temperature was restored, no significant differences were highlighted between the microbial communities in the control digester and the ones in the test digester. Microbial dynamics were monitored with a multi-omics approaches (metataxonomics, metagenomics, metabolomics) to decipher the influence of temperature modifications.

C.5 EXPLOITATION DES RESULTATS

Concerning task 1, we have been able to show that Zeolite could be a strategy to prevent inhibition by ammonia and to understand the reasons behind this effect. This strategy could be tested at the industrial level. Concerning task 3, the results support the application of a low temperature AD strategy when it comes to the operation of under-loaded digesters to improve plant energy balance. Indeed, the biogas reduction induced by temperature decrease seems to be counterbalanced by an increase in the amount of biogas available for valorization, thus potentially increasing resulting income. The fact that no permanent detrimental effect was observed on the microbial communities shows that temperature can be seen as an operational lever.

C.6 DISCUSSION

The different initial objectives presented in C2 have been achieved. 1 st Scientific objective Different meta-omic approaches have been used (metataxonomics, metagenomics and metabolomics) to create an AD microbiome diagnostic as planned. 16S metabarcoding and metabolomics approaches were found to be the best suited methods to monitor anaerobic digesters, as they provide high-throughput data that can be used to rapidly create extensive databases and design AD diagnostic warning bioindicators.

2 nd Scientific objective Innovative biostatistics data mining methods have been employed (PLS-DA, ComDim, …) and developed (methods for integration of metabolomics and metataxonomics data, specific framework for the analysis of longitudinal data from high-throughput approaches that are not limited to but applied to metataxonomics and metabolomics) to analyze the generated data for operational purposes 3 rd Scientific objective Using the meta-omic approaches, we have been able to evaluate the importance of microbial ecological parameters to maintain functional stability and robustness of bioprocesses. For example in task 3, after restoring the initial mesophilic temperature, the bacterial community manifested resilience in terms of microbial structure and functional activity. This characteristic of the microbial community is essential for process stability.

1 st Industrial objective The project gives (i) relevant indications for feeding strategies to overcome dysfunction during co-digestion process; (ii) promising results for the improvement of the plant energy balance by reducing the digester temperature without a permanent detrimental effect either on the microbial communities or the biogas production; (iii) better description of the relation between ammoniac concentration, digester microbiome and biogas production performances.

2 nd Industrial objective Several microbial indicators of optimal performance and early warning indicators have been proposed. For example, microorganisms specific to the ammonia accumulation were identified and could be used as potential bio-indicators to forecast digester inhibition. The results obtained during the Digestomic project open several perspectives both from a scientific and an industrial point of view. For example, based on the results obtained, some microbial management strategies of anaerobic digesters should be investigated and implemented on-site; for example, using reactive material to overcome ammonia inhibition or using temperature as a lever to improve plant energy balance. One other important perspective would be to develop biological diagnostic tools to rapidly assess the correct functioning of the digesters and prevent strong inhibitions or failures. Until recently, the use of high-throughput techniques was limited to selected lab studies as they were expensive, time-consuming and the results were not easy to use for operational purpose. Methodological developments now allow more feasible and faster approaches that are able to capture the biological particularities of the AD systems and to develop fast diagnostic tools. This is the objective of the methadiag project that we have submitted to the ANR AAPG.

C.7 CONCLUSIONS

The results of this project highlight the importance of the operational parameters, even at a small-scale, on the microbial activity. Innovative statistical frameworks for the analysis of the microbiome were proposed to go further into the description of the microbiome. For example, key phylotypes specifically sensitive to inhibition were identified. Some of them could be used as bio-indicators to monitor digester functioning. Moreover, this work highlights the value of developing metabolomic analysis in the bioprocess fields, as it enables monitoring the degradation of the organic matter and infers the potential role of the microorganisms in the degradation process. Some microbial management strategies of anaerobic digesters have also been proposed (use of reactive materials to counteract ammonia inhibition, using temperature as a lever to improve plant energy balance…)

D LISTE DES LIVRABLES

E.2 LISTE DES PUBLICATIONS ET COMMUNICATIONS

Publications :

 Cardona, L., Levrard, C., Guenne, A., Chapleur, O., & Mazéas, L. (2019)



  2017-15 th IWA World Conference on Anaerobic Digestion (Beijing, China) -Low temperature sewage sludge anaerobic digestion: full-scale proof of interest and study of microbial adaptation. Conteau D, Franchi Morales O, Chapleur O, Gaval G, Traba Lago G, Mazéas L, Araya Kroff P, Barillon B  2018 -2 nd International Conference on Anaerobic Digestion Technology (Chiang Mai, Thailand) -Impact of anaerobic codigestion on microbial community and associated degradation pathways. Cardona L, Madigou C, Bureau C, Rouillac L, Mazéas L and Chapleur O  2017 -3 rd International Conference on Biogas Microbiology (Wageningen, Netherlands) -Understanding the effect of ammonia on anaerobic microbiota during biowaste anaerobic digestion. Cardona L, Poirier S, Madigou C, Bouchez T, Mazéas L, Chapleur O  2018 -17 th International Symposium on Microbial Ecology (Leipzig, Deutschland) -Multi-omics data integration to decipher the impact of feeding composition on the microbiota of anaerobic digestion. Cardona L, Lê Cao K-A, Bureau C, Madigou C, Rouillac L, Mazéas L, Chapleur O  2018 -17 th International Symposium on Microbial Ecology (Leipzig, Deutschland) -Effect of ammonia on the dynamics of anaerobic digestion microbiome: omics data integration in a time course context. Chapleur O, Poirier S, Lê Cao K-A  2019 -8 th Congress of European Microbiologists (Glasgow, Scotland) -Data integration to correlate microbial activity to molecules degradation during anaerobic digestion. Cardona L, Lê Cao K-A, Bureau C, Madigou C, Mazéas L, Chapleur O  2019 -8 th IWA Microbial Ecology and Water Engineering Specialist Conference (Hiroshima, Japan) -Omics integrative analysis to improve the anaerobic digestion of wastewater sludge. Cardona L, Lê Cao K-A, Bureau C, Madigou C, Mazéas L, Chapleur O  2020 -CHIMIOMÉTRIE 2020. Chemometric analysis of a metagenomics dataset to decipher the functional role of the microbiota from anaerobic digesters. (Liège, Belgium). Puig-Castellví F, Midoux C, Cardona L, Chapleur O, Conteau D, Cordella C, Gaval G, Jouan-Rimbaud Bouveresse D, Kroff P, Mazeas L, Rutledge DN  2020 -#RSCPoster Twitter Conference. (Online) -Title: "Understanding waste degradation with Omics". Puig-Castellví F  2020 -European RFMF / Metabomeeting 2020 (Toulouse, France). Assessment of the substrate biodegradability in anaerobic co-digestion using a metabolomics approach. Puig-Castellví F, Cardona L, Bouveresse D, Cordella C, Mazéas L, Rutledge DN, Chapleur O Conférences nationales :  2019 -Chimiométrie XX (Montpellier). Unraveling the microbial community interactions in anaerobic digesters with Common Components Analysis. Puig-Castellví F, Cardona L, Chapleur O, Cordella C, Jouan-Rimbaud Bouveresse D, Mazeas L and Rutledge DN  2021 -Réseau National de Spectrométrie de Masse FT-ICR à très Haut Champ, journée utilisateur (videoconference). Time-course trajectories of metabolomic response in anaerobic digesters inhibited by ammonia. Wang X, Latapy J, Nicol E, Cardona L, Bouchonnet S, Mazéas L, Chapleur O  2021 -3 rd RFMF Webinar. Can we monitor waste degradation using metabolomics? (Invited). Puig-Castellví F, Midoux C, Guenne A, Conteau D, Franchi O, Bureau C, Madigou C, Bouveresse D, Kroff P, Mazéas L, Rutledge DN, Gaval G, Chapleur O

RESUME CONSOLIDE PUBLIC B.1 RESUME CONSOLIDE PUBLIC EN FRANÇAIS

	Acronyme du projet	DIGESTOMIC
	Titre du projet	Elaboration de nouvelles stratégies opératoires
		pour lever les verrous de la digestion anaérobie
		et élargir ses domaines d'application à l'aide
		d'approches méta-omiques
	Coordinateur du projet	INRAE
	(société/organisme)
	Période du projet	1/10/2016 -31/08/2021
	(date de début -date de fin)
	Site web du projet, le cas échéant
	Rédacteur de ce rapport
	Civilité, prénom, nom	M. MAZEAS Laurent
	Téléphone	01 40 96 60 61
	Adresse électronique	Laurent.mazeas@inrae.fr
	Date de rédaction
	Si différent du rédacteur, indiquer un contact pour le projet
	Civilité, prénom, nom
	Téléphone
	Adresse électronique
	Liste des partenaires présents à la	INRAE : Laurent MAZEAS
	fin du projet (société/organisme et	AgroParisTech : Douglas RUTLEDGE
	responsable scientifique)	SUEZ : Gilberte GAVAL
	B

IMPACT DU PROJET E.1 INDICATEURS D'IMPACT Nombre de publications et de communications (à détailler en E.2) Publications multipartenaires Publications monopartenaires International Revues à comité de lecture 6

	Date de	N°	Titre		Nature (rapport,	Partenaires	Commentaires
	livraison				logiciel, prototype,	(souligner le
					données, …)	responsable)
	M9	1	inhibition D1.1: Effect of ammonia on	Livrable	IRSTEA
			microbiome
	M21	2	M1.1: microbial management Choice of	Jalon	IRSTEA, AGROPARISTECH SUEZ,
			strategy for task 1.
	M9	3	keep M3.1: opportunities to decreasing	Jalon	IRSTEA, SUEZ
			temperature
	M12	4	increase temperature M3.2: opportunity to	Jalon	IRSTEA, SUEZ
	M24	5	D3.1: Adaptation of microbiome to	Livrable	IRSTEA, AGROPARISTECH SUEZ
			temperature decrease
	M15	6	M2.1: Choice of optimal sewage sludge/ co-	Jalon	IRSTEA, AGROPARISTECH SUEZ
			substrate ratio for task
			2.2
	M27	7	D1.2: microbial management Strategies of	Livrable	IRSTEA, AGROPARISTECH SUEZ,
			for ammonia inhibition
			mitigation.
	M45	8	D3.2: temperature Evaluation as	of an	Livrable	IRSTEA, AGROPARISTECH SUEZ,
			operational lever
	M30	9	D2.1: Effect of co-substrate composition	Livrable	IRSTEA, AGROPARISTECH SUEZ,
			variation on microbiome
	M30	10	M2.2: microbial management Choice of	Jalon	IRSTEA, AGROPARISTECH SUEZ,
			strategy for task 2.3
	M43	11	D2.2: microbial management Strategies of	Livrable	IRSTEA, AGROPARISTECH SUEZ,
			during co-digestion.
	E Ouvrages ou chapitres
			d'ouvrage
			Communications		3
			(conférence)
			Revues à comité de
			lecture
	France		Ouvrages ou chapitres
			d'ouvrage
			Communications		2
			(conférence)
			Articles vulgarisation
	Actions de		Conférences
	diffusion		vulgarisation
			Autres
	Référence du formulaire : ANR-FORM-090601-01-01			13/17

Autres valorisations scientifiques (à détailler en E.3) Nombre, années et commentaires (valorisations avérées ou probables) Brevets internationaux obtenus Brevet internationaux en cours d'obtention Brevets nationaux obtenus

  Prix de thèse pour Laëtitia Cardona : Médaille d'argent-Dufrenoy délivrée par l'Académie d'agriculture.

	Brevet nationaux en cours
	d'obtention
	Licences d'exploitation
	(obtention / cession)
	Créations d'entreprises ou
	essaimage
	Nouveaux projets
	collaboratifs
	Colloques scientifiques
	Autres (préciser)

  . Co-digestion of wastewater sludge: Choosing the optimal blend. Waste Management, 87, 772-781. https://doi.org/10.1016/j.wasman.2019.03.016 Puig-Castellví, F., Cardona, L., Bureau, C., Bouveresse, D., Cordella, C. B. Y., Mazéas, L., Rutledge, D. N., Chapleur, O. (2020). Effect of ammonia exposure and acclimation on the performance and the microbiome of anaerobic digestion.anaerobic digester after a severe ammonia shock. Soumise à Bioresource Technology  Puig-Castellví, F.,Midoux, C., Guenne, A., Conteau, D., Franchi, O., Bureau, C., Madigou, C., Jouan-Rimbaud Bouveresse, D., Kroff, P., Mazéas, L., Rutledge, DN., Gaval, G., Chapleur, O. Metataxonomics, metagenomics, and metabolomics analysis of the influence of temperature modification in full-scale anaerobic digesters. Soumise à Bioresource Technology

	Communications :
	Conférences internationales :
	 Cardona, L., Mazéas, L., & Chapleur, O. (2020). Zeolite favours propionate syntrophic degradation during anaerobic
	digestion	of	food	waste	under	low	ammonia	stress.	Chemosphere,	594(Iii),	127932.
	https://doi.org/10.1016/j.chemosphere.2020.127932
	 Cardona, L., Cao, K. A. L., Puig-Castellví, F., Bureau, C., Madigou, C., Mazéas, L., & Chapleur, O. (2020). Integrative
	analyses to investigate the link between microbial activity and metabolite degradation during anaerobic digestion. Journal
	of Proteome Research, 19(10), 3981-3992. https://doi.org/10.1021/acs.jproteome.0c00251
	Bioresource Technology Reports, 11, 100488. https://doi.org/10.1016/j.biteb.2020.100488
	 Puig-Castellví, F., Bouveresse, D., Mazéas, L., Chapleur, O., Rutledge, D. (2021). Rearrangement of incomplete multi-
	omics datasets combined with ComDim for evaluating replicate cross-platform variability and batch influence.
	Chemometrics and Intelligent Laboratory Systems, 104422 https://dx.doi.org/10.1016/j.chemolab.2021.104422
	 Cardona L., Faire progresser les biotechnologies environnementales par le biais d'approches d'écologie moléculaire : de
	la description à la gestion. Thèse de doctorat.
	Publications soumises:

 Puig-Castellví, F.,

Cardona, L., Bouveresse, D., Cordella, C. B. Y., Mazéas, L., Rutledge, D. N., Chapleur, O. (2020)

.

Assessment of substrate biodegradability improvement in anaerobic Co-digestion using a chemometrics-based metabolomic approach. Chemosphere, 254, 126812. https://doi.org/10.1016/j.chemosphere.2020.126812  Puig-Castellví, F., Cardona, L., Bouveresse, D., Cordella, C. B. Y., Mazéas, L., Rutledge, D. N., Chapleur, O. (2020). Assessment of the microbial interplay during anaerobic co-digestion of wastewaer sludge using common components analysis. Plos One, 15(5), 1-17. https://doi.org/10.1371/journal.pone.0232324   Cardona, L., Mazéas, L., Chapleur, O. Deterministic processes drive the microbial assembly during the recovery of an Référence du formulaire : ANR-FORM-090601-01-01 14/17

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extend its fields of application by using meta-omic approaches

Appel à projets générique 2016

Illustration

Legend: Digestomic project concept

Factual information

The DIGESTOMIC project is an industrial research project coordinated by INRAE. It also associates AgroParisTech and SUEZ. The project started in October 2016 and lasted 58 months. It received ANR aid of € 446,565 for a total cost of around € 1,700,000.

E.3 LISTE DES ELEMENTS

E.4 IMPACT AU NIVEAU DES PARTENAIRES INDUSTRIELS (LE CAS ECHEANT)

Entreprise xxx SUEZ Xxx Gilberte GAVAL gilberte.gaval@suez.com SUEZ contribution to the project can be wrap-up in three main points: (i) industrial contact facilitating the access to full-scale digesters or substrate/inoculum samples; (ii) feedback on the alignment of the trials with on-field reality; (iii) management and realization of the full-scale trial for the assessment of the impact of low temperature on anaerobic digestion. For SUEZ, the main interest of the current project consists in the opportunity to get access to a deeper understanding of the anaerobic digestion microbiome and thus to be able to move from a "black-box" management towards a microbial-based management.

The project highlighted several relevant outcomes with potential industrial applications: (i) relevant indications for feeding strategies to overcome dysfunction during co-digestion process;

Référence du formulaire : ANR-FORM-090601-01-01

16/17

(ii) promising results for the improvement of the plant energy balance by reducing the digester temperature without a permanent detrimental effect either on the microbial communities or the biogas production; (iii) better description of the relation between ammoniac concentration, digester microbiome and biogas production performances. On this basis, SUEZ intends, if be possible, to propose new operational strategies to overcome technical barriers in anaerobic digestion leading to an increase of biogas production flow rate, quality and stability. In addition, from an economical point of view, these new strategies could then be related either to augmented revenues or reduced capital expenditures by increasing the initial plant capacity.