Raja-Sekhar Guthi
email: raja-sekhar.guthi@inrae.fr

Katharina Tondera

Sylvie Gillot

Pierre Buffière

Mathieu Boillot

Florent Chazarenc

A-Stage processchallenges and drawbacks from lab to full scale studies: a review

In response to the growing global resource scarcity, wastewater is increasingly seen as a valuable resource to recover and valorise for the benefit of the society rather than another waste that needs treatment before disposal. Conventional wastewater treatment plants (WWTPs) oxidises most of the organic matter present in wastewater, instead of recovering it as a feedstock for biomaterials or to produce energy in the form of biogas. In contrast, an A-Stage is capable of producing a concentrated stream of organic matter ready for valorisation, ideally suited to retrofit existing large plants. This technology is based on the principle of highrate activated sludge process that favours biosorption and storage over oxidation. In this paper, we summarize peer-reviewed research of both pilot-scale and full-scale studies of A-Stage process under real conditions, highlighting key operational parameters. In the majority of published studies, the Sludge Retention Time (SRT) was identified as a key operational parameter. An optimal SRT of 0.3 days seems to maximize the redirection of influent CODup to 50% to the sludge flux, while simultaneously keeping mineralization under 25% of total influent COD. Other key optimal parameters are a hydraulic residence time of 30 min and dissolved oxygen levels of 0.5 mg⋅L -1 . In addition, nutrient removal efficiencies of 15-27% for total nitrogen and 13-38% for total phosphorus are observed. Influence of mixing on settling efficiencies remain largely underexplored, as well as impact of wet weather flow and temperature on overall recovery efficiencies, which hinders to provide recommendations on these aspects. Evolution of modelling efforts of A-Stage process are also critically reviewed.

The role of extracellular polymeric substances remain unclear and measures differs greatly

can hence be considered as a resource. Approximately 6.9 kJ of free energy is contained in 1 L for wastewater with an average concentration of 500 mg COD⋅L -1 [START_REF] Mccarty | Domestic wastewater treatment as a net energy producer-can this be achieved?[END_REF].

Traditionally, primary sedimentation tanks (PSTs) redirect some of the influent organics to produce primary sludge, which is then anaerobically digested to generate biogas to offset the energy needs of the WWTP. However, PSTs have high space requirements and are quite inefficient in recovering this resource without chemical addition. Recovering organic matter prior to its oxidation is doubly advantageous as it minimizes aeration (energy) demand and simultaneously increases the methane produced [START_REF] Wan | COD capture: A feasible option towards energy self-sufficient domestic wastewater treatment[END_REF]. Among the best available process technologies that maximises recovery potential is a process based on high rate activated sludge (HRAS), also called A-Stage, which is the focus of this review. Main advantage of A-Stage over other technologies for recovery such as Chemically Enhanced Primary Treatment (CEPT) is relatively lower operational costs as usually no reagent is utilised [START_REF] Wan | COD capture: A feasible option towards energy self-sufficient domestic wastewater treatment[END_REF].

The A-Stage is part of the A/B (Adsorption-Belebung) process that has originally been developed in the 1970s and 1980s in the Technical University of Aachen [START_REF] Böhnke | Das Adsorptions-Belebungsverfahren[END_REF] to maintain nitrification process stability. The A/B process is a two-stage process consisting of two activated sludge processes in series, each with its own reactor, clarifier and independent sludge recirculation loops as shown in Figure 1. Essentially, a PST is replaced by the A-Stage process and can hence be considered a "biologically-enhanced" primary treatment. At the time of its development, the B-Stage was operated only for ammonia removal as a low-rate activated sludge process. [START_REF] Versprille | the a-B Process : a Novel Two[END_REF] A-Stage is characterised by its high organic loading rate (F/M ratio) coupled with low sludge age and short hydraulic residence times. There is no universally accepted definition of operating parameters of A-Stage; however, most authors use a limit solid residence time (SRT) of two days. A comparison of some key process parameters between the A-Stage and conventional processes is presented in Table 1. The A/B process did not establish itself as a widely used process configuration because full mineralisation of nitrogen became an additional treatment requirement in the beginning of the 1990s. The increased carbon removal in the A-Stage hinders heterotrophic denitrification in the B-Stage due to lack of sufficient organic carbon, and most A/B plants have been converted

to conventional activated sludge (CAS) processes. Compared to CAS, the extended aeration process variant applies very long sludge ages in order to achieve high organic matter removal and aerobic stabilisation of the sludge.

However, two driving forces inspire the renewed interest in the A/B process in recent years.

Firstly, the development in autotrophic N removal in the last two decades led to new approaches in coupling A-Stage and autotrophic N removal strategies such as Partial Nitritation / Anammox (PNA) [START_REF] Dai | High-rate contact stabilization process-coupled membrane bioreactor for maximal recovery of organics from municipal wastewater[END_REF][START_REF] Gu | Bioresource Technology A review on mainstream deammoni fi cation of municipal wastewater : Novel dual step process 299[END_REF][START_REF] Gu | Mainstream anammox in a novel A-2B process for energy-efficient municipal wastewater treatment with minimized sludge production[END_REF]. Secondly, the impacts of climate change [START_REF] Mata | IPPC citation[END_REF] incites all sectors of human activity to minimize its contribution to the greenhouse effect. Developing waste valorisation technologies that can offset fossil fuel use has clear environmental benefits [START_REF] Modin | Nonoxidative removal of organics in the activated sludge process[END_REF].

Recent reviews from [START_REF] Sancho | New concepts on carbon redirection in wastewater treatment plants: A review[END_REF] and [START_REF] Guven | Towards sustainable and energy efficient municipal wastewater treatment by up-concentration of organics[END_REF] compiled the relevant technologies for recovering influent organic carbon, focussing on a comparison of the main available carbon redirection technologies, highlighting the importance of A-Stage process. The review proposed by [START_REF] Rahman | Moving forward with A-stage and high-rate contact-stabilization for energy efficient water resource recovery facility: Mechanisms, factors, practical approach, and guidelines[END_REF] gives an interesting evaluation of A-Stage and high rate contact stabilisation processes for carbon capture and highlights the interest of such processes to remove a significant amount of N and P in waste activated sludge (WAS).

However, a detailed analysis of peer-reviewed studies on A-Stage studies, discussing its mechanisms as well as its challenges and drawbacks is lacking. This study aims to fill this gap, and additionally summarises the experience of full-scale implementations as well as attempts to model and simulate this process. Specific combinations of keywords were chosen for a search on webofknowledge.com and sciencedirect.com for studies on HRAS or A-Stage processes, the results of which are succinctly summarized in Table S1 (supplementary material). The search was continuously updated; the most recent update was performed end of December, 2021. Only original research articles were included. In addition, to be considered, the experimental conditions of the studies had to be clearly described, e.g. the size of the treatment units, duration and analytical procedures. The keyword search yielded in a total of 116 publications. The titles of studies were then screened according to their relevance to the topic, and, in case of a positive outcome, the abstract and full text were consulted. Additional screening was conducted to select only papers related to the scope of our investigations.

Thirty-eight publications dealt with HRAS or A-Stage processes. Two studies had to be excluded from further evaluation since crucial information was lacking (e.g. size of the experimental setups). In the end, 36 publications met the defined criteria, of which nine described investigations of "

A-Stage Process: Current knowledge

During the initial development of A/B process technology, no particular attention was given to A-Stage technology and the focus was mainly on nitrogen removal in B-Stage as maintaining nitrification process stability was seen as a prime advantage [START_REF] Versprille | the a-B Process : a Novel Two[END_REF]. As this technology is being revisited in view of its ability to redirect influent COD to a sludge stream, a better understanding of the mechanisms will be useful in design and operation. The current knowledge on the A-Stage process, starting with the driving mechanisms are summarized in the following sections. Then a section presenting results from laboratory-scale and demonstration-scale experiments is proposed including major influencing factors on carbon and nutrient capture. Finally, a section presenting most of the A-Stage or HRAS full-scale experiments published will be proposed before the discussion with the aim of highlighting the main challenges associated to the implementation of A/B process into WRRFs.

Mechanisms of Organic Matter Capture/Interactions

Organic matter present in the influent wastewater interacts with the return sludge in the A-

Stage reactor in many complex mechanisms. Return sludge contains activated sludge flocs, which are loosely bound amorphous structures that contain microorganisms embedded in a matrix of extracellular polymeric substances (EPS) [START_REF] Ni | Microbial Products of Activated Sludge in Biological Wastewater Treatment Systems: A Critical Review[END_REF]. The major mechanisms of interaction are assimilation, biosorption and settling, which are conceptually represented in [START_REF] Modin | Nonoxidative removal of organics in the activated sludge process[END_REF] Assimilation is a biological mechanism that removes part of the soluble, biodegradable fraction (biodegradable sC -org ) of the organic carbon molecules that are small enough to pass through the cell membrane. This fraction of biodegradable organic carbon serves both as carbon source and electron donor for the microorganisms for cell growth, storage of polymers, or production of Extracellular Polymeric Substances (EPS), and for cell maintenance. Hence, a fraction of biodegradable sC -org is oxidised to carbon dioxide and water by aerobic heterotrophic microorganisms. The ratio of microbial growth rate and oxidation rate is called yield (Y) [START_REF] Sperling | Activated Sludge and Aerobic Biofilm Reactors[END_REF].

Microorganisms can store soluble biodegradable organic compounds in the form of intracellular inclusions. The soluble compounds after penetrating the cell walls are converted to insoluble polymers (such as polyhydroxyalkanoates (PHA), triacylglycerides (TAG), wax esters, etc.) to avoid build-up of high osmotic pressure across the cell membrane. Volatile Fatty Acids (VFAs) such as acetate will result in storage of PHAs whereas carbohydrates such as glucose will result in storage of glycogen. The bacteria are capable of reutilising the stored carbon for cell functions when no external carbon source is available, i.e., during a famine phase. Dynamic process configurations such as Sequencing Batch Reactors (SBRs) in which the activated sludge is exposed to periods of feast and famine, induce storage behaviour [START_REF] Coats | Toward Polyhydroxyalkanoate Production Concurrent with Municipal Wastewater Treatment in a Sequencing Batch Reactor System[END_REF].

Biosorption is a combination of physico-chemical interactions that bind colloidal and particulate fraction (collC -org and pC -org ; biodegradable or not) of organic matter to activated sludge flocs by weak adhesive forces. There is no universally accepted definition of biosorption and various other terms are used to refer to this phenomenon such as sorption, enmeshment, bioflocculation, agglomeration, extra-cellular adsorption, etc. EPS may assist with this agglomeration, depending on its quantity, composition and surface properties (Kinyua et al., 2017a). In the sense of this article, biosorption describes an extra-cellular phenomenon, distinguished from storage, which is an intra-cellular phenomenon. Biosorption has been recognized as the key mechanism contributing to carbon capture and driving the interest in A-Stage systems.

Finally, settling is a physical phenomenon where agglomerates form in the A-Stage reactor and sufficiently large organic matter (pC -org ) settle to the bottom of the separation unit, typically a surface clarifier. The settled sludge is either removed from the system, or recirculated back to the reactor.

Overall A-Stage performances: pilot-and demonstrationscale investigations

From the identified papers, nine articles at pilot and demonstration scale performed with real wastewater were selected. Information on experimental conditions of the reactor and influent-effluent characteristics are summarized in Table 2. There is a diversity in the scale of studies from laboratory scale to demonstration scale, but most common HRT was set to 30 min and SRT varied from 0.3 days to up to 2 days. Dissolved oxygen (DO) level in the reactor was set to 0.5 mg L -1 in all studies except two, where DO set point was of 2 mg⋅L -1 . Information on the influent and effluent COD was compiled when available. All considered studies used raw wastewater with total COD ranging from a low 300 mg⋅L -1 to more than 600 mg⋅L -1 .

Likewise, information on fractions was compiled with colloidal fractions being measured following the protocol of [START_REF] Mamais | A rapid physical-chemical method for the determination of readily biodegradable soluble COD in municipal wastewater[END_REF] when available. This protocol of COD fractionation yields three fractions of COD, namely particulate COD (pCOD), colloidal COD (cCOD) and flocculated-filtered COD (ffCOD). Some studies followed an older and simpler fractionation that involves direct filtration at 0.45 µm resulting in only two fractionsparticulate COD and filtered COD (fCOD). It can be seen that all studies achieved a tCOD removal of > 50%, with one study reaching up to 90% [START_REF] Taboada-Santos | Comprehensive comparison of chemically enhanced primary treatment and high-rate activated sludge in novel wastewater treatment plant configurations[END_REF], which can be considered an outlier. Examining the COD fractions of the effluent, most COD removal is due to the removal of pCOD, followed by ffCOD and lastly cCOD.

Influence of SRT on COD mass balance

COD mass balance is visually represented in Figure 3. It shows three possible transformations of influent COD upon leaving the A-Stage system.

Figure 3 Visualisation of COD mass balance

It can be calculated based on daily performance data of the process in steady conditions from the following equation (considering no accumulation in the reactor):

COD_in = COD_eff + COD_min + COD_valo ( 1 )
Where:

• COD_in is the COD of influent;

• COD_eff is the COD of the A-Stage effluent;

• COD_min is the portion of COD that is lost as CO 2 ;

• COD_valo is the COD embedded in the waste sludge.

An optimized A-Stage process maximizes the portion of COD_in that is diverted to the sludge matrix (COD_valo), while simultaneously minimizing the mineralized fraction (COD_min), which is lost as CO 2 to the atmosphere. Table 3 presents the mineralization rate reported in literature on A-Stage. The ratio of COD_valo to COD_in is the measure of the efficiency of the A-Stage process, which is referred to as carbon recovery efficiency (Cao et al., 2020) or carbon harvesting efficiency [START_REF] Rahman | Bioflocculation management through high-rate contactstabilization: A promising technology to recover organic carbon from low-strength wastewater[END_REF]. COD_eff is the fraction of influent COD that leaves A-Stage to the next unit treatment process. Typically, COD_min is calculated using this equation, i.e., to close the mass balance, whereas the rest of the parameters are measured.

All studies that reported mineralization rates are presented in Table 3. [START_REF] Jimenez | High-rate activated sludge system for carbon management -Evaluation of crucial process mechanisms and design parameters[END_REF] concluded that the operational parameter with the highest influence on mineralization is the SRT, as longer SRT resulted in more mineralisation of COD. However, even under comparable SRTs, there is a substantial variation across different studies of the influent COD lost to the atmosphere as CO 2 . These variations are possibly due to varying composition of influent COD, MLSS concentration, temperature in the reactor and other uncontrolled process operational parameters (Kinyua et al., 2017a). Influent COD fractions can play a major role on carbon redirection. If we consider that 1) the particulate COD fraction is mostly removed by primary settling linked to the efficiency of the settling system in the A-Stage process, 2) the soluble fraction is mostly linked to the bacterial assimilation and growth, and 3) the colloidal matter fraction is removed by biosorption on bacterial material and EPS, therefore the carbon redirection of an influent rich in particulate COD will be limited by the settling capacity, a effluent rich in colloidal COD will be limited by EPS production and collision probability with biomass and an effluent rich in soluble COD by the bacterial growing conditions. (2017a), much like for the opposing trends between the two studies observed for mineralisation rates. In the majority of published studies, SRT of 0.3 days seems to be the optimal SRT to maximise the capture of influent COD as sludge, even though it is largely variable and most likely dependant on other operational parameters and nature of organic matter present in influent wastewater. The optimal recovery value reaches 50% of the influent COD. By considering the different pathway of COD in A-Stage, several studies concluded that there is a threshold with an SRT ranking between 0.2 and 0.4 enabling to maximize carbon capture [START_REF] Jimenez | High-rate activated sludge system for carbon management -Evaluation of crucial process mechanisms and design parameters[END_REF][START_REF] Rahman | Moving forward with A-stage and high-rate contact-stabilization for energy efficient water resource recovery facility: Mechanisms, factors, practical approach, and guidelines[END_REF].

Influence of DO and HRT on COD mass balance

DO has a key role in bacterial metabolism and settling efficiency. With regard to carbon mineralisation, several studies have shown that a balance has to be found between low DO level (< 0.5 mg⋅L -1 ) where bacterial growth, EPS production and settling efficiency are limited and high DO conditions (>1.5-2 mg⋅L -1 ) where mineralisation level and energy consumption are too high (Kinyua et al., 2017a, b;[START_REF] Jimenez | High-rate activated sludge system for carbon management -Evaluation of crucial process mechanisms and design parameters[END_REF].

Far fewer studies reported the impact of HRT on the COD recovery potential of A-Stage processes. [START_REF] Jimenez | High-rate activated sludge system for carbon management -Evaluation of crucial process mechanisms and design parameters[END_REF] investigating HRTs from 10 min to 60 min, concluded that a HRT of 30 min as optimal beyond which negligible increase in tCOD and sCOD removal were recovery is considerable: if too short (less than 30 min) the contact time between the biomass and the substrate reduce the production of EPS and the overall COD removal decreases, if is too long (generally larger than 1-1.5 hour), the carbon capture is not maximized and mineralisation becomes too high.

Nutrients removal in A-Stage

Capturing nutrients represents another benefit of an A-Stage process and is mostly linked to assimilation during bacterial growth combined with capture into WAS. Average removal rate ranking from 15-27% TN and 13-38% TP have been reported [START_REF] Rahman | Moving forward with A-stage and high-rate contact-stabilization for energy efficient water resource recovery facility: Mechanisms, factors, practical approach, and guidelines[END_REF]. This could represent an interesting energy reduction in the downstream nutrient removal system. The SRT was observed to be a major factor influencing N removal with an increase of TN removal going from 22% at 0.5 d up to 49% at a 3 d [START_REF] Ge | Nutrient removal and energy recovery from high-rate activated sludge processes -Impact of sludge age[END_REF]. The same pattern was observed for TP removal going from ~15% at SRT< 1d up to 34 % at a 3 d SRT [START_REF] Ge | Nutrient removal and energy recovery from high-rate activated sludge processes -Impact of sludge age[END_REF].

Increasing the SRT improves the yield and therefore assimilation of nutrients but at the same time increases the mineralisation of COD (> 60% at a 3 d SRT). Therefore, the overall mass balance has to be considered to target the treatment strategy by either favouring carbon capture or nutrients removal. Typically, full-scale plants add metal salts in A-stage to achieve additional TP removal (see Section 2.3).

Role of EPS

EPS are microbial secretions and products of cellular lysis or hydrolysis. They are negatively charged and make up 50-80% of organic fraction in activated sludge. Proteins (PN)

and polysaccharides (PS or carbohydrates) are its main constituents, making up 78-80% of EPS with the rest being other organic fractions such as humics, nucleic acids, lipids, etc., [START_REF] Dignac | Chemical description of extracellular polymers: Implication on activated sludge floc structure[END_REF]. The EPS concentration present in the sludge is naturally a function of various operating parameters including SRT and DO levels as well as influent characteristics [START_REF] Faust | Effect of dissolved oxygen concentration on the bioflocculation process in high loaded MBRs[END_REF]. .

It is hypothesized that both quantity and nature of EPS can play an important role in biosorption of colloidal and particulate matter into the activated sludge floc matrix in A-Stage systems. Table 4 presents the quantity of total EPS reported in different A-Stage systems as a function of SRT at comparable DO levels. Two studies [START_REF] Jimenez | High-rate activated sludge system for carbon management -Evaluation of crucial process mechanisms and design parameters[END_REF][START_REF] Rahman | Bioflocculation management through high-rate contactstabilization: A promising technology to recover organic carbon from low-strength wastewater[END_REF] used the cation exchange resin [START_REF] Frølund | Extraction of extracellular polymers from activated sludge using a cation exchange resin[END_REF] method for EPS extraction.

Whereas the rest of the studies, which are more recent, used the heat extraction method [START_REF] Li | Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge[END_REF]. As of yet, no standard method or protocol for EPS measurement in activated sludge exists, making it challenging to compare and interpret the results meaningfully.

Settling characteristics

Settling is most commonly characterized by a measurement of Sludge Volume Index (SVI) following APHA (2005) which considers settling over 30 minutes. (2021) show a sharp peak SVI of around 550 mg/L for an SRT of approx.. 3 days, with SVIs below 100 mg/L for SRTs below 2 days. However, a low SVI alone is not a sufficient criterion to judge the clarifier performance in an A-Stage: the TSS concentrations of the supernatant can still be unusually high, as observed by [START_REF] Guven | Retrofitting of Five Preliminary Wastewater Treatment Plants in Istanbul (Turkey) to High-Rate Activated Sludge System and/or Post Oxidation[END_REF] who reported an SVI < 50 mL⋅ g TSS -1 , but effluent TSS concentrations higher than 150 mg⋅L -1 . This represents particulate and colloidal COD that could have been redirected to the waste sludge, but is instead "lost" in the effluent. This might be a consequence of the characteristics of the A-Stage mixed liquor where very small flocs are formed and sometimes not settling leading to high TSS concentration in the supernatant, the settled sludge could be made of particulate COD mostly. all reported SVI between 50 and 90 mL⋅g TSS -1 , which is lower than that of conventional activated sludge, where SVIs lower than 150 mL/g is considered a fair value (Von [START_REF] Sperling | Activated Sludge and Aerobic Biofilm Reactors[END_REF]. This contrasts with the findings of the review of [START_REF] Sancho | New concepts on carbon redirection in wastewater treatment plants: A review[END_REF], where poor settling of A-Stage sludge is mentioned as the drawback that prevented a wider adaptation of A/B processes worldwide. In Kinyua et al. (2017a), overall lower SVI values at shorter SRT (0.3 days) could be due to the washout of filamentous organisms, the impact of DO was pronounced at higher SRT, presumably due to differences in types of filamentous organisms.

Table 5 Settling properties of A-Stage sludge SVI30 [mL⋅ g TSS

Research on conventional activated sludge indicated links between the composition of EPS of the sludge and its settling characteristics [START_REF] Li | Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge[END_REF]. This method further characterizes EPS as Loosely Bound (LB) fraction and Tightly Bound (TB) fraction by stepwise extraction. The fractions are then analysed for their COD, protein content (PN) and polysaccharide content (PS). Two studies (Kinyua et al., 2017a;[START_REF] Van Winckel | Overcoming floc formation limitations in high-rate activated sludge systems[END_REF] adopting this method for EPS extraction and characterization, explored a relation between settling and various A-Stage systems but did not find a conclusive relation between settling performance and EPSneither total quantity nor its quality (such as PN/PS ratio). This could be intrinsically linked to the role of EPS in the capture of colloidal and particulate matter in the A-Stage reactor, as discussed in 2.2. (non-Stokesian) settling, while TOF is the concentration limit to induce floc formation and consequently transition from discrete to flocculent settling. TOF is hence a measure of particle collision efficiency. It is hypothesized that if the mixed-liquor solids concentration is between LOSS and TOF, the clarifier performance is optimized with minimal TS loss in the effluent.

Developing on this approach, very recent research (Ngo et al., 2021;Ngo and Massoudieh, 2021) proposes a relation between TOF and effluent suspended solids (via flocculent settling coefficientr p ) and a second relation between EPS and TOF. This marks a first attempt in describing a relation between EPS content and effluent suspended solids, albeit indirectly. As these parameters and approaches were only recently introduced, little information is available in literature to validate the hypotheses.

In conclusion, it is challenging to predict the settling properties and effluent quality of A-Stage and certainly more research is needed to explore the links between quantity and properties of EPS and settling characteristics. Apart from TOF, LOSS and EPS measurement, other investigations could be performed to evaluate sludge settleability, including microscopic ones, measure of floc resistance to shear stress and measure of zeta potential of the flocs. To our knowledge, these investigations have never been applied to A-Stage mixed liquor.

Valorisation potential of A-Stage sludge

The most common valorisation pathway of the produced sludge is via anaerobic digestion to produce biogas, which is mainly a mixture of CH 4 and CO 2 . The produced energy can offset the plant's own energy requirement or it can be sold. Biochemical Methane Potential (BMP) is a measure of cumulative methane produced as the organic matter in the sample degrades under anaerobic conditions. It is crucial in getting an insight into design parameters for anaerobic digesters [START_REF] Holliger | Towards a standardization of biomethane potential tests[END_REF].

Table 6 presents the BMP from various A-Stage studies expressed as produced volume of methane under normal conditions of temperature and pressure, normalized to the amount of volatile solids introduced (NmL CH 4 ⋅g VS -1 ). Choo-kun ( 2015) compared the BMP of sludge from primary settling, A-Stage, and a long sludge age process and found that A-Stage sludge had the higher BMP of 335 NmL CH 4 /g VS, compared to 290 NmL CH 4 g VS -1 and 220 NmL CH 4 g VS -1 for primary sludge and secondary sludge from extended aeration, respectively.

Hence, the BMP of A-Stage sludge is not so different from primary sludge. Other studies have reported BMP values in the same range, but comparison between studies is difficult as the influent wastewater characteristicsboth quantity and quality of organic mattercan vary widely. The wide range reported by [START_REF] Trzcinski | Characterization and biodegradability of sludge from a high rate A-stage contact tank and Bstage membrane bioreactor of a pilot-scale AB system treating municipal wastewaters[END_REF] shows the influence of intraday variations and high oil and grease content in the influent. Few energy recovery indices were proposed, such as Methane Recovery Index and Energy Recovery Index [START_REF] Rey-Martínez | Comparing continuous and batch operation for high-rate treatment of urban wastewater[END_REF]. These indices are useful in comparing the overall recovery potential of different A-stage systems, however not enough information is available to calculate these indices for studies summarized in Table 6.

Table 6 Bio-Methane Potential of A-Stage sludge

Reference

2016) 460

Pilot scale (42 m 3 •h -1 ) with SRT 0.5 day, DO = 0.3 mg•L -1 and 30 min HRT; 500 mL bottles used for BMP.

Another emerging valorisation pathway of the concentrated organics stream is the production of high value biopolymers such as Polyhydroxyalkanoates (PHA) that can be used to produce bioplastics. However, regulatory barriers and public acceptance of such products are of concern. Further research is required to make biopolymer production from waste sludge economically viable compared to plastics produced from fossil fuels [START_REF] Gherghel | A review on wastewater sludge valorisation and its challenges in the context of circular economy[END_REF].

Full-Scale Performance

In the 1970s and 1980s, A/B process has been successfully implemented in full-scale plants in some European countries especially in the Netherlands, Germany and Austria. As mentioned in introduction, the emergence of more stringent effluent requirements on nitrogen led to a decline in A/B systems, with some of them being reconfigured to CAS due to lack of organic carbon for achieving the required denitrification. The information that was found on existing full-scale A-Stage/HRAS implementation is summarized in Table 7. It can be noted that A-Stage has been implemented in large to very large treatment plants.

In the case of the Nieuwveer plant which was originally built in 1976 and refurbished to A/B process in 1992, its original primary settlers were used as A-Stage settlers. The Vienna WWTP was undergoing an upgrade (at the time of publication of Kroiss and Klager 2018), however the existing PSTs were retained in the process scheme, and A-Stage treats PST effluent. The Vienna strategy is not a conventional A/B process as defined in our review but presents an interesting strategy by using a two-stage activated sludge with an HRAS receiving nitrates.

Likewise, the Blue Plains plant also utilises A-Stage (HRAS) technology after a primary treatment step, in this case a CEPT process. All full-scale plants dose metal salts for phosphorus precipitation in the aeration tank, but concentrations used are not provided for any of them in the available literature. Without the concentrations of metal salts used, it is difficult to evaluate the effect of metal salt addition on COD valorisation efficiency and on settling. [START_REF] Wett | Operational and structural A-stage improvements for high-rate carbon removal[END_REF] observed no improvement in COD removal or settling performance up to a molar ratio of metal/P of 0.5.

Most of the publications presenting full-scale investigations focus on A-Stage and carbon

removal strategy, few elements are provided on the B-Stage. The strategy presented at the Vienna plant rely on a full nitrification/denitrification process and an optimisation of carbon capture enabling enough redirection to perform full denitrification. Few options are available and rely on either full nitrification (in case no restriction are applied) or PNA processes. Recent reviews have shown that PNA processes are not completely mature for mainstream application; to our knowledge, no full-scale success stories have been reported. Challenges remains at several levels including implementing an intelligent control of partial nitrification, exploring the molecular biological mechanism of NOB selective inhibition and strengthening of pre-treatment process for partial nitrification [START_REF] Wang | A review of partial nitrification in biological nitrogen removal processes: from development to application[END_REF].

Energy consumption data on A-Stage is not available for most plants, but it was reported as 0.17 and 0.10 kWh⋅ kg COD removed -1 for Dokhaven and Utrecht, respectively [START_REF] De Graaff | Full-Scale Highly-Loaded Wastewater Treatment Processes (A-Stage) to Increase Energy Production from Wastewater: Performance and Design Guidelines[END_REF]. It is higher than the 0.08 kWh⋅ kg COD removed -1 (calculated) reported by [START_REF] Wett | Operational and structural A-stage improvements for high-rate carbon removal[END_REF]. Little knowledge is available on the dynamic performance of the A-Stage process and how the drop in performance during rain events, or even sludge washout can be mitigated, which is a major drawback of A-Stage processes [START_REF] Diamantis | Performance of the biosorptive activated sludge (BAS) as pre-treatment to UF for decentralized wastewater reuse[END_REF].

Discussion

Knowledge synthetized in models

Numerical models are made for representing reality in a more or less simplified way and are very useful when comparing different treatment configurations. In addition, they embed the current knowledge associated to a specific process. As far as short residence time processes such as A-Stage are concerned, modifications to 'historical' activated sludge models (ASM)

were required. Widely used models like ASM1 are not suitable for systems with short residence times (< 2 days) as first recognized by [START_REF] Haider | The effect of low sludge age on wastewater fractionation (SS, SI)[END_REF], since the biodegradable fractions of organic matter was modelled as a single substrate and by a single removal kinetic (one Monod term), which is a valid assumption for sludge ages longer than 5 days. Moreover, these early models do not explicitly account for neither EPS production nor biosorption mechanisms, which play a key role in A-Stage process. These physico-chemical mechanisms were indeed not considered in the original ASMs, as they were not limiting for the considered sludge ages (>3 days in ASM1).

To model high-rate processes without increasing the complexity of the model, Smitshuijzen was found in the aerobic reactors at SRT of 0.28 days and DO of 1.0 mg•L -1 with a corresponding PHA yield of 1.20 g PHA COD gVFA -1 . PHA concentration were always higher at the lower SRT in this study. Literature on storage product formation in A-Stage is scare and more research is perhaps needed to further understand the contribution of storage and its influence on other operating parameters.

The new plant-wide model has the advantage of being valid in other biological process units with different operating conditions, without employing interface equations. It predicted low mineralization rates found in A-Stage systems. Colloidal concentrations in the effluent were also predicted thanks to explicit modelling of flocculation as a function of EPS content: the greater the quantity of EPS, the higher is the portion of captured colloids to form particulates.

However, the bio-kinetic model can be quite sensitive to influent characterization, as it demands many fractions that are generally not measuredsuch as VFA and biomass concentration.

Moreover, in all the models, settling is still considered as an empirical process whereas due to the weakness of A-Stage flocs, floc disturbance at the inlet structure of A-Stage clarifier might have a big impact on its efficiency [START_REF] Solon | Resource recovery and wastewater treatment modelling[END_REF]. Recent research by Ngo et al.

(2021) attempts at predicting effluent concentrations by means of an empirical function using experimentally determined TOF value. They further developed this approach to link EPS characteristics and TOF (Ngo and Massoudieh, 2021). As EPS had already been included in the model as a function of operating parameters, the authors were able to predict effluent solids concentrations as a function of process conditions. Since this approach is very recent, there are no comparable studies in literature. Overall, more effort is needed to gain a clearer insight into floc formation processes, their dependence on temperature and mixing intensity within A-Stage systems and finally settling phenomena of A-Stage sludge. Implementing a strategy to improve floc densification in A-Stage could represent an interesting research topic such as hydrocyclone-based wasting to achieve SVI improvements [START_REF] Regmi | Combining continuous flow aerobic granulation using an external selector and carbon-efficient nutrient removal with AvN control in a full-scale simultaneous nitrification-denitrification process[END_REF] and/or use of bio-sourced polymeric substances.

Design recommendations

Based on the present review, optimal ranges for operating parameters of an A-Stage system are presented in Table 8. The parameters suggested may need to be adjusted based on the influent conditions, reactor configurations and other site-specific conditions, but provide a starting point based on consensus values in the available literature. Optimal operating parameters ultimately depend on the objective of A-Stage, which is most often to maximise COD_valo, but depending on the downstream processes, it could have an additional constraint on COD/N ratios of A-Stage effluent [START_REF] Miller | Combining high-rate activated sludge and shortcut nitrogen removal for efficient carbon and energy utilization[END_REF]. To maintain the DO at 0.5 mg/L, most often aeration is provided by compressed air through diffusers at the bottom of the reactor. The ratio of oxygen transfer efficiency under process conditions compared to fresh water is referred to as α-factor, which is an important design parameter.

Based on long-term studies, α-factor of A-Stage was determined to be 0.45 [START_REF] Schwarz | Oxygen transfer in two-stage activated sludge wastewater treatment plants[END_REF] under dry weather flow, which is similar to the factor suggested by [START_REF] Kroiss | How to make a large nutrient removal Plant energy self-sufficient. Latest upgrade of the Vienna Main wastewater treatment plant (VMWWTP)[END_REF] or for non-nitrifying CAS systems [START_REF] Gillot | Prediction of alpha factor values for fine pore aeration systems[END_REF]. Typical reported α-factor CAS is in the range 0.60 to 0.75 for nitrifying systems, higher α-factor of 0.8 was found for B-Stage [START_REF] Schwarz | Oxygen transfer in two-stage activated sludge wastewater treatment plants[END_REF], which is attributed to the removal of surfactants and other inhibitors in the A-Stage. In high-loaded systems such as A-Stage reactors, constituents that hamper oxygen transfersurfactants in particular -are indeed less degraded than in CAS, thus reducing oxygen transfer efficiency (Gillot and Heduit, 2008;[START_REF] Bencsik | Dynamic alpha factors: Prediction in time and evolution along reactors[END_REF].

A sludge extraction control based on maintaining a constant MLSS concentration in the reactor was found to be optimal to buffer diurnal variations in influent COD concentrations [START_REF] Miller | Controlling the COD removal of an A-stage pilot study with instrumentation and automatic process control[END_REF]. In the matter of separation unit design or sizing, little to no information is available in the literature. The clarifier to reactor volume ratio varies widely from 0.5 to 7 in the studies reviewed (see Table S2 supplementary material).

Conclusions and need for further research

The growing interest in recovering resources in municipal wastewater led to a resurgence in the interest in the A-Stage process technology, owing to its capacity to recover influent organic carbon and ease of integration into existing WWTPs. Recent research summarized in this review recognised EPS production and biosorption, including intracellular storage, as the dominant bio-chemical mechanisms involved in this technology. Key operational parameters that drive the process efficiency were also pointed out, as well as the observed limitations:

-To maximise the redirection of influent COD to the sludge flux, SRT was found to be the key parameter with an optimal value of 0.3 days. HRT and DO levels are also important operational parameters, with target values of 30 min and 0.5 mg•L -1 , respectively.

-Optimal performances show a redirection of up to 50% of the influent COD to the sludge flux, while the mineralization can be kept below 25% of the influent COD.

-Published full-scale applications show a great variability in the observed performance that cannot be explained through the current knowledge on bio-chemical mechanisms.

Figure 1

 1 Figure 1 Simplified flow diagram of A/B process[START_REF] Versprille | the a-B Process : a Novel Two[END_REF]

Figure 2 .

 2 Figure 2. These mechanisms are a function of the properties of influent organic matter

Figure 2

 2 Figure 2 Schematization of interactions between bacteria and different fractions of organic carbon (Corg) (inspired by[START_REF] Modin | Nonoxidative removal of organics in the activated sludge process[END_REF]

  observed. A similar conclusion was reached by De Graaff et al. (2016) carrying out aerated jar tests on A-Stage sludge from full-scale plants. The authors observed that 15 min contact time (including the return stream) is sufficient for most sCOD removal. Guven et al. (2017) measured the impact of HRTs of 130, 95 and 60 min and found that the shortest HRT resulted in highest tCOD recovery in the sludge and lowest effluent TSS. The effect of HRT on COD

  of HRT,[START_REF] Guven | Effect of Hydraulic Retention Time on the Performance of High-Rate Activated Sludge System: a Pilot-Scale Study[END_REF] concluded that in addition to improving the % COD recovered as sludge, the SVI also improved from HRT of 60 min compared to that of 130 min. Similarly,[START_REF] Rahman | A-stage and high-rate contact-stabilization performance comparison for carbon and nutrient redirection from high-strength municipal wastewater[END_REF],[START_REF] Van Winckel | Overcoming floc formation limitations in high-rate activated sludge systems[END_REF] and[START_REF] Wett | Operational and structural A-stage improvements for high-rate carbon removal[END_REF]

  4. New metrics to characterize settling such as Limit of Stokesian settling (LOSS) (Mancell-Egala et al., 2016) and Threshold of Flocculation (TOF) (Mancell-Egala et al., 2017) were developed to overcome the limits of established solids flux theory. LOSS is the solids concentration of the sludge at which flocculent settling (Stokesian) transitions into hindered

  et al. (2016) modified ASM1 by introducing a factor (f settler ) that accounts for the fraction of biodegradable particulate matter that is captured by the A-Stage system. This factor accounts for both the efficiency of biosorption and the efficiency of separation in the settler in one term.It was estimated from data from the full-scale plant Dokhaven (Rotterdam, The Netherlands) including daily and seasonal variations to investigate the impact of rain events and changes in water temperature. The results show that the model was able to predict effluent total COD with a 10% error under steady-state conditions. The model was also able to describe the drop in COD removal in A-Stage during winter months (temperatures of 10-12°C) via dynamic simulations, which according to[START_REF] Smitshuijzen | A simple model to describe the performance of highly-loaded aerobic COD removal reactors[END_REF] is due to the decrease in heterotrophic activity at low temperatures rather than decrease in the efficiency of biosorption.Examining the data from another full-scale A-Stage plant,[START_REF] Wett | Operational and structural A-stage improvements for high-rate carbon removal[END_REF] found the same relation of decreasing COD removal efficiency with decreasing temperatures. Contrarily, they argue that it cannot be explained by the Arrhenius effect on microbial growth and instead is rather by temperature sensitivity of biosorption reactions. It is clear that more research effort is needed to fully understand this relation. It can be however hypothesized that the decrease of temperature might reduce the frequency of collision between the colloidal matter and the floc / Tightly Bound EPS thus reducing biosorption. Lastly, it was reported that the Dokhaven plant uses iron salts for P-removal in A-Stage reactor[START_REF] De Graaff | Full-Scale Highly-Loaded Wastewater Treatment Processes (A-Stage) to Increase Energy Production from Wastewater: Performance and Design Guidelines[END_REF], which was not mentioned in the context of the model development.This simplified yet effective A-Stage model of[START_REF] Smitshuijzen | A simple model to describe the performance of highly-loaded aerobic COD removal reactors[END_REF] was further integrated into whole-plant modelling by[START_REF] Jia | Model-based evaluation of an integrated high-rate activated sludge and mainstream anammox system[END_REF] in order to study the benefits and drawbacks of a coupled A-Stage-PNA (Partial Nitritation Anammox) system compared to a CAS system. The simulation results showed a trade-off between maximising COD capture for energy recovery and minimizing residual COD entering the autotrophic PNA reactor with operating conditions being an SRT of 0.3 to 0.5 days and DO of 0.3 to 0.5 mg/L. The study also found the A-Stage/PNA configuration led to an increase of 50% of COD recovery as sludge compared to a CAS plant, and 60% lower overall aeration energy, all the while meeting the EU discharge limits (EU Urban wastewater directive 91/271/EEC of 21 May 1991). In A-Stage/PNA configuration, the COD recovery is mostly linked to the A-Stage sludge, contribution of B-Stage sludge has been estimated as to a maximum of 10-12 % of COD redirection[START_REF] Wan | COD capture: A feasible option towards energy self-sufficient domestic wastewater treatment[END_REF][START_REF] Nogaj | Modeling of organic substrate transformation in the high-rate activated sludge process[END_REF] developed a more mechanistic approach to model high-rate processes by introducing additional state variables to ASM1 model. On a single biomass population two substrates were consideredfast and slow biodegradable organic matter with fast biodegradable fraction being considered as VFA concentration in the influent. Influent COD was split into three subparts with colloidal COD being considered explicitly as an additional state variable, which is an upgrade compared to ASM1. EPS was also added as a state variable, along with storage products and sorption of colloidal substrate.[START_REF] Hauduc | Colloids, flocculation and carbon capture -A comprehensive plant-wide model[END_REF] built upon this model by adding a new fast growing biomass and a further fractionation of fast biodegradable organic matter into VFAs and other organic compounds. Flocculation of colloidal particles is a rate limiting process in A-Stage systems and thus was modelled explicitly as a function of EPS present in the biomass. Furthermore, as flocculation is sensitive to temperature and mixing intensities, an empirical factor (range 0-1) on flocculation rate was introduced to account for deflocculation processes. Finally, storage products are modelled to reflect lower mineralization rates observed in A-Stage, and it is supported by experimental observations byKinyua et al. (2017b). This study measured PHA production yields at different SRTs (0.28 day and 0.56 day) and DO concentrations (0.5, 1.0 and 1.5 mg•L -1 ) without a dynamic feast-famine configuration. A maximum PHA concentration of 42 mg COD•gVSS -1

Table 1 :

 1 Key operating parameters of A-Stage and other processes

Process variant SRT (days) HRT (hours) F/M ratio (kgBOD⋅kgMLVSS -1 ⋅day -1 ) (Versprille et al., 1984)

		A-Stage	< 2	0.5-1	3 -6
		B-Stage	15-20	2-4	0.15 to 0.30
		CAS	4 to 10	6-8	0.25 to 0.50
	(Von Sperling,
	2015)	Extended aeration	18 to 30	16-24	0.07 to 0.15

  classical" A-Stage setups. The remaining articles were either HRAS-variants (such as High Rate Contact Stabilization (HiCS) and High Rate Sequencing

Batch Reactors (HRSBR)), investigations conducted under static conditions, focusing on specific parameters (e.g. EPS) or in very small pilot scale (A-Stage reactor volume smaller than 1L), or review articles without any experimental data.

Table 2 :

 2 Pilot and demonstration scale investigations: operating conditions and removal efficiencies

	Reference	Reactor vol. (L)	Study period (d)	SRT (d)	HRT (h)	DO conc. (mg⋅L -1 )	MLSS (g⋅L -1 )	Inflow COD* (mg⋅L -1 )	Removal efficiency (%)	Inflow nutrients (mg⋅L -1 )	Nutrients Removal efficiency (%)
								t: 375-750	87
	(Taboada-Santos et	2	65	1	2	3-3.5	2-3	s: 80-150	N/A	NH 4 -N: 45
	al., 2020)
								p: 295-600	N/A	PO 4 -P: 2.3
								t: 238	52	TKN: 55
	(Diamantis et al., 2014)	3	36	0.3-0.5	0.9	2.0	2.1	s: 80	25	NH 4 -N: 33
								p: 155	66	PO 4 -P: 3.8
								t: 675	68
	(Koumaki
	et al.,	15	14	0.25	0.6	>2	2.3
	2021)
								t: 480	75
	(Jimenez et al., 2015)	260	365	0.1-2	0.1-1	0.0-2.0	2.6	ff: 110 c: 80	90 55
								p: 290	75
								t: 619	68	TKN: 45
	(Rahman et al., 2019)	510	N/A	0.3	0.5	0.5	3.7	ff: 157 c: 67	62 25	NH 4 -N: 35 TP: 6.1
								p: 395	77	PO 4 -P: 4
								t: 575	70
	(Kinyua et al., 2017a, 2017b)	510	63	0.3-0.6	0.5-1	0.5-1.5	2.6-3.5	ff: 149 c: 85	25 37
								p: 345	75

*t: total COD, s: filtered COD, ff: flocculated-filtered COD, c: colloidal COD, p: particulate COD

Table 3

 3

	Reported mineralization and recovered sludge (as % of influent COD) in pilot-and
		demonstration-scale investigations
	SRT (days)	0.1	0.3	0.5	1.0	2.0
		Mineralization / recovered sludge (as % of influent COD)
	(Jimenez et al., 2015)	14 / 31	N/A / 48	37 / 42	N/A / 29	67 / 23
	(Kinyua et al., 2017a)		23 / 46	22 / 56
	(Cao et al., 2020)			24 / 41
	(Rahman et al., 2019)		23 / 43
	(Guven et al., 2017)		23 / 38

Table 3

 3 presents also the recovery rate of the sludge reported in the original research studies on classical A-Stage.[START_REF] Jimenez | High-rate activated sludge system for carbon management -Evaluation of crucial process mechanisms and design parameters[END_REF] reported a trend of decreasing recovery as SRT increased from 0.3 days to 2 days, which is in contradiction with the study ofKinyua et al.

Table 4

 4

	: EPS measured in A-Stage sludge, varying SRT and DO conditions (Total EPS mg
			COD⋅ g VSS -1 )
					SRT (days)
		DO (mg⋅L -1 )	0.16	0.3	0.5	1.5	2.0
	(Jimenez et al., 2015) a,1	1.0		50	105		125
	(Rahman et al., 2016) a,2	1.0	230		308		367
	(Van Winckel et al., 2019) a,1	N/A.				90
	(Kinyua et al., 2017) b,1	1.0		227	200
	(Rahman et al., 2019) b,1	0.5	165	123

a: Cation Exchange Resin method; b: Heat extraction method; 1: Raw wastewater as influent; 2: CEPT effluent; N/A: not available

Table 4 presents

 4 EPS measured in the mixed liquor in A-Stage. In addition to the large heterogeneity in the total EPS found across the studies, its trend with respect to varying SRTs is also different depending on studies.[START_REF] Jimenez | High-rate activated sludge system for carbon management -Evaluation of crucial process mechanisms and design parameters[END_REF] observed increasing EPS concentrations with increasing SRTs, which concurs with[START_REF] Rahman | Bioflocculation management through high-rate contactstabilization: A promising technology to recover organic carbon from low-strength wastewater[END_REF]. However, capture through biosorption. EPS production is believed to improve carbon capture through colloidal COD capture mostly, although contradictory results have been published. Maybe the following hypothesis can be formulated: Tightly Bound (TB) fraction to

	Kinyua et al. (2017a) and Rahman et al. (2019) observed the opposite trend studying two SRTs
	of 0.28 and 0.56 days, as well as 0.16 and 0.3 days respectively. Apparent differences in the
	reported values may be linked to measurement protocols as well as influent characteristics,
	MLSS concentrations, F/M ratio and other uncontrolled parameters. With regards to the

capture of pCOD and cCOD,

[START_REF] Jimenez | High-rate activated sludge system for carbon management -Evaluation of crucial process mechanisms and design parameters[END_REF]

reported increasing EPS concentration increased their capture, whereas

Kinyua et al. (2017a)

found no correlation. More research is therefore needed to establish the part played by EPSits total quantity and its nature, in aiding organic matter

Table 7

 7 Summary of full-scale A-Stage installations and carbon capture full-scale plants

			Design
	Location	Year Operational	max Capacity	Details on A-Stage	Reference
			(P.E)
	Nieuwveer, NL	1992	440 000	3 500 m 3 plug flow tank
	Dokhaven, NL	1988	564 000	4 800 m 3 plug flow tank	(De Graaff et al.,
	Utrecht, NL	1976	480 000	3 750 m 3 CSTR**, square	2016)
	Garmerwolde, NL	2006	375 000	2 760 m 3 CSTR round
	Strass, AT	1999	200 000	644 m 3 CSTR	(Wett et al., 2020)
	Vienna, AT	2020	4 000 000	N/A*	(Kroiss and Klager, 2018)
	Blue Plains, Washington D.C., US	1969	8 700 000 #	N/A	(Rahman et al., 2016)

* N/A = not available ** CSTR = Continuous-flow Stirred Tank Reactor # calculated based on 0.2 L per P.E

Table 8

 8 Suggested operating and design parameters based on this review

	Parameter	Suggested value based on this review
	HRT	30 min
	SRT	0.3 -0.5 days
	DO	0.5 mg•L -1
	MLSS	2-3 g•L -1
	Recirculation rate	0.5 -1
	Waste strategy / Sludge extraction	Target MLSS based control

Being a short retention time biological process, A-Stage is quite sensitive to fluctuating concentrations of COD in the influent. Especially in the case of a municipal plant receiving influent from a combined or a semi-separative sewer system, rain-induced dilution can destabilize the process that leads to a drop in performance and can even lead to sludge washout.

Challenges remain over the wide spread adoption of A-Stage technology despite its natural advantage over other similar processes such as CEPT that require expensive chemicals to operate. The following have been principally identified:

-A better understanding of A-stage performances requires further research on the operating parameters affecting them; especially temperature and mixing that are overlooked in the literature.

-Continuing the effort to better understand liquid-solid separation efficiency is also mandatory, starting with a better description of separation units in A-stage related publications. The relationship between biosorption and settling phenomena should be further investigated in a holistic manner.

-Conceptualisation of biosorption mechanism in numerical models can help accelerate research in this domain. This necessitates the characterization of new state variables and a finer influent fractionation.

-Observed impact on influent dilution calls for mitigation or avoidance strategies that might be useful to define the operating range of the system and especially to handle wet weather flows.

Acknowledgements

This work resulted from the collaborative project CAPTURE led by INRAE REVERSAAL.

We thank the Agence de l'Eau Rhône Méditerranée Corse for their funding under the grant number 2018 0685. This work was performed within the framework of the EUR H2O'Lyon


Georgi Georgiev
email: ggeorgiev.fri@gmail.com

Jérôme Rousselet

Mathieu Laparie

Christelle Robinet

Margarita Georgieva

Gergana Zaemdzhikova

Alain Roques

Alexis Bernard

Laura Poitou

Maurane Buradino

Georgi Georgiev1

Jérôme Rousselet2

Mathieu Laparie2

Christelle Robinet2

Margarita Georgieva1

Gergana Zaemdzhikova1

Alain Roques2

Alexis Bernard2

Laura Poitou2

Maurane Buradino3

Carole Kerdelhue4

Jean-Pierre Rossi4

Maria Matova1

Peter Boyadzhiev5

Plamen Mirchev1

Comparative studies of egg parasitoids of the pine processionary moth (Thaumetopoea pityocampa, Den. & Schiff.) in historic and expansion areas in France and Bulgaria

Keywords: Thaumetopoea pityocampa, range expansion, survival, egg parasitoids, France, Bulgaria

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The pine processionary moth, Thaumetopoea pityocampa (Denis & Schiffermüller, 1775) (Lepidoptera: Notodontidae) is one of the most important forest pests in the Mediterranean region. Due to climate change, and particularly winter warming [START_REF] Battisti | Expansion of geographic range in the pine processionary moth caused by increased winter temperatures[END_REF][START_REF] Battisti | A rapid altitudinal range expansion in the pine processionary moth produced by the 2003 climatic anomaly[END_REF][START_REF] Robinet | Modelling the effects of climate change on the potential feeding activity of Thaumetopoea pityocampa (Den. & Schiff.)[END_REF], an altitudinal and latitudinal expansion of its range has been observed over the last few decades [START_REF] Hoch | Range expansion of the pine processionary moth in Europe II: Activity and survival of Thaumetopoea pityocampa (Lep.: Thaumetopoeidae) during the winter months in an Alpine valley[END_REF][START_REF] Imber | Expansion d'un ravageur forestier sous l'effet du Rechauffement climatique: la processionnaire du pin affecte-t-elle la biodiversité entomologique dans les zones nouvellement colonisées? Thèse pour obtenir le grade de[END_REF]Mirchev et al., 2011a;[START_REF] Netherer | Potential effects of climate change on insect herbivores in European forests-General aspects and the pine processionary moth as specific example[END_REF][START_REF] Roques | Range expansion of the pine processionary moth in Europe. I. Mechanisms underlying PPM expansion in France in relation to global warming[END_REF][START_REF] Toffolo | Climate change and expansion of the Pine Processionary Moth, Thaumetopoea pityocampa[END_REF]. In its current range, T. pityocampa lives in different habitats both in its historical range and newly colonized areas.

In France, five egg parasitoids of pine processionary moth have been recorded: Baryscapus servadeii (Domenichini, 1965) (Hymenoptera: Eulophidae), Ooencyrtus pityocampaе (Mercet, 1921) (Hymenoptera: Encyrtidae), Anastatus bifasciatus (Geoffroy, 1785), Eupelmus sp. (Hymenoptera: Eupelmidae) and Trichogramma sp. (Hymenoptera: Trichogrammatidae) [START_REF] Auger-Rozenberg | Ecological responses of parasitoids, predators and associated insect communities to the climate-driven expansion of the pine processionary moth[END_REF]. In addition, Baryscapus transversalis Graham, 1991, Pediobius bruchicida (Rondani, 1872) (Hymenoptera: Eulophidae) and Eupelmus (Macroneura) vesicularis (Retzius, 1783) (Hymenoptera: Eulophidae), have been established in Bulgaria (Mirchev et al., 2011b;Tsankov et al., 1996a). The performance and distribution of the pine processionary moth and its insect enemies are highly dependent on climatic conditions. Higher temperatures in winter favour larval survival of the pine processionary moth [START_REF] Battisti | Expansion of geographic range in the pine processionary moth caused by increased winter temperatures[END_REF], whereas summer temperatures above 32° С are considered lethal for the development of T. pityocampa eggs and young larvae [START_REF] Roques | Climate Warming and Past and Present Distribution of the Processionary Moths (Thaumetopoea spp.) in Europe, Asia Minor and North Africa[END_REF]. The climatic conditions in a particular biotope comprehensively influence the variety of egg parasitoid species as well. Indeed, [START_REF] Masutti | Ricerche sui parassiti oofagi della Thaumetopoea pityocampa (Schiff.)[END_REF] reported that O. рityocampae is sensitive to high temperatures and biotopes with prolonged periods above 30° С are not suitable for its survival. In contrast, another hymenopteran species, B. servadeii, is more tolerant and develops successfully even at high temperatures. The parasitoid complex in newly colonized areas may also depend on the pace of the range expansion of T. pityocampa. The egg parasitoid assemblage of T. pityocampa is an indicator of two different types of pest expansion in new habitats: i) when T. pityocampa penetrates into new regions, relatively remote from the primary habitats where the specific B. servadeii is absent [START_REF] Mirchev | Long-term studies on egg parasitoids of pine processionary moth (Thaumetopoea pityocampa) in a new locality in Bulgaria[END_REF]; ii) when the pine processionary moth gradually enlarges its range in adjacent stands at the rate of 2-3 km per year, the host is followed by B. servadeii [START_REF] Mirchev | Enlargement of the pine processionary moth (Thaumetopoea pityocampa) range in Bulgaria[END_REF].

The aim of this study was to compare the egg parasitoids complex of T. pityocampa in historic and expansion areas in France and Bulgaria, the westernmost and easternmost countries where the northward expansion of the pest is observed.

Materials and methods

During the period 2016-2017, a total of 206 egg-batches of T. pityocampa were collected from France and Bulgaria between the end of August and November (Table 1). In Bulgaria, all samples were collected in Austrian pine (Pinus nigra Arn.) plantations. In France, samples were collected in P. nigra, maritime pine (Pinus pinaster Aiton) and Monterey pine (Pinus radiata D.Don) stands (Table 1). In France, 97 egg-batches were collected in nine localities from three regions corresponding to different colonization dates (Fig. 1): -Ré Island, a small island in the Atlantic Ocean situated about 4 km away from mainland France, which is within the historical range (there is data from the 1970s);

-Orléans, in central France, which has been colonized in the 1990s; -Fréhel, in Brittany, which has been newly colonised in the 2010s. In Bulgaria, 109 egg-batches were collected in two 'historical' and two 'newly colonized' localities from two distinct regions (Fig. 2): -Southwest Bulgaria -Struma valley (town of Sandanski) and Ograzhden Mt. (Gega vill.). In that area, the pine processionary moth has been present since 1949, considered historical range.

-Central Bulgaria -in the land of Sladak kladenets vill. In Sredna gora Mt. (the pest was first recorded in 2012) and in the region of Maglizh (newly colonized by T. pityocampa in 2015). Upon collection, all egg batches were transported to the laboratory of entomology of Forest Research Institute in Sofia. The scales of egg-masses were removed, and the samples were analysed according to the protocol described in Tsankov et al. (1996). The egg-batches were put singly in test tubes, covered with cotton stoppers, and kept at room temperature (20-22° C).

During the laboratory studies, the samples were checked five times and the parasitoids that emerged were separated in plastic capsules for identification. During November-December 2018, the eggs without exit holes were dissected, and the meconia and remains of emerged or dead insects were determined under a stereomicroscope (40×). Parasitoids that emerged before collection were determined by their meconia and remains according to [START_REF] Schmidt | Identification by meconia of two egg parasitoids of Thaumetopoea wilkinsoni[END_REF], [START_REF] Tanzen | Identification by meconia of four species egg parasitoids Thaumetopoea pityocampa (Den. & Schiff.) (Insecta Lepidoptera Thaumetopoeidae)[END_REF], Schmidt et al. (1997a) and Tsankov et al. (1996[START_REF] Tsankov | Distribution of egg parasitoids of the pine processionary moth Thaumetopoea pityiocampa (Den. et Schiff.) (Lep., Thaumetopoeidae) in the southwestern region of Bulgaria[END_REF].

The data was analysed using descriptive statistic of MS Excel 2013. The Clopper-Pearson method [START_REF] Hollander | Nonparametric statistical methods[END_REF] was used to compare relative share of the specific B. servadeii in parasitoid assemblages of T. pityocampa in old and newly colonized areas.

Results

Viability of the pine processionary moth in the egg stage

The number of eggs in T. pityocampa batches varied from 185.7±47.7 to 232.2±43.5 in France and from 216.4±41.0 to 251.3±31.3 in Bulgaria (Table 2). In Bulgaria, the average number of eggs in a batch (226.6±43.2) was higher than that obtained from France (194.3±50.1). The highest average egg production per female (251.3±31.3) and the smallest dispersion between samples (204-299 eggs/batch) was recorded in Maglizh. In this locality, the proportion of hatched larvae was almost half (49.8%) of all analysed eggs. A significant share (41.5%) of parasitized and destroyed by predators eggs was registered. In the rest of the localities, the rate of parasitized and destroyed eggs varied between 10.3% (Sladak kladenets) and 19.2% (Sandanski).

The lowest average number of eggs per batch (185.7±47.7) and a large dispersion between samples (55-278) were recorded in the recently infested by the pine processionary moth region of Fréhel. The highest proportion of hatched larvae was established in this region (95.7%), and the rate of eggs parasitized and destroyed by predators was only 2.9%. In the rest of the French localities the impact of parasitoids and predators reached up to 13.6% (Ré Island) and 24.5% (Orléans) (Table 2).

Table 2.

Another factor that also affected the rate of successful hatching was the abortion of eggs, which varied between 1.4% (Fréhel) and 10.8% (Sandanski) (Table 2). The number of dead larvae in the eggs (without exit holes) varied between 0.7% and 2.5% in France, and between 2.1% and 4.7% in Bulgaria. The proportion of sterile, non-fertilized eggs recorded in all samples varied between 0.6% (Fréhel) and 5.0% (Sandanski).

The predatory effect in most localities was negligible (0.02-2.5%) with the exception of Maglizh in Bulgaria (12.5%) (Table 2).

Complex of egg parasitoids in different localities

In this study, the egg parasitoids were the main factor causing pine processionary moth mortality. The mortality rate in France varied between 2.9% (Fréhel) and 24.4% (Orléans) (Table 2). In Bulgaria, the parasitoids affected between 7.8% (Sladak kladenets) and 29.0% (Maglizh) of host eggs.

Four primary egg parasitoids were identified in both France and Bulgaria: Ooecyrtus pityocampae, Baryscapus servadeii, Anastatus bifasciatus, Trichogramma sp. (Table 3).

Table 3.

In a recently colonized area in France (Fréhel), only Trichogramma sp. and a single specimen of O. pityocampae represented the complex of parasitoids (Table 3). In areas of historical range (Orléans and Ré Island), B. servadeii was dominant with 84.7% and 97.3%, respectively. A minor presence of O. pityocampae and Trichogramma sp. was also recorded in them, whereas in Orléans, four specimens of A. bifasciatus were detected.

In the historical range of T. pityocampa in Bulgaria, the most numerous was the polyphagous O. pityocampae in Sandanski (72.1%) and the oligophagous B. servadeii in Gega (75.4%). In the newly colonized sites, the polyphagous parasitoids prevailed. In Sladak kladenets, only O. pityocampae and A. bifasciatus were identified, with a significant prevalence of the former species. In Maglizh, four primary parasitoids were reared from host eggs but the specific species B. servadeii occupied only 2.1% of the parasitoid complex.

In general, B. servadeii is common at all sites where the pine processionary moth has occured for a long time (Ré Island, Orléans, Sandanski, Gega) and it is insignificant in all sites where the pest has penetrated recently (Fréhel, Sladak kladenets, Maglizh) (Table 3). This regularity of B. servadeii distribution has been confirmed in all studied areas. The Clopper-Pearson method determined the confidence interval [59,100] of the percentage of cases in which the rule is confirmed with at a confidence level of 0.95. This suggests that the spread of B. servadeii is related to the old habitats of pine processionary moth and exceptions to this rule are rare (maximum 31%).

The hyperparasitoid B. transversalis was found only in Bulgaria at Sandanski and Gega, both within the historical range of the pest.

Bioecological characteristics of egg parasitoids

A difference in the phenology of O. pityocampae was observed in France and Bulgaria. In samples from Ré Island and Orléans, most of the individuals emerged after collection of egg batches -77.8% and 67.0%, respectively (Тable 4). In contrast, in three Bulgarian localities (Gega, Sladak kladenets and Maglizh), the predominant part of O. pityocampae adults (60.6-73.6%) had emerged before collection of egg batches, and only in Sandanski, most of them (55.8%) emerged after collection. The proportion of established dead O. pityocampae adults in host's eggs was the lowest in Orléans (3.3%) whereas it ranged from 10.2 to 32.2% in Bulgarian localities. The average survival rate of the parasitoid in French and Bulgarian localities was 96.0% and 85.4%, respectively.

Table 4.

High average survival rate of B. servadeii 97.3% was recorded for both countries, and the mortality was low in both French and Bulgarian sites (1.5-3.4%). There was no difference in phenology for this parasitoid in both countries.

The polyphagous A. bifasciatus was established in one locality in France (Orléans) and all studied localities in Bulgaria (Table 3). In Bulgaria, the majority of adults emerged after hibernation. The polyembrionic Trichogramma sp. was found in 9.5-13.2% of T. pityocampa egg batches from the historical range localities in France and Bulgaria, and in 38.5-39.1% from the newly colonized areas (Table 4). A high mortality (up to 70.0%) of adults was observed in the eggs. The hyperparasitoid B. transversalis parasitized 4.8-6.2% of B. servadeii and O. pityocampae. A high mortality (16.7-21.4%) of the hyperparasitoid was recorded in Sandanski and Gega (Тable 4).

In this study, all emerging adults of B. servadeii and O. pityocampae were female, unlike A. bifasciatus where the specimens recorded were only male.

Discussion

Female moths of T. pityocampa usually lay only one egg batch (Douma-Petridou, 1990), and therefore the average number of eggs in batches corresponds to the fertility of the species. The average number of eggs per batch collected in Bulgaria (226.6) was higher than that collected in France (194.3). The fertility of T. pityocampa depends on climatic conditions, host plant quality and population dynamics [START_REF] Masutti | Thaumetopoea pityocampa (Den. & Schiff.) in Italy. Bionomics and perspectives of integrated control[END_REF]. It is higher in the northern parts of its range [START_REF] Mirchev | Egg parasitoids on pine processionary moth Thaumetopoea pityocampa[END_REF] and increases with altitude [START_REF] Özkazans | Cam keseböcegi (Thaumetopoea pityocampa Schiff.) (Lep.: Thaumotopoeidae) 'nin yumurta birakama dayranislari üzerinde incelemeler[END_REF][START_REF] Tiberi | I parassitoidi oofagi di Thaumetopoea pityocampa (Den. & Schiff.) su Pinus halepensis Mill. Nel Gargano, Pugli (Hymenoptera, Chalcidoidea; Lepidoptera, Thaumetopoeidae)[END_REF]. In contrast, [START_REF] Masutti | Thaumetopoea pityocampa (Den. & Schiff.) in Italy. Bionomics and perspectives of integrated control[END_REF] reported lower fertility in colder areas. The biochemical composition of the host plant is one of the significant environmental factors affecting T. pityocampa [START_REF] Carvalho | Caracterização do ataque de Thaumetopoea pityocampa num povoamento de Pinus pinaster por SPME e GC-MS[END_REF][START_REF] Tiberi | The role of the monoterpene composition in Pinus spp., needles in host selection by the pine processionary caterpillar, Thaumetopoea pityocampa[END_REF][START_REF] Moura | Estudo da distribuição espacial da processionária (Thaumetopoea pityocampa) num povoamento de Pinus pinaster[END_REF][START_REF] Roussis | Pine secondary metabolites as perceived by the mother Thaumetopoea pityocampa (Den. & Schiff.) and experienced by the offspring[END_REF]. In mixed stands of Pinus radiata and Pinus pinaster, the pine processionary moth prefers the former species on which caterpillars grow faster, reach a larger size and pupate earlier [START_REF] Buxton | The influence of host tree species on timing of pupation of Thaumetopoea pityocampa Schiff. (Lep., Thaumetopoeidae) and its exposure to parasitism by Phryxe caudata Rond. (Dipt., Larvaevoridae)[END_REF]. The parasitoid assemblage of T. pityocampa and its impact on the pest may also be affected by the identity of the host plant. According to [START_REF] Mirchev | Egg parasitoids on pine processionary moth Thaumetopoea pityocampa[END_REF], the rate of parasitism in different pine species varies greatly between years and localities: in Turkish pine (Pinus brutia Tenore) stands in Turkey, the average parasitism in Köycegiz was 9.8%, but in Iskenderun -34.9%; in Bulgaria, in P. nigra plantations, the parasitism varied from 4.4% (Kurtovo) to 43.7% (Ploski), and in Scots pine (Pinus sylvestris L.) -from 0% (Kyustendil) to 23.5% (Satovcha); in Greece, the lowest and highest rates of parasitism in Aleppo pine (Pinus halepensis Mill.) stands were 13.3% (Fteri-Egio) and 39.6% (Hydra Island), respectively.

Egg parasitoids are considered the most important regulator of T. pityocampa egg stage in both Bulgaria and France, that was found in many previous studies [START_REF] Battisti | Field studies on the behaviour of two egg parasitoids of the pine processionary moth Thaumetopoea pityiocampa[END_REF][START_REF] Biliotti | Les parasities et predateurs de Thaumetopoea pityocampa Schiff. (Lepidoptera)[END_REF][START_REF] Biliotti | Une virose de Thaumetopoea wikinsoni et ses rapports avec la polyédrie cytoplasmatique de T. pityocampa[END_REF][START_REF] Cadahia | Tratamientos de la 'Processionaria del Pino', en la campaña 1963/64[END_REF][START_REF] Ceballos | Predatores de Thaumetopoea pityocampa y posibilidades de utilizacion[END_REF][START_REF] Demolin | Les Ephippigeres (Orthopteres, Tettigonidae) predateurs occasionnels, mails importants de, Thaumetopoea pityocampa Schiff[END_REF][START_REF] Harapin | A note on the parasite complex of Thaumetopoea pityicampa in Yugoslavia[END_REF][START_REF] Jamaâ | Factors of natural death of the forest caterpillar: Thaumetopoea pityocampa Schiff. (Lepidoptera: Thaumetopoeidae) at the egg stage in Morocco[END_REF][START_REF] El-Yousfi | La processionaria del cedro, Thaumetopoea bonjeani (Powell)[END_REF][START_REF] Kitt | Parasitism of egg-batches of the pine processionary moth Thaumetopoea wilkinsoni Tams (Lep., Thaumetopoeidae) in the mountains of Lahav (Israel)[END_REF][START_REF] Tiberi | I parassitoidi oofagi di Thaumetopoea pityocampa (Den. & Schiff.) su Pinus halepensis Mill. Nel Gargano, Pugli (Hymenoptera, Chalcidoidea; Lepidoptera, Thaumetopoeidae)[END_REF][START_REF] Tsankov | Egg parasitoids of the pine processionary moth, Thaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumotopoeidae ) in Bulgaria[END_REF][START_REF] Szeczepaňski | Materialy do znajności pasożytóv jaj korowódki śródziemnomarskiej Thametopoea pityicampa Schiff. (Lepidoptera, Thaumetopoeidaea) w Bulgarii[END_REF]. Percent parasitism is an indirect indicator of parasitoid effects on host populations, although life table studies that consider stagespecific effects of parasitoids and other mortality factors provide a more accurate picture of the role of different parasitoids [START_REF] Van Driesche | The meaning of percentage parasitism revisited: solutions to the problem of accurately estimating total losses from parasitism[END_REF]. In the present study, a higher rate of parasitized eggs was detected at the localities of historical range of the species in France -Ré Island and Orléans, compared to a newly colonized one, Fréhel. In Bulgaria, the host mortality caused by parasitoids in historical localities (Sandanski and Gega) was 19.2-17.3%. In one of the new localities, Sladak kladenets, a typical low parasitism rate was recorded. In contrast, an unusually high parasitism rate (29.0%) was established in the second newly colonized locality, Maglizh. The high value of parasitism in Maglizh is due to the polyphagous O. pityocampae which is associated with a wide range of hemipteran and lepidopteran hosts [START_REF] Noyes | Universal Chalcidoidea Database[END_REF]. As this parasitoid is less specialized and adapted to T. pityocampa (the parasitoid's emergence occurs in late autumn or spring), survival on other hosts is probably responsible for its high impact on the pest.

Baryscapus servadeii and O. pityocampae are widespread across the distribution range of T. pityocampa [START_REF] Mirchev | Egg parasitoids on pine processionary moth Thaumetopoea pityocampa[END_REF]. The oligophagous B. servadeii parasitizes not only pine processionary moth, but also two other species of Thaumetopoea genus -T. bonjeani [START_REF] Demolin | Intensification de la protection phytosanitaire des for ts[END_REF] and T. wilkinsoni [START_REF] Halperin | Natural enemies of Thaumetopoea spp. (Lep., Thaumetopoeidae) in Israel[END_REF]. The number of B. servadeii and O. pityocampae may be limited by different adaptability to environmental conditions. Baryscapus servadeii is more adaptive to thermal stressors [START_REF] Masutti | Ricerche sui parassiti oofagi della Thaumetopoea pityocampa (Schiff.)[END_REF]. It successfully infests T. pityocampa eggs from their deposition to larval hatching whereas O. pityocampae cannot develop after 32 days of host incubation period [START_REF] Halperin | Natural enemies of Thaumetopoea spp. (Lep., Thaumetopoeidae) in Israel[END_REF]. The number of egg parasitoids of T. pityocampa is also influenced by the presence of scales on egg batches [START_REF] Biliotti | Les parasities et predateurs de Thaumetopoea pityocampa Schiff. (Lepidoptera)[END_REF]. This is obviously much more relevant for small-sized parasitoids such as Trichogramma sp. since females cannot overcome the scales and can only parasitize uncovered eggs in the ends of batches [START_REF] Tsankov | Egg parasitoids of the pine processionary moth, Thaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumotopoeidae ) in Bulgaria[END_REF].

The hyperparasitoid B. transversalis was found in Bulgarian samples. It is distributed in Southeast Europe: Albania, Bulgaria, Bosnia and Herzegovina, Greece and Turkey [START_REF] Noyes | Universal Chalcidoidea Database[END_REF]. Another representative of the genus, Baryscapus endemus (Walker, 1839) (syn. Tetrastichus tibialis Kurdjumov, 1913) was reported as a secondary parasitoid in France [START_REF] Biliotti | Les parasities et predateurs de Thaumetopoea pityocampa Schiff. (Lepidoptera)[END_REF]. It was also found on T. pityocampa in Spain [START_REF] Cadahia | Tratamientos de la 'Processionaria del Pino', en la campaña 1963/64[END_REF] and North Macedonia [START_REF] Roques | Climate Warming and Past and Present Distribution of the Processionary Moths (Thaumetopoea spp.) in Europe, Asia Minor and North Africa[END_REF]. B. endemus is a polyphage of many insect hosts from Coleoptera, Hemiptera, Hymenoptera and Lepidoptera orders [START_REF] Noyes | Universal Chalcidoidea Database[END_REF].

Abiotic factors (high temperature, low humidity, etc.) are the main causes for embryonal mortality of T. pityocampa [START_REF] Mirchev | Egg parasitoids on pine processionary moth Thaumetopoea pityocampa[END_REF]. When the egg batches are exposed to direct sunlight, their temperature might rise by 14° C [START_REF] Milani | The temperature of the egg masses of Thaumetopoea pityocampa (Den. & Schiff.) (Lepidoptera, Thaumetopoeidae)[END_REF]. Sterile, unfertilized or empty eggs in T. pityocampa batches were reported in Greece [START_REF] Bellin | Structure, ooparasitoid spectrum and rate of parasitism of egg-batches of Thaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumetopoeidae) in Greece[END_REF], Morocco (Schmidt et al., 1997b), Portugal [START_REF] Mirchev | Parasitism of egg-batches of pine processionary moth Thaumetopoea pityiocampa (Den. et Schiff.) (Lepidoptera: Thaumetopoeidae) collected in Portugal[END_REF] and other countries.

The proportion of eggs destroyed by predators is usually negligible, but in Maglizh, the regulating effect reached high values (>10%). In Bulgaria, Ephippiger ephippiger (Fiebig, 1784) (Orthoptera: Bradyporidae) and Pterolepis germanica (Herrich-Schäffer, 1840) (Orthoptera: Tettigoniidae) were observed to destroy T. pityocampa egg batches (Tsankov et al., 1996b). The first species was reported as a predator of the pine processionary moth in France as well [START_REF] Demolin | Les Ephippigeres (Orthopteres, Tettigonidae) predateurs occasionnels, mails importants de, Thaumetopoea pityocampa Schiff[END_REF]. [START_REF] Halperin | Natural enemies of Thaumetopoea spp. (Lep., Thaumetopoeidae) in Israel[END_REF] reported Monomorium dentigerum (Roger, 1862) (Hymenoptera: Formicidae) and tetigonid species as predators of T. wilkinsoni in Israel, and Monomorium destructor (Jerdon, 1851) (syn. Myrmica gracillima Smith, F., 1861) in Cyprus.

In conclusion, parasitoids were the most important regulating factor of T. pityocampa at the egg stage in both France and Bulgaria. In newly colonized areas, the impact of the parasitoids on the host is significantly lower. This suggests that parasitoids lag behind the expansion of the pine processionary moth. In one of the most important species, O. pityocampae, the temperature conditions and the presence of alternative hosts are essential factors for its effectiveness. The hyperparasitoid B. transversalis caused relatively low mortality (4.8-6.2%) of primary parasitoids at sites within the historical range of T. pityocampa in Bulgaria.

Fig

  Fig. 1.

Fig

  Fig. 2.

Table 1 .

 1

Table 1 .

 1 Main characteristics of sample plots in France and Bulgaria

	Region Ré Island Fréhel Orléans Sandanski Ograzhden Mt. Historical Range Historical Newly colonized Camp de Fréhel Locality Portes-en-Ré Ars-en-Ré St.-Martin-en-Ré N46º11'13.4'' Coordinates Altitude, m France N46º15'11.3'' 14 W01º30'54.0'' N46º13'15.1'' 0 W01º31'49.9'' 14 W01º22'06.8'' N48º39'15.9'' 14 W02º21'22.5'' Camp de Fréhel N48º39'15.9'' 14 W02º21'22.5'' Plurien N48º38'02.5'' 8 W02º24'37.0'' Newly colonized Saran N47º56'58.5'' 121 E01º50'16.0'' Orléans N47º49'22.3'' 108 E01º55'29.5'' Châteaudun N48º03'34.4'' 127 E01º23'39.0'' Bulgaria Historical Sandanski N41º34'22.0'' 325 Е23º17'14.9'' Gega N41º27'09.7'' 856 Е22º59'54.0'' St. Zagora Newly colonized Sladak kladenets N42º24'19.8'' 403 Е25º22'58.5'' Maglizh Newly colonized Maglizh N42º36'08.6'' Е25º30'31.9'' 374	Date of collection 21.11.2017 21.11.2017 21.11.2017 22.11.2017 22.11.2017 22.11.2017 23.11.2017 23.11.2017 23.11.2017 20.09.2017 31.08.2017 28.10.2016 03.10.2017	Egg batches 15 2 4 40 14 10 8 2 2 22 38 36 13	Host tree Pinus nigra Pinus pinaster Pinus pinaster Pinus radiata Pinus pinaster Pinus pinaster Pinus nigra Pinus nigra Pinus nigra Pinus nigra Pinus nigra Pinus nigra Pinus nigra

Table 2 .

 2 Viability of Thaumetopoea pityocampa in egg stage in different localities

			France			Bulgaria
	Parameters	Ré Island	Fréhel	Orléans	Sandanski	Gega	kladenets Sladak	Maglizh
	Generation of PPM	2017	2017	2017	2017	2017	2016	2017
	Total number (n) of egg batches	21	64	12	22	38	36	13
	Larvae died without opening, n (%)	30 (0.7)	84 (0.7)	70 (2.5)	232 (4.7)	242 (2.9)	173 (2.1)	112 (3.4)
	Larvae died with opening, n (%)	9 (0.2)	13 (0.1)	3 (0.1)	49 (1.0)	115 (1.4)	26 (0.3)	35 (1.1)
	Undeveloped eggs with dried-up yolk, n (%)	76 (1.7)	74 (0.6)	72 (2.6)	245 (5.0)	226 (2.7)	149 (1.8)	97 (3.0)
	Eggs totally empty, without any remains, n (%)	7 (0.2)	-	11 (0.4)	4 (0.1)	99 (1.2)	26 (0.3)	41 (1.3)
	Total number of aborted eggs, n (%)	122 (2.8)	171 (1.4)	156 (5.6)	530 (10.8)	682 (8.3)	374 (4.5)	285 (8.7)
	Number of larvae hatched, n (%)	3642 (83.6)	11198 (95.7) 1947 (69.8) 3452 (70.0) 6120 (74.4) 7052 (85.2) 1626 (49.8)
	Impact of parasitoids, n (%)	593 (13.6)	332 (2.9)	681 (24.4)	947 (19.2) 1392 (16.9)	650 (7.8)	947 (29.0)
	Impact of predators, n (%)	1 (0.02)	2 (0.02)	3 (0.1)	-	32 (0.4)	203 (2.5)	409 (12.5)
	Total number of eggs, n (%)	4358 (100)	11703 (100) 2787 (100) 4929 (100) 8226 (100) 8279 (100) 3267 (100)
	Range of eggs per egg batches	106-287	55-278	172-305	105-284	94-277	92-306	204-299
	Mean eggs per egg batches±Std	207.5±50.5	185.7±47.7	232.2±43.5 224.0±41.8 216.4±41.0 230.0±48.8 251.3±31.3

Table 3 .

 3 Percentage of egg parasitoids of Thaumetopoea pityocampa in different localities

	France
	Parasitoids

Table 4 .

 4 Characteristics of egg parasitoids of T. pityocampa in different localities

	Species and parameters	France

Acknowledgements

This study was supported by RILA Project DTNS France 01/8/09.05.2017 'Standardizing Automated large-scale Monitoring' to understand atypic phenologies at both ends (France and Bulgaria) of the climate-driven expansion front of a European urticating pest, the Pine Processionary Moth (SAM3PM) financed by the National Scientific Fund. The research was funded by the French Ministry of Europe and Foreign Affairs (MEAE) and the French Ministry of Higher Education, Research and Innovation (MESRI).

Funding

This work was supported by the Bulgarian National Science Fund (RILA Project DTNS France 01/8/09.05.2017), the French Ministry of Europe and Foreign Affairs (MEAE) and the French Ministry of Higher Education, Research and Innovation (MESRI).


domain: spi sdv

text:
Roche

Y Gillot

Implementing hydrocyclones at existing plants -A need to revisit historical design & operation guidelines

Keywords: activated sludge densification, continuous flow system, design rules

The impact of sludge densification was studied through side-by-side full-scale experiments on the WRRF of Dijon (France). The comprehensive analysis of the results pointed out a need to substantially change the rules usually used to size and operate activated sludge systems equipped with hydrocyclones. Among others, the maximum clarifier surface overflow rates and the apparent sludge retention times for complete nitrification need modification. The impact on main equipment (pumping and mixing power, oxygen transfer efficiencies) remains to be evaluated.

Background and objectives

Among the possibilities to improve sludge settleability in activated sludge systems, selective wasting of flocs by means of hydrocyclones has been demonstrated to be a very promising candidate [START_REF] Regmi | Impact of Hydrocyclone based wasting on settling characteristics and nutrient removal[END_REF][START_REF] Willoughby | Wasting through the spin cycle: a full-scale demonstration of WAS hydrocyclones to provide capacity improvements[END_REF]Avita et al., 2021;[START_REF] Roche | Biological process architecture in continuous-flow activated sludge by gravimetry: Controlling densified biomass form and function in a hybrid granule-floc process at Dijon WRRF, France[END_REF]. This cost effective solution has shown its capacity to improve process stability and for process intensification.

Hydrocyclones technology consists of imposing a gravimetric selection pressure to retain in the system dense flocs and granules. To this aim, hydrocyclone skids are installed on the wasted sludge line. Denser material contained in the underflow of the hydrocyclones are sent back to the biological reactor, whereas the overflow containing poor settling sludge is directed to the sludge line.

This paper scrutinises the performance obtained on a full-scale implementation of hydrocyclones in one of the four 100 000 p.e. secondary treatment trains of the Dijon WRRF. The main objectives were to identify potential impacts on the operation of the plant, in order to point out the modifications of classical design and operation rules.

Material & methods

During the period under study, the Dijon WRRF (400 000 p.e.) was operated at 44% of its design organic load. One of the biological trains was equipped with a skid of 8 hydrocyclones (10 m 3 /h) fed with a portion of return activated sludge.

From October 2019 to June 2021, the performance of this (test) train was compared to the one of a conventional activated sludge (control) train. As detailed in [START_REF] Roche | Biological process architecture in continuous-flow activated sludge by gravimetry: Controlling densified biomass form and function in a hybrid granule-floc process at Dijon WRRF, France[END_REF], main influent and effluent physicochemical parameters were measured (e.g. COD, BOD5, TSS, N, P), and the sludge was characterised in terms of settleability and particle size distribution.

Results and discussion

Sludge settleability. As shown in Figure 1, the implementation of the cyclones helped to maintain a constant and low dSVI, even during the winter period usually associated with hampered sludge settling due to growth of filamentous bacteria (as seen for the control train). In addition to stabilising this important operation parameter, enhancing the sludge settleability allowed to reduce the return flow (RAS) down to 40 % of the inflow. The drastic reduction of dSVI may allow to increase the maximum clarifier SOR well above the usual 0.6-0.8 m 3 /m 2 /h.

Accumulation of inorganic matter. The mass split of ISS is presented in Table 1 for three different stabilised periods (loading rate doubled in comparison to test train during Periods 2&3). As seen in Figure 2, the sludge of the densified train became less organic than the control one. Nevertheless, ISS accumulation stabilised after a few months of operation.

Apparent vs weighted SRT. In order to specify the functioning of the system, the sludge retention times of the granular biomass (fraction > 200 µm) and of the flocculent biomass were determined (Figure 3). A weighted SRT was calculated from the granular and flocculent SRTs, weighted by the respective mass fraction of granular and flocculent sludge in the bioreactor. This weighted SRT may be considered to determine nitrification limits (Figure 3 and Table 1).

Sludge viscosity. Preliminary measurements (Figure 4) indicate a different rheological behaviour of the densified sludge in comparison to the control sludge. This in turn can have an impact on pumping and mixing power, as well as on aeration performances that are currently under investigation.

Conclusion and significance

The main impacts of the implementation of the hydrocyclones are schematised in Figure 5. Among the design and operation practices that require modifications, the increase of the maximal clarifier surface overflow rate is the most obvious one, while redefining mixing power and aeration demand calls for further investigations.

Figure 1 Figure 2

 12 Figure 1 dSVI in the conventional activated sludge (control) train and in the densified (test) train Figure 2 MLVSS/MLSS and ISS variations in the conventional activated sludge (control) train and in the densified (test) train

Figure 3 Figure 4 Figure 5

 345 Figure 3Granular and flocculated SRTs. The apparent SRT corresponds to the usual SRT. The weighted SRT is calculated from the granular and flocculated SRTs, weighted by their corresponding fractions of sludge.

Table 1

 1 Flow and mass split along the studied period

		F/M ratio	Ratio weighted SRT/	Flow split	ISS flux split
		kg BOD5/kg VSS/d	apparent SRT	(UF/OF)	(UF/OF)
		Control	Test
	Period 1	0.07	0.09	1.08	18/82	51/49
	Period 2	0.05	0.10	1.22	17/83	67/33
	Period 3	0.06	0.11	1.42	20/80	73/27


Fruit industry in France (Lamine et al. 2015) A territorial approach to agrifood systems agroecological transitions

The territorial scale …  Allows to consider food systems' initiatives, components and actors in their diversity  Scale of direct interactions between ecological and social processes, and at which these could be articulated in order to support the reconnection of agriculture, food, environment, and health  Scale of shared responsibility and mutual commitments towards a common future/community of fate

• montaneous region

• a diversified agriculture with some niche productions (chestnut, wine)

• the decline of a formerly successful sector (fruit production) for "lack of competitiveness"

A multi-actors action-research in order to build a collective understanding of transition mechanisms

Collective multi-actors work on the past and current trajectory

  Usually sociologists are asked what are farmers' motivations for (transition) adoption of new techniques or models? … from the scale of agricultural practices to that of the wider agrifood system Lock-ins and path-dependency effects Intensification trajectories -> need to combine a socio-historical analysis of lock-in mechanisms and a comprehensive analysis of change mechanisms at both scales of farmers and

  To address transitions, need for a systemic but also a critical perspective, looking at power reconfigurations  In an action-research perspective, need to include not only representative but also concerned actors  Combine localised approaches and larger translocal exchange networks (bringing together academic and practicioners)

	Transitions rely on power Conclusion
		reconfigurations in the
		agrifood system
	Analysing key public
	policies at different
	scales over time
	Alternative
	Mapping the diversity initiatives
	of initiatives and networks
	can influence the « system » when
	they simultaneously act on its different components	MERCI
			Lamine C., Bui S., Ollivier
	Identification of key mechanisms Eg. lasting role of neo rurals in the	G., 2015. Pour une approche systémique non réductionniste de la
	AE transition (OF, marketing etc.)	transition écologique des
	With agroforestry as an attractive model for them today (also recognised by the AE policy)	systèmes agri-alimentaires, Cahiers de recherche sociologique, 58, 95-117

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