Ulysse Brémond

Aude Bertrandias

Jean-Philippe Steyer

Nicolas Bernet

Hélène Carrere
email: helene.carrere@inrae.fr

A vision of European biogas sector development towards 2030: Trends and challenges

Keywords: Renewable energy, Anaerobic digestion, Decarbonization, Biogas, Biomethane, Agricultural sector

The future European "Green Deal" will set ambitious greenhouse gas emissions reduction by 2030. Reaching these targets will partly rely on the decarbonization of the gas sector and the development of the biomethane. In this context, this article aims to provide a vision of the European biogas sector development from 2020 towards 2030 and beyond. It appears that the biogas sector is facing a shift in its development paradigm. Indeed, this sector is still mainly dominated by a model based on energy crops, high feed-in tariffs and local electrical production via combined heat and power units. However, biogas sector is now moving towards a different model, where organic wastes, agricultural by-products as well as sequential crops are mainly used as feedstocks, biogas is upgraded to biomethane for various applications (transportation, chemical production, heat, etc.) and subsidy schemes are progressively reduced. Overall, current European policies are pushing the sector to increase its sustainability and to reduce biogas production costs. To answer these challenges, the main improvement tracks are identified and discussed. In the future, it is believed that

the biogas sector should address these aspects to ensure its growth towards 2030 and beyond.

Introduction

According to the Intergovernmental Panel on Climate Change (IPCC) Special Report Global Warming of 1.5°C, the world needs to limit the rise in global temperatures to 1.5°C to avoid unacceptable consequences on ecosystems and populations. This objective is still doable but it means reaching worldwide net-zero emissions by 2050 and implies a quick, strong and long-term international climate action (IPCC, 2018). The Paris agreement adopted in 2015 by 195 countries and its rulebook decided in the COP24, early 2019, are the first steps towards a global energy transition. In the wake of the IPCC Special Report, the European Union (EU) presented its strategic long-term vision to reach a climate-neutral Europe by 2050, in which the EU expresses its willingness to be a global leader in the fight against climate change and to be among the first to reach net-zero greenhouse gases (GHG) emissions by 2050 (European Commission, 2018). The European "Green Deal", recently announced by the European Commission President Ursula von der Leyen, aims GHG emissions reduction of at least 50% by 2030 in comparison to 1990. To reach this target, further decarbonization of the energy system will be essential (European Commission, 2019a).

To reach such targets, recent scenarios agreed on the fact that renewable electricity will be essential to fully decarbonize the energy system. Thus, it is forecasted that the share of electricity in final energy demand will at least double (European Commission, 2018).

Renewable electrical production will increase mainly relying on a strong growth in the number of solar as well as on-shore and off-shore wind facilities (EURELECTRIC, 2018).

However, for Dr. Klaus-Dieter Borchardt, Europe's Director of the Internal Energy Market Integration, pathways towards a decarbonized economy only relying on renewable electricity will not be cost-effective and will be technologically impossible mainly due to its storage issue using batteries (Florence School of Regulation, 2019). The optimum solution may lie in a "dual" gas-power network [START_REF] Bowden | EU sees dual gas-power energy system as best bet to reach zero carbon by 2050[END_REF].Indeed, according to Dr. Borchardt : "Gas infrastructure should become the batteries of the future European energy system" [START_REF] Brun | European regulation process -GRT gaz[END_REF]. The link between the power and gas networks will be ensured by the emerging power-to-gas technologies. They allow converting renewable electricity via an electrolysis process to hydrogen, a high gravimetric energy density carrier, which can be stored, directly used as a fuel [START_REF] Gür | Review of electrical energy storage technologies, materials and systems: Challenges and prospects for large-scale grid storage[END_REF]. Hydrogen can even be further converted through a methanation process into methane, that can be injected and stored on natural gas grid [START_REF] Götz | Renewable Power-to-Gas: A technological and economic review[END_REF]. As current high cost for these technologies should be reduced in the future, they are expected to become one of the best options for long-term electricity storage [START_REF] Thema | Power-to-Gas: Electrolysis and methanation status review[END_REF]. It could allow decarbonization of sectors that would be hard to electrify such as long-distance heavy transportation or industrial processes using high-temperature heat (Gas for Climate, 2019a).

Integration and decarbonization of the gas sector are currently at the top of the political agenda in Brussels with notably the preparation of the "gas decarbonization package" (Euractive, 2019). This package should set, by the end of 2020 or early 2021, a clear vision on the role of gas up to 2050. It is notably expected to propose a framework (e.g. targets, certificate, etc.) for a "green" gas [START_REF] Olczak | What to expect from the 2020 gas package[END_REF]. Developments of hydrogen, biomethane and syngas production through power-to-gas, anaerobic digestion (AD) and gasification as well as Carbon Capture & Storage (CCS) are envisioned as decarbonization vectors of the gas sector (Gas for Climate, 2019b).

Power-to-gas [START_REF] Thema | Power-to-Gas: Electrolysis and methanation status review[END_REF], CCS [START_REF] Reiner | Learning through a portfolio of carbon capture and storage demonstration projects[END_REF] and gasification [START_REF] Sansaniwal | Recent advances in the development of biomass gasification technology: A comprehensive review[END_REF] sectors are still in early stage development, while anaerobic digestion is currently most advanced in terms of industrialization (Scarlat et al., 2018a). AD consists in a biological process that allows the conversion of organic matter/waste into biogas (a mix of methane and carbon dioxide). In Europe, AD industrial development is historically based on power generation from biogas trough combined heat and power (CHP) units. After a quick growth up to 2015, the sector has slowed down and is currently at a turning point in its history. Indeed, biomethane recovery through biogas upgrading technologies (splitting biogas into biomethane and carbon dioxide gas streams) and its subsequent broader end-use applications can be envisioned as the next growth driver for the sector. At the end of 2018, annual production of biomethane from AD in the EU corresponded to 2.3 billion (10 9 ) m 3 (EBA, 2019). By 2050, combining realistic manure biomethane potential (Scarlat et al., 2018b) and methane potentials of other feedstocks provided in the Gas for Climate study (Gas for Climate, 2019a), it can be estimated that it could reach 64.2 billion m 3 in the case of an optimized gas scenario. If integration of the gas sector into the EU decarbonization strategy is validated, AD will continue to grow, but it is clear that the sector should undergo a strong shift in its development paradigm.

The aim of this article is to provide a vision of the European biogas sector development from 2020 towards 2030 and beyond. This vision is based on the identification of (i) the main current market trends and sector challenges from an overview of biogas sector past development and current situation in some representative European countries; (ii) the potential improvement tracks that tackle these challenges and may allow biogas sector a sustainable growth.

Methods

This paper is the result of a literature review. In order to provide to the reader a synthetic vision on the topic only scientific articles, reports and web pages that were identified as particularly relevant for understanding the recent evolution of the biogas sector were selected. Additionally, five European countries, presented thereafter, were selected as support cases for this paper. Search was performed using Google, Web of science, Google Scholar and Science Direct. A combination of the following search terms were used to find the selected articles: "anaerobic digestion", "bioenergy", "biogas", "biorefinery", "biomethane", "climate", "digestate", "digester", "energy", "Europe", "European Union", "feedstock", "gas", "greenhouse gases", "sequential crops", "life-cycle analysis", "manure", "mixing", "monitoring", "nutrient", "perspectives", "pilot", "plant", "policies", "power", "pretreatment", "renewable", "review", "socio-economic", "storage", "strategy", "technoeconomic", "upgrading". The names of the selected countries were also included in the search terms. The three following precepts were applied:

(i) Scientific papers published in peer-reviewed journals in English language and reports published in English and French languages from recognized organizations were selected for this review.

(ii) A particular attention was dedicated to identify and select works for the relevance of their data (notably for sector/techno-economic reports), for the quality of the analysis and insights provided (notably for the reviews), or for the identified high potential of the presented practices/technologies (in the case of more technical scientific articles related to the anaerobic digestion).

(iii) Finally, a priority was given to most recent works. Thus, 85% of the literature provided in this review (93 references) was published between 2017 and 2020.

Besides, to draw lessons from European biogas sector recent development, while remaining synthetic, only five European countries with an existing biogas industry on their territory were selected. Selection criteria were not based on countries having the highest numbers of biogas plant or biogas production. Instead, they were based on diversity in terms of policies, historical and forecast developments both in biogas and biomethane in order to provide a representative vision of the current trends and challenges that exist in Europe. The selected countries are: Germany, as a pioneer and leader in biogas production performing mainly CHP valorization, Denmark and Sweden, as countries among the most advanced in biomethane development and finally, France and Italy, two of the most active European countries on the path of CHP valorization shift towards biomethane.

To support this choice and bring some comparison elements with others European countries, it can be added that: (i) situations in Austria and the Czech Republic are close to Germany state of affairs [START_REF] Banja | Support for biogas in the EU electricity sector -A comparative analysis[END_REF]; (ii) United Kingdom and the Netherlands are already significant biomethane producers, with respectively 3.3 TWh and 2.2 TWh in 2018 [START_REF] Regatrace | Mapping the state of play of renewable gases in Europe[END_REF]. They should continue to develop this sector, similarly to France, for heating & cooling and transportation applications [START_REF] Schmid | Biogas upgrading: A review of national biomethane strategies and support policies in selected countries[END_REF]. However, contrary to France, current lack of biomethane targets in these countries should make the development pace less dynamic; (iii) Switzerland, Poland, Finland, Republic of Ireland, Spain and Belgium are in early stages of biomethane development. Support systems as well as targets in their legislation for 2030 are still being defined, therefore envisioned biomethane strategy remains for the moment unclear (REGATRACE, 2020).

Lessons from European biogas sector development and current situation

The European biogas production

Over the last 20 years, biogas production worldwide from AD has experienced a strong growth. Between 2000 and 2017, global biogas production was more than quadrupled, from 78 to 364 TWh, which corresponds to a global yearly volume of 61 billion m 3 biogas: it is shared mainly between Europe (54%), Asia (31%) and Americas (14%) (WBA, 2019). Biogas production and its utilization display great disparities between developing and developed countries. For the former, biogas is mainly produced from small-scale low-tech household plants in order to provide gas for cooking and lighting and reduce firewood consumption [START_REF] Mccabe | Integrated Biogas Systems: Local applications of anaerobic digestion towards integrated sustainable solutions[END_REF]. While, for the latter biogas is produced from large scale automated and monitored plants and valorized under the form of electricity or biomethane, which are generally injected in national grids [START_REF] Vasco-Correa | Anaerobic digestion for bioenergy production: Global status, environmental and techno-economic implications, and government policies[END_REF]. With 18,202 biogas plants (EBA, 2019) and 12.6 GW installed in 2018, Europe is the world leader in biogas electricity production, far ahead the USA (2.4 GW) and China (0.6 GW) [START_REF] Irena | Renewable Capacity Statistics[END_REF].

European biogas electricity production represents 68% of the global electricity capacity from biogas plants estimated at 18.1 GW. This last value represents only 0.7% share of the worldwide renewable electricity production (2,351 GW) mainly dominated by hydropower (52%), onshore wind (23%) and solar (20%), as represented in Fig. 1 [START_REF] Irena | Renewable Capacity Statistics[END_REF].

In 2018 in the EU, it can be estimated that about 88.5% of the biogas is valorized directly onsite, through CHP units (total primary energy production from biogas was 192 TWh and biomethane upgrading capacity was 22 TWh) [START_REF] Eurobserv | The State of Renewable Energies in Europe[END_REF]. This is the result of energy policies, set between 2000 and 2015, from several state members based on subsidies incentives for this technology [START_REF] Vasco-Correa | Anaerobic digestion for bioenergy production: Global status, environmental and techno-economic implications, and government policies[END_REF]. However, the recent decline in subsidy levels can explain the slowdown of the biogas industry in the last few years (EurObserv'ER, 2018). To better understand the possible future of AD in the EU and current market driving trends, it is relevant to look at its recent history in some European countries. 3.2. Market driving trends and challenges in the biogas sector from relevant European countries Germany, the European biogas leader with 9,500 biogas plants at the end of 2018, has built its biogas sector on the basis of the Renewable Energies Act that started in 2000 (EBA, 2019). This act forced energy supply companies to buy electricity generated from renewable sources at guaranteed feed-in tariffs (FIT) over a period of 20 years. Combined with strong incentives in 2004 and 2009 for energy crops cultivation, the biogas industry boomed between 2000 and 2012. It was mainly technologically based on a "standard" wet digestion, i.e. continuously stirred tank reactor (CSTR), plant using energy crops (especially maize) coupled with cattle slurry that allows ensuring high and resilient biogas production valorized via a CHP unit [START_REF] Theuerl | The future agricultural biogas plant in Germany: A vision[END_REF]. From this industry scheme, several drawbacks have been pointed out:

(i)
The extensive use of energy crops has led to the "maizification" phenomenon. It corresponds to the wide spreading of maize monoculture over 2.1 million ha with inherent adverse side effects such as nitrate leaching, soil degradation, pesticide use, increased pressure of soil-borne diseases and weed problems, low biodiversity and high water consumption [START_REF] Markard | Institutional dynamics and technology legitimacy -A framework and a case study on biogas technology[END_REF]. This phenomenon diminished beneficial environmental impact of biogas production as well as public acceptance of the technology and created agricultural distortions such as the increase of land rental prices, the increase of energy crops prices (also used for cattle feeding) and therefore the difficulty for smaller farms or biogas exploitations to thrive [START_REF] Appel | Effects of the German Renewable Energy Act on structural change in Agriculture -The case of biogas[END_REF]. (iii) A lack of efficient utilization of the heat produced from the CHP as local heat sink is missing, especially in rural areas. It was thus estimated that at least a third of the biogas produced was subject to such lack of heat recovery [START_REF] Strauch | Biomethane Guide for Decision Makers[END_REF]. This is strongly impacting CHP valorization efficiency [START_REF] Pöschl | Evaluation of energy efficiency of various biogas production and utilization pathways[END_REF] and biogas plant economic viability [START_REF] Lantz | The economic performance of combined heat and power from biogas produced from manure in Sweden -A comparison of different CHP technologies[END_REF]. Besides, this reduces its interest over biogas upgrading valorization to biomethane [START_REF] Lee | Evaluation the financial feasibility of biogas upgrading to biomethane , heat , CHP and AwR[END_REF].

These drawbacks have led to regulatory changes for new biogas plants since 2012. Energy crops intake is limited (the maize-cap), FIT progressively reduced and replaced (except for small biogas plants below 150 kW) in 2017 by auctions [START_REF] Theuerl | The future agricultural biogas plant in Germany: A vision[END_REF]. The aim was to control sector development following a "deployment corridor" set at 150 MW annually from 2017-2019 and 200 MW annually from 2020-2022 and only allow construction of the most economically competitive plants that are generally the larger ones (> 1 MW) (IEA, 2020). All these shifts have led to cease the growth of the biogas sector and auctions in 2017 and 2018 did not reach the targeted power set by the German government (only 76.5 MW in 2018) [START_REF] Tsanova | Low interest, 76.5 MW of awards in 2nd German biomass tender[END_REF]. This is the proof that electricity production from biogas is suffering competition notably from solar PV [START_REF] Hill | Solar Dominates Latest German Renewable Auction[END_REF]. Even flexible biogas plants, using specific equipment (storage facilities, larger generators) that enable them to provide their electricity production offset from wind and solar production, have seen their premium FIT, initially set in 2012, gradually decline, so that today the concept of producing power from biogas flexibly is no longer economically viable [START_REF] Lauven | Estimating the revenue potential of flexible biogas plants in the power sector[END_REF]. By 2020, biogas plants commissioned in 2000 will be the first to see their guaranteed FIT expired and the question of plant shutdown will be raised if no new policy for biogas power generation is undertaken soon [START_REF] Theuerl | The future agricultural biogas plant in Germany: A vision[END_REF].

Biogas upgrading to biomethane has been identified as a good alternative to power and heat generation as it can be stored in the natural gas grid. It can then be used for clean transportation or in urban CHP units, where heat is efficiently valorized (Scarlat et al., 2018a). While the biomethane sector remains minor in Germany and has stalled at around 200 biogas plants since the deletion of the biomethane upgrading bonus in 2014, other EU state members have already based their biogas strategy on biomethane instead of power generation [START_REF] Eyl-Mazzega | Biogas and Biomethane in Europe Lessons from Denmark[END_REF]. Table 1 displays a short summary of current situations and applied biogas policies in Germany and countries that are now favoring biomethane. , 2019). This is because the sector started its expansion later than power generation, due to a lack of associated subsidies and a higher cost for upgrading units than CHP units, especially for small plants [START_REF] Kampman | Optimal use of biogas from waste streams -An assessment of the potential of biogas from digestion in the EU beyond[END_REF]. However, between 2011 and 2019 the number of plants in the EU has more than tripled going from 187 to more than 660 mainly due to the implementation of incentive policies in several state members (EBA, 2019). Historically Sweden and more recently Denmark have been leaders in the development of the biomethane sector.

Sweden has developed biomethane mainly for the transport sector via a network of filling stations as there is no comprehensive gas grid infrastructure in the country. In 2016, 64% of the 2 TWh annual biogas production were used in transportation under the form of compressed or liquefied biomethane provided to a fleet of 55,000 gas vehicles (Sverige Energigas, 2018). Instead of applying FIT, Sweden has historically supported biomethane for transportation via investment grants and exemptions from fuel taxes that make the sector competitive against gasoline and diesel [START_REF] Larsson | Upgraded biogas for transport in Sweden -Effects of policy instruments on production, infrastructure deployment and vehicle sales[END_REF]. These funding plans are running until 2020, but the Swedish government, despite the new climate policy framework that aims to achieve a 70 percent reduction in emissions from transport by 2030, is still unclear on how the support system will evolve. A proposal for the biogas production goal for 2030 was recently submitted to the Swedish government with an objective set at 10 TWh [START_REF] Westlund | Mer biogas! För ett hållbart Sverige[END_REF]. To reach this objective it is proposed a prolongation of the tax exemption system and notably the introduction of feed-in-premiums for biomethane upgrading/liquefaction. This would resume biogas sector growth in Sweden, however, at that moment, adoption of this proposal by the government remains to be done. injected) at condition that biogas sector decrease even further its cost production. All of these aspects call for both a limited growth of the sector and a necessary decrease of the biomethane production cost (Ministère de la transition écologique et solidaire, 2020).

Potential positive externalities of anaerobic digestion (Fig. 2), which are estimated to represent an additional value comprised between 55 and 75€ per MWh produced in 2030,

were not taken into account by the French government despite lobbying of biomethane sector on these points (Enea Consulting, 2019). However, these positive externalities may be considered to a greater extent in other European countries. What emerges from Sweden, Denmark, France and Italy strategies is that, as for power generation, biomethane upgrading is strongly dependent on subsidies as production cost remains high and makes it currently approximately four times more expensive than natural gas [START_REF] Eyl-Mazzega | Biogas and Biomethane in Europe Lessons from Denmark[END_REF]. A recent scenario with a moderate penetration of biomethane technology has estimated 2030 EU potential to reach 18 billion m 3 per year (~180 TWh/year), corresponding to 9 times 2017 production [START_REF] Prussi | Review of technologies for biomethane production and assessment of Eu transport share in 2030[END_REF]. Achieving such an objective for the biomethane sector will depend on the energy market, supporting initiatives from state members, full demonstration of its positive environmental impacts (notably on agriculture), higher acceptation by local population, a better recognition of positive externalities and a reduction of biogas and biomethane production costs. Identified challenges for the biogas sector are gathered in Table 2.

Improvement tracks for the anaerobic digestion biogas sector

To answer the production cost challenge as well as to demonstrate its sustainability, the biogas sector will have to improve all the parts of the industrial value chain and potentially monetize co-benefits. These improvements will be important to make AD process a key technology in the future energy system and bio-based circular economy.

Biogas sector upstream

AD can treat numerous organic materials. Sewage sludge, food waste, organic fraction of municipal solid waste, unsorted municipal solid waste, industrial waste (e.g. from food industry), animal by-products and agricultural feedstocks are the main groups of feedstocks that can be transformed into biogas. It is important to underline that the AD process is unable to convert woody biomass consisting of lignocellulose, as it is a very dry and insoluble organic matter and that lignin molecules cannot be degraded by microorganisms forming AD consortia. Gasification process would thus be preferred for such types of feedstocks.

In 2018, based on the primary energy production from biogas in the EU (EurObserv'ER, 2019), Europe biogas production share by feedstock (IEA, 2020) and average substrate mix for biogas production in the different European countries [START_REF] Bahrs | Status quo and perspectives of biogas production for energy and material utilization[END_REF], it can be estimated that energy crops are still the dominant feedstock used (around 35-38% of the net primary energy produced from the EU biogas), in front of livestock manure (33%), municipal solid waste (17%), agricultural residues as well as sequential crops (around 6-9%)

and sewage (6%) . Such shares can be mainly explained by the weight of the German biogas sector at European scale and the remaining high use of energy crops in this country (50% of total feedstocks mass in 2018) (EBA, 2019).

In the case of an optimized development scenario of the sector, by combining realistic manure biomethane potential (Scarlat et al., 2018b) 2019b). This percentage illustrates that development of biomethane from AD represents only a small fraction of the total effort required to reach carbon neutrality by 2050. In the Green Deal, increase in energy efficiency, better energy and transport infrastructure, development of renewables in the power sector, additional transformation in the gas sector (power to gas, CCS, syngas) and development of a circular economy notably for consumer goods are envisioned to be the other pillars to reach this objective (European Commission, 2019a).

Among organic materials, agricultural feedstocks, that are already dominating the biogas production (above 70% of the biogas energy share), are the largest deposit available in the future. Therefore, the market for biomethane plants using these feedstocks has the highest potential growth. As depicted in Fig. 3, it was estimated that agricultural feedstocks, in case of optimized development of the AD sector, have a potential in the EU of 62 billion m 3 biomethane produced annually by 2050, thirty times higher than potential from food waste or sludge (2.2 billion m 3 ). It is estimated that 1,200 million tons (wet weight) of livestock manure are produced every year in the EU and 860 million tons (wet weight) could be collected. From that, a biomethane potential in the EU from manure was assessed at 16 billion m 3 per year (Scarlat et al., 2018b). Crop related feedstocks have an estimated potential of 46 billion m 3 of biomethane per year by 2050. Due to their low sustainability, energy crops are envisioned to be progressively replaced by sequential crops (147 million tons in 2050 to produce 41 billion m 3 of biomethane) and crop residues (30 million tons in 2050 to produce 5 billion m 3 of biomethane) as agricultural practice should evolve concomitantly with the biogas sector growth (Gas for Climate, 2019a). Indeed, life-cycle analysis studies on GHG emissions from agricultural biogas plants, despite various methodologies and systems (feedstocks/region), have made analogous conclusions on the most sustainable practices [START_REF] Ingrao | Investigating energy and environmental issues of agro-biogas derived energy systems: A comprehensive review of Life Cycle Assessments[END_REF]. Here are some of the most important points:

(i) Plants using energy crops are producing biogas with the highest GHG emissions, notably due to their competition with feed production and the phenomenon of indirect land-use change (iLUC) that adds up the carbon footprint of the displaced food production [START_REF] Hijazi | Review of life cycle assessment for biogas production in Europe[END_REF].

(ii) Negative GHG emissions are mainly driven by avoided emissions from livestock manure or other by-products/residues that would instead decay and release GHGs into the atmosphere without delivering any energy services [START_REF] Bartoli | Coupling economic and GHG emission accounting models to evaluate the sustainability of biogas policies[END_REF].

(iii) Sequential cropping developed with the Biogasdoneright™ practice can solve energy crop issues and allow additional agricultural benefits [START_REF] Dale | Biogasdoneright TM : An innovative new system is commercialized in Italy[END_REF]. It consists, for a given agricultural land, to produce two crops within one year instead of only one. Indeed, the main crop occupies soil only a fraction of the year, leaving in conventional practice, the soil uncovered the rest of the year. In this remaining time slot, the newly cultivated and harvested crop is called sequential crop (or also catch crop/intermediate crop). Higher carbon soil sequestration due to continuous soil coverage as well as additional biomass produced and left in the field (roots and residues), soil protection against erosion and lower nutrients leaching are the main advantages of this practice [START_REF] Valli | Greenhouse gas emissions of electricity and biomethane produced using the Biogasdoneright TM system : four case studies from Italy[END_REF]. It can be added that to even further increase the sustainability of this practice, similarly to other biomass used for energy purpose, the use of fertilizer produced from petrochemical sources should be reduced as much as possible [START_REF] Sherwood | The significance of biomass in a circular economy[END_REF]. Therefore, additional good practices can be applied for this purpose such as optimized soil preparation (with minimum tillage, direct seeding on previous culture residues), finer dosing in time, space and quantity of digestate and fertilizer (precision farming, fertigation) or increase use of leguminous crops to fix additional nitrogen [START_REF] Pellerin | How can French agriculture contribute to reducing greenhouse gas emissions? Abatement potential and cost of ten technical measures -Short summary of the study report conducted by INRA[END_REF].

From these observations, upstream enhancements will be based on using local, sustainable and low-cost organic residues combined with improved logistics. Sequential crops, manure, residues from landscape management such as grass [START_REF] Bedoić | Green biomass to biogas -A study on anaerobic digestion of residue grass[END_REF], perennial crops cultivated on marginal agricultural lands [START_REF] Cossel | Prospects of bioenergy cropping systems for a more social-ecologically sound bioeconomy[END_REF], agricultural lignocellulosic residues (straw, etc.), as well as food waste are envisioned to be increasingly used (Gas for Climate, 2019a). From these feedstocks, GHG neutral to negative biogas can be produced but it also implies that anaerobic co-digestion will be increasingly used in the future.

Anaerobic digestion process

Mastering anaerobic digestion process will be necessary to reduce operational costs and maximize gas incomes. This will be possible via better management of the preparation, the co-digestion and the digestion completion steps that will allow to recover most of the methane from a wide variety of feedstocks:

(i)

The preparation step will firstly rely on an efficient storage when feedstocks are not directly anaerobically digested to preserve over time the methane potential of feedstocks [START_REF] Peyrelasse | SAM -Bonnes pratiques pour le stockage de matiere[END_REF]. This is notably based for agricultural feedstocks on optimized ensiling strategies and livestock manure storage practices. Secondly, on the application of additional adequate pretreatments (mechanical, thermal, chemical, biological) to fully unlock the methane potential of feedstocks [START_REF] Carrere | Review of feedstock pretreatment strategies for improved anaerobic digestion: From lab-scale research to full-scale application[END_REF]. It is important to highlight that adequate pretreatment will vary from a feedstock to another and there is not an optimal solution that will suit best for all feedstocks.

(ii)

The anaerobic co-digestion process has inherent positive aspects such as the use of low-cost/waste feedstocks or the potential synergies between feedstocks (e.g. adjustment of C/N ratio, inflow of nutrients and water that would be missing in some feedstocks, etc.) that can occur during AD and increase biogas recovery, but also some technological issues coming from a higher AD process complexity [START_REF] Hagos | Anaerobic co-digestion process for biogas production: Progress, challenges and perspectives[END_REF]. To handle this complexity, better and cheaper monitoring based on modelling and finer understanding of the process will be required to avoid operational issues and to develop optimal strategies to recover most of the methane from feedstocks (e.g. optimized feedstocks supply) [START_REF] Wu | Anaerobic digestion: A review on process monitoring[END_REF].

The use of chemical or biological additives to improve AD performance [START_REF] Romero-Güiza | The role of additives on anaerobic digestion: A review[END_REF] and the reduction of biogas plant operational expenditure, based for instance on the optimization of mixing strategy [START_REF] Singh | State of the art on mixing in an anaerobic digester: A review[END_REF], are two other key axes of improvement. Finally, regular leakage control surveys on digesters and the use of a flare to avoid methane discharge in some particular occasions (for instance maintenance) are required to ensure high sustainability of the AD process [START_REF] Liebetrau | Ecoefficiency assessment of farm-scaled biogas plants[END_REF].

(iii) The digestion completion step, which consists in a proper digestate management, is very important to reduce uncontrolled methane, carbon dioxide and nitrogen emissions and increase biogas plant efficiency (Lijó et al., 2017). Strategies to handle optimally solid and liquid digestates have to be determined. For instance, coverage and heating of storage tank containing liquid digestate [START_REF] Bacenetti | Agricultural anaerobic digestion plants: What LCA studies pointed out and what can be done to make them more environmentally sustainable[END_REF], further energy recovery from solid digestate via various post-treatment [START_REF] Monlau | New opportunities for agricultural digestate valorization: Current situation and perspectives[END_REF], were identified as potential good practices.

Combination of these three steps, described in Fig. 4, should lead to high methane recovery from co-digested feedstocks, increase biogas plant sustainability (reduced GHG emission from digestate) and potentially decrease biogas production costs, as long as each step remains cost-effective. Development and wider adoption of these AD process optimization strategies at full scale are expected in the future as the biogas sector is gaining in theoretical and practical know-how. Finally, the possible side recovery of biohydrogen and biochemicals from the AD process such as acids or alcohols, which have a higher added value than biogas, is a very interesting track to enhance the profitability of biogas plants [START_REF] Moscoviz | The environmental biorefinery: State-of-the-art on the production of hydrogen and value-added biomolecules in mixed-culture fermentation[END_REF]. This might be possible in the future by coupling AD to other existing technologies such as dark fermentation [START_REF] Bundhoo | Coupling dark fermentation with biochemical or bioelectrochemical systems for enhanced bio-energy production: A review[END_REF], electrochemical systems [START_REF] De Vrieze | Interfacing anaerobic digestion with (bio)electrochemical systems: Potentials and challenges[END_REF] or membrane separation and solvent extraction systems [START_REF] Aghapour Aktij | Feasibility of membrane processes for the recovery and purification of bio-based volatile fatty acids: A comprehensive review[END_REF]. Their implementation will rely on feedstock features (for instance solids content, acid concentrations), on profitable operation and on the mastery of higher processes complexity.

Currently, all these kinds of couplings are still in their early stages of development and further improvement, scale up or finer economic studies are required to fully validate their interest for large-scale applications.

Biogas sector downstream

Downstream processes are also likely to experience great changes that should provide additional revenues to the sector. The current lack of specific legal framing for digestate use is placing it into the "waste" classification, which is rendering its commercialization difficult [START_REF] Guilayn | First fertilizing-value typology of digestates: A decision-making tool for regulation[END_REF]. Adequate legislation, as well as an enhancement in digestate posttreatment (dewatering, nitrogen or phosphorus recovery, etc.), may procure additional revenues from its commercialization and/or extraction of its by-products [START_REF] Vaneeckhaute | Nutrient Recovery from Digestate: Systematic Technology Review and Product Classification[END_REF]. On this point, a decision-support guide was created that can help biogas plant operators aiming to implement N & P recovery strategies. Based on local fertilizer demands/constrains and digestate features an adequate and optimized recovery strategy can be determined [START_REF] Vaneeckhaute | Nutrient recovery from digested waste: Towards a generic roadmap for setting up an optimal treatment train[END_REF].

Concerning biogas valorization, several tracks are foresighted. The number of local CHP with no or low heat valorization should be progressively reduced as they are too strongly dependent on subsidies. Instead, large-scale biogas plants that perform upgrading to biomethane should develop, especially in countries that are highly relying on natural gas imports. For smaller or remote biogas plants without a close gas grid access, mobile upgrading and storage systems [START_REF] Gil-Carrera | Feasibility study of an off-grid biomethane mobile solution for agri-waste[END_REF] or small-scale bio-liquefied natural gas (bio-LNG) units [START_REF] Baccioli | Small scale bio-LNG plant: Comparison of different biogas upgrading techniques[END_REF] were recently identified as potential alternatives.

Biomethane obtained can be either used on-site in filling stations, similarly to Sweden, or injected into the grid. Grid connection can represent large investment costs, especially when a biogas plant is remote from the gas grid (190 € per meter of pipeline), and system for their coverage varies from a country to another (CEGIBAT, 2018). For instance, in Denmark, Italy or the UK, biomethane plant owners should entirely pay for it, while in France or Germany cost is shared with gas grid operators (REGATRACE, 2020). Once biomethane is injected, it can be stored and used at other points of the grid, thanks to guarantees of origins (also called biomethane certificates) which certify to the company/consumer buying it that the gas used from the grid corresponds to biomethane. Currently, each country has its own policy on the topic and cross-border trade are limited to some countries (for instance between Denmark and Sweden) (REGATRACE, 2020). However, European Gas package is likely to introduce a system of EU-wide certification or guarantees of origin for biomethane that would favor larger and easier cross-border trade [START_REF] Olczak | What to expect from the 2020 gas package[END_REF].

In the future, biomethane could be used in combined cold, heat and power (CCHP) units to allow cold applications [START_REF] Angenent | Integrating electrochemical, biological, physical, and thermochemical process units to expand the applicability of anaerobic digestion[END_REF], as a C1 carbon source to produce microbial proteins that could be used to feed animals and humans [START_REF] Peyrelasse | SAM -Bonnes pratiques pour le stockage de matiere[END_REF], or to produce biochemicals and fuels [START_REF] Verbeeck | Upgrading the value of anaerobic digestion via chemical production from grid injected biomethane[END_REF]. On this last aspect, a recent study has shown a promising way to produce biochemicals based on super-dry reforming (SDR). This process combines one molecule of biomethane with three CO2 molecules produced from industry to form four CO molecules that can be further transformed into chemicals such as methanol.

Additional carbon capture and its positive economics without subsidies, in the current context, make this solution particularly attractive. However, the bottleneck remains in the upscaling of the SDR process and more generally of CO2 to CO technologies [START_REF] Verbeeck | Upgrading the value of anaerobic digestion via chemical production from grid injected biomethane[END_REF]. Finally, technologies that allow higher flexibility in biomethane injection on the gas grid are emerging, such as liquefaction storage (e.g., the LiliBox system by Azola) or backfeeding installations that make gas networks bidirectional (e.g., the West Grid Synergy project conducted by GRTgaz in France). These should enable biogas plant operators, in the future, to bypass network constraints and to perceive more interest towards the optimization of the whole chain of the biogas production process in order to increase plant efficiency as well as biogas production.

Second generation upgrading technologies are likely to bring additional economic benefits to the sector in the near future. They are represented in Fig. 5 and can be defined as techniques valorizing and possibly trapping the CO2 contained in the biogas [START_REF] Villadsen | The Potential of Biogas; the Solution to Energy Storage[END_REF]. Biogas can be subject to high temperature catalytic reforming in order to produce syngas [START_REF] Chen | Energy-efficient biogas reforming process to produce syngas: The enhanced methane conversion by O2[END_REF]. From this syngas, hydrogen can be recovered [START_REF] Hajjaji | Life cycle assessment of hydrogen production from biogas reforming[END_REF] or it can subsequently be used to synthesize liquid organic molecules [START_REF] Hernández | Optimal Process Operation for Biogas Reforming to Methanol: Effects of Dry Reforming and Biogas Composition[END_REF]. Several types of biogas reforming strategies that convert CH4 and CO2 into CO and H2 have been explored: dry reforming, dual reforming (addition of water) and trireforming (addition of water and oxygen) [START_REF] Zhao | Biogas Reforming to Syngas: A Review[END_REF]. The last two processes would be preferred for subsequent synthesis of methanol or liquid fuel (for instance diesel fuel via Fischer-Tropsch process) as syngas obtained as a high H2 to CO ratio (comprised between 1.1 and 2.7). Biogas reforming pathway is still in its early stage of development and its cost requires to be further reduced working notably on the improvement of catalysis (selectivity, lifespan), reduction of process temperature, and further synergy with syngas conversion technology [START_REF] Zhao | Biogas Reforming to Syngas: A Review[END_REF]. Alternatively, biogas can be directly sent to a methanation process that uses hydrogen (green ideally) to produce additional methane. Catalytic methanation via the Sabatier reaction or biological upgrading via in-situ, ex-situ or hybrid strategies are the two existing processes [START_REF] Angelidaki | Biogas upgrading and utilization: Current status and perspectives[END_REF]. The former process has notably been running for long-term operation (> 1000 hours) with promising results (no to low catalyst deactivation and outlet gas with 88-99% of CH4) in two recent pilot studies using packed [START_REF] Dannesboe | Catalytic methanation of CO2 in biogas: Experimental results from a reactor at full scale[END_REF] or fluidized bed reactors [START_REF] Witte | Demonstrating direct methanation of real biogas in a fluidised bed reactor[END_REF]. Drivers for the development of methanation will be the improvement of process conversion efficiency and above all the price as well as the availability of hydrogen [START_REF] Thema | Power-to-Gas: Electrolysis and methanation status review[END_REF]. Profitability and industrial development of such technologies will be dependent on cost reduction of electrolysis. Thus, looking at cost development scenarios for electrolysis, positive business cases for methanation technologies might not be expected before 2030 [START_REF] Schulze | Power-To-Gas in a Decarbonized European Energy System Based on Renewable Energy Sources[END_REF]. Finally, carbon dioxide can also be recovered at the outlet of the upgrading unit, after being separated from the biomethane, and used in the methanation process but also for greenhouse or microalgae cultivation enhancement [START_REF] Theuerl | The future agricultural biogas plant in Germany: A vision[END_REF]; dry ice or chemical production (notably methanol, formic acid and longer chain hydrocarbons via catalytic hydrogenation) [START_REF] Li | A short review of recent advances in CO2 hydrogenation to hydrocarbons over heterogeneous catalysts[END_REF]; or food applications (carbonation of beverages, etc.) [START_REF] Esposito | Simultaneous production of biomethane and food grade CO2 from biogas: An industrial case study[END_REF].

Research and industrial outlooks

From this overview of the current situation in Europe and the full value chain of the biogas process, several outcomes can be perceived:

-Agricultural feedstocks will remain prominent in the future. Farming of sequential crops appears as a potential solution to conciliate food and energy production.

However, it is important that their productions remain sustainable. Thus, further development should occur on the determination of appropriate local practices (varieties to be used, sowing and harvesting dates, fertilization), the diffusion of this practical knowledge within the agricultural community and the supervision to avoid deviations in their use (WWF France, 2020). Such an approach, federating farmers, agronomists, biogas stakeholders and governments are likely to be a long-term learning movement in Europe as anaerobic digestion and sequential cropping for energy production are relatively new concepts in most countries.

-Sustainability analysis (including LCA and social LCA) should continue to develop and be performed to fully support the ecological interest of the sector and identify best practices. Use of primary data (concerning for instance biomass cultivation practice or digestate emissions), reflecting specific local context, should be favored in the future to strengthen analysis reliability and allow comparison between different biogas systems [START_REF] Ingrao | Investigating energy and environmental issues of agro-biogas derived energy systems: A comprehensive review of Life Cycle Assessments[END_REF].

-Pretreatments and additives are promising tracks to increase biogas plant energy efficiency. If well described in the literature, their impact on AD have been mainly evaluated using batch biomethane potential tests that have some limitations [START_REF] Koch | Power and Limitations of Biochemical Methane Potential (BMP) Tests[END_REF]. For instance, observed increased in methane yield under batch conditions following a thermochemical pretreatment could not be observed under a long-term trial. This is likely due to the adaptation over time of the inoculum rendering the pretreatment inefficient [START_REF] Janke | Pretreatment of filter cake for anaerobic digestion in sugarcane biorefineries: Assessment of batch versus semi-continuous experiments[END_REF]. In parallel, direct scale-up to full scale might be difficult to evaluate as many parameters can vary at such process size (e.g. ration, maintenance). Thus, development of semi-continuous pilot studies (from several hundred litters to a few dozen cubic meters), where all parameters are controlled and monitored (feedstocks, pH, temperature, agitation, gas flows and potentially other parameters depending on the experiment specificity), would be valuable to clearly evaluate the most efficient solutions to improve methane yield and degradation kinetics of feedstocks. For instance, a recent study on a 1000 L pilot digester validated positive impact found at lab scale of biochar addition (15 g/L) on food waste AD [START_REF] Zhang | Biochar enhanced thermophilic anaerobic digestion of food waste : Focusing on biochar particle size , microbial community analysis and pilot-scale application[END_REF]. Thus, average methane yield was increased by 37% following biochar addition. Besides, based on recent methodologies, environmental impact of these additional treatments should be better considered when evaluating them in the future [START_REF] Fan | Anaerobic digestion of lignocellulosic waste: Environmental impact and economic assessment[END_REF]. For a given AD system, optimal pretreatment or additive solution will be the one with the best trade-off in terms of cost and environmental performances.

-Existing and upcoming biogas and biomethane plants must be attentive to local opportunities to create additional revenue streams and territorial synergy. Thus, CO2

sinks (e.g. greenhouse, factories) or potential markets for the valorization of nutrients/biomolecules appear as potential tracks. Spreading of such strategies must go hand in hand with the improvement or upscaling of the associated technologies (e.g. CO2 upgrading, nutrient recovery, biomolecule production).

-Regarding European countries with no to small biogas sector, political incentives will be key. Growth of a resilient biogas industry will be based on: i) Determination of national and territorial specificities in terms of biomass and organic waste production, product demand (biomethane, CO2, nutrients, others) and existing infrastructure (e.g. gas grid); ii) Subsequent creation of adapted support systems at national scale that are well relayed by territorial authorities [START_REF] Bourdin | The role of a local authority as a stakeholder encouraging the development of biogas: A study on territorial intermediation[END_REF]; iii) Development of sustainable AD systems with high local acceptance (based on technical and social feedbacks from more advanced countries on the topic).

European union next steps forward, with the "gas package", may trigger sector development in these countries.

Conclusions

Current market trends show that the European AD sector is undergoing a change from a model still dominated by energy crops and local electrical production via combined heat and power units towards a model where organic wastes, agricultural by-products as well as sequential crops are mainly used as feedstocks and biogas is upgraded to biomethane for various applications (transportation, chemical production, heat, etc.). Current main challenges for the sector are: (i) to decrease biogas production costs as the golden era of high feed-in tariffs for biogas production appears to be over. Instead, observed trends in biogas policies are regulation of sector deployment and progressive reduction of subsidy schemes; (ii) to increase AD process sustainability and associated agricultural practices. To tackle these challenges, the following tracks were notably identified: (i) using cheaper and more sustainable feedstocks; (ii) improving efficiency of the AD co-digestion process and the upgrading step; (iii) developing additional sources of revenues (commercialization of digestate/nutrients/biomolecules, CO2 valorization, monetization of co-benefits, etc.); (iv) identifying and diffusing good agricultural practices concerning sequential crops cultivation and digestate management. In the coming decade, research, industries and farmers need to focus on these improvement tracks to increase biogas economic competitiveness and ensure sustainable development of the European AD sector in the long-term.

List of table captions

Figure 1 :

 1 Figure 1: Worldwide renewable electricity capacity and share of biogas sector in 2018 (IRENA, 2019)

  from biogas remains high in comparison to other types of renewable energies. Indeed, solar photovoltaics (PV) and onshore wind have known since 2010 a strong diminution in their leveled cost of electricity (LCOE) that may reach by 2020 respectively 0.06 and 0.05 USD per kWh, while biogas LCOE has remained quite constant over time close to 0.08-0.09 USD per kWh (IRENA, 2018);

Figure 2 :

 2 Figure 2: Identified positive externalities of the development of the biomethane sector (Enea Consulting, 2019)

Figure 3 :

 3 Figure 3: Share of feedstocks in an optimized scenario for biomethane production in the EU in 2050

Figure 4 :

 4 Figure 4: Main stages of an optimized anaerobic co-digestion process

Figure 5 :

 5 Figure 5: Potential pathways for second generation biogas upgrading technologies -CO2 valorization

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 1234512345 Figure 1: Worldwide renewable electricity capacity and share of biogas sector in 2018 (IRENA, 2019) Figure 2: Identified positive externalities of the development of the biomethane sector (Enea Consulting, 2019) Figure 3: Share of feedstocks in an optimized scenario for biomethane production in the EU in 2050

Table 1 : Biogas sector and applied biogas policies in selected European countries

 1 As previously given, in 2018, biomethane contributed to only 11.5% of generated energy from biogas in the EU (EurObserv'ER

  Denmark has a much more developed gas network and had mainly based its strategy on injecting biomethane into the grid that is subsequently used in town-located CHP with district heating systems. The sector has boomed between 2014 and 2018 due to the introduction of 20-year Feed-in premiums for biomethane injection. Annual biomethane capacity has bounced from almost zero in 2014 to 2.7 TWh and now accounts for more than 10% of volumes transported in the natural gas grid (Eyl-Mazzega and Mathieu, 2019).However, this has been accompanied by a massive increase in subsidies granted that reached 215 million euros in 2017. Therefore, a new subsidy scheme part of the Energy Agreement has been adopted in 2018. It will start in 2021 and is based on a yearly 32 million Nm 3 /h, will be reduced by 40% in 2028 (from a current average of 102€/MWh to an average of 60€/MWh) and even more for smaller capacity plants. A second scenario is envisioned, where injected biomethane could reach 10% in total gas demand (22 TWh

	Energy Lab, 2019). Besides, national studies from ADEME (ADEME, 2018) and the négaWatt
	association (Association Négawatt, 2017) demonstrated, in several scenarios, the future
	importance of anaerobic digestion and biomethane to reach both neutral emissions by 2050
	and a 100% renewable gas system. However, the recent adoption of the Energy Strategy
	Plan for 2030 (Programmation Pluriannuelle de l'Énergie also called PPE) has lowered in its
	main scenario the initial target set to only 7% (14 TWh injected) of biomethane in total gas

euros pool that will be assigned to new projects in tenders with price ceilings (Ministry of Climate Energy and Utilities, 2018). Danish biomethane sector growth potential will thus be limited from 2020, similarly to the "deployment corridor" in Germany.

In France, future energy policies may also challenge and contain biomethane sector development. Until now, biomethane has known a strong rise with 107 operating plants mid 2019 (+143% increase versus 2017) due to FIT introduced in 2014 (SDES, 2020). The plants are mainly connected to the gas grid and produce a yearly 1.3 TWh in 2018 (Sia Partnersdemand (instead of 10%), has introduced yearly auctions of 700 GWh and has sharply decreased FIT to be implemented in 2023 and 2028. For instance, FIT of an agricultural plant injecting 200

Table 2 : Identified challenges for the European biogas sector

 2

Table 1 : Biogas sector and applied biogas policies in selected European countries Table 2: Identified challenges for the European biogas sector List of figure captions

 1

Table 1 : Biogas sector and applied biogas policies in selected European countries

 1 [START_REF] Eyl-Mazzega | Biogas and Biomethane in Europe Lessons from Denmark[END_REF]; 2 (Danish Energy Agency, 2018); 3 (Sverige Energigas, 2018a); 4 (SDES, 2019a); 5 (SDES, 2019b); 6 (EurObserv'ER, 2019);

	Country	Germany	Denmark	Sweden	France	Italy
	Biogas sector and policies
	Main biogas valorization pathway	CHP	Upgrading	Upgrading	CHP	CHP
	Sector evolution	Stagnant sector	Sector slowdown since	Stagnant sector	Growth	Sector stagnant since FIT
			2018			reductions in 2013
	Biomethane policy	FIT deleted in 2014 -No	Premiums tariffs -	Fiscal incentives -	FIT since 2014 -	Favorable quota system
		biomethane incitation	Replaced by auctions in	Potential positive	Dynamic growth but	set in 2018. Forecasted
		scheme	2020	evolution of the	auctions/reduced FIT	dynamic growth
				support system	from 2020
	Biomethane dominant end-use	CHP with high heat	CHP with high heat	Transportation	Heat & cooling and	Transportation
		valorization	valorization		transportation
	Envisioned strategy	Uncertain	Slower extension of	Resume of upgrading	Shift from CHP toward	Shift from CHP toward
			upgrading development	development	upgrading	upgrading
	Associated figures
	Number of biogas plants in 2018	9,500 1	172 2	279 3	712 4,5	1,555 1
	Primary energy production from	88.7	3.7	2.1	10.2	21.9
	biogas (TWh/year) in 2018 6
	Evolution of the primary energy	-1.2%	+21%	-1,1%	+7%	0%
	production from biogas between
	2017 and 2018 5
	Number of biomethane plants in	213 7	25 7	69 3	76 4,5	1 1
	2018
	Injected biomethane (TWh) in 2018	10.2 7	2.7 1	1.3 7	1.2 5	0.3 1
	Percentage of total biogas energy	11.5%	73%	63%	11.8%	1.5%
	upgraded in 2018

1 7

[START_REF] Regatrace | Mapping the state of play of renewable gases in Europe[END_REF]

Acknowledgment

National Research and Technology Association (ANRT) is gratefully acknowledged for the PhD grant allocated to Ulysse Brémond (reference CIFRE N° 2016/0617).

Declaration of interest

Declarations of interest: none


Tran Duc

Anh Ly

Van Thuan Hoang

Meriem Louni

Thi Loi Dao

Sekene Badiaga

Herve Tissot-Dupont

Philippe Brouqui

Philippe Colson

Dr Philippe Gautret
email: philippe.gautret@club-internet.fr

Epidemiological serosurvey and molecular characterisation of sexually transmitted infections among 1,890 sheltered homeless people in Marseille: cross-sectional one day-surveys (2000-2015)

Keywords: Homeless, hepatitis, HBV, HCV, Treponema pallidum, syphilis

Objectives. We observed the prevalence and distribution of potential risk factors for sexually transmitted infections (STIs) among Marseille homeless population.

Methods

. Over the 2000-2015 period, we enrolled 1890 sheltered homeless adults and collected serum samples. Markers of hepatitis B and C viruses (HBV, HCV) and Treponema pallidum were searched using the CMIA testing. Positive HBsAg or anti-HCV samples underwent sequencing; positive anti-T. pallidum sera were subjected to the RPR test.

Results. The overall prevalence of HBsAg, anti-HBs, anti-HBc, anti-HCV and anti-T. pallidum (by CMIA and RPR) was 4.1 %, 22.9%, 35.5%, 5.3% and (6.8%, 1.0%), respectively. We found a significantly higher prevalence of HBsAg and anti-T. pallidum among individuals born in sub-Saharan Africa (or Asia) compared to those born in Europe.

Being older (>42 years), toxicomania status, cannabis use and underweight status (compared to normal status) were independent factors associated with HCV seropositivity. Using sequencing, we obtained a substantial diversity of HBV and HCV genotypes. One HCV sequence harbouring a L31M substitution in the NS5a protein may be associated with reduced drug sensitivity.

Conclusions.

The positive relationship between toxicomania and HCV suggests the need for effective prevention programmes including health education activities and addiction treatment.

Introduction

Homelessness is an increasing social and public health concern in both developing and developed countries. Given their lack of health insurance coverage leading to a limited access to healthcare, homeless people are particularly exposed to infectious diseases which are more severe than in the general population [START_REF]Homelessness, Health, and Human Needs[END_REF] . In addition, homeless people are disproportionately affected by substance and alcohol abuse and the consequences of this on their health negatively impacts their general well-being and mental health [START_REF]Homelessness, Health, and Human Needs[END_REF] .

As blood-and sexually-transmitted pathogens, the hepatitis B virus (HBV), hepatitis C virus (HCV), and Treponema pallidum can be diffused via blood transfusion, vertical transmission and sexual practice [START_REF] Cao | Prevalence of hepatitis B virus, hepatitis C virus, human immunodeficiency virus and Treponema pallidum infections in hospitalized patients before transfusion in Xiangya hospital Central South University, China from 2011 to 2016[END_REF] . Homeless people are at high risk of these diseases as the result of injected drug use, unprotected sexual activity and financial and nutritional poverty [START_REF] Vahdani | Prevalence of HBV, HCV, HIV and syphilis among homeless subjects older than fifteen years in Tehran[END_REF][START_REF] Robertson | Health status and access to health services among the urban homeless[END_REF] .

Globally, epidemiological surveys carried out among homeless populations have shown a prevalence of 2.2%-36.2% for HCV infection [START_REF] Beijer | Prevalence of tuberculosis, hepatitis C virus, and HIV in homeless people: a systematic review and meta-analysis[END_REF][START_REF] Cardona-Arias | Prevalence of hepatitis B/C viruses and associated factors in key groups attending a health services institution in Colombia, 2019[END_REF] . The prevalence of past HBV infection (positive anti-HBc antibodies) ranged from 10.4% to 80.3% and that of ongoing infection (positive HBsAg) ranged from 0.4% to 4.7% [START_REF] Cardona-Arias | Prevalence of hepatitis B/C viruses and associated factors in key groups attending a health services institution in Colombia, 2019[END_REF][START_REF] Ly | Vaccine-preventable diseases other than tuberculosis, and homelessness: A systematic review of the published literature, 1980 to 2020[END_REF] . Syphilis was observed in approximately 0.5% of homeless people in Iran (2012), in 1.1% in England (2004), in 7% in Brazil (2000-2011) [START_REF] Jahanbakhsh | Prevalence of HAV Ab, HEV (IgG), HSV2 IgG, and Syphilis Among Sheltered Homeless Adults in Tehran, 2012[END_REF][START_REF] Grimley | Sexually transmitted infections among urban shelter clients[END_REF] , and in 33.2% of Brazilian female prisoners with a history of homelessness (2014-2015) [START_REF] Da | Lifetime Syphilis Prevalence and Associated Risk Factors Among Female Prisoners in Brazil[END_REF] . Human immunodeficiency virus (HIV) infection is frequent in the homeless population and has been extensively studied [START_REF] Beijer | Prevalence of tuberculosis, hepatitis C virus, and HIV in homeless people: a systematic review and meta-analysis[END_REF] .

Homelessness affects more than 1,500 persons on any given night in Marseille, including 800 who sleep on the city"s streets and approximately 600 who are living temporarily in municipal shelters, which have a high turnover [START_REF]Investigation of endemic and emerging diseases in populations of homeless households in Marseille[END_REF] . Medical care of sheltered homeless people, who are a highly mobile population and mostly of migrant origin, often remains fragmented across multiple inpatient and outpatient settings. The screening and treatment of infectious diseases, notably due to sexually-transmitted pathogens (STPs), in the homeless population is thus necessary. The purpose of the current study was, therefore, to investigate the seroprevalence of several STPs (other than HIV infection) and their risk factors among homeless people living within shelters in Marseille, France, between 2000 and 2015.

Materials and methods

Ethics

The Marseille Institutional Review Board-Ethics Committee approved and reviewed the design of this retrospective study (Protocol: CCPCRB 99-76 [period 2000-2009] and 2010-A01406-33 [after 2010]). Eligible participants were confidentially interviewed and gave their informed consent. Data were anonymised and de-identified prior to analysis.

Patient and sample design

In our cross-sectional one-day surveys, adult homeless people who presented at two shelters (A and B) in Marseille, France, were recruited on a voluntary basis during the winter in 2000, 2001, 2002, 2003, 2005, 2006, 2010, 2011, 2013, and 2015. The inclusion criteria were being 18 or over and being willing to participate. Participants were asked to complete a specially designed questionnaire to provide information on demographics and substance abuse, and they were physically examined by medical doctors. Trained medical personal collected a blood sample for serological testing from each patient. Those who tested positive for these infections were counselled about their infections and referred to primary care.

Serological assays

A 5 ml blood sample was drawn from each subject using a sterile vacutainer tube (Becton Dickinson, Rutherford, NJ, USA) and the serum was separated using centrifugation (at 2900 g for 15 minutes). All sera were screened for the presence of antibodies against HBV, HCV and syphilis. The fully automated chemiluminescent microparticle immunoassay (CMIA, Abbott, Diagnostics Division, Wiesbaden, Germany) was used to detect the following serological markers: hepatitis B surface antigen (HBsAg), hepatitis B surface antibodies (anti-HBs), antihepatitis B core total antibodies (anti-HBc), antibodies to hepatitis C virus (anti-HCV), and antibodies to T. pallidum (anti-T. pallidum), according to the manufacturer"s instructions. The criteria for seropositivity and seronegativity for each marker are detailed in Supplementary Table 1. Samples which were positive for HBsAg underwent additional testing for HBsAg positivity using a neutralisation assay (Abbott, Diagnostics Division) according to the manufacturer"s guidance. Depending on the results of different combinations of HBV serological markers, patients were defined as: (i) chronically or acutely infected, (ii) susceptible (absence of immunity), (iii) immune due to natural infection, (iv) immune due to hepatitis B vaccination, or (v) presenting a past resolved infection (Supplementary Table 2).

For HCV, patients testing positive for anti-HCV antibodies were considered as having been infected during their lifetime. Subsequently, samples showing seropositivity for HBsAg and anti-HCV antibodies were investigated using molecular assays to test for replication and ongoing infection, respectively. For T. pallidum, samples which tested positive using CMIA were further tested using the rapid plasma regain test (RPR) (RPR Latex Test Kit, Diagnostic Automation/Cortez Diagnostics, Inc., USA) according to the manufacturer"s instructions.

DNA and RNA extraction

Total DNA and RNA were automatically extracted from 200 μL of serum using the EZ1 Advanced XL (Qiagen, Hilden, Germany) with the Virus Mini Kit v2.0 (Qiagen) according to the manufacturer"s recommendations. The obtained extracts were stored at -80°C.

Conventional PCR amplification and DNA sequencing

We characterised the full length HBsAg and reverse transcriptase (HBsAg/RT) genes (for HBV) and large fragments of NS4B and NS5A genes (for HCV). Only samples with seropositive results for HBV and/or HCV were further tested using standard PCR (as described in Supplementary Data). The purified PCR products were sequenced using specific primers and the BigDye Terminator® version 1.1 cycle sequencing ready reaction mix (Applied Biosystems, Foster City, CA). All primers and in-house protocols used in this study have previously been described (as shown in Supplementary Table 3). The sequencing reactions were purified with SephadexG-50 Superfine on MAHVN 45-50 plates (Millipore, Molsheim, France) and then sequenced on the Applied Biosystems 3130 platform (ABI PRISM, PE Applied Biosystems, USA). For each pathogen, the sequences obtained were edited and assembled using Chromas Pro1.7.7 software (Technelysium Pty Ltd, Australia). HBV (or HCV) genotypes were determined by phylogenetic analysis using a set of reference HBV (or HCV) sequences available from the NCBI GenBank or from our institute"s local HBV (or HCV) sequence bank of the population of Marseille and the Max-Planck-Institut für Informatik [START_REF]Max planck institut informatik[END_REF] sequence databases, using the Mega 7.0 software (https://www.megasoftware.net). In these websites, the nucleotide sequences obtained were simultaneously translated into amino acid (aa) sequences then compared with reference HBV (or HCV) sequences of the same genotype in order to determinate aa substitutions previously described in association with reduced antigenicity or susceptibility to anti-HBV (or HCV) drugs. These sequences are available in GenBank (GenBank accession nos. MK840522 to MK840537 [for HBV] and MK816407 to

MK816412 [for HCV]).

Statistical analysis

Statistical procedures were performed using STATA 11.1 software (StataCorp LLC, USA).

Statistical differences in baseline characteristics were evaluated by Pearson"s chi-square or

Fisher"s exact tests as categorical variables. A two-tailed p-value <0.05 was considered as statistically significant. Three separate models (HBV, HCV and T. Pallidum) were created. A separate multivariate logistic regression analysis was used to identify independent risk factors for sero-prevalence of each pathogen. Only variables with a prevalence ≥5.0% by descriptive analysis were used to examine associations between multiple factors and sero-prevalence of each pathogen. The results were presented by percentages and odd ratio (OR) with 95% CI estimations. The initial model included variables presenting a p-value <0.2. The stepwise regression procedure and likelihood-ratio tests were applied to determine the final model.

Results

Participant characteristics (Table 1)

The demographic characteristics of the study sample are presented in Table 1. The study included a total of 1,890 individuals who completed questionnaires; of them, 1,718 (90.9%) provided blood samples. There were 1,779 (95.2%) men and 90 (4.8%) women with a mean age (± standard deviation [SD]) of 43.1 (±16.4) years. Homeless people were mainly born in North Africa (n=873, 46.5%), Europe (n=853, 45.5%), and sub-Saharan Africa (n=115, 6.1%). The average duration of homelessness (±SD) was 3.9 (±6.9) years and about 45% had experienced homelessness for more than one year (defined as chronic homelessness). For migrants, their mean duration of residence in France (±SD) was 13 (±16.7) years. Frequent alcohol abuse and tobacco smoking was reported by 38% and 78% of individuals, respectively. Current cannabis use was reported by 115 individuals (of the 610 who were asked, 18.9%). Drug addiction status, was uncommon, being reported by 47 individuals (6.1%). A history of blood transfusion was reported by 102 individuals (of the 756 who were asked, 13.5%); of those, 13 participants (1.7%) had received a transfusion before 1992. About 63.5% of patients showed normal weight, 75 (6.4%) were underweight, 278 (23.8%) were overweight and 74 (6.3%) were obese.

Serological patterns and risk factor analysis: multivariate models (Table 1-3 &

Supplementary Figure 12

)

The overall prevalence of HBsAg, anti-HBs, anti-HBc, anti-HCV, and anti-T. pallidum during the study period was 4.1% (54/1310), 22.9% (301/1312), 35.5% (379/1059), 5.3% (90/1601) and 6.8% (106/1564) via CMIA testing, respectively. Of the 1053 samples tested for all three HBV markers, more than half showed an absence of immunity against HBV. Only 69 individuals (6.5%) had acquired immunity through vaccination, whereas 19 (17.9%) had immunity due to natural HBV infection, 189 (17.8%) had a past resolved infection, and 54 (4.1%) had an ongoing (chronic or acute) infection. Of the 106 patients considered as seropositive for anti-T. pallidum via CMIA testing, 15 (of 1564, 1%) tested positive via RPR testing. The most frequent dual infection was observed with T. pallidum and HCV in seven cases (0.5%).

The sero-prevalence of these pathogens did not significantly vary over time. In multivariate analysis, being born in sub-Saharan Africa (or Asia) continued to be associated with higher HBsAg and anti-T. pallidum seropositivity rates when compared to those born in Europe. Being over the age of 42, addicted to drugs, smoking cannabis and being underweight remained associated with anti-HCV seropositivity.

Genotypic patterns of HBV (Figure 1 & Supplementary Table 4)

HBV genotyping was performed on 44 available sera from 44 HBsAg-positive subjects. We succeeded in amplifying the gene from 16 subjects (36%). The phylogenetic tree showed that HBV sequences of our homeless people clustered with genotype A (HBV-A), D (HBV-D) or E (HBV-E) reference sequences. Thus, we detected the HBV-A in 2 cases (12.5%; A2=2), HBV-D in 9 cases (56.2%; D1=3, D2=1, D3=1, D4=2, D7=2), and HBV-E in 5 cases (31.2%). Overall, the mean (±SD, range) nucleotide identity between our 16 HBV sequences characterised and their best match available in the GenBank and in our local sequence database was 98.7% (±0.8%, range 96.8%-99.7%) and 97.1 (±1.0%, range 95.8%-98.7%), respectively. The HBV sequences recovered from homeless people were scattered across the phylogenetic tree, and were neither clustered between each other (with one exception) nor more frequently clustered with locally-acquired sequences compared to GenBank sequences.

The deduced HBsAg aa sequences showed that aa substitution T127I/P was most frequently encountered, being present in nine sequences (of 16, 56.2%) when compared to reference sequences. Patient SDF097 had 10 substitutions (of 226 HBsAg aa, 4.4%). Only one patient showed a substitution (D144E) known to confer altered antigenicity of the HBsAg protein.

However, no substitution was found that is described to decrease susceptibility to HBV antiviral drugs.

Genotypic patterns of HCV (Figure 2 & Supplementary Table 5)

HCV RNA could be amplified from six of the 30 (20%) sera available from 30 anti-HCVpositive subjects. Two of six (33.3%) HCV sequences belonged to subtype 1a (HCV-1a), two (33.3%) to subtype 1b (HCV-1b), one (16.6%) to subtype 3a (HCV-3a), and one (16.6%) to subtype 4d (HCV-4d). Overall, the mean (±SD, range) nucleotide identity between the six HCV sequences characterised in this study and their best match available in the GenBank and in our local sequence database was 96.2% (±1.5%, range: 93.6%-97.8%) or 94.2% (±1.9%, range: 92.3%-96.7%), respectively. The HCV sequences recovered from homeless people were scattered across the phylogenetic tree and were neither clustered close to one another nor more frequently clustered with locally acquired sequences than with GenBank sequences.

As regards specific aa substitutions, the L31M mutation that was previously described to be associated with reduced susceptibility to Daclatasvir, Elbasvir or Ledipasvir drugs was present in one patient (SDF108). This patient received a blood transfusion in 1975 and reported no history of HCV infection at the time of recruitment.

Discussion

Our study documented the prevalence of sexually transmitted infections (STIs) among sheltered homeless adults in Marseille du the period 2000-2015 and their risk factors. The key strengths of this study are its long duration and large population. We found a high prevalence of past or current HBV infection (39.8%). The overall HBsAg prevalence was 4.1% in the homeless population studied, which is higher than the 1.1% prevalence in the general local population over the same period [START_REF] Cire Provence-Alpes-Cote | Bulletin de veille sanitaire[END_REF] . We found that originating from Asia or sub-Saharan Africa was an independent risk factor for HBsAg positivity. However, positivity rates were still high in those originating from North Africa (3.2%) and Europe (4.0%). Such a high rate of active infection may be due to the low proportion of homeless people immunised through vaccination (6.5%), compared to 40% of the general French population in 2000 [START_REF]The Global Advisory Committee on Vaccine Safety rejects association between Hepatitis B vaccination and multiple sclerosis (MS)[END_REF] . High rates of HBsAg positivity have been reported among homeless people in the New Haven, USA (4.7%), while lower rates were observed in Iran (1.0%-2.6%), Canada (1.6%), Brazil (0.6%-3.3%), Colombia (0.4%) [START_REF] Cire Provence-Alpes-Cote | Bulletin de veille sanitaire[END_REF][START_REF] Raoult | Infections in the homeless[END_REF][START_REF] Altice | Adherence to hepatitis B virus vaccination at syringe exchange sites[END_REF][START_REF] Doosti-Irani | Prevalence of HIV, HBV, and HCV and Related Risk Factors amongst Male Homeless People in Lorestan Province, the West of Iran[END_REF][START_REF] Carvalho | Prevalence, risk factors and hepatitis B immunization: helping fill the gap on hepatitis B epidemiology among homeless people, Goiânia, Central Brazil[END_REF] .

The seroprevalence rate of HCV infection in Marseille homeless people (5.3%) was higher than that of the general local population whose anti-HCV antibodies prevalence decreased from 1.3% in 2004 [19] to 0.9% in 2015 [START_REF] Cire Provence-Alpes-Cote | Bulletin de veille sanitaire[END_REF] . High rates of positive anti-HCV antibodies were observed in many other homeless populations ranging from 2.2% to 36.2% [START_REF] Beijer | Prevalence of tuberculosis, hepatitis C virus, and HIV in homeless people: a systematic review and meta-analysis[END_REF][START_REF] Cardona-Arias | Prevalence of hepatitis B/C viruses and associated factors in key groups attending a health services institution in Colombia, 2019[END_REF] with the highest rate being reported in Dublin, Ireland, in 2005. The strongest independent determinant for anti-HCV seropositivity in Marseille homeless population was addiction to injected and inhaled drugs or the use of drug substitutes, followed by being over the age of 42 years, consuming cannabis and being underweight. An association between injected and noninjected drug use and HCV infection has been already reported in homeless populations [START_REF] Ferreira | Exposure to hepatitis C virus in homeless men in Central Brazil: a cross-sectional study[END_REF] . By contrast, there was no association between cannabis consumption and HCV infection in these previous reports [START_REF] Ferreira | Exposure to hepatitis C virus in homeless men in Central Brazil: a cross-sectional study[END_REF][START_REF] Fuster | Community Screening, Identification, and Referral to Primary Care, for Hepatitis C, B, and HIV Among Homeless Persons in Los Angeles[END_REF] , although such an association has been reported in the general population [START_REF] Alter | The prevalence of hepatitis C virus infection in the United States, 1988 through 1994[END_REF][START_REF] Scheinmann | Non-injection drug use and hepatitis C virus: a systematic review[END_REF] . Although higher than in the overall French population [START_REF]Observatoire Français des Drogue et des Toxicomanies[END_REF] , the proportion of homeless individuals reporting drug injection in our survey (2.1%) was lower that reported among other homeless populations [START_REF] Buresh | Recent fentanyl use among people who inject drugs: Results from a rapid assessment in Baltimore, Maryland[END_REF][START_REF] Akiyama | Low Hepatitis C Reinfection Following Direct-Acting Antiviral Therapy Among People Who Inject Drugs on Opioid Agonist Therapy[END_REF] .

We found a high prevalence of previous exposure to T. pallidum (6.8%) including recent or ongoing syphilis infection (1.0%) in Marseille homeless population. No data on the seroprevalence of syphilis is available regarding the general French population; however, a 35% increase in early syphilis cases was observed in 2014-2015 with the vast majority presenting among men who have sex with men [START_REF] Ngangro | Bacterial sexually transmitted infections in France: recent trends and patients" characteristics in 2016[END_REF] . High rates of syphilis infections, ranging from 0.3 to 33.2%, were reported in homeless populations from Brazil, Iran and England [START_REF] Jahanbakhsh | Prevalence of HAV Ab, HEV (IgG), HSV2 IgG, and Syphilis Among Sheltered Homeless Adults in Tehran, 2012[END_REF][START_REF] Grimley | Sexually transmitted infections among urban shelter clients[END_REF][START_REF] Da | Lifetime Syphilis Prevalence and Associated Risk Factors Among Female Prisoners in Brazil[END_REF] . Similar to HBV infection, we identified Asian and sub-Saharan African origins as being independent risk factors for syphilis infection.

The strength of our study was that it confirmed active infection through detection and genotyping of HBV and HCV nucleotides. Identifying the sub-genotype and predictive aa mutations associated with drug resistance is of critical importance as these mutations are associated with transmission, response to therapy and treatment outcome. We showed a high genetic diversity of HBV but a low congruence between the origin of homeless individuals and that of the sequences most similar to that of their viruses (seen in one case of a Tunisian individual, the sequence was from the same origin). The data on HBV genotypes are in agreement with previous studies conducted in several African countries, showing that HBV-E is the most prevalent serotype circulating in western sub-Saharan Africa including Senegal, Burkina Faso, Nigeria [START_REF] Brah | Molecular characterization of hepatitis B virus from chronically-infected patients in Niamey, Niger[END_REF] . The HBV-D (sub-genotype D1-D4) is prevalent worldwide and is predominant in Mediterranean area and in the Middle East [START_REF] Norder | Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HBsAg subtypes[END_REF] , and HBV-A is spread equally across Europe and Africa. For HCV-1, a genotype known to be the most common in all anti-HCV infections and to be spread globally [START_REF] Sy | Epidemiology of hepatitis C virus (HCV) infection[END_REF][START_REF] Gower | Prevalence of serological blood-borne pathogens Abbreviations: CMIA; chemiluminescent microparticle immunoassay; HBsAg, hepatitis B surface antigen, anti-HBs, antibody to hepatitis B surface antigen; anti-HBc, anti-hepatitis B core total antibodies; antibodies to hepatitis C virus; anti-T. pallidum, antibody to Treponema pallidum. Blood-borne pathogens N (%) HBV (CMIA test) HBsAg 54/1310 (4.1) Anti-HBs 301/1312 (22.9) Anti-HBc 379/1059 (35.5) Combinations of HBV serologic markers Absence of immunity (HBsAg [neg], anti-HBc [neg], anti-HBs [neg]) 557/1059 (52.6Immune due to vaccination (HBsAg [neg], anti-HBc [neg], anti-HBs [pos]) 69/1059 (6.5) Immune due to natural infection (HBsAg [neg], anti-HBc [pos], anti-HBs [pos]) 190/1059 (17.9) Recently resolved infection or inactive chronic infection (HBsAg [neg], anti-HBc [pos], anti-HBs [neg]) 189/1059 (17.8) Chronically or acutely infected (HBsAg [pos], anti-HBc [pos], anti-HBs [neg],) 54/1310 (4.1) HCV (CMIA test) 90/1601 (5.3) Treponema pallidum CMIA test[END_REF] accounted for four of our six sequences. HCV-3, predominately described in Asia, Pakistan and India, and HCV-4 which was principally reported in North Africa and the Middle East [START_REF] Gower | Prevalence of serological blood-borne pathogens Abbreviations: CMIA; chemiluminescent microparticle immunoassay; HBsAg, hepatitis B surface antigen, anti-HBs, antibody to hepatitis B surface antigen; anti-HBc, anti-hepatitis B core total antibodies; antibodies to hepatitis C virus; anti-T. pallidum, antibody to Treponema pallidum. Blood-borne pathogens N (%) HBV (CMIA test) HBsAg 54/1310 (4.1) Anti-HBs 301/1312 (22.9) Anti-HBc 379/1059 (35.5) Combinations of HBV serologic markers Absence of immunity (HBsAg [neg], anti-HBc [neg], anti-HBs [neg]) 557/1059 (52.6Immune due to vaccination (HBsAg [neg], anti-HBc [neg], anti-HBs [pos]) 69/1059 (6.5) Immune due to natural infection (HBsAg [neg], anti-HBc [pos], anti-HBs [pos]) 190/1059 (17.9) Recently resolved infection or inactive chronic infection (HBsAg [neg], anti-HBc [pos], anti-HBs [neg]) 189/1059 (17.8) Chronically or acutely infected (HBsAg [pos], anti-HBc [pos], anti-HBs [neg],) 54/1310 (4.1) HCV (CMIA test) 90/1601 (5.3) Treponema pallidum CMIA test[END_REF] were identified in two of our patients. A match between the origin of homeless individuals and that of the most similar sequences was found in five of six people (exception for sequence Seq5).

The only L31M substitution found in one HCV reverse transcriptase sequence in this study has been shown to confer resistance to the anti-HCV drug Elbasvir, Ledipasvir and to reduced susceptibility to Daclatasvir, which is widely available in many countries such as the USA, Japan and Tunisia [START_REF]Max planck institut informatik[END_REF] . Such resistance was demonstrated in treatment-naïve patients infected with HCV-1b, the sequence of which was close to our result.

Our study has some limitations. The bias or measurement errors can be subjectively created because of self-reported data. In questionnaires, no question addressed sexually risky behaviours.

Notwithstanding these limitations, in this paper we summarise our surveillance work over fifteen years. We evidenced a high prevalence of sexually transmitted infections in homeless people that remained relatively constant over the time period of the study. Findings such as this argue the case for systematic screening among this at-risk population and effective prevention programmes including health education activities, the provision of condoms and vaccination against HBV infection where appropriate. 2 Median of the variable is used for analysis. [START_REF] Vahdani | Prevalence of HBV, HCV, HIV and syphilis among homeless subjects older than fifteen years in Tehran[END_REF] Low prevalence was not entered into multivariate model [START_REF] Robertson | Health status and access to health services among the urban homeless[END_REF] Based on the WHO classification, "underweight" is defined as having a body mass index (BMI) below 18.5, "normal" corresponds to a BMI between 18.5 and 25, "overweight" corresponds to a BMI ≥25, and "obese" refers to those with a BMI ≥30. Source: WHO. Abbreviations: aOR: adjusted odds ratio; CI: confidence interval; HBsAg: hepatitis B surface antigens, anti-HCV, antibodies to hepatitis C virus, anti-T. pallidum, antibodies to Treponema pallidum.

Figure 1 .Figure 2 .

 12 Figure 1. Maximum-likelihood phylogenetic tree highlighting the position of the hepatitis B virus (HBV) identified in body lice of homeless people in Marseille ( and in black text highlight colour) compared to other HBV available in the GenBank database ( ) or local reference sequences in Marseille general population ( ), and based on partial nt126-nt1213 polymerase (P) gene with HBV clone 13 (Gen Bank accession no. NKY881832.1) used as outgroup. Note: Isolate name/country of origin/Gen Bank accession number Sequence and patient code/ country of origin/Gen Bank accession number Sequence code in local sequence bank

Table 1 .

 1 Univariate analysis of seropositivity result of each blood borne disease as a dependent variable.

	Parameters	Total N (%)	n(%)	HBsAg positivity OR [95%CI], p-value	n(%)	Anti-HCV seropositivity OR [95%CI], p-value	Anti-Treponema pallidum seropositivity n(%) OR [95%CI], p-value
	Total	1890	54 (4.1)		90 (5.3)		106 (6.8)
	Year of recruitment (1890) 1			N/A			N/A		N/A
	2000	261 (13.8) 9 (3.5)		22(8.8)		(6.9)
	2001	165 (8.73)	N/A		7 (4.6)		(7.9)
	2002	291 (15.4) 12 (5.0)		10 (3.5)		(3.8)
	2003	198 (10.5)	N/A		9 (4.6)		(6.1)
	2005	213 (10.3) 8 (4.0)		11 (5.5)		6 (3.2)
	2006	147 (7.8)	1 (0.8)		8 (6.4)		N/A
	2010	149 (7.9) 10 (12.2)		4 (5.0)		(12.0)
	2011	162 (8.5)	4 (2.9)		9 (6.5)		26 (18.9)
	2013	182 (9.6)	7 (4.5)		6 (3.8)		5 (3.2)
	2015	122 (6.5)	3 (3.2)		4 (4.2)		6 (6.4)
	Shelter (1890)
	B	816 (43.2) 25 (4.6)	REF	45 (6.0)	REF	(6.0)	REF
	A	1074 (56.8) 29 (3.8)	0.83 [0.48-1.44], 0.5	45 (4.9)	0.78 [0.51-1.20], 0.26	(7.4)	1.24 [0.83-1.87], 0.28
	Genre (1869)			N/A			N/A		N/A
	Female	90 (4.8)	8 (12.9)		3 (4.0)		(11.7)
	Male	1779 (95.2) 46 (3.7)		87 (5.4)		(6.6)
	Age (1869)
	Mean age (±SD) (years)	43.1±14.6
	Age range (years) 2	18-86
	≤42 years of age	959 (51.3) 33 (4.7)	REF	41 (4.7)	REF	(4.2)	REF
	>42 years of age	910 (48.7) 21 (3.5)	0.73 [0.42-1.27], 0.27	49 (6.1)	1.32 [0.86-2.01], 0.19	(9.7)	2.47 [1.62-3.8], <0.001
	Birthplace (1877)
	Europe	873 (46.5) 23 (4.0)	REF	53 (6.7)	REF	(4.4)	REF
	North-Africa	853 (45.5) 10 (3.2) 0.79 [0.43-1.47], 0.47	28 (3.7)	0.53 [0.33-0.86], 0.01	(8.0)	1.91 [1.22-3.00], 0.004
	Sub-Saharan Africa	115 (6.1)	9 (9.0)	2.4 [1.04-5.55], 0.04	6 (5.9)	0.87 [0.37-2.08], 0.76	10 (11.2)	2.77 [1.31-5.7], 0.007
	Asia	32 (1.7)	4 (17.4) 5.1 [1.61-16.3], 0.006	2 (7.2)	1.07 [0.24-4.65], 0.92	(25.3)	7.67 [3.0-19.5], <0.001
	America	4 (0.2)	0	N/A	0		N/A	1 (25)	N/A
	Mean duration of residence in France for	12.9 (0-9.5)						N/A

5.41 [2.29-12.75],

						p<0.001	9 (9.2)	0.91 [0.42-1.97], 0.8
	Drug addiction (771)
	Never	724 (93.9) 27 (5.0)	REF	25 (4.3)	REF	46 (8.1)	REF
	Yes	47 (6.1)	1 (3.2)	0.69 [0.09-5.27], 0.72	10 (26.3)	8.03 [3.51-18.33], p<0.001	2 (5.3)	0.63 [0.15-2.70], 0.53
	Injecting illicit substances	16 (2.1)	0	N/A	4 (36.4)	N/A	2 (18.1)	N/A
	Snorting illicit substances	27 (3.5)	0	N/A	6 (27.3)	N/A	1 (4.5)	N/A
	Using opioid	22 (2.9)	1 (7.1)	N/A	8 (44.5)	N/A	1 (7.3)	N/A

Abbreviations: SD, standard deviation; BMI, Body mass index; N/A, not applicable; REF, Reference category.

[START_REF]Homelessness, Health, and Human Needs[END_REF]

Number of individuals for whom data was available.

  Body mass index -BMI http://www.euro.who.int/en/health-topics/disease-prevention/nutrition/ahealthy-lifestyle/body-mass-index-bmi; (2019). Bold lines indicate the variables recruited in initial multivariate model.

	agonist treatment
	Blood transfusion (Yes versus No) (102)	102 (13.5) 7 (9.0)	2.2 [0.9-5.34], 0.08	7(9.0)	2.1 [0.88-5.07], 0.1	8 (10.7)	1.51 [0.68-3.39], 0.3
	Before 1992	13 (1.7)	2 (20.0) 5.1 [1.05-25.4], 0.044 3	3(30)	8.84 [2.17-36.04], 0.002 3	1(10.0)	N/A
	In and after 1992	89 (11.8)	5(5.6)	REF	4(4.5)	REF	7 (7.9)	N/A
	BMI (1170) 4
	Mean BMI (SD) (kg/m2)	23.5±4.0
	Range of BMI (kg/m2)	(13.5-50)
	Normal weight	743 (63.5) 28 (5.2)	REF	35 (5.3)	REF	34 (5.3)	REF
	Underweight	75 (6.4)	1 (1.8)	0.32 [0.04-2.42], 0.27	6 (8.8)	1.74 [0.71-4.31], 0.23	4 (6.1)	1.17 [0.40-3.38], 0.78
	Overweight	278 (23.8) 8 (4.0)	0.75 [0.33-1.68], 0.49	2 (0.8)	0.15 [0.03-0.64], 0.01	25 (10.6)	2.12 [1.23-3.6], 0.006
	Obesity	74 (6.3)	2 (4.0)	0.75 [0.17-3.25], 0.7	0	N/A	1 (1.6)	0.30 [0.04-2.19], 0.23

Table 2 . Parameter estimates for the final weighted model for HBsAg, anti-HCV, and anti-T. pallidum antibodies

 2

Acknowledgments

We are grateful to our colleagues for their technical assistance.

Funding

This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the French National Research Agency under the "Investissements d"avenir" programme [ANR-10-IAHU-03], the Région Provence Alpes Côte d"Azur and European FEDER PRIMI funding.

Potential conflicts of interest

No reported conflicts of interest.

Author Contributions Statement

TD, PC and PG contributed to experimental design, data analysis, statistics, interpretation and writing. VT, TL, ML, SB, HTD, PB administered questionnaires, examined patients and collected samples. VT, ML provided technical assistance. PC contributed to critically reviewing the manuscript. PG coordinated the work.