Biodegradation https://doi.org/10.1007/s10532-023-10017-6 ORIGINAL PAPER Evaluating the suitability of granular anammox biomass for nitrogen removal from vegetable tannery wastewater C. Polizzi · T. Lotti · A. Ricoveri · G. Mori · D. Gabriel · G. Munz Received: 31 August 2022 / Accepted: 6 February 2023 © The Author(s) 2023 Abstract In the present study, the potential inhibi- test with non-acclimated biomass (CT); saline control tory effect of biologically pre-treated vegetable tan- test with acclimated biomass (SCT); vegetable tan- nery wastewater (TW) on anammox granular biomass nery wastewater test with acclimated biomass (TWT). was evaluated. Beside high organic and chemicals Compared with non-saline CT, the specific anammox load, vegetable TW are characterised by high salin- activity in tests SCT and TWT showed a reduction of ity and high tannins concentration, the latter belong- 28 and 14%, respectively, suggesting that salinity, at ing to a group of bio-refractory organic compounds, conductivity values of 10 mS/cm (at 25 °C), was the potentially inhibitory for several bacterial species. main impacting parameter. As a general conclusion, Recalcitrant tannin-related organic matters and salin- the study reveals that there is no technical limitation ity were selected as the two potential inhibitory fac- for the application of the anammox process to veg- tors and studied either for their separate and com- etable TW, but preliminary biomass acclimation as bined effect. Parallel batch tests were performed, with well as regular biomass activity monitoring is recom- biomass acclimated and non-acclimated to salinity, mended in case of long-term applications. To the best testing three different conditions: non-saline control of our knowledge, this is the first work assessing the impact of vegetable TW on anammox biomass. Supplementary Information The online version Keywords Anammox granular biomass · Vegetable contains supplementary material available at https://​doi.​ org/​10.​1007/​s10532-​023-​10017-6. tannery wastewater · Salinity · Tannins · Inhibition · Nitrogen removal C. Polizzi · T. Lotti · A. Ricoveri · G. Munz Department of Civil and Environmental Engineering, University of Florence, Via di S. Marta, 3, 50139 Florence, Introduction Italy G. Mori Tannery industry produces significant amounts of Consorzio Cuoiodepur S.p.A, Via Arginale Ovest, 81, highly polluted wastewaters and industrial facili- 56020 San Romano, Pisa, Italy ties are present all over the world, in developed and D. Gabriel (*) developing countries. Leather processing comprises GENOCOV Research Group, Department of Chemical, multiple steps during which raw leather undergoes Biological and Environmental Engineering, Escola chemical-physical treatments aimed at stabilizing the d’Enginyeria, Universitat Autònoma de Barcelona, organic matter and conferring the required physical 08193 Bellaterra, Spain e-mail: david.gabriel@uab.cat properties to the final product. Two types of tanning Vol.: (0123456789) 13 Biodegradation process are usually adopted: Chromium(III)-based regulation for discharge. A schematic of the current and vegetable-tannin-based, the former covering the plant configuration is presented in figure S.1 (sup- vast majority of leather processing worldwide. The plementary material). In the perspective of WWTP derived wastewaters are characterized by high con- rethinking towards energy self-sustainability and centrations of nitrogen (as ammonium and organic resource valorisation, anaerobic and autotrophic pro- nitrogen), organic compounds, sulphur (as sulphide cesses are gaining attention as alternatives to TW and sulphate), salts and chemicals (Mannucci et al. conventional activated sludge systems. As an exam- 2010; Saxena et al. 2016). The high content in chlo- ple, innovative treatment lines might integrate Car- rides is due to the fact that salted flesh remains the bon (C), Nitrogen (N) and Sulphur (S) cycles in a most cost-effective manner to preserve the raw mate- newly conceived synergy exploiting anaerobic treat- rial prior to its processing. ments, anammox process and autotrophic denitrifi- Depending on national regulations on effluent cation (Kalyuzhnyi et al. 2006). Specifically, COD quality, nutrient removal can be required and bio- load could be valorised through anaerobic diges- logical treatments are typically adopted in order to tion and the N-rich downstream digestate could be achieve stringent discharge limits on nitrogen and treated through the anammox process (AMX); nitrite carbonaceous compounds. Primary and tertiary treat- required for AMX could be supplied by partial auto- ments are also crucial for the removal of suspended trophic denitrification achieving simultaneous sul- and recalcitrant/colloidal matter, respectively (Saxena phide oxidation (Polizzi et al. 2022a, 2022b). Alter- et al. 2016). The high salinity of TW is an important natively, the reduced forms of sulphur present in the aspect that needs to be considered when biological biogas could further be exploited for the removal of treatments are adopted. Its adverse effect on biomass nitrate produced in a partial nitritation/anammox step activity is further exacerbated by the fact that waste- (PN/A), through autotrophic denitrification (Grubba water characteristics may vary significantly over the et al. 2022). Possible alternatives for innovative treat- year according to the industrial activity (e.g. vacation ment lines are presented in figure S.2 (supplementary periods and market fluctuations), exposing activated material). sludge biomass to salinity and load gradients. When The anammox process is considered a mature vegetable or synthetic tannins are used in the pro- technology especially for highly N-loaded streams, duction process, further drawbacks may arise in the but the effect of compounds present in industrial biological treatment unit, due to their bio-recalcitrant streams should be assessed prior to its application, and potentially inhibitory nature. Tannins inhibition since AMX activity could be dramatically affected on bacterial activities has been reported by Munz (Carvajal-arroyo et al. 2013; Lackner et al. 2014). A et al. (2009) and Nelson et al. (1997), among others. successful implementation of PN/A process on Chro- In conventional activated sludge systems treating veg- mium-based tannery wastewater was presented by etable TW, nitrifying bacteria have been reported as Frijters et al. (2007), reporting the pioneering treat- the most sensitive microbial population to high salin- ment line implemented in the Lichtenvoorde (NL) ity and tannins gradients and low maximum growth wastewater treatment facility. Specifically, in the rate are reported as an aggregate result of the inhibi- mentioned plant, an anaerobic IC® reactor allows for tory effect of compounds related to the tanning pro- COD removal, sulfate reduction and biogas produc- cess (Moussa et al. 2006; Munz et al. 2009; Szpyrko- tion; an aerobic sulphide oxidation reactor provides wicz and Kaul 2004). sulphide removal coupled with elemental sulphur The wastewater treatment plant managed by recovery and last, a PN CIRCOX® reactor is cou- Consorzio Cuoiodepur S.p.A. (San Romano, PI) is pled with an Anammox reactor for autotrophic N a large facility treating municipal wastewater and removal. An important milestone of their work was industrial wastewater generated by the vegetable- the achievement of stable operation in the anaerobic tannery district located in the Italian region of Tus- treatment unit, since the effectively treatment of the cany, representing one of the most important district complex COD mixture present in tannery wastewater for vegetable-tanned leather production in Europe. was not obvious. Conventional primary, secondary and tertiary treat- In the newly conceived treatment alternatives, ment are currently adopted to comply with Italian AMX process emerges a key process. To the best of Vol:. (1234567890) 13 Biodegradation our knowledge, there are no studies on the suitabil- almost 1:1 dilution is, in fact, a practical way to atten- ity of the anammox process for nitrogen removal in uate the potential inhibitory effect of high salinity and vegetable tannery wastewaters and the present study tannins levels, as well industrial load fluctuations. is intended to provide an evaluation of possible Currently, the actual salinity content of water entering inhibitory effects on the anammox biomass. Recal- the biological unit is 2–3 gCl/l and 1–1,5 ­gSO42−/l, citrant tannin-related organic matters and salinity are with an average electrical conductivity of 10 ± 2mS/ selected as the two potential inhibitory factors and cm (at 25 °C), whereas these parameters are almost studied both for their separate and combined effect. double in the raw tannery influent, as presented in Parallel batch tests with synthetic non-saline and table 1 (see fig. S.1). saline solutions as well as real tannery wastewater were performed in order to assess the sole contribu- Batch activity tests tion of salinity and the aggregate effect of salinity and recalcitrant compounds. The objective of the experiment was to assess the effect of salinity and of (potentially) inhibitory organic compounds, such as tannins, separately. Material and methods The manometric batch test was implemented according to the general procedure reported by Lotti Cuoiodepur WWTP et al. (2012). Six OxiTop® bottles (each with a total volume of 350 ml, WTW, Germany) were run for a In Cuoiodepur WWTP, more than 95% of the polluted total of 8 days. Manometric tests allow for the esti- load in terms of nitrogen, organic carbon, sulphur mation of anammox activity (i.e. nitrogen removal) and salts is related to the industrial stream; whereas through the monitoring of the headspace pressure in terms of flowrate, the municipal stream accounts increase due to ­N2 production. Three conditions were for around 40% of the total incoming flowrate. The tested in duplicates: (i) fresh biomass suspended in synthetic medium, herein Control Test, CT; (ii) fresh biomass acclimated to saline conditions and Table 1   Average characteristics of tannery wastewaters as reported in literature and observed in Cuoiodepur WWTP suspended in synthetic saline medium, herein Saline Control Test, SCT; (iii) fresh biomass acclimated to Parameter Reported ­TW1 Cuoiodepur ­TW2 (mg/l) saline conditions and suspended in pre-treated tan- nery wastewater, herein TWT. The experimental COD 3000–23,000 12,800 ± 1370 conditions are summarised in Table 2. Fresh AMX TSS 2000–3000 5630 ± 1260 granular biomass was withdrawn from an AMX gas- N–NH4+ 120–250 320 ± 60 lift reactor as reported in Polizzi et al. 2022a, 2022b, S2− 50–130 240 ± 100 after 245 days of operation. AMX biomass used in Cl− 2000–7000 6030 ± 1120 SCT and TWT tests was preliminary acclimated to SO4−− 1700–2700 12,800 ± 1370 saline conditions, in order to avoid transient saline 1 Mannucci et al. (2010); Zhao and Chen (2019) shock effect in the assessment of biomass activ- 2 Industrial influent, average values over 2015–2019 (curtesy of ity. The procedure used for biomass acclimation is Consorzio Cuoiodepur S.p.a) described in Sect “Biomass acclimation procedure to Table 2  Experimental Condition tested Biomass Liquid medium Conduc- conditions tivity [mS ­cm−1 @25 °C] *To saline conditions according to the fast Control Test, CT Non-acclimated Non-saline Synthetic Medium 4.3 acclimation procedure Saline Control Test, SCT Acclimated* Saline 12 (Sect “Biomass acclimation Synthetic Medium procedure to saline Tannery WW test, TWT​ Acclimated* Tannery WW 12 conditions”) Vol.: (0123456789) 13 Biodegradation saline conditions”. Prior to the test, a mixture of ­N2/ presented in Eq. 1, was used to perform N balances CO2 (95% and 5%, respectively) was sparged in the for the anammox reaction. headspace for 5 to 10 min, in order to create anoxic conditions. Bottles were then placed in a pre-heated NH + 4 + 1.225NO− 2 + 0.073HCO3 + 0.024H − + (1) incubator at 30 °C. Continuous mixing was provided → 1N2 + 0.21NO− 3 + 0.073CH1.74 O0.31 N0.2 + 1.95H2 O by an orbital shaker set at 150–180 rpm. Tests were run at 30 °C for three main reasons: (i) it is an opti- Finally, in order to confirm the statistical signifi- mum temperature for AMX activity; (ii) the biomass cance of the data, paired t-test was performed on the used in the test was grown at 30 °C; (iii) influent TW paired SAA outcome sets (CT- SCT; CT-TWT; SCT- in Cuoiodepur WTTP typically exhibits warm tem- TWT). T-test on paired means was considered appro- peratures and values of 25–35 °C are observed, on priate since it allows to assess whether the mean dif- average, in the biological unit of the plant, throughout ference in the pairs is different from zero (McDonald the year. Fresh biologically pre-treated tannery waste- 2014). The resulting p-value was calculated through water, herein tannery WW (TW), was withdrawn the data analysis tool pack of Microsoft Excel (t-test, at the exit of the secondary effluent of Cuoiodepur paired, double tale) and the significance level, α, set WWTP and used in test TWT. Such a pre-treated at 0.05. wastewater was deemed the most suitable stream to study potential inhibitory effect on anammox biomass Biomass acclimation procedure to saline conditions since the salt and tannin content is the same of the stream currently entering the biological unit and no The procedure described here was adopted in order to readily biodegradable COD is present. avoid saline shock effects on biomass activity. Tannery Tannery WW was filtered with paper filters in wastewater was withdrawn from the outflow of the bio- order to remove residual activated sludge in suspen- logical unit (figure S.1) and characterized in terms of sion. The amount of biomass placed in each bot- conductivity, chloride, sulphate, metals, COD, ammo- tle was set in order to achieve a complete nutrient nium, nitrite and nitrate. The conductivity observed in removal within 7–12 h, since preliminary biomass tannery WW was as high as 10 mS/cm (at 25 °C) and activity tests results were available from Polizzi et al. mainly related to chlorine and sulphate ions. Synthetic (2022a, 2022b). Volatile Suspended Solids (VSS) mineral medium used to feed the source AMX gas-lift analysis were done at the end of the test. The entire reactor held a background conductivity of 4.3 mS/cm liquid content was filtered and incinerated. Aver- (at 25 °C). A saline synthetic medium was prepared by age VSS concentration was 1.1 ± 0.3 g/l. Concen- adding chloride (as NaCl and KCl, in a 1:1 ratio) and trated pulses of ammonium and nitrite were provided sulphate (as ­Na2SO4) to the synthetic mineral medium through addition of 1 M ­(NH4)2SO4 and 1 M ­NaNO2, in order to achieve the same electrical conductivity in order to achieve 40–80 mgN/l both as ammonium of tannery WW. The ratio C ­ l−/SO42− ions was kept and nitrite in the bulk liquid. A minimum of 1:1 ratio as close as possible to the one observed in the real of ammonium:nitrite was ensured in order to obtain WW. A total of 10 saline solutions (around 300 ml nitrite-limiting conditions. A total of 6 consecutive each) were prepared by diluting the saline synthetic pulses were provided. Between spike 4 and 5, a mix- medium with different ratio of distilled water. Saline ture of ­N2 and ­CO2 (95% and 5%, respectively) was solutions had increasing conductivity levels starting sparged in the headspace in order to avoid ­CO2 limi- from 5 mS/cm to 10 mS/cm (targeted conductivity, tation drawbacks. Intermediate supernatant samples i.e. real wastewater conductivity), with a progressive were taken between one spike and the subsequent in increase of 0.5 mS/cm. All the solutions were kept order to analyse ammonium, nitrite and nitrate con- at room temperature (21–24 °C). Fresh granular bio- centration. pH was not controlled, but monitored mass was withdrawn from the anammox reactor and throughout the tests. COD was also analysed in TWT. acclimated to room temperature in a 300 ml flask with Analyses on the nitrogen components were used to (non-saline) synthetic medium. A step-wise exposure perform nitrogen mass balances, comparing removed procedure towards increasing saline conditions was nitrogen (as N–NO2−) and N ­ 2 estimated by pressure implemented. At each step, supernatant was removed increase. The stoichiometry from Lotti et al. (2014), Vol:. (1234567890) 13 Biodegradation and granular biomass poured into the saline solutions Table 3  Tannery (pre-treated) wastewater used in the TWT at increasing conductivity. The maximum conductivity test increase at each step was around 0.5 mS/cm and bio- Parameter Concentration mass was left to acclimate for a minimum of 20 min [mg/l] prior to be poured in the saline solution with higher CODtot 451 conductivity. Once the biomass was exposed for more CODfiltered 400 than 20 min to the last saline solution (10 mS/cm) the N–NH4+ 0.6 acclimation procedure was considered concluded. The N–NO2− < 0.1 overall acclimation procedure lasted around 5.5 h. N–NO3− 2.2 The aliquot of the withdrawn biomass used to seed Cl− 2750 the non-saline control test was left at room tempera- SO4− 1370 ture in non-saline synthetic medium during the dura- Cu 0.02 tion of the acclimation procedure, in order to avoid Cr 0.31 activity discrepancies due to temperature gradi- Ni < DL* ents, between the acclimated and the not-acclimated Pb 0.5 biomass. B 0.5 Cd < DL* Analytical methods Al 0.11 Zn 0.17 Ammonium, nitrite, nitrate and COD were analysed Fe 1.51 spectrophotometrically through commercial kits (Dr. P 0.47 Hach Lange). Further analysis on Chloride, Sul- phate and Phosphate ions were conducted through *Below Detection Limit (DL) Ionic Chromatography, IC (Dionex ICS-1100, Ther- moScientific). Metals in TW were analysed through inductively coupled plasma optical emission spec- one of the tannery WW, but sufficient to achieve the trometry, ICP-OES (Optima 2100 DV ICP-OES, conductivity threshold of 10 mS/cm since the back- PerkinElmer), after sample digestion with nitric acid ground conductivity of the synthetic medium was as (one-hour digestion at 100 °C, followed by filtration high as 4 mS/cm. Analysis on metals concentration at 0.45 μm). TSS and VSS analysis were performed showed that none of the element presented inhibi- according to standard methods (APHA, 2005). Elec- tory levels (Graaf et al. 1996; Huang et al. 2022). trical conductivity was measured through a port- Note that the COD concentration of the pre-treated able electrical conductivity meter (CM 35, Crison TW is related to recalcitrant colloidal organic instrument), equipped with a temperature sensor (Pt compounds, mainly tannins, removed in the plant 1000); the instrument provides conductivity values at through tertiary chemical-physical treatments. 25 °C and all values reported in the present work are Figure 1 shows the results of the maximum SAA referred to this reference temperature. values observed in the three tests, at each of the six consecutive spikes. Average values and standard deviation are reported for each duplicate. Results The nitrogen balance closed within a 10% of error; pH remained at 7.3 ± 0.2 throughout the Batch activity tests experiment, in all the tests. It can be observed that the control test (CT) keeps the highest activ- Table 3 reports the analytical composition of the ity throughout the experiment. SAA in CT showed pre-treated wastewater; pH was 7.5 and conduc- its maximum values after spikes 3, 4 and 6, during tivity 10 mS/cm. The saline synthetic solution which nitrite and ammonium concentrations were achieved a chloride and sulphate concentration of around 75 mgN/l, higher than in the other spikes 2100 ­mgCl−/l and 1400 ­ mgSO4−−/l, respectively. where they reached around 40–50 mgN/l. Chloride concentration was slightly lower than the Vol.: (0123456789) 13 Biodegradation Fig. 1  SAA results from 0.5 six consecutive spikes of nutrients 0.4 gNNO2-/gVSS/d 0.3 Control Test Saline Control Test 0.2 Tannery Wastewater Test 0.1 0.0 Spike 1 Spike 2 Spike 3 Spike 4 Spike 5 Spike 6 Fig. 2  Box and Whisker plot of SAA activities for the three tested conditions (median, internal line; mean, internal cross) In Fig. 2, Box and Whisker plot of the SAA values Table 4  Observed stoichiometry in the inhibition experiment obtained in each of the three series of data is reported. Test NO3−prod/NH4+rem NO2−rem/NH4+rem Considering average values and comparing to non- mgN/mgN mgN/mgN saline control test, SAA in saline control test and tan- nery WW test showed a decrease of 28% and 14%, CT 0.15 ± 0,06 1.44 ± 0.32 respectively. The stoichiometry observed in each test SCT 0.13 ± 0,02 1.42 ± 0.31 is reported in Table 4 (values estimated for spike 2, 4 TWT​ 0.21 ± 0,13 1.61 ± 0.47 and 6). The statistical analysis confirmed the statisti- cal significance of the experimental outcomes, since the p-values fell in the range of 0.01–0.04, lower matrix of TW. COD analyses, in the TWT dupli- than the threshold α-value of 0.05 (0.0110, 0.0395 cate, were performed prior to the 5th pulse and at and 0.0069 for the pairs CT-SCT, CT-TWT and SCT- the end of the test in order to assess possible hetero- TWT, respectively). ­ ODfilt resulted in 372 ± 4 and trophic denitrification. C As it can be observed, TWT presents the high- 364 ± 4 mg/l in the intermediate and final analysis, est variation either in N­ O3−/NH4+ ratio and in the respectively. As the initial ­CODfilt was 400 mg/l, ca. − + ­NO2 /NH4 one. Possibly, interference in the colori- 30 mg/l of ­CODfilt were removed at the initial and metric analyses might have caused by the coloured intermediate time, whereas COD remained quite Vol:. (1234567890) 13 Biodegradation stable from the 5th spike until the end of the test. Fil- through ammonification of organic nitrogen present tered COD of biologically pre-treated tannery WW in the TW and not accounted in the ratios’ calcula- is mainly related to dispersed or colloidal organic tion. Likely, a partial contribution of the two phenom- fractions, mainly tannins, a recalcitrant fraction that ena of heterotrophic denitrification and organic nitro- exits the biological unit, even under extensive aera- gen ammonification occurred. tion conditions (SRT in Cuoiodepur WWTP is as Results from the inhibition experiments indicate high as 70–80 d). In Cuoiodepur WWTP, such a high that tannery wastewater does not show any severe content in filtered COD is removed through chemi- inhibition effect on anammox granular biomass. cal precipitation prior to effluent discharge. Accord- ing with personal communication with plant process Discussion engineer, sudden COD depletion has been observed several times when pre-treated WW was dosed in The results from the inhibition experiment suggest activated sludge lab-scale reactors. Such a fast deple- that the application of the anammox process to veg- tion has been ascribed to adsorption phenomena more etable tannery wastewater has no technical limitation. than to fast biodegradation due to the recalcitrant According to the outcomes, salinity more than tan- nature of the suspended organic matter as well as the nins seems to show a potential inhibitory effect. physical properties of colloidal particles. The evi- The effect of salinity on anammox biomass has dence that ­CODfilt remains almost constant in the last been extensively studied in the last decades, in order two days of the test, is in line with such an assump- to assess its feasibility with saline N-rich wastewa- tion. Anyway, the evidence that average ­NO2−/NH4+ ters such as those generated by seafood, leather and ratio in test TWT was higher than in test CT and textile dying industries, leachate as well as diges- SCT, suggests a possible impact of heterotrophic tate from anaerobic digestion of organic fraction of denitrification, leading to higher ­NO2− removal than municipal solid waste (OFMSW). Two main fac- the stoichiometric one. Nevertheless, nitrate was not tors are reported to be crucial for stable treatment consumed but rather produced and N ­ O3−/NH4+ ratio of saline wastewaters: (i) microbial population shift in TW was also higher than in the other tests. Deni- towards halophilic or more saline-tolerant species, trification in tannery wastewater has been reported to such as “Ca. Scalindula” and “Ca. Kuenenia”; (ii) require COD/TKN ratio as high as 8–12 gCOD/gN, proper biomass acclimation to saline conditions. due to the presence of slowly biodegradable organic High salt concentrations may have detrimental effect fractions (Carucci et al. 1999; Szpyrkowicz and Kaul on biological activity due to cascade mechanisms 2004). Thereby, it can be speculated that 2–5 gCOD/ related to osmotic pressure on the cell wall, enzy- gN-NO2− would be required for denitritation only— matic inhibition and, ultimately, cellular lysis (Zhang considering the different electron requirement for et al. 2020). Stable anammox activity at saline condi- nitrite reduction compared to complete nitrate reduc- tions has been reported after biomass acclimation. A tion. According to such a ratio and the observed concentration of 30 gNaCl/l is pointed out by many COD reduction, 5 to 8% of the nitrite dosed in the authors as the salinity threshold for stable process first fourth spikes could have been removed by het- performance (Dapena-Mora et al. 2010; Kartal et al. erotrophic denitritation. The corresponding rate of 2006; Li et al. 2018; Ma et al. 2012). In some cases, heterotrophic denitrification rate, cautiously calcu- a conducive effect on SAA has been shown in case lated over the time of maximum ­N2 production rate, of gradual increase in salinity concentration until 20 would have accounted for 6–9% of the observed rate, gNaCl/l (Dapena-mora et al. 2010; Jin et al. 2011) confirming that its possible impact in the calculation or moderate salinity shock (Ma et al. 2012). On the of the MSAA was marginal. The evidence that SAA contrary, in case of intense salinity shock events, a observed in the last two spikes, when no significant transient (and typically reversible) inhibition in anam- COD removal was observed, was comparable to the mox activity has been observed together with a pro- one of the first spikes, further supports this hypoth- gressive process recover, the recovery period being esis. On the other hand, the higher N ­ O2−/NH4+ and related to the saline gradient (Ma et al. 2012). Experi- − + ­NO3 /NH4 ratios observed in TWT could have ences with AMX granular biomass treating digestate been also a result of additional N­ H4+ made available of OFMSW showed 50% inhibition at 6 mS/cm (at Vol.: (0123456789) 13 Biodegradation 25 °C), i.e. ­IC50, for non-acclimated biomass (Scagli- present work, the fast acclimation procedure proved one et al. 2017) and 70% inhibition at 11.2 mS/cm to be an effective solution to avoid transient responses (25 °C) for acclimated biomass (Lotti et al. 2019), to saline shocks. As presented in Polizzi et al. (2022a, yet allowing for stable performance in a continuous 2022b), the inoculum presented a relative abundance AMX reactor. As reported by the authors, these val- of 25% for “Ca. Brocadia” and 8% for “Ca. Kuene- ues are lower than the estimated threshold of 11.6 and nia”, resulting in a planctomycetes population com- 22.6 mS/cm (25 °C), corresponding to the I­C50 salt position of 68% for Ca. Brocadia and 13% for Ca. concentrations of 5.4 gNaCl/l reported by Carvajal- Kuenenia. The presence of the salinity-tolerant genus arroyo et al. (2013) and 13.5 gNaCl/l reported by of “Ca. Kuenenia”, could have also favoured the posi- Dapena-mora et al. (2010). However, it should be tive response observed in the TWT. noticed that ­IC50 values are affected by several fac- Another interesting aspect of long-term salin- tors, such as biomass adaptation and salinity exposure ity exposure is the impact on granules morphology (gradual/shock). Tannery wastewater considered in and composition. Fang et al. (2018) and Zhang et al. the present study presented a chloride concentration (2020) recently published two comprehensive studies of around 3 g­ Cl−/l (equivalent NaCl concentration of aimed at untangling mechanisms of anammox gran- ca 5 gNaCl/l), sulfate concentration of 1.47 g/l and ulation response to high salinity levels. Both stud- conductivity of 10 mS/cm and the observed inhibition ies agreed in the general conclusion that high salin- ranged from 14 to 28%. In the scenario of anammox ity levels (15 and 30 gNaCl/l) are not beneficial for process applied to treat only the industrial wastewa- granulation, due to many factors that can be synthe- ter collected to Cuoiodepur WWTP (no dilution with tized as follows: (1) EPS composition is affected by municipal WW), chloride concentration would be salinity and, specifically, high salinity leads to higher around 6 ­gCl−/l, equivalent to ca 10 gNaCl/l. Dapena- production of the hydrophilic water-retaining polar Mora et al. (2010) and Kartal et al. (2006) reported molecules of polysaccharides (PS), and lower produc- a conducive effect of moderate salt concentrations tion of proteins (PN), showing an hydrophobic behav- (5–6 gNaCl/l) on not acclimated biomass, whereas iour; (2) Ionic strength due to metal cations is usually Jin et al. (2011) reported the same positive effect on reported as conducive to biomass aggregation into acclimated biomass and around 70% SAA reduction granules up to 0.1–0.2 M, though an opposite effect in not acclimated biomass after shock exposure of has been observed at higher values, possibly due to few days to the saline condition. In the present work, ­Na+ substitution to ­Ca2+ ion, the latter being involved a moderate 28% SAA reduction was observed in the in bridging mechanism within PS molecules. SCT, holding a comparable saline concentration as The salinity levels considered by the two studies the mentioned studies. It owns to be mentioned that are much higher than the one considered in the tan- the biomass used in the present study was withdrawn nery WW in Cuoiodepur WWTP, either pre-treated from a reactor operating at a moderate conductivity of (and diluted) or not. Nevertheless, such evidences and 4 mS/cm and this factor likely contributed to the low possible mechanisms should be considered in case of inhibition observed. Moreover, the acclimation proce- real-scale implementation in order to ensure stable dure adopted in the present work appeared effective process performance. to prevent severe and transient SAA reduction due to saline shocks and that the observed reduction is likely to be further attenuated in case of long-term exposure Conclusions to the moderate saline level of pre-treated tannery wastewater as reported by several works (Dapena- Results from batch tests on fresh biomass, acclimated Mora et al. 2010; Ma et al. 2012). A longer adaptation to saline conditions, indicate that there is no evident period would have been conducive for a better bio- inhibition of anammox biomass due to exposure to mass activity response in the inhibition experiment, vegetable tannery wastewater. Salinity more than the since the acclimation time was much shorter than the mix of bio-refractory organic compounds is likely to one reported in similar studies (5.5 h versus 12 h to be the impacting parameter for anammox biomass few days). Since the shock effect of sharp gradients activity, since a 28% and 14% decrease in biomass in salinity concentration was not the objective of the activity was observed in the saline test and tannery Vol:. (1234567890) 13 Biodegradation WW test, compared to the non-saline control test. References Nevertheless, biomass acclimation is widely reported to be effective to increase anammox activity under Carucci A, Chiavola A, Majone M, Rolle E (1999) Treatment of tannery wastewater in sequencing batch reactor. Water saline conditions and high anammox activities are Sci Technol 40:253–259 likely to be achieved. 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