Pierre Albina
email: pierre.albina@ouàook.fr

Nadège Dm Ban

Alexandra Bertron
email: bertron@insa-toulouse.fr

Achim Albrecht C ' ' ' '

Jean-Charles Robinet

Benjamin Erable
email: benjan1in.erable@toulouse-inp.fr

Nitrate and nitrite bacterial reduction at alkaline pH and high nitrate concentrations, comparison of acetate versus dihydrogen as electron donors

Denitrification

Activated sludge Heterotrophy Hydrogenotrophy pH modelling Nitrite inhibition This study assesses bacterial denitrification at alkaline pH, up to 12, and high nitrate concentration, up to 400 mM. Two types of electron donors organic (acetate) and inorganic (dihydrogen) were compared. With both types of electron donors, nitrite reduction was the key step, likely to increase the pH and lead to nitrite accumulation. Firsày, an acdin1ation process was used: nitrate was progressively increased in three cultures set at pH 9, 10, or 11. This method allowed to observe for the first tinle nitrate reduction up to pH 10 and 100 mM nitrate with dihydrogen, or up to pH 10 and 400 mM nitrate with acetate. Ni trate reduction kinetics were faster in the presence of acetate. To investigate further the inlpact of the type of electron donor, a transition from acetate to dihydrogen was tested, and the pH evolution was modelled. Denitrification with dihydrogen strongly increases the pH while with acetate the pH evolution depends on the initial pH. The main difference is the production of acidifying CO 2 during the acetate oxidation. Finally, the use of long duration cultures with a highly alkaline pH allowed a nitrate reduction up to pH 11.5 with acetate. However, no reduction was possible in hydrogenotrophy as it would have increased the pH further. Instead, bacteria used organic marrer from inoculum to reduce nitrate at pH 11.5. Therefore, considering bacterial denitrifi cation in a conrext of alkaline pH and high nitrate con centration an organic electron donor such as acetate is advantageous.

Introduction

Nitrate pollution is a global environmental issue in which industiial effluents represent a major source of pollution. Bacterial deniti•ification may be the most economical and efficient way to deal with these ef fluents compared with other physicochemical nitrate removal tech niques [START_REF] Kapoor | Nitrate removal from drinking water-review[END_REF][START_REF] Mohsenipour | Removal techniques of nitrate from water[END_REF]. Bacterial deniti•ification is usually studied for nitrate removal in wastewater ti•eatment context, where authors seek to optimize the per formance of denitrification working at neuti• al pH values and relatively moderate niti • ate concentrations (0.1-10 mM). However, some indus niai wastes are characterized by higher pH and nitrate concenti•ation ranges. For exan1ple, in a radioactive waste reposito1y, niti•ate concen ti• ations are expected to reach 1000 mM [START_REF] Francis | Biological denitrification of high-nitrates wastes generated in the nuclear industry[END_REF][START_REF] Glass | Denitrification of high-nitrate, high-salinity wastewater[END_REF]. Other effluents, such as those produced dming stainless steel production or the explosives industiy, may contain several tens (Femândez-Nava et al., 2008) to hundreds of mM of niti• ate [START_REF] Marecik | Phytoremediation of industrial wastewater containing nitrates, nitroglycerin, and nitroglycol[END_REF]. Nitrate pollution may also occur in alkaline en vironments. Effluents from a stainless steel factory may have a pH of 9.6 (Femândez-Nava et al., 2008). In the context of radioactive waste re positories, alkaline pH between 9 and 13, generated by the presence of cementitious materials, are expected [START_REF] Albrecht | Microbial catalysis of redox reactions in concrete cells of nuclear waste repositories: a review and introduction[END_REF][START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF]Bertren et al., 2014;Durban et al., 2018). In addition, the question of the electron donor type (organic or inorganic) reducing ni ti•ate is relevant when considering industi•ial contexts. Usually, in do mestic wastewater ti•eatment processes, the presence of organic matter provides electi• ons for nitrate reduction. However, in some industiial context, the organic matter could be missing, leaving inorganic com pounds as the only alternative for electi• on source. For instance, in a radioactive waste reposito1y, dihydrogen generated in situ and/or organic acids are expected depending on the waste type [START_REF] Gales | Molecular hydrogen from water radiolysis as an energy source for bacterial growth in a basin containing irradiating waste[END_REF]Grebliunas and Peny, 2016;Libeit et al., 2011;Pedei•sen, 1997).

Bacterial denitiification is a respiration process in which niti•ate is reduced to niti• ite, nitric oxide, niti• ous oxide and finally diniti• ogen. The (1)

2 NO 3 + 5 H 2 → 4 H 2 O + N 2 + 2 OH
(2)

These two particular electron donors were compared because they are widely used in bacterial denitrification contexts [START_REF] Dhamole | Denitrification of high strength nitrate waste[END_REF][START_REF] Fernández-Nava | Denitrification of wastewater containing high nitrate and calcium concentrations[END_REF][START_REF] Karanasios | Hydrogenotrophic denitrification of potable water : a review[END_REF], and are likely to be present in the industrial contexts mentioned above. Usually, denitrification is studied with organic electron donors to optimize nitrate removal yields. Indeed, during denitrification with dihydrogen (hydrogenotrophic metabolism), slower growth and nitrate reduction kinetics are generally observed [START_REF] Albina | Influence of hydrogen electron donor, alkaline pH, and high nitrate concentrations on microbial denitrification: a review[END_REF][START_REF] Ergas | Hydrogenotrophic denitrification of drinking water using a hollow fibre membrane bioreactor[END_REF][START_REF] Mateju | Biological water denitrification -a review[END_REF]. Authors explain it with: (i) the low solubility of dihydrogen which makes it less accessible, (ii) the need for hydrogenotrophic (autotrophic) bacteria to assimilate inorganic carbon [START_REF] Chang | Hydrogenotrophic denitrification with immobilized Alcaligenes eutrophus for drinking water treatment[END_REF][START_REF] Ghafari | Improvement of autohydrogenotrophic nitrite reduction rate through optimization of pH and sodium bicarbonate dose in batch experiments[END_REF][START_REF] Liu | Study on Heterotrophic-Autotrophic Denitrification Permeable Reactive Barriers (HAD PRBs) for in Situ Groundwater Remediation[END_REF]. In addition, alkaline pH and high nitrate concentration also strongly affect bacterial denitrification. High nitrate concentration inhibits bacterial activity [START_REF] Banihani | Nitrate and nitrite inhibition of methanogenesis during denitrification in granular biofilms and digested domestic sludges[END_REF][START_REF] Dhamole | Denitrification of high strength nitrate waste[END_REF][START_REF] Glass | Denitrification of high-nitrate, high-salinity wastewater[END_REF]. As a result, only a few studies have evaluated bacterial denitrification in environments with nitrate concentrations greater than 100 mM [START_REF] Denariaz | A halophilic denitrifier, Bacillus halodenitrificans sp[END_REF][START_REF] Dhamole | Denitrification of high strength nitrate waste[END_REF][START_REF] Miao | Metagenomic insights into salinity effect on diversity and abundance of denitrifying bacteria and genes in an expanded granular sludge bed reactor treating high-nitrate wastewater[END_REF][START_REF] Napier | In-situ biodenitrification of the S-3 ponds[END_REF]. High nitrate concentrations tend to cause considerable nitrite accumulations [START_REF] Albina | Influence of hydrogen electron donor, alkaline pH, and high nitrate concentrations on microbial denitrification: a review[END_REF]. Nitrite has been described as an inhibitor of bacterial activity with known toxicity [START_REF] Baumann | Inhibition of denitrification activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH[END_REF][START_REF] Cua | Effects of nitrite on ammonia-oxidizing activity and gene regulation in three ammonia-oxidizing bacteria[END_REF][START_REF] Yarbrough | Bacterial inhibitory effects of Nitrite : inhibition of active transport , but not of group translocation , and of intracellular enzymes[END_REF]. Nitrite could, for instance, form free radical compounds with oxygen within cells [START_REF] Richardson | The Prokaryotic Nitrate Reductases[END_REF][START_REF] Titov | Nitrite -Catalase Interaction as an Important Element of Nitrite Toxicity[END_REF][START_REF] Van Der | Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite: a potential additional mechanism of nitric oxide-dependent toxicity[END_REF]. The pH generally affects the activity of enzymes; the pH limits tolerated by bacteria are between 11.5 and 12 [START_REF] Albina | Adaptation of neutrophilic Paracoccus denitrificans to denitrification at highly alkaline pH[END_REF][START_REF] Janto | The genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4[END_REF][START_REF] Shapovalova | Heterotrophic denitrification at extremely high salt and pH by haloalkaliphilic Gammaproteobacteria from hypersaline soda lakes[END_REF][START_REF] Sorokin | Is there a limit for high-pH life?[END_REF]. In alkaline medium, denitrifying activity slows down and nitrite accumulation is generally observed [START_REF] Albina | Influence of hydrogen electron donor, alkaline pH, and high nitrate concentrations on microbial denitrification: a review[END_REF][START_REF] Tang | A pH-control model for heterotrophic and hydrogen-based autotrophic denitrification[END_REF][START_REF] Vasiliadou | A kinetic study of hydrogenotrophic denitrification[END_REF].

Accordingly, the management of wastes and effluents in an environment that combines high pH and high nitrate concentration is problematic, the bacterial activity will likely be severely slowed down. The issue is even more challenging when there is only an inorganic source of electron donors. As a result, the review of the literature revealed a lack of information regarding bacterial denitrification at alkaline pH or with high nitrate concentrations, and this lack is even greater when both conditions are considered simultaneously. To date, denitrification has not yet been tested for a combination of pH above 9 and nitrate concentration above 120 mM [START_REF] Albina | Influence of hydrogen electron donor, alkaline pH, and high nitrate concentrations on microbial denitrification: a review[END_REF]. However, the nitrate depollution issue needs to be addressed even in a difficult environment. For instance, high nitrate concentrations could impact the speciation and mobility of radionuclides in radioactive waste [START_REF] Albrecht | Microbial catalysis of redox reactions in concrete cells of nuclear waste repositories: a review and introduction[END_REF][START_REF] Francis | Biological denitrification of high-nitrates wastes generated in the nuclear industry[END_REF].

Therefore, the objective of this work is to evaluate the bacterial denitrification possibilities in environments characterized simultaneously by alkaline pH and high nitrate concentrations. Denitrification was assessed by monitoring nitrate and nitrite reductions. The experiments were carried out with either organic acetate or inorganic dihydrogen. A new approach, based on predictive pH modelling allowed to assess the strong impact of the electron donor type on the pH evolution. To maximize the probability of observing bacterial activity in this context, long culture times and acclimation procedures (gradual increases in pH or nitrate concentration) were used [START_REF] Albina | Adaptation of neutrophilic Paracoccus denitrificans to denitrification at highly alkaline pH[END_REF]. Activated sludge from urban wastewater treatment plant was used to match with industrial contexts and to maximize the inoculum survivability. Indeed, activated sludge communities can adapt and reduce nitrate at high pH [START_REF] Durban | Nitrate and nitrite reduction activity of activated sludge microcosm in a highly alkaline environment with solid cementitious material[END_REF], high nitrate concentrations [START_REF] Miao | Metagenomic insights into salinity effect on diversity and abundance of denitrifying bacteria and genes in an expanded granular sludge bed reactor treating high-nitrate wastewater[END_REF] and in hydrogenotrophy [START_REF] Rezania | Kinetics of hydrogen-dependent denitrification under varying pH and temperature conditions[END_REF].

Materials and methods

Reactor medium

The reactor medium was cement leachate, a mineral medium representative of an aqueous system in a concrete dominated environment as in a radioactive waste repository. The cement leachate was manufactured from crushed, hydrated CEM V/A cement pastes. One gram per litre of crushed cement was poured into water and mixed at 900 rpm for 24 h. The cement leachate was filtered and then stored in hermetic bottles saturated with dinitrogen. The average composition of the cement leachate is shown in Table 1.

In the heterotrophic reactors, sodium acetate was added about three times a week to maintain its concentration at 20 mM. In the hydrogenotrophic reactors, dihydrogen (1 bar) was bubbled at 80 mL min -1 . The leachate was supplemented with sodium nitrate at various concentrations (10-400 mM) and 50 mM sodium bicarbonate as a pH buffer (and as the inorganic carbon source for hydrogenotrophic bacteria). The pH was adjusted by additions of NaOH and HCl (10 M) to the reactors.

Inoculum

Activated sludge was collected in the aeration basin of the wastewater treatment plant at Castanet near Toulouse, France. The collected sample was characterized by a nitrate concentration of 0.7 ± 0.4 mM, a pH of 7.6 ± 0.01, and 6.5 ± 1.5 g/L of suspended solids. The sample was centrifuged (4610 g, 10 min, 25 • C), then 1 g/L of the activated sludge was added to the fresh medium to initiate precultures. The precultures were carried out for about a week to adapt the bacteria to cement leachate at pH adjusted to 9-10 and nitrate utilization (10-50 mM) with acetate or dihydrogen depending on the experiment that was to follow. The reactors were then inoculated with a 50/50 (v/v) mixture of preculture and fresh cement leachate.

Reactor set-up

The reactors were glass bottles (volume 1 L) agitated at 300 rpm (magnetic stirrer) and maintained at a temperature of 30 • C. They were kept anaerobic by a constant flow of dinitrogen (1 bar, 80 mL min -1 ) in heterotrophy and of dihydrogen (1 bar, 80 mL min -1 ) in hydrogenotrophy. The sequential batch reactors were operated in successive stages: a first batch period, during which nitrate reduction was monitored, and a second period of partial renewal by fresh medium 50/50% (v/v) containing a higher concentration of nitrate. These two periods were repeated in cycles. During each stage of medium renewal, the agitation was stopped for a few hours before changing the medium. This operation allowed to keep the majority of the biomass decanted in the bottom volume of the reactor. The majority of experiments were conducted in sequential batch reactors and were therefore subdivided into several batch periods; others were conducted with only one batch period. All the experimental set-ups are summarized in Table 2. The experiments involving dihydrogen had shorter duration for operational use of organic versus inorganic matter to provide electron for nitrate reduction results in fundamental bacterial activity differences. For instance, the reactions with organic acetate and inorganic dihydrogen are presented in reactions (1) and ( 2) respectively. Acetate is a potential source of electrons and inorganic carbon. Furthermore, during denitrification with acetate (heterotrophic metabolism) there is a generation of CO 2 which has a strong impact on pH. • SBR stands for sequential batch reactors. and safety reasons.

Analycical cechniques 2.4.1. pH monicoring

The pH was rnonitored using FisherBrand gelled electrolyte probes connected to a Consort data acquisition system (mode! C3060). co�

and HCO 3 concenn• ations were detemlined together by tin• ation with hydrochloric acid (HCI).

Biomass analysis

Bacte1ial poptùation diversity was analysed by sequencing 16S RNA. At the end of the experirnent, the biomass was centrifuged (4610 g, 10 IllÎn, 4 °C), the total DNA present in the solid was extracted using QIAGEN DNeasy PowerBiofilm kits. The sarnples were sent to the Research and Testing Laboratory (RTL, Texas, USA), where the DNA was sequenced by PCR with the bacterial primers (515F GTGCCAGCMGCCGCGGTAA -806R GGACTACHVGGGTWTCTAAT) according to RTL protocols. Subsequent data analyses of the DNA quality, DNA sequence alignment, clustering in operational taxonomie units, and assignment were also perfom1ed by RTL according to their

Chemical analysis

After filtration at 0.2 µm by cellulose acetate filters (Minsisart PES, Fisher Scientific) acetate, nitrate, and nitt• ite concentrations were determined by high-precision ion chromatography (Dionex ICS-2000 and ICS-3000); the method is described in [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF].

Results and discussions

3.1. Denirrificari. on at high nicrace concencrari. on and alkaline pH

Deniuificalion in the presence of acemce

This experirnent was carried out in three sequential batch reactors set at pH 9, 10 and 11. Three successive batch periods (vertical lines in Fig. 1) allowed to increase the initial 50 mM nitrate conceritration to 200 and 400 mM respectively on day 14 and 49. At the start of each batch period, the pH was readjusted to 9, 10 or 11. In Fig. 1 (A), the coloured numbers indicate the average nitrate reduction kinetics in mM.d-1 , in Fig. 1 (B), they indicate the nitrite concentration that was reduced during each batch period as calculated from equation ( 3).

(3)

Drning the batch period at 50 rnM nin• ate, the reactor initially set at pH 9 had higher nitrate reduction rate. However, its nitrite reduction was low: only 15 mM of nittite was reduced building a 40 mM nin'ite accumulation, Fig. 1 (B). NB: The non-zero nicrice concencralions observed at the beginning of each bacch period were caused by the possible presence of nicrice in the inoculum In reactors at pH 10 and 11, the nitrate was not fully consurned, therefore the nitrite accumulation was lower.

Regarding the pH, it decreased in both reactors at initial pH 10 and 11, while it decreased then increased in the reactor at initial pH 9. Thus, the pH evolution was dependant of the initial pH. During the batch period at 200 rnM nitrate, nitrate was reduced at the sarne rate regardless of the initial pH. The higher nitrite accumulation in the reactor at pH 9 during the former batch period probably led to a subsequent slowdown in bacterial activity. While the reactor at initial pH 11.0 acidified to 9.9, which could explain why the nitrate reduction kinetics in the reactor at pH 11 was not very different frorn those of reactors at initial pH 9 and 10. In the batch period at 400 mM nitrate, nitrate was reduced at the (B) same rate at initial pH 9 or 1 O. Nitrite was only reduced in the reactor at initial pH 9. Jt is difficult to draw a definitive conclusion at pH 11 given the ve1y low concentration of nitrate reduced compared to the initial concentration. Nitrate reduction is therefore possible at least up to pH 10 and 400 mM nitrate. High nitrate concentrations slow down bacteiial activity [START_REF] Bollag | Effects of nitrite toxicity on soil bacteria aerobic and anaerobic conditions[END_REF][START_REF] Dhamole | Denitrification of highly alkaline nitrate waste using adapted sludge[END_REF][START_REF] Glass | Inhibition of denitrification in activated sludge by nitrite[END_REF][START_REF] Glass | Denitrification of high-nitrate, high-salinity wastewater[END_REF]. Among other things, high nitrate concentration seems to generate nitrite accumulation. Nitrite can inhibit bacterial activity at relatively low concentrations, of the order of 10 mM [START_REF] Albina | Influence of hydrogen electron donor, alkaline pH, and high nitrate concentrations on microbial denitrification: a review[END_REF][START_REF] Bollag | Effects of nitrite toxicity on soil bacteria aerobic and anaerobic conditions[END_REF][START_REF] Yarbrough | Bacterial inhibitory effects of Nitrite : inhibition of active transport , but not of group translocation , and of intracellular enzymes[END_REF]. For example, nitrite can form cytotoxic free-radical species or block various metabolic activities [START_REF] Van Spanning | Introduction to the biochemistry and molecular biology of denitrification[END_REF][START_REF] Yarbrough | Bacterial inhibitory effects of Nitrite : inhibition of active transport , but not of group translocation , and of intracellular enzymes[END_REF]. In these experiment during the batch period at 400 mM nitrate, the nitrite accumulation increased from 40 to 80 mM which could explain the low nitrate reductions. In the reactor at pH 9, the ni trite accumulation in the first batch period slowed the subsequent ni trate reduction in the second batch period. This can explained by a denitrification reg ulation in order to limit internai accumulations of nitrite [START_REF] Chen | Denitrification and aerobic respiration, hybrid electron transport chains and co-evolution[END_REF][START_REF] Van Spanning | Introduction to the biochemistry and molecular biology of denitrification[END_REF]. A genetic regulation by transcription factors of the fumarate and nitrate reductase regulatoiy family has been observed in bacteria (Arai et al., i 1997;[START_REF] Crack | Biochemical properties of Paracoccus denitrificans FnrP : reactions with molecular oxygen and nitric oxide[END_REF][START_REF] Kuroki | Fine-tuned regulation of the dissimilatory nitrite reductase gene by oxygen and nitric oxide in Pseudomonas aeruginosa[END_REF]Silvestini et al., 1994). Various forms of metabolic regulation also have been observed. For example, competition for electrons between denitrification reductases was shown (Komaros et al., 1996;[START_REF] Kucera | The influence of pH on the kinetics of dissimilatory nitrite reduction in Paracoccus denitrificans[END_REF][START_REF] Thomsen | Mass spectrometric studies of the effect of pH on the accumulation of intermediates in denitrification by Paracoccus denitrificans[END_REF]. The nitrate reduction could also be controlled by the bacterial population evolution. In bacterial communities, rather than considering an uniform reaction, the denitrification is segmented by partially deni trifying bacteria that share the reduction steps between them [START_REF] Lycus | Phenotypic and genotypic richness of denitrifiers revealed by a novel isolation strategy[END_REF][START_REF] Roco | Modularity of nitrogen-oxide reducing soil bacteria: linking phenotype to genotype[END_REF]. Thus, in denitrifying communities, rather than performing ail four stages of denitrification, most bacteria will express only one or a few reduction steps. Since nitrite accumulation is a key parameter in denitrification, several authors have simply catego rized the bacterial communities into two types: "nitrate respiring" bac teria that only reduce nitrate to nitrite and cause nitrite accumulation, and "true denitrifier" bacteria that are capable of reducing nitrate and nitrite to dinitrogen [START_REF] Dhamole | Denitrification of high strength nitrate waste[END_REF][START_REF] Glass | Denitrification kinetics of high nitrate concentration Water : pH effect on inhibition and nitrite accumulation[END_REF][START_REF] Liessens | Mixed culture hydrogenotrophic nitrate reduction in drinking water[END_REF][START_REF] Szekeres | Microbial population in a hydrogen-dependent denitrification reactor[END_REF][START_REF] Wilderer | Competition in denitrification systems affecting reduction rate and accumulation of nitrite[END_REF]. Growth kinetics observed for "nitrate respiring" bacteria can be three times higher than for "true denitrifier'' bacteria [START_REF] Turk | Benefits of using selective inhibition to remove nitrogen from highly nitrogenous wastes[END_REF]. Consequently, it is generally reported that, as long as nitrate is present, nitrate respiring bacteria dominate the community and nitrite accu mulation occurs. When nitrate becomes limiting, true denitrifier bacte ria dominate the population and nitrite accumulation disappears [START_REF] Glass | Denitrification kinetics of high nitrate concentration Water : pH effect on inhibition and nitrite accumulation[END_REF]. Therefore, in the reactor at pH 9 during batch phase at 50 mM nitrate, it was assumed that at day 7 the nitrite accu mulation and nitrate limitation caused an evolution of the denitrifying community towards "true denitrifier'' bacteria. In the following batch period at 200 mM, the lower proportion of "nitrate respiring" bacteria in the reactor at initial pH 9 finally slowed the reduction of nitrate.

�9 v� •• •• •• ....... . .. •-

Deniuification in the presence of dihydrogen

In this dihydrogen-fed experiment, the initial 10 mM nitrate con centration was increased to 50 mM on day 5 and then to 100 mM on day 19, in three sequential batch reactors at pH 9, 10 or 11, Fig. 2. In the reactor set at pH 11, acetate was added on day 75 in an attempt to restart bacterial activity.

In the batch period at 10 mM nitrate, in the reactors at initial pH of 9 and 10, the nitrate was completely reduced, Fig. 2 (A). In the reactor at initial pH of 11, only 5 mM nitrate was reduced. Nitrate and nitrite reduction kinetics were correlated to the initial pH of the reactor. In the batch period at 50 mM nitrate, nitrate was completely reduced at pH 9 with reduction kinetics comparable to those for heterotrophy under the same conditions. Nitrite was only reduced in the reactor at pH 9. At pH 10, the reduction of nitrate slowed down. At pH 11, nitrate was not reduced at ail. During the batch period at 100 mM, the nitrate reduction was still going on at pH 9 and 10. Nitrate reduction was therefore possible up to pH 10 and 100 mM nitrate in hydrogenotrophy. No bacterial reduction was going on at pH 11 and higher nitrate concen tration than 10 mM. However, the addition of acetate into the reactor at pH 11 on day 75 initiated bacterial activity: 40 mM nitrate was suddenly reduced and nitrite accumulated. Analyses of the bacterial communities were carried out on the final biomass of the three reactors. In reactors at pH 9 and 10, the genus Thauera sp., including several hydrogenotrophic denitrifiers species, was oveiwhelmingly represented [START_REF] Fida | Biodegradation of isopropanol and acetone under denitrifying conditions by Thauera sp. TK001 for nitrate-mediated microbially enhanced oil recovery[END_REF][START_REF] Macy | Thauera selenatis gen. Nov., sp. nov., a member of the beta subclass of proteobacteria with a novel type of anaerobic respiration[END_REF]. However, in the reactor at initial pH 11, the final addition of acetate resulted in the dominance of a heterotrophic denitrifying bacteria: Halomonas sp., [START_REF] Alquier | Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals[END_REF][START_REF] Rafrafi | Use of a continuous-flow bioreactor to evaluate nitrate reduction rate of Halomonas desiderata in cementitious environment relevant to nuclear waste deep repository[END_REF]. Therefore, heterotrophic denitrification was advantageous at pH 11. This conclusion can be explained by the pH evolution. lndeed in reactors at pH 9 or 10, regular manual addition of HCl was necessary to readjust the pH which was constantly increasing. These additions are indicated by the pH steps (dotted lines) in Fig. 2 (B). ln the reactor at initial pH 11, the pH remained constant as bacterial activity was low.

After the addition of acetate, the medium quickly acidified to pH 10.2 during nitrate reduction. There was an opposite pH evolution as soon as the denitrification used acetate as the electron donor. lt is quite clear that in these alkaline pH ranges, the more the pH is acidified, the more the bacterial activity is facilitated.

According to these results, hydrogenotrophic denitrification seems to tolerate lower pH and nitrate concentration compared to heterotrophic denitrification. Studies on heterotrophic denitrification tested pH from 7.5 to 9.5 and nitrate concentrations from 10 to 100 mM and observed high nitrate reduction rate around 60-600 mM d-1 (Fernândez-Nava et al., 2008;[START_REF] Glass | Denitrification kinetics of high nitrate concentration Water : pH effect on inhibition and nitrite accumulation[END_REF][START_REF] Thomsen | Mass spectrometric studies of the effect of pH on the accumulation of intermediates in denitrification by Paracoccus denitrificans[END_REF]. Sorne specific operating conditions, such as biomass concentration, even allowed nitrate reduction kinetics to reach 5000 mM d-1 [START_REF] Kucera | The influence of pH on the kinetics of dissimilatory nitrite reduction in Paracoccus denitrificans[END_REF]. ln this present study, the nitrate reduction rates were globally lower. Experimental conditions, designed to approach the context of radioactive waste repositories, such as the low density of the inoculum, or the poor cementitious medium, slowed down the nitrate reduction rates. Under hydrogenotrophy, studies report nitrate reduction kinetics between 1 and 50 mM d-1 . The pH and nitrate concentration ranges generally tested are from pH 6.5 to 9.5 and nitrate concentrations from 1 to 10 mM [START_REF] Chang | Hydrogenotrophic denitrification with immobilized Alcaligenes eutrophus for drinking water treatment[END_REF][START_REF] Lee | Effects of pH and precipitation on autohydrogenotrophic denitrification using the hollow-fiber membrane-biofilm reactor[END_REF][START_REF] Rezania | Kinetics of hydrogen-dependent denitrification under varying pH and temperature conditions[END_REF][START_REF] Vasiliadou | A kinetic study of hydrogenotrophic denitrification[END_REF]. These huge differences between the two types of electrons donors were explained by the low dihydrogen solu bility [START_REF] Karanasios | Hydrogenotrophic denitrification of potable water : a review[END_REF], and the need for (autotrophic) hydro genotrophic bacteria to assimilate inorganic carbon [START_REF] Chang | Hydrogenotrophic denitrification with immobilized Alcaligenes eutrophus for drinking water treatment[END_REF][START_REF] Ghafari | Improvement of autohydrogenotrophic nitrite reduction rate through optimization of pH and sodium bicarbonate dose in batch experiments[END_REF][START_REF] Liu | Study on Heterotrophic-Autotrophic Denitrification Permeable Reactive Barriers (HAD PRBs) for in Situ Groundwater Remediation[END_REF]. Despite these differences, some authors have achieved kinetics of the order of 300 mM d-1 under hydrogenotrophic conditions by increasing dihydrogen partial pressure which increased its solubility and its availability to bacteria [START_REF] Epsztein | High-rate Hydrogenotrophic Denitrification in a Pressurized Reactor[END_REF]. lt is also possible to increase dihydrogen solubility using specifically designed reactors i.e. porous membrane, hollow fibre, and pH pKa + Log(

[CO 2 3 ] final [HCO 3 ] final ) 10.32 + Log( [CO 2 3 ] initial ± [CO 2 3 ] [HCO 3 ] initial ± [HCO 3 ] ) (4) pH 10.32 + Log( [CO 2 3 ] initial + 3 8 [NO 3 ] [HCO 3 ] initial + 7 8 [NO 3 ] ) (5) pH 10.32 + Log( [CO 2 3 ] initial + [NO 3 ] [HCO 3 ] initial [NO 3 ] ) (6)
Equation ( 6) applies only when the initial bicarbonate concentration is higher than the denitrified nitrate concentration ([HCO 3 -]initial >

[NO 3 -]). Otherwise, HCO 3 -is depleted and carbonate species cannot act as a buffer. According to equations ( 5) and ( 6), in a reactor with 50 mM carbonate, different pH evolution curves are plotted as a function of the initial pH and the reduced nitrate concentration, Fig. 4.

If the electron donor is acetate, the pH tends towards 10 during denitrification regardless of the initial pH. With dihydrogen, the pH increases regardless of the initial pH. Therefore, there is a fundamental difference in the pH evolution during denitrification between an organic or inorganic electron donor. The major reason is the production of CO 2 by the acetate oxidation, which as the potential to acidify the solution. Thus the hydrogenotrophic denitrification causes rapid alkalinization. In addition, in hydrogenotrophy, another phenomenon must be taken into account. Indeed, in the absence of organic carbon for growth, bacteria have to reduce and assimilate inorganic carbon. The consumption of inorganic carbon, in the CO 2 form [START_REF] Blombach | CO 2 -intrinsic product, essential substrate, and regulatory trigger of microbial and mammalian production processes[END_REF] or HCO 3 -form [START_REF] Chollet | Phosphoenolpyruvate carboxylase: a ubiquitous, highly regulated enzyme in plants[END_REF][START_REF] Tong | Structure and function of biotin-dependent carboxylases[END_REF] unbalances the carbonate buffer in favour of the CO 3 2-form, which increases the pH of the medium. Therefore, an additional explanation of the differences between heterotrophic and hydrogenotrophic denitrification is presented in this study. As the pH evolution is opposite between the two metabolisms, it strongly impacts the denitrification, especially in an alkaline environment.

The evolution of the pH has been further investigated by considering the key step of nitrite reduction. It can be stated from reactions ( 7) and ( 8) that the nitrite influence on pH could be a cause of its adverse effect. Indeed by comparing the OH -generation during nitrite reduction to nitric oxide, reactions ( 7) and ( 8), with the OH -generation during the whole denitrification, reactions (1) and ( 2), it appears the nitrite reduction is the key step which produces OH -and generate alkalinity.

H 2 + 2 NO 2 → 2 NO + 2 OH (7) CH 3 COO + 8 NO 2 + 3 H 2 O → 2 CO 2 + 16 NO + 9 OH (8)
Consequently, nitrite generally accumulates, so that denitrification takes place in two stages: reduction from nitrate to nitrite then reduction from nitrite to dinitrogen. Therefore, in presence of acetate, using the method of [START_REF] Albina | Influence of hydrogen electron donor, alkaline pH, and high nitrate concentrations on microbial denitrification: a review[END_REF], the pH evolution during the reduction of nitrate to nitrite and then of nitrite to dinitrogen can be respectively expressed with equations ( 9) and ( 10); in the pH range between 8 and 12. The pH evolution curves in Fig. 5 were plotted from these equations. In the presence of dihydrogen, the pH evolution during the denitrification is only caused by the nitrite reduction. Thus, the pH evolution during nitrite reduction is similar to Fig. 4 (B).

NO 3 → NO 2 pH 10.32 + Log( [CO 2 3 ] initial 2 8 [NO 3 ] [HCO 3 ] initial + 6 8 [NO 3 ] ) (9)
NO 2 → N 2 pH 10.32 + Log(

[CO 2 3 ] i + 5 8 [NO 2 ] [HCO 3 ] i + 1 8 [NO 2 ] ) (10)
NB: The final pH 10 during denitrification in Fig. 4 (A), is different from the final pH 11 during nitrite reduction in Fig. 5 (B). Indeed in Fig. 5 (B), the acidifying effect of nitrate reduction is not considered. To compare both figures, the final pH of Fig. 5 (A) and the carbonate generated during nitrate reduction must be considered.

Therefore, with acetate during the reduction of nitrate to nitrite, the pH decreases regardless of its initial value, Fig. 5 (A). Then, during the reduction of nitrite to dinitrogen, the pH equilibrates towards 11, Fig. 5 silicone tube reactors [START_REF] Karanasios | Hydrogenotrophic denitrification of potable water : a review[END_REF]. In this present study, the limited solubility of dihydrogen allowed only 0.74 mM of dihydrogen to be dissolved at 30 • C and 1 bar. Even though dihydrogen was supplied continuously, adding 20 mM soluble acetate provided an electrons donor 27 times more concentrated, thus dihydrogen was a limiting kinetic factor. In addition, dihydrogen provides 2 electrons when oxidized to protons while acetate provides 8 electrons when oxidized to CO 2 , so there are potentially 108 times more electrons available in the presence of 20 mM acetate. This limitation of electron availability surely slowed down bacterial activity.

.2. Transition from acetate and dihydrogen to dihydrogen only

In this experiment, the reactors were continuously fed with dihydrogen gas, and 20 mM acetate was added only on the first day of the experiment. Four batch periods were successively carried out with an initial nitrate concentration about 50 mM, the fifth batch period was initiated at 100 mM nitrate, Fig. 3. During the fourth batch period the reactors were adjusted at pH 9, 9.5, and 10. During the last batch period pH was adjusted at pH 9, 10 and 11, the reactors were named after this last batch period. The dotted pH jumps represent manual pH adjustments.

The rapidly consumed acetate reached negligible concentrations on day 25, from that day the remaining bacteria were considered using mainly hydrogenotrophic metabolism (Fig. 3). During the two initial batch periods, when acetate was still present, high nitrate reduction rates were observed despite the initial pH at 10.3. That can be explained by the pronounced acidification of 2.5 pH units, Fig. 3 (C). As the durations of the batch periods were short, denitrification was incomplete and nitrite accumulated up to 60 mM. Between day 10 and day 25, the acetate concentration became limiting and, as a result, the nitrate reduction kinetics were slowed down. From day 25, during hydrogenotrophic metabolism, the pH was adjusted manually to finally reach pH 9, 10 and 11 in the last batch period, at the same time nitrate concentration was increased from 50 mM to 100 mM in the last batch period. Therefore, the nitrate reduction was possible up to pH 10 and 100 mM nitrate, which confirms the previous findings in hydrogenotrophy, Fig. 2. It appears that nitrate reduction was strongly affected by pH rather than the nitrate concentration. Indeed, when the reactor at pH 9 had an increase from 50 to 100 mM its nitrate reduction rate did not change. On the opposite increasing pH above 9.5 in the reactors strongly decreased the reduction rates. The relatively low pH sensitivity in heterotrophy and high pH sensitivity in hydrogenotrophy can again be explained by the pH evolution during denitrification. When acetate was still present the denitrification caused acidification, which allowed high nitrate reduction rate, as in Fig. 1. On the opposite, as in Fig. 2, in the presence of dihydrogen the denitrification caused alkalinization.

.. pH evolution during denitrification

This opposite pH evolution can be explained through calculation. In a medium buffered by carbonate, the Henderson-Hasselbalch equation allows determining the pH from carbonate concentration [START_REF] Bhagavan | Water, acids, bases, and buffers[END_REF][START_REF] Davies | Carriage OF gases BY the blood and acid/base balance[END_REF] according to equation (4). The final concentration of CO -and HCO - 3 species can be expressed by their initial concentration together with the produced or consumed concentration of CO -and HCO - 3 , which can be expressed in nitrate equivalents according to equations (1) or (2). The calculation, further detailed in [START_REF] Albina | Influence of hydrogen electron donor, alkaline pH, and high nitrate concentrations on microbial denitrification: a review[END_REF] results in equations ( 5) and ( 6). These equations express the final pH according to the nitrate concentration reduced to dinitrogen. The equations are applicable in the pH range between 8 and 12. reactors, the acidifications were correlated to the nitrate reduction.

Then, when the nitrite accumulated started to be reduced there was pH evolution shift, as explained in section 3.2. During nitrite reduction acidification stopped, and in the reactor at initial pH 12 the medium even started to alkalinize. ln Fig. 6, assuming that ail the nitrate and the nitrite were reduced to dinitrogen it is possible to confront the results with the model section 3. able to survive for about 200 days at pH 12 and then initiate their denitrifying activity when the environmental conditions became more favourable. These results confinn conclusions from the bibliography in which the maximum physiologically tolerated pH limit for bacteria is reported to lie between pH 11 and 12 [START_REF] Rizoulis | The potential impact of anaerobic microbial metabolism during the geological disposal of intermediate-level waste[END_REF][START_REF] Sorokin | Is there a limit for high-pH life?[END_REF][START_REF] Watts | Treatment of alkaline Cr (VI)-contaminated leachate with an alkaliphilic metal-reducing bacterium[END_REF]. The pH has a strong impact on bacterial ac tivity as the pH affects ail enzyme activity, for instance, denitrification reductases have a functional optimum pH between 7.5 and 9.0 [START_REF] Kucera | The influence of pH on the kinetics of dissimilatory nitrite reduction in Paracoccus denitrificans[END_REF][START_REF] Thomsen | Mass spectrometric studies of the effect of pH on the accumulation of intermediates in denitrification by Paracoccus denitrificans[END_REF]. Therefore, using long time culture allowed to observe bacterial activity at a highly alkaline pH given the optimal pH for denitrification. Experimentation in highly alkaline en vironments requires rigorous medium pH control [START_REF] Sorokin | Is there a limit for high-pH life?[END_REF]. Indeed, the medium pH can be quickly modified by bacterial activity, in particularly denitrification products as OH-and C02 strongly impact

••••••••••-0' • • • • • •• • • •◊ ••••, • ••<> . . ••--••

Conclusion

• Using acclimation protocols and long culture time, it was possible to observe bacterial denitrification at high pH and high nitrate concentration simultaneously. • High nitrate concentration or high pH causes nitrite accumulation due to the difficulty for bacteria to reduce nitrite as it is toxic and increases the pH. • Denitrification strongly affects the pH of the medium. During denitrification with acetate, the pH tends to 10 while during denitrification with dihydrogen the pH increases. • Heterotrophic denitrification resulted in faster nitrate reduction kinetics and larger ranges of nitrate and pH tolerated by bacteria, 400 mM nitrate or pH 11.5. • Hydrogenotrophic denitrifying bacteria tolerated concentrations of nitrate up to 100 mM or pH up to 11. • Hydrogenotrophic metabolism is affected by (i) the low solubility of dihydrogen, (ii) the need for inorganic carbon assimilation, and (iii) the adverse pH evolution. • Therefore, as soon as the nitrate concentration is greater than 50 mM or the pH is greater than 9, an organic electron donor such as acetate is advantageous for bacterial denitrification.

the pH of the environment.

.4.2. Denitrification in the presence dihydrogen

Two reactors adjusted to pH 11.5 and 12 were monitored in batch mode with 50 mM nitrate under a constant dihydrogen flow (1 bar, 80 mL min -1 ), Fig. 7.

In the reactor initially at pH 11.5, only 10 mM nitrate was reduced while pH was acidified. The pH decreased despite the hydrogenotrophic conditions, which was contradictory with the above findings. The nitrate reduction started at day 105, after the initiation of the acidification when pH reached 11.2. Nitrite was not reduced and accumulated. The concentration of soluble carbonate (HCO - 3 + CO -) increased by 11 mM in the reactor at initial pH 11.5. In an airtight reactor with a medium initially devoid of organic matter, the increase in inorganic carbon can only have come from one source: the mineralization of the inoculum biomass. Therefore, it was hypothesised that a part of the biomass was oxidized into inorganic carbon by some of the bacteria, which were thereby able to reduce nitrate. Analyses of the bacterial communities were carried out on the final biomass of the reactor. The reactor popu-lation was composed of 93% of Bacillus sp. The genus Bacillus is described as denitrifying, heterotrophic, and haloalkaliphilic [START_REF] Denariaz | A halophilic denitrifier, Bacillus halodenitrificans sp[END_REF][START_REF] Preiss | Alkaliphilic bacteria with impact on industrial applications , concepts of early life forms , and bioenergetics of ATP synthesis[END_REF]. More importantly, some species, such as Bacillus subtilis, are described as saprophytes i.e. capable of utilizing organic matter from other bacteria by releasing digestive enzymes [START_REF] Ochiai | Plant cell wall degradation by saprophytic Bacillus subtilis strains: gene clusters responsible for rhamnogalacturonan depolymerization[END_REF]. The presence of a dominant alkalophilic hetero-trophic bacteria capable of oxidizing the biomass of other bacteria gives credence to the hypothesis that at pH 11.5 nitrate was reduced by heterotrophic bacteria using the biomass provided by the inoculum. Therefore, hydrogenotrophic denitrification was not possible at pH 11.5 in these experimental conditions. As for the reactor at pH 12, the nitrate was not reduced and the pH remained almost constant despite 185 days of experiment; the pH was too high for both heterotrophic and hydro-genotrophic metabolisms.

  Fig. 1. Infl uence of a nitrate increase from 50 to 400 mM on the dynamics of nitrate reductions (A), and the evolurion of pH and nitrite concentrations (B) in heterotrophic reactors maintained at 20 mM acetate and supplemented with 50 mM bicarbonate. The coloured numbers above the cwves in graph (A) indicate the average nitrate reduction kinetics in mM.d 1 • Those above the curves in graph (B) indicate the nitrite concentration reduced (mM) during each batch period.

  Nitrate at pH 9 -+-Nitrate at pH 10 -+-Nitrate at pH 11

  Fig. 2. Influence of a nitrate increase from 10 co 100 mM on the dynamics of nitrate reduccions (A), and the evolucion of pH and nitrite concentrations (B) in hydrogenotrophic reactors fed with a constant flow of dihydrogen (1 bar, 80 mL min 1 ) and 50 mM bicarbonate. The coloured numbers above the curves in graph (A) indicate the average nitrate reduction kinetics in mM.d 1 . Those above the curves in graph (B) indicate the nitrite concentration reduced (mM) during each batch petiod.

  at pH 9 -+-Nitrate at pH 10 ---+-Nitrate at pH 11 •-•◊• .. Acetate at pH 9 .. -o ... Acetate at pH 10 -.. f:s ... Acetate at pH 1 pH (pH final 9) -+-pH (pH final 10) -+-pH (pH final 11)

Fig. 3 .

 3 Fig. 3. Influence of a transition from acecace co dihydrogen on the dynamics of nitrate reductions (A), on the evolucion of niO'ite concentration (B), and pH (C). Reaccors fed wich a constant flow of dihydrogen (1 bar, 80 mL min 1 ), 50 mM bicarbonate and 20 mM acecace only ac the initial day. The coloured numbel'S above the curves in graph (A) indicate the average niO'ate reduccion kinetics in mM.d 1 . Those above the curves in graph (B) indicate the niO'ite concentration reduced (mM) during each batch period.

FigFig. 5 .Fig. 6 .

 56 Fig. 4. pH evolucion during the microbial denill' ification process as a funccion of the initial pH and type of elecn•on donor (acecace (A) or dihydrogen (B)), according co equations (5) and (6).

  2. Starting with 50 mM nitrate at pH 11.5 or pH 11.0 (the reactor at initial pH 12 was manually acidified to pH 11) the experimental final pH were respectively 10.5 and 10.3. According to Fig. 4 (A), the theoretical pH are respectively 10.5 and 10.4. Theoretical and experimental pH values were, therefore, really close.Therefore, bacterial denitrification at 50 mM nitrate is possible at pH 11.5 but impossible in these conditions at pH 12 and it seems that pH 11 is a threshold value for bacterial activity. However, the bacteria were

Fig. 7 .

 7 Fig. 7. Influence of highly alkaline pH with 50 mM nitrate on the evolution of nitrate and nitrite concentrations (A), and of pH and carbonate concentration (B) in hydrogenotrophic reactors fed wich a constant flow of dihydrogen (1 bar, 80 mL min 1 ), and 50 mM bicru•bonace. The coloured numbers above the nin-ace curves in graph (A) indicate the average nitrate reduction kinetics in mM.d 1 , Those above the nitrite cwves in graph (A) indicate the final nitrite concentration reduced in mM.

Table 1

 1 Average chemical composition of the cement leachate.

	Concentration (mM)						pH
	Na	K	Ca	Mg	Al	Fe	Si
	0.53	0.14	0.97	0.01	0.04	<0.01	0.23	11.4
	±0.11	±0.01	±0.26	±0.00	±0.01		±0.07	±0.2

Table 2

 2 Summary of the experimental set-ups reviewed in this srudy.

	Experimental set-

up Reactor Electron Nitrate pH Duration set-up donor (mM)

			(days)
	High nitrate	SBR•	Acetate

�00 9, 10, 108 concentration (20 mM)

					11
	with acetate
	Highly alkaline pH	Batch	Acetate	50	11.5,	234
	with acetate		(20 mM)		12
	High nitrate	SBR•	H2	1�100	9, 10,	83
	concentration		(1 bar,		11
	with dihydrogen		3o•q
	Highly alkaline pH	Batch	H2	50	11.5

, 186 with dihydrogen (1 bar, 12 3o•q Transition from SBR• Acetate �100 9 -11 48 acetate to (20 mM) dihydrogen only to H2 (1 bar, 30 °C)

Funding

This work was supported by Andra (Agence nationale pour la gestion des déchets radioactifs), France.

Credit author statement

Pierre Albina: Conceptualization, Methodology, Investigation, Writing Nadège Durban: Validation, Methodology Alexandra Bertron:

Funding acquisition, Corrections, Supervision Achim Albrecht: Ressources, Supervision Jean-Charles Robinet: Funding acquisition, Project administration Benjamin Erable: Corrections, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.jenvman.2020.111859.