Chemosphere 295 (2022) 133936 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Successful sulphide-driven partial denitrification: Efficiency, stability and resilience in SRT-controlled conditions☆ Cecilia Polizzi a, David Gabriel b, *, Giulio Munz a a Department of Civil and Environmental Engineering, University of Florence, Via di S. Marta, 3, 50139, Firenze, Italy b GENOCOV Research Group, Department of Chemical, Biological and Environmental Engineering, Escola D’Enginyeria, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain H I G H L I G H T S G R A P H I C A L A B S T R A C T • Highly efficient and stable nitrite accu­ mulation was achieved. • Influent S/N < 0.6 g/g allowed for complete inhibition of N2 production. • Thiobacillus outcompeted Sulfurimonas under strict S-limiting conditions. • Nitrate uptake rate was not affected by high nitrite levels. A R T I C L E I N F O A B S T R A C T Handling Editor: A Adalberto Noyola Partial denitrification is emerging as a valuable solution for NO2− supply in Anammox systems. When reduced sulphur compounds are used as electron donors, S-driven Partial Autotrophic Denitrification (PAD) can also be Keywords: achieved, allowing for an integrated autotrophic nitrogen (N) and sulphur (S) removal from liquid and gaseous Sulphide-driven Partial Autotrophic streams. The aim of the present work was to maximise NO3− reduction to NO2− coupled with complete HS− Denitrification (PAD) oxidation, by the selective control of influent S/N ratio and sludge retention time (SRT). A 2.5-L chemostat was Nitrite accumulation operated for 115 days and three operational phases were tested at decreasing SRT of 40, 23 and 13 h, testing S/N Sulphur oxidizing biomass (SOB) Influent S/N ratios in the range of 0.5–1 gS/gN. Successful sulphide-driven PAD was achieved and lead to average NO2− conversion efficiencies as high as77±17% at all the conditions tested, with the highest value of 99% at the lowest S/N of 0.58 gS/gN and SRT of 23 h. Respirometric tests showed that NO3− uptake rate was stable at 90±10 mgN/ gVSS/h, when NO3− was present as sole electron acceptor or at NO2− levels as high as 120 mgN/l; on the contrary, NO2− uptake rates were very sensitive to the applied conditions. Metabarcoding analyses revealed that the microbial community was highly enriched in Sulphur Oxidizing Bacteria (SOB>80%) and stable S-limiting conditions appeared to favour Thiobacillus over Sulfurimonas genus. A preliminary assessment of N2O potential emission was also performed. To the best of our knowledge, this is the first work evaluating the synergic effect of SRT and influent S/N ratio on nitrite accumulation in highly SOB-enriched systems and the NO2− conversion efficiencies achieved are among the highest reported in literature. ☆ All the authors contributed to the revision of the work and agreed with the final version of the article. * Corresponding author. Via di S. Marta, 3, 50139, Firenze, Italy. E-mail addresses: cecilia.polizzi@unifi.it (C. Polizzi), David.Gabriel@uab.cat (D. Gabriel). https://doi.org/10.1016/j.chemosphere.2022.133936 Received 30 September 2021; Received in revised form 30 December 2021; Accepted 7 February 2022 Available online 8 February 2022 0045-6535/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). C. Polizzi et al. Chemosphere 295 (2022) 133936 1. Introduction The multiple intermediate steps occurring both in the oxidation and reduction reactions and the high chemical reactivity of sulphide signif­ The anammox process has been conventionally combined with Par­ icantly increase process complexity. As a consequence, the selective tial Nitritation (PN) as their synergy allows for a significant reduction in control of one catabolic step over the others might be a challenging task oxygen requirement and sludge production compared with conventional since multiple combinations of e-donors and e-acceptors can occur. On nitrification-denitrification systems. Nevertheless, PN long-term stabil­ the one hand, the application of the novel PAD/A process would be ity is challenged by the successful and stable suppression of nitrite limited to wastewaters baring proper sulphide/nitrate/ammonia ratios, oxidizing bacteria (NOB) and partial denitrification is emerging as an as required by the two processes. On the other hand, the remarkable alternative route for nitrite supply in anammox systems (Zhang et al., versatility of the PAD process allows for a wide combination of S/N 2019). In case reduced sulphur compounds (such as sulphide, thio­ loads, ranging from 0.6 to 2.3 gS/gN (eqs. (2)) and (4)), widening its sulphate or sulphur) are used as electron donors, Partial Autotrophic application to different types of wastewaters and/or gaseous streams, as Denitrification (PAD) can also be achieved. Some industrial wastewaters above mentioned. from chemical and food processing as well as petroleum and leather The selective control of the sole denitratation step is referred to as processing are highly rich in nitrogen (N) and sulphur (S) compounds, Partial Denitrification. According to equations (1) and (2), 1.43 and 0.57 the latter being present also in gaseous streams, typically in the form of gS/gN are required to drive complete sulphide oxidation coupled with hydrogen sulphide (Cristovao et al., 2015; Jain et al., 2020; Mannucci complete and partial denitrification, respectively. In S-driven denitrifi­ et al., 2010). Thereby, the combination of PAD and Anammox (PAD/A) cation, nitrite accumulation is reported in continuous or batch opera­ appears particularly appealing for an integrated S and N removal tions as a transient or undesired consequence of e− donor limitation or through anaerobic and autotrophic processes, offering a sustainable overload conditions (Campos et al., 2008; Manconi et al., 2007). Nitrite alternative also for sulphide-rich gaseous streams such as biogas. Despite can be potentially toxic for a wide range of microorganisms; there is the fact that the link between sulphur-based denitrification and anam­ general consensus on the fact that the actual cytotoxic role is played by mox lays at the very beginning of the anammox story (Mulder et al., its conjugated acid, the free nitrous acid, FNA (Zhou et al., 2011). 1995), little attention has been paid on the feasibility of achieving stable Although the inhibition mechanisms on denitrifiers are not fully un­ nitrite accumulation over sulphide oxidation as a possible pre-treatment derstood, it is often reported that the major mechanism is due to enzy­ for anammox (Chen et al., 2018; Liu et al., 2017). Recently, higher matic inhibition of NO2− reductase, as well as on N2O reductase, the attention has been devoted to the one-stage PAD/A. Successful one-stage latter being responsible for possible N2O emissions (Wang et al., 2018; PAD/A operation are reported by Chen et al. (2018), Deng et al. (2021) Zhou et al., 2011). Thereby, when targeting high NO2 accumulation, and Kalyuzhnyi et al. (2006), among others. The promising results show high FNA concentration might favour denitritation inhibition and the key role of influent S/N ratio, short hydraulic retention time (HRT) further nitrite accumulation. Yet, drawbacks on N2O release must be and high loading rates (the latter two often linked). When sulphide is addressed as well. To the best of our knowledge, the role of sludge used as electron (e− ) donor in simultaneous PAD/A, its potential retention time (SRT) in nitrite accumulation efficiency is not addressed toxicity on the anammox biomass should be properly assessed, since IC50 in literature, since systems with no SRT control are often reported for are reported in the range of 0.1–5 mgS/l (Dapena-mora et al., 2007; Russ PAD or PAD/A applications. Moreover, the possible role of different et al., 2014). A two-stage process could provide stable and controlled functional strains has been speculated by some authors, but the evi­ nitrite accumulation through partial autotrophic denitrification for a dences on microbial diversity are not exhaustive to clearly depict the subsequent anammox treatment, limiting the risk of (irreversible) sul­ actual impact on process performance (Cui et al., 2019b; Moraes and phide toxicity (Huang et al., 2021). Colourless Sulphur Oxidizing Bac­ Foresti, 2012). teria (SOB) are a wide group of bacteria capable of using reduced forms In the present work, stable nitrite accumulation over sulphide of sulphur as e− donors and oxygen or nitrate/nitrite as terminal oxidation was challenged at different influent S/N ratios and SRT values, e− acceptors. Even though the metabolic pathways proposed for sul­ in chemostatic conditions. Monitoring of microbial population, N2O phide oxidation are quite complex (Ghosh and Dam, 2009), its oxidation emissions as well as respirometric tests were performed for a more to sulphate is typically considered as a two-step process, elemental comprehensive understanding of the system response to the applied sulphur (S0) being the intermediate product. Autotrophic denitrifica­ conditions. tion, i.e. SOB anoxic respiration, follows the 4-step reduction chain (NO3− → NO2− → NO→ N2O→ N2), but nitrite is typically considered the 1.1. Material and methods main intermediate, in a two-step denitrification assumption. The cata­ bolic reactions of denitratation (NO3− → NO2− ) and denitritation 1.1.1. Reactor operation (NO2− → N2) coupled with complete or partial sulphide oxidation are A 2.5-L glass completely stirred tank reactor (CSTR) was operated for presented in the following equations: 115 days. The reactor was seeded with SOB-enriched suspended sludge, withdrawn from a pilot-scale denitrifying CSTR treating hydrogen sul­ HS− + 1.6NO−3 + 0.6H + →SO2−4 + 0.8N2 + 0.8H2 O phide absorbed from biogas (source reactor conditions: Sulphide / (1) Loading Rate, SLR, up to 2.4 gS/l/d, 2000–3000 ppmv of H2S; ΔG = − 744.4 Kj molS 1000–3000 mgN/l). Completely mixing conditions were provided by mechanical stirring (100–120 rpm) and temperature was maintained at HS− + 4NO−3 →SO2−4 + 4NO−2 + H + 30±1 ◦ C by continuously recirculating tempered water in the steel / (2) bottom jacket of the reactor. Mineral medium was prepared according to ΔG = − 480.2 Kj molS Mora et al. (2014) apart from nitrate and sulphur concentration. Nitrate was added in the mineral medium as KNO3. A concentrate sulphide HS− + 0.4NO−3 + 1.4H + →S0 + 0.2N2 + 1.2H2 O solution (0.1–0.2 M) was prepared with Na2S⋅9H2O salt, fed separately / (3) and kept in an air-tight browned bottle, connected to a N2 gas pocket, in ΔG = − 196.4 Kj molS order to prevent air inlet and compensate inner depression caused by pump suction. The two solutions were fed in continuous by means of HS− + NO−3 + H + →S0 + 0.2NO−2 + H2 O peristaltic pumps (Gylson Minipuls 3). A moderate flow of 0.1 l/min of a / (4) mixture of N2–CO2 (95% and 5%, respectively) was regulated by mass ΔG = − 130.3 Kj molS flow controllers (Low-Δp-flow mass meter, Bronkhorst) and bubbled at the bottom of the reactor in order to strip away any residual/undesired 2 C. Polizzi et al. Chemosphere 295 (2022) 133936 dissolved oxygen (DO). Reactor pH was controlled by adding acid/base NO−2 out solutions (1 M HCl and 1 M NaOH, respectively) and set at 7.3±0.1 for NiAE = ⋅100 (6) NO−3 in the first 15 days of operation to accommodate the inoculum and then increased to 7.6±0.1. Pumps operation, temperature, pH control and N2out flowmeters were controlled by a centralised PLC. The reactor was dNPE = ⋅100 (7) NO−3 in operated as an ideal chemostat, without biomass recirculation; thereby, SRT was directly determined by dilution rates. Regular cleaning of re­ Where NO3− in is the nitrate concentration in the influent and NO3− out actor’s internal walls and submersed mechanical stirrer and tubes was and NO2− out the nitrate and nitrite concentration in the effluent (all performed in order to remove any possible biofilm formation that could expressed as mgN/l). have altered the actual SRT. As a matter of fact, biofilm formation was Considering the rate of the two denitrification steps, in case of un­ never relevant but cleaning operations were performed anyway as a balance between the denitratation rate (r_dNO3) and the denitritation precautionary measure. rate (r_dNO2), the resulting nitrite accumulation rate (r_NiA) can be Influent S/N was always kept below 1.43 gS/gN (eq. (1)), while a calculated according to equation (8): minimum of 0.57 gS/gN was ensured to provide sufficient e-donor to rNiA = rdNO3 − rdNO2 (8) convert all the influent nitrate to nitrite (eq. (2)). In this work, sulphide limiting conditions are referred to influent conditions with S/N ratio the rates expressed as gN/gVSS/h. below the theoretical ratio required by complete denitrification equa­ At steady-state conditions, biomass yield was calculated according to tion (eq. (1)). Slightly limiting conditions are referred to S/N values equation (9). slightly below the ratio required in equation (1) and falling in the range of 0.80–1.40; strict limiting conditions are referred to S/N values close VSSout − VSSin YX/S = (9) or equal to the ratio required in eq. (2), falling in the range of 0.57–0.79. HSin− − HSout − Applied operational conditions are summarised in Table 1. Phase 1, 2 Since the reactor was fed with synthetic wastewater along the whole and 3 were planned to test decreasing SRT of 40 to 23 to 13 h and a range experimental period, VSSin was zero and VSSout was the concentration of S/N ratio, from slightly limiting to strictly limiting conditions for the measured in the CSTR. e-donor. The first 22 days of operation (phase 1a) were characterized by highly variable influent S/N, due to technical problems mainly related to 1.1.3. Respirometric tests the fine tuning of the concentrated sulphide solution pump. Stable The respirometric equipment and procedure described by Mora et al. conditions were maintained from phase 1b on. SRT reduction at each (2014) were adopted. Biomass was centrifuged at 10000 rpm and phase was achieved by doubling influent flowrate; nitrate concentration concentrated approximately 4 times, by resuspension in a nutrient-free in the mineral medium was kept at 200 mgN/l throughout the experi­ medium, except for test A, in which the reactor supernatant was used mental period whereas sulphide concentration in the concentrated so­ for biomass resuspension. Tests were run at day 54, 94, 102 and 113 of lution was set according to the required S/N ratio. Therefore, each phase the CSTR operation. Pulses of concentrated solutions (1 M KNO3, 0.5 M change implied a concomitant doubling in Nitrogen Loading Rate, NLR NaNO2, 0.2–0.4 M Na2S⋅9H2O) were added, according to the targeted (from 0.1 to 0.2 to 0.4 gN/l/d). concentrations. After each pulse, samples were taken every 10–40 min, Moreover, the potential inhibition effect of high nitrite level was also depending on the test. TSS and VSS were analysed at the end of the test addressed. FNA concentrations were calculated according to the by filtering the entire liquid volume. The initial concentrations of following equation (Anthonisen et al., 1976): e-acceptor and e-donor, for each test, are reported in Table 2. NO−2 Uptake rates were calculated for the N and S species by linear FNA = (3) ka *10pH regression of concentrations trends observed in the test and, as a general rule, only value with R2 > 0.95 were considered as reliable estimations 2300 where ka = e(− 273+T) , FNA and NO2− are expressed as mgN/l and T as ◦ C. (if not specified otherwise). For each test, two steps were distinguished: step 1 referring to sulphide consumption and step 2, for S0 consumption. 1.1.2. Process performance assessment Endogenous activity was neglected in agreement to previous works Nitrate removal efficiency (NRE) was expressed as the net nitrate removal between influent and effluent concentration; nitrite conversion efficiency (NiCE) as the nitrite production over the removed nitrate; Table 2 nitrite accumulation efficiency (NiAE) as the nitrite production over the Initial concentrations of respirometric tests. influent nitrate and the dinitrogen gas production efficiency (dNPE) as NO3− NO2− HS− Aim of the test the gaseous nitrogen produced over the influent nitrate. The mathe­ mgN/ mgN/ mgS/ matical formulations for efficiencies calculations are as follows: l l l NO−3 in − NO−3 out Test A 85 30 20 e-acceptor competition (preliminary) NRE = ⋅100 (4) Test B 35 5 40 Maximum NUR NO−3 in Test C – 55 10 Maximum NiUR Test D 10 120 30 NiUR and NUR at high NO2− NO−2 out NiCE = ⋅100 (5) concentrations NO−3 in − NO−3 out Table 1 Applied operation conditions and main results. Phase Days SRT S/N NLR NRE NiCE NiAE dNPE h gS/gN gN/l/d % % % % Phase 1a 1–22 44±2 1.76±1.65 0.10±0.02 76±25 7±2 5±2 72±24 Phase 1b 23–51 40±2 0.67±0.16 0.11±0.01 83±4 76±6 63±7 20±5 Phase 2 52–93 23±1 0.65±0.12 0.20±0.01 74±7 95±7 70±6 4±5 Phase 3 94–115 13±1 0.96±0.20 0.38±0.05 82±8 73±17 57±9 25±13 3 C. Polizzi et al. Chemosphere 295 (2022) 133936 (Mora et al., 2014). analysed once or twice a week, according to standard methods (APHA, 2005). N2O concentration in the reactor headspace was analysed with 1.1.4. Analytical methods Unisense N2O microsensor; the sensor was placed in the headspace of the Samples were filtered with 0.22 μm disposable syringe and analysed reactor and measurements recorded for 3–5 h. for nitrate, nitrite, sulphate and thiosulphate by ionic chromatography (Dionex ICS-2000, ThermoScientific). Sulphide was analysed with a 1.1.5. Microbial community analysis Silver/Sulphide ion selective electrode (VWR International Eurolab, S. Biomass samples were collected at day 0 (inoculum) and at the end of L). Sulphide concentration was analysed on the fresh feedstock solutions each phase (days 49, 80, 114). Samples were centrifuged and concen­ and right before replacing it with a new one; intermediate checks were trated multiple times. DNA was extracted with commercial kit (Norgen, also performed, if deemed necessary. Elemental sulphur was assessed Biofilm-kit), according to the manufacturer’s instructions. DNA con­ according to sulphur mass balance (Mora et al., 2015). VSS and TSS were centration and purity were checked by analyses on NanoDrop 1000 Fig. 1. Experimental results on reactor operation and performance: a) influent and effluent nitrogen concentrations; b) nitrogen conversion efficiencies; c) influent sulphide and produced sulphate concentration; d) applied SRT and S/N, horizontal continuous lines indicate S/N ratios required by equations (1) and (2). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4 C. Polizzi et al. Chemosphere 295 (2022) 133936 Spectrophotometer (Thermo Fisher Scientific). Extracted DNA samples accumulation recovered within three days. In phase 3, a S/N ratio of were delivered to the UAB Genomics and Bioinformatic Service (Uni­ 0.96±0.2 gS/gN was applied, in fact a value slightly higher than phase 2, versitat Autonoma de Barcelona). PCR amplification targeted the V3 and and restored N2 production, successfully inhibited in phase 2. Despite V4 region of 16s gene and amplicon sequencing was conducted on that, a satisfactory nitrite concentration of 119±15 mgN-NO2-/l was Illumina MiSeq according to the Illumina MiSeq protocol for library ensured. On day 108, a S/N ratio of 1.39 gS/gN was accidently applied. preparation (support.illumina.com/content/dam/illuminasupport/doc­ As a consequence, nitrite concentration dropped again, but high nitrite uments/documentation/chemistry_documentation/16s/16s-metageno levels were restored within one day, as the S/N returned S-limited from mic-library-prep-guide-15,044,223-b.pdf” title="https://support.illum day 109 on. Phase 3 was characterised by more unstable operational ina.com/content/dam/illuminasupport/documents/documentation/ch conditions, but conversion efficiencies were maintained as high as emistry_documentation/16s/16s-metagenomic-library-prep-guide 73±17% and 57±9% for NiCE and NiAE, respectively. Sulphate pro­ -15,044,223-b.pdf">https://support.illumina.com/content/dam/ill duction in phase 3, closed sulphur balance with a 10% error, indication uminasupport/documents/documentation/chemistry_documentation/ that the inlet sulphide was completely oxidized to sulphate. Elemental 16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). Taxonomic analysis conducted on day 106 and 107 confirmed that only 2.3±0.8% classification was based on Greengenes database. Raw reads and taxa of the dry matter were actually accounted as sulphur. abundance data are available in the NCBI GEO database (accession VSS concentration in the reactor remained very low with average number GSE192713). concentrations of 13±5, 13±2 and 36±10 mgVSS/l, in phase 1, 2 and 3, respectively. VSS/TSS ratio remained stable at 83 ± 5%. The higher 1.2. Results and discussion value observed in phase 3 was crossed with the elemental analysis and it did not seem to be related to significant elemental sulphur accumula­ 1.2.1. Successful NO2− accumulation tion, as discussed above. Biomass yield estimated in phase 1, 2 and 3 In Fig. 1, results on the reactor operation are reported. The main were 0.11±0.05, 0.12±0,04 and 0.19±0.09 gVSS/gS, respectively. The findings at each phase are summarised in Table 1. Since inoculum observed yields are in line with the values of 0.13 gVSS/gS reported by biomass was not diluted nor washed prior to inoculation, initial con­ Can-dogan et al. (2010) and lower than those reported by Campos et al. centrations of nitrite, nitrate and sulphate in phase 1a were one order of (2008) and Mora et al. (2014) ranging values of 0.33–0.36 gVSS/gS. To magnitude higher than those achieved at steady-state conditions in the the best of our knowledge, none of the available yields in literature is following phases, due to higher loading conditions in the source reactor. estimated from a continuous system operating partial denitrification as In phase 1a, S/N ratio and nitrate removal were highly variable and the main process. no significant nitrite was observed in the effluent. In phase 1b, influent SRT as low as 12 h did not cause any biomass washout, indicating an S/N was set stably within the targeted range (continuous lines in actual biomass growth rate higher than the applied dilution rate of 0.08 Fig. 1d), ensuring S-limiting conditions at a dilution rate of 0.025 h− 1 h− 1, i.e. 1.8 d− 1, in line with the reported value of maximum growth rate (SRT of 40 h). According to the catabolic stoichiometry of equations (1) of denitrifying SOB, ranging from 1.2 to 3 d− 1 (Claus and Kutzner, 1985; and (2), the sulphide provided in the first week of phase 1b was suffi­ Mora et al., 2015; Oh et al., 2000). A further increase in dilution rates is cient either to a full denitrification of 30% of the influent nitrate load or considered an interesting development of the present work, since it for an almost complete conversion to nitrite (assuming sulphide com­ would allow to study both the effect of higher loading rates and the plete oxidation to sulphate). From day 22 to day 29, a 30% of nitrate maximum biomass growth rate, at high nitrite levels. removal efficiency was, in fact, observed and accomplished by complete According to the main outcomes, successful nitrite accumulation was conversion of nitrate to dinitrogen gas (no nitrite was detected). After a achieved by ensuring strict sulphide-limiting conditions, i.e. S/N ratios few days of stable and strict sulphide limiting conditions, a gradual ni­ less than 0.8 gS/gN, in SRT-controlled conditions. The closer the S/N trite accumulation was observed, until a stable effluent concentration of ratio to the value of 0.57 gS/gN, required by equation (2), the higher the 125±9 mgN-NO2-/l was maintained, resulting in a NiAE of 63±5% and a conversion efficiency. NiCE higher than 70% were obtained at all the NiCE of 76±6%. In this phase, sulphate production closed sulphur bal­ SRT tested. It is speculated that SRT control contributed to system sta­ ance within a 10% error, indicating that all the influent sulphide was bility: the low values applied might have promoted a highly selected converted to sulphate. At day 52, SRT was halved to 23 h (phase 2). The SOB culture, conferring the remarkable system resilience observed. sudden increase of loading rates and dilution rate (0.04 h− 1), deter­ Similarly to the present study, Deng et al. (2019) and Huang et al. (2021) mined a net drop in nitrite concentration down to 22 mgN-NO2-/l. challenged nitrite accumulation over nitrate reduction in S-driven Nevertheless, in three days, the concentration raised back to values of denitrification system by maintaining S-limiting conditions, i.e. low S/N 136±13% mgNNO2-/l, kept throughout phase 2. A NiCE of 95±7% was values. The maximum nitrite conversion efficiencies obtained in the achieved with a concomitant NiAE of 70±6%. On days 61–71, the lowest mentioned studies are 50 and 55.3%, respectively, whereas an average S/N of 0.58 gS/gN was maintained and resulted in the maximum and value of 75% has been stably maintained in the present work with a basically complete nitrite conversion with NiCE of 99±2%. Thereby, maximum 99% conversion obtained at the lowest S/N ratio of 0.58 complete inhibition of dinitrogen gas production was obtained, i.e. the gS/gN at SRT of 23 h. Other studies report successful NO2− accumula­ denitritation rate was almost zero. During phase 2, sulphate production tion even though S/N is either not specified or significantly over stoi­ was exceeding the influent sulphide by a 10–30%. It is assumed that chiometric for the e− donor (Chen et al., 2018; Liu et al., 2017), showing thiosulphate or sulphate were present in the concentrated sulphide so­ other possible strategies for nitrite accumulations, e.g. using S0 instead lution as a result of sulphide chemical oxidation. Since only 4 e− are of sulphide or high Sulphide loading rates. donated per mole of S–S2O32− oxidized to SO4− , compared to 8 e-mol per molS-HS- oxidized to SO42− , the catabolic S/N ratio required for 1.2.2. N2O emission and FNA role complete or partial denitrification over thiosulphate are as high as 2.9 Nitrous oxide was measured in the headspace at the end of phase 1 and 1.1 gS/gN, respectively. Thereby, in the worst case of sole thio­ (days 47 and 50) and phase 2. No measurements could be performed sulphate (and not sulphate) presence, the ultimate effect on nitrogen during phase 3. A summary of the measured N2O concentrations is conversion is deemed as low as 15%. In order to limit abiotic sulphide available in table S1, in the supplementary material. An average con­ oxidation products, sulphide solution in phase 3 was prepared with centration of 2.1±0.6 μmol/l and 3.7±0.2 μmol/l (as N2O) was esti­ freshly opened sulphide salt. In phase 3, the applied dilution rate was mated for phase 1 and phase 2, respectively. Since no measurements increased to 0.08 h− 1 (SRT of 13 h). As in the transition from phase 1 to were conducted in the liquid phase nor estimates of the kLa of the system phase 2, the sudden flowrate and load increase lead to a drop in nitrite were available, N2O emissions were estimated according to mass bal­ concentration down to 49 mgN-NO2-/l. Successfully, nitrite ances over the gas phase and the derived considerations are considered 5 C. Polizzi et al. Chemosphere 295 (2022) 133936 as illustrative more than exhaustive. N2O emissions were estimated as accumulation, but its role in maintaining stable PAD cannot be 2.0±0.5 and 2.4% of the influent N–NO3- load in phase 1 and phase 2, excluded. On the contrary, preliminary results warns on its possible respectively. In the mini-review presented by Cui et al. (2019a), S-based impact in N2O emission and further studies on pH-based mitigation autotrophic (complete) denitrification systems often exhibit lower strategies are recommended. emission factors than the heterotrophic ones, with values of 0.01–0.8% vs 2.3–13%, respectively. In the present work higher N2O emissions 1.2.3. Microbial diversity were estimated. However, direct comparison of emission factors might Fig. 2 reports the results on microbial diversity at class and genus be deceptive due to differences in mixing conditions. Differently from level. Operational taxonomic units showing a relative abundance lower the reported studies adopting UASB configurations or static batch tests than 1% are not presented. A clear shift in SOB population was observed (Liu et al., 2016; Yang et al., 2016), continuous mixing and nitrogen gas at class and, consistently, at genus level. At genus level, Sulfurimonas bubbling were provided in the present work, likely promoting higher (ε-proteobacteria) exhibited a relative abundance of 90% in the seeding liquid-gas transfer efficiencies. Yet, the sulphide limiting strategy sludge whereas its relative abundance dropped to 4% and 1% on days 49 adopted might have promoted the production of denitrification in­ and 80, respectively, and then raised back to 53% on day 114. On the termediates other than nitrite. N2O concentration increased with contrary, the relative abundance of Thiobacillus genus (β-proteobacteria) increasing FNA in the bulk (tab. S1). High pH are reported to be effective was lower than 1% in the seeding sludge, but increased to 47% on day 49 for N2O emission attenuation in presence of high nitrite levels (Wang and then reached 83% on day 80, prior to decrease to 38% on day 114. et al., 2018; Zhou et al., 2007) and might be considered for mitigation. It Day 49 and 80 are representative of the stable and strict S-limiting can be claimed that FNA inhibition was not crucial for triggering NO2− conditions applied during phase 1b and 2. Day 115, on the contrary, was Fig. 2. Microbial diversity in the inoculum and across the three operational phases. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 6 C. Polizzi et al. Chemosphere 295 (2022) 133936 characterised by a slightly higher S/N. Interestingly, Thiobacillus deni­ environmental conditions compared to the membrane-bond nitrate trificans has been reported to have a higher efficiency in terms of growth reductase is considered of relevance for defining the theoretical frame­ bioenergetics compared to Sulfurimonas denitrificans (Klatt and Pole­ work behind successful PAD systems. A 30% relative abundance of recky, 2015). The proposed reasons are related to the fact that Chryseobacterium, belonging to the class of Flavobacterium, was also T. denitrificans adopts membrane-bond nitrate reductase (Nar) and detected on day 49. Their presence is not fully understood since these S. denitrificans periplasmic nitrate reductase (Nap) instead. Indeed, the bacteria are typically reported as heterotrophic and the only organic activity of Nar is directly linked to proton motive force generation as matter available was the organic compounds deriving from endogenous protons are driven across the cytoplasmic membrane and energy is decay of biomass. directly harvested during its action, while Nap activity does not deter­ The nitrite drop episodes and subsequent nitrite accumulation re­ mine any direct proton motion, since its action occur directly in the covery reveal a remarkable functional resilience of the system, against periplasmic environment (Sparacino-Watkins et al., 2014). Moreover, daily S/N variations, and suggest that successful nitrite accumulation the activity of Nar is less influenced by environmental conditions due to was not due to the selection of biomass lacking of the enzymatic pool for the fact that the reduction reaction occurs in the cytoplasm, differently complete denitrification, as speculated in other studies (Chen et al., from the Nap which operates in the periplasm, more susceptible to 2018; Cui et al., 2019b), since the accumulated nitrite was promptly environmental conditions, such as pH (Glass and Silverstein, 1998). reduced in presence of abundant electron donor. Cell-signalling mech­ Thiobacillus is also reported to encode both the enzymatic pools required anisms, such as Quorum Sensing (QS), are speculated to have played a in the Sox system and in the branched pathway for S-oxidation, whereas role in the first catabolic shift observed on days 29–33, when complete Sulfurimonas only relies on the Sox system, resulting in a competitive denitrification was gradually replaced by partial denitrification, pro­ advantage of the more versatile genus of Thiobaicllus (Klatt and Pole­ vided stable S-limiting conditions. QS molecules have been, in fact, re­ recky, 2015). It can be speculated that strict and stable energy limited ported to affect denitrification activity likely acting at the conditions, resulting from e− donor limitation, lead to a competitive transcriptional level of the reduction-chain enzymes (Cheng et al., advantage of the genus with higher energy efficiency. The role of the 2017). periplasmic nitrite and nitrate reductases, more sensitive to Fig. 3. Results from respirometric tests A, B, C and D. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 7 C. Polizzi et al. Chemosphere 295 (2022) 133936 1.2.4. Respirometric tests Authors’ contributions Results of respirometric tests are reported in Fig. 3. In test A, a preliminary NUR estimation was 100±35 mgN/gVSS/h, as an overall C. Polizzi: Conceptualization, wet laboratory, Supervision, writing. value in step 1 and 2, and confirmed in the subsequent tests. D. Gabriel: Supervision, discussion, fund acquisition. G. Munz: Super­ In test B, thiosulphate formation was observed at the beginning of vision, discussion, fund acquisition. the test and it is believed to result from chemical interactions between dissolved sulphide and other chemically forms S intermediates (Van Den Declaration of competing interest Bosch et al., 2007). Thiosulphate uptake is considered as representative of sulphide uptake since it is considered interchangeable with sulphide The authors declare that they have no known competing financial for chemolithotrophic biomass and is often used for SOB kinetic interests or personal relationships that could have appeared to influence assessment (Mora, 2014). During step1, uptake rates were 80 the work reported in this paper. mgN-NO3-/gVSS/h, 200 mgS-HS-/gVSS/h and 125 mgS-S2O32− /gVSS/h. A small but constant accumulation of nitrite was also observed at a rate of 12 mgN-NO2-/gVSS/h (R2 = 0,94). According to eq. (8), Acknowledgements nitrite consumption rate resulted in 65 mgN-NO2-/gVSS/h. NUR uptake over S oxidation was not clearly profiled. In test C, a temporary delay in The present work was supported by a financial contribution from nitrite consumption was observed. It can be speculated that it could Consorzio Cuoiodepur S.p.a. (Pisa, IT) and was partially developed result from an enzymatic delay since biomass was grown on nitrate only within the Project Recycles (GA: 872053 funded by the European and nitrite was predominantly accumulated rather than consumed. Commission within the Horizon 2020 Mary Sklodowska Curie Rise 2019 Uptake rates resulted in 190 mgN-NO2-/gVSS/h; 140 mgS-HS-/gVSS/h programme. The authors would like to thank professor Matteo Ram­ and 90 mgS-S2O32− /gVSS/h. NiUR in step2 was as low as 27 azzotti from the Department of Biomedical, Experimental and Clinical mgN-NO2-/gVSS/h, 7 times lower than the value observed in step1. Test Sciences “Mario Serio” of the University of Florence for his support in D was conducted at nitrite levels as high as those observed in the CSTR, NGS data handling. in order to assess whether nitrite inhibition was playing a significant role in the reactor performance and the estimated FNA was 4–5 μgN/l. When Appendix A. Supplementary data sulphide was present as sole e-donor, test D exhibited the following rates: 85 mgN-NO3-/gVSS/h; 84 mgN-NO2-/gVSS/h (R2 = 0,9) and 299 Supplementary data to this article can be found online at https://doi. mgS-HS-/gVSS/h. NUR at FNA of 4–5 μgN/l was in fact comparable with org/10.1016/j.chemosphere.2022.133936. the value observed in absence of nitrite. No clear nitrite consumption could be gauged from step2. References Summarising respirometric results, an average NUR of 90±10 mgN/ gVSS/h was observed in all the tests, irrespective of nitrite or FNA Anthonisen, A.C., Loehr, R.C., Prakasam, T.B., Srinath, E.G., 1976. 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