Science of the Total Environment 777 (2021) 145969 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Sustained effect of zero-valent iron nanoparticles under semi-continuous anaerobic digestion of sewage sludge: Evolution of nanoparticles and microbial community dynamics Raquel Barrena a,⁎, María del Carmen Vargas-García b, Georgina Capell a, Maja Barańska a, Victor Puntes c,d,e, Javier Moral-Vico a, Antoni Sánchez a, Xavier Font a a GICOM research group, Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de Barcelona, Edifici Q, Carrer de les Sitges, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain b Department of Biology and Geology, CITE II-B, Universidad de Almería, Agrifood Campus of International Excellence ceiA3, 04120 Almería, Spain c Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, 08193 Barcelona, Spain d Vall d'Hebron Institut de Recerca (VHIR), 08035 Barcelona, Spain e Institució Catalana de Recerca i Estudis Avançats (ICREA), P. Lluís Companys 23, 08010 Barcelona, 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 • Dossing of nZVI improved methane con- tent in the anaerobic digestion. • Punctual doses every 5–7 days sustained positive effects with higher methane content. • Magnetic retention of nZVI in reactors showed the loss of effect along time. • TEM-EELS analysis showed the oxida- tion of nZVI during anaerobic digestion. • Changes in microbial community struc- ture can be related to nZVI use. a r t i c l e i n f o a b s t r a c t Article history: The effects of adding zero-valent iron nanoparticles (nZVI) on the physicochemical, biological and biochemical Received 17 November 2020 responses of a semi-continuous anaerobic digestion of sewage sludge have been assessed. Two sets of consecu- Received in revised form 12 February 2021 tive experiments of 103 and 116 days, respectively, were carried out in triplicate. nZVI were magnetically Accepted 13 February 2021 retained in the reactors, and the effect of punctual doses (from 0.27 to 4.33 g L−1) over time was studied. Available online 19 February 2021 Among the different parameters monitored, only methane content in the biogas was significantly higher when Editor: Damia Barcelo nZVI was added. However, this effect was progressively lost after the addition, and in 5–7 days, the methane con- tent returned to initial values. The increase in the oxidation state of nanoparticles seems to be related to the loss Keywords: of effect over time. Higher dose (4.33 g L−1) sustained positive effects for a longer time along with higher meth- nZVI ane content, but this fact seems to be related to microbiome acclimation. Changes in microbial community struc- Anaerobic digestion ture could also play a role in the mechanisms involved in methane enhancement. In this sense, the microbial Methanothrix consortium analysis reported a shift in the balance among acetogenic eubacterial communities, and a marked in- Oxidation state crease in the relative abundance of members assigned to Methanothrix genus, recognized as acetoclastic species Magnetic nanoparticles showing high affinity for acetate, which explain the rise in methane content in the biogas. This research demon- strates that biogas methane enrichment in semicontinuous anaerobic digesters can be achieved by using nZVI nanoparticles, thus increasing energy production or reducing costs of a later biogas upgrading process. © 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). ⁎ Corresponding author. E-mail address: raquel.barrena@uab.cat (R. Barrena). https://doi.org/10.1016/j.scitotenv.2021.145969 0048-9697/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). R. Barrena, M.C. Vargas-García, G. Capell et al. Science of the Total Environment 777 (2021) 145969 1. Introduction and also that their presence change the microbial profile towards more methane-generating microbial communities. Specifically, this work High world energy demand and waste generation conflict with bio- aims to assess the effects of nZVI punctually dosed at different concen- sphere preservation. Thus, the transition from fossil fuels to renewable trations and different interval in a semi-continuous operation and es- energy sources, along with the improvement of solid waste treatment, tablish an operation mode that sustains the positive effects but avoids becomes a necessary changeover for the balance between humankind a continuous and extra NPs addition. With that purpose, nZVI were and the environment. In this context, anaerobic digestion (AD) is a magnetically retained in the reactors, and the effect of punctual doses well-implemented technology to treat a wide range of organic waste over time was studied. Different doses were tested to study both the im- streams, such as livestock manure, domestic and industrial wastewa- pact on methane production and their persistence over time. The oxida- ters, food waste, or fat and oils (Lora Grando et al., 2017) while produc- tion state of nZVI and the microbial community dynamics after nZVI ing green energy. Through the implementation of AD, an energetically addition was also addressed to understand: i) the loss of nZVI effect optimal product, biogas, a mixture of methane (CH4) and carbon dioxide on methane production over time ii) the acclimation and adaptation (CO2), is obtained. Nevertheless, the AD process still needs to be opti- of microorganisms to nZVI. mized to bring more and better-quality energy. The use of additives to increase biogas production and the improvement of biogas upgrading 2. Materials and methods are among the aspects that can still promote the advancement of AD (Lora Grando et al., 2017). 2.1. Synthesis and characterization of nZVI Iron is recognized as a potential energy source for bacteria. It is an es- sential redox element (between Fe2+ and Fe3+ forms) fundamental for nZVI were synthesized following the protocol proposed by Choe biological processes that functions as a versatile cofactor for numerous et al. (2001). To obtain 6.5 g L−1 of Fe0, 7.04 g of NaBH4 (≥98%) were dis- proteins in biological processes. It plays a fundamental role in biological solved in 200 mL of deoxygenated ultrapure water and then added processes such as DNA replication and repair among others, but also as dropwise to 200 mL of a deoxygenated FeCl3 (≥98%) 7.54 g L−1 solution. an electron donor and acceptor in many microorganisms such as Archae After 25 min of vigorous stirring under an N2 atmosphere, the Fe0 pre- and Bacteria (Folgosa et al., 2015). Although iron is one of the most cipitate was rinsed three times with deoxygenated ultrapure water to abundant elements on earth, its bioavailability is not as extensive as it remove residual acidity or impurities, redispersed in 400 mL of ultra- would be expected, since at neutral pH its solubility is 0.1 M and pure water and finally stored in a 500 mL glass bottle after flushing 10−8 M, for Fe2+ and Fe3+, respectively. The optimal proportion of N2. NPs suspension was ultrasonicated for 30 min before every use to trace metal could be a crucial aspect of the AD process (Baniamerian avoid agglomeration. All chemicals were purchased from Sigma- et al., 2019), and thus, their supplementation can be a powerful tool. Aldrich and used as received. Casals et al. (2014) indicated that the key to maintaining the increased nZVI were characterized in terms of size, distribution and morphol- biogas production was to preserve an optimal iron concentration in an ogy by scanning electron microscopy (SEM), using a Quanta FEI 200 anaerobic digester over time. FEG-ESEM instrument, equipped for energy-dispersive X-ray analysis The use of nanoparticles (NPs) as new additives in AD is an emerging (EDX). Electron energy-loss spectroscopy in a transmission electron mi- technology, especially explored in the last years (Baniamerian et al., croscope (TEM-EELS) analysis for the determination of the oxidation 2019). Nanoscale zero-valent iron (nZVI) can be found among the stud- state of NPs was performed on an FEI Tecnai G2 F20 microscope ied nanomaterials. Thanks to their characteristics, nanoscale zero-valent equipped with a Gatan Imaging Filter (GIF) Quantum SE 963 operating metals are expected to have more promising applications compared to at 200 kV. their bulk counterparts (Baniamerian et al., 2019). Recent studies have proven that nZVI could be an effective additive to enhance AD of sewage 2.2. Inoculum and substrate sludge, especially the increase of methane content in the biogas (Córdova et al., 2019; Su et al., 2013). The positive results of nZVI addi- Anaerobic sludge used as inoculum and was obtained from the tion have been related both, to the enhancement of enzyme activities in mesophilic anaerobic digester of Riu Sec municipal wastewater treat- hydrolysis and acidification steps (Zhang et al., 2020) even during the ment plant (WWTP) (Sabadell, Barcelona, Spain). Sewage sludge fed removal of persistent organic substances in waste activated sludge daily to the reactors was also collected from the Riu Sec WWTP. The (Zhou et al., 2020) and antibiotics (Zhou et al., 2021); and the growth sludge was collected periodically, twice for Experiment 1 (3.7% total of H2-utilizing microorganisms including homoacetogens and solids TS); 2.7% volatile solids (VS) and 3.3% TS; 1.8% VS and once for Ex- hydrogenotrophic methanogens by providing electrons directly or pro- periment 2 (3.7% TS; 2.9% VS) and stored in 0.5-L plastic bottles at a tem- ducing hydrogen through the iron anaerobic oxidation (Zhen et al., perature of −24 °C and thawed at room temperature when used. 2015; Xu et al., 2019). However, adverse effects on the methanogenesis were also reported at high doses (Dong et al., 2019). Some references 2.3. Anaerobic digestion experimental set-up and procedure using powder and scrap Fe in AD experiments have shown positive ef- fects in biogas yield. However, although Fe0 has been reported to im- The experimental set-up consisted of six fermentation glass vessel prove hydrolysis–acidification and the CH4 yield, its efficiency was low reactors used in two sets of semi-continuous operation of 103 days (Ex- compared with nZVI (Wang et al., 2018) because it is directly propor- periment 1) and 116 days (Experiment 2). The reactors were operated tional to its specific surface area. Instead, other authors did not observe at 37 °C with intermittent stirring: agitated for 15 min every 45 min any effect using micro-scale zero-valent iron (200 μm) (Xu et al., 2019). using a mixing device located on the top. The total volume for each re- In a moment when some research efforts are focused on the actor was 2 L (1.8 L working volume). The hydraulic retention time upgrading of biogas of broaden its applications, the use of nanoparticles (HRT) was set at 21 days, as it was in Riu Sec WWTP, following a can be a promising way to have enriched methane streams of biogas. In semi-continuous feeding strategy, according to which the reactors this case, the residual CO2 of these streams can be further removed were fed manually once a day, 5 days a week (from Monday to Friday) using novel and consolidated techniques (adsorption, absorption, mem- replacing the same volume that was previously extracted from them. branes, etc.). Although some studies have evaluated the effects of nZVI The substrate feeding/exchange was done as follows: during the agita- on AD of sewage sludge, their application in a more realistic continuous tion period, 80 mL of digestate were extracted by using a graduated process has been scarcely explored, a fact that could imply changes from 50 mL syringe through the outlet pipe. Then, 80 mL of new feed was in- those effects observed in batch conditions. It is expected that the addi- troduced in the reactors through the inlet pipe. After feeding, all reactors tion of nZVI increases methane production in (semi)realistic conditions were flushed with N2 and then sealed. 2 R. Barrena, M.C. Vargas-García, G. Capell et al. Science of the Total Environment 777 (2021) 145969 The different doses of nanoparticles were added to three reactors filtered supernatant sample using a pH-meter (pH 50+ DHS, XS Instru- (nZVI reactors) while the remaining three were used as control reactors ments, Italy). TS and VS were analysed two–three times per week. VFA, (control reactors). When added, nZVI were fed at the same time that specifically formic, acetic, propionic, iso-butyric, butyric, iso-valeric and sewage sludge. It is important to note that reactors were fed daily but valeric acids, were measured once a week. VFA samples, filtered nZVI were added punctually. Thus, during the daily substrate feeding, through a 0.22 μm polyethersulfone syringe filter (Millipore Millex- nZVI (added the previous days) were retained in the reactors by using GP), were quantified by liquid chromatography (HPLC, Dionex Ultimate a neodymium permanent magnet (NdFeB magnet). The strong mag- 3000) equipped with a 210 nm ultraviolet detector and a Transgenomic netic power of the NdFEB maintains in the reactor the main part of ICE-COREGEL 87H3 column (300 mm × 4.6 mm). The mobile phase nZVI added. The volume of biogas produced was measured by consisted of an acid solution of 320 μL L−1 of sulphuric acid. The total connecting to each reactor a Ritter MilliGascounter (Ritter, Germany). time for each run was 150 min. A volatile free acid mix solution The biogas production was weekly averaged and calculated in reference (CRM46975, Sigma-Aldrich) was used as standard. to the reactor working volume. The process was started-up in the 6 re- Biogas composition was analysed by gas chromatography (GC, actors feeding only the sewage sludge during approximately two HRTs Agilent 7820) equipped with a thermal conductivity detector (TCD) before adding the first dose of nZVI. and two columns (Agilent G3591-81136 and G3591-80017) connected with a valve and activated with synthetic air. A sample of 100 μL was 2.4. nZVI dosing collected from a valve in the headspace reactors with a gas-tight syringe and injected. Nitrogen was used as carrier gas. Injector and detector Different nZVI doses were tested during the semi-continuous AD op- temperatures were 200 °C and 250 °C, respectively. The initial oven eration. Table 1 summarizes the punctual doses studied and the day of temperature was 70 °C for 2 min, with a subsequent temperature addition for both experiments. Two sets of experiments were carried ramp of 20 °C per minute, up to 40 °C until the end of the analysis. out: for Experiment 1, the first dose of nZVI was 0.27 g L−1 (equivalent The total time for each run was 6 min. An external standard (100% to 10.0 g kg−1 SV for Experiment 1 and 9.3 g kg−1 SV for Experiment CH4) was used for the quantification of CH4 content. 2) based on previous works (Casals et al., 2014) and results from a series of batch experiments (Fig. S1) and added on day 38. The second dose 2.6. Microbial community analysis was set at 0.54 g L−1, doubling the previous one. Later, four more doses of 0.27 g L−1 were added to the reactors approximately every The microbiome was analysed in Experiment 1 from samples 7 days from day 74 until 98. Lastly, the dose was increased up to extracted from each reactor (total six samples: three from nZVI and con- 0.81 g L−1 to check the system response to higher doses and was sup- trol reactors respectively) at end of the study (day 99). Sequencing and plied on day 99. After the results obtained in Experiment 1, a new pro- bioinformatics were performed by Life Sequencing S.A. (Valencia, cess (Experiment 2) was carried out in order to evaluate the effects of Spain). PowerSoil™ DNA Isolation kit (MoBio Laboratories Inc., incremental doses of nZVI on methane production. Thus, experiment 2 Carlsbad, CA, USA) was used for the extraction of DNA according to corresponds to a new experiment where the reactors were started-up the manufacturer's instructions and extracts were tested to ensure con- again with a new inoculum, and after approximately two HRTs the centration and quality using a NanoDropTM 2000 spectrophotometer first dose of nZVI was added on day 38. Here, the first punctual dose ap- (Thermo Fisher Scientific, Waltham, MA, USA) for the determination plied was also 0.27 g L−1 and then the dose was doubled every time: of A260/A280 and A260/A320 ratios. Genomic libraries from the V3- 0.54, 1.08, 2.17, and 4.33 g L−1 to see the overall performance of the sys- V4 hypervariable region 16s rRNA gene were constructed using the uni- tem under an exponentially increasing dose of nZVI to have preliminary versal primers Pro314F (CCTACGGGNBGCASCAG) and Pro805R (GACT values of the limits of nZVI application. ACNVGGGTATCTAATCC), designed for the simultaneous detection of Bacteria and Archea (Takahashi et al., 2014). Sequencing data obtained 2.5. Analytical methods from the Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA) were analysed according to a 300 bp fragments paired-end protocol Alkalinity, pH, and volatile fatty acids (VFA) were also periodically and processed using PEAR software v.0.9.1, CUTADAPT software evaluated to assess the stability of the process. Total alkalinity (TA) v.1.8.1 and Chimera Uchime (Edgar et al., 2011) for the construction of and partial alkalinity (PA), total (TS) and volatile (VS) solids content the overlapped sequences, the removal of adapters and chimeric se- and pH were determined according to standard methods (APHA, quences, respectively. The remaining sequences were examined to se- 1999). The intermediate alkalinity (IA) was calculated as the difference lect those displaying quality scores higher than Q20 and length > 200. between TA and PA. Alkalinity and pH were measured twice a week in a Finally, the CD-HITt software v.4.6.8 was applied for clustering, using a Table 1 Summary of the doses applied and main results from semi-continuous anaerobic digestion processes in Experiment 1 and 2. Different letters in the same column indicate significant dif- ferences between the evaluated doses (p < 0.05). Dose (g L−1) Day of addition Averaged CH4 (control) (%) Averaged CH4 (nZVI)⁎ (%) Maximum CH4 (nZVI) (%) Persistence of improvement (days)⁎ Experiment 1 0.27 38 66.1 ± 1.8 a 68.2 ± 3.0 a 72.6 5 0.54 57 64.6 ± 1.7 a 66.0 ± 2.3 a 69.3 7 0.27 74 65.1 ± 2.1 a 67.9 ± 2.8 a 72.1 All (5) 0.27 79 65.9 ± 1.6 a 72.1 ± 2.6 b 75.1 All (6) 0.27 85 66.0 ± 1.9 a 72.7 ± 1.7 b 75.4 All (6) 0.27 92 66.4 ± 1.0 a 71.5 ± 1.8 b 74.0 All (7) 0.81 99 65.1 ± 1.5 a 75.9 ± 2.8 c 79.2 All (4) Experiment 2 0.27 38 62.1 ± 1.4 a 64.8 ± 0.8 a 65.3 5 0.54 54 63.2 ± 1.8 a 67.3 ± 2.1 b 70.1 5 1.08 65 63.8 ± 2.6 a 69.4 ± 2.3 b 72.8 9 2.17 85 64.0 ± 1.7 a 68.4 ± 1.9 b 72.9 9 4.33 108 63.6 ± 1.4 a 71.0 ± 0.9 c 72.9 9 ⁎ When differences in methane content in biogas between control and nZVI reactors were significant (p < 0.05). 3 R. Barrena, M.C. Vargas-García, G. Capell et al. Science of the Total Environment 777 (2021) 145969 97% cut-off, and resulting OTUs were phylogenetically classified to the production of 18.1 but only 6.9% in the methane content. Su et al. maximum taxonomical level possible using the BLAST tool for Local (2013) reported improved production of biogas and methane by 30.4 Alignment (NCBI, http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the asso- and 40.4%, respectively. Nevertheless, all of the reported works dealing ciated GenBank database, as well as EzBioCloud 16S database. with the effects of nZVI in AD have been performed in batch conditions, Shared and unshared OTUs among samples were represented which could be the reason behind the differences observed with through Venn diagram, while biodiversity was estimated by calculating this work. Shannon-Wiener, Chao 1 and Simpson indices at species taxonomic level (α-biodiversity), using a 97% sequence similarity threshold for 3.2.1. Biogas composition: methane enrichment after nZVI dosing the establishment of OTUs. Sørensen-Dice qualitative (presence/ab- In Experiment 1, the first dose of 0.27 g L−1 of nZVI was supple- sence data) and quantitative (relative abundance data) indices provided mented on day 38. Immediately after the addition, methane content in knowledge about community similarity among samples (β-biodiver- biogas was statistically different (p < 0.05) from the mean in control re- sity). Finally, evolutionary relationships between species were actors (66.1%), reaching maximum methane content of 72.6% (Table 1). established using the Neighbor-Joining method with Mega version 6.0 This effect was previously reported and attributed to several possible software. causes (Suanon et al., 2017). Low CO2 production has been related to the partial conversion of produced CO2 to methane via electron transfer. 2.7. Statistical analysis Also, the addition of nZVI could contribute to the production of H2 via the microbial corrosion and surface oxidation of nZVI and the produced Statistical analysis was based on one-way ANOVA (p < 0.05 confi- H2 could directly be used by hydrogenotrophic methanogens to convert dence) with the Tukey test. Significant differences were analysed com- CO2 to CH4. Also, homoacetogenic bacteria could use CO2 and paring nZVI reactors with control reactors (without nZVI) using the overproduced H2 for the formation of acetate. Besides, Xu et al. (2019) software Minitab 16 (Minitab Inc.). Values were reported as the average reported that nZVI addition contributes to stable and favourable condi- of replicates and the corresponding standard deviation. tions for methanogens by lowering the oxidation–reduction potential. However, the effect was only sustained for some days after dosing, 3. Results and discussion since there were no significant differences between the two groups of reactors six days after nZVI addition. Methane composition was moni- 3.1. Overall performance of the reactors tored during one HRT before adding a new dose. No significant differ- ences were observed between both groups of reactors during this The effect of nZVI addition was studied through the evolution of typ- time. The day 57, the next dose, doubling the first one (0.54 g L−1), ical operational parameters. Data corresponding to pH, alkalinity and was added. Again, the difference between both groups of reactors VFA are presented in Supplementary material. VS removal ranged be- started to be statically different immediately after the addition. In this tween 50% and 60% for all the reactors, with small fluctuations due to case, the enhancement was significantly higher for seven days. The in- substrate variability (Fig. S2). Some authors have reported an improve- crease of methane in the biogas per day was similar for both doses ment in solid removal when nZVI were added, such as Córdova et al. (Table 1). After the addition of these two punctual doses, the results (2019). The latter observed an increment on VS removal of 19.6% on suggested that a minimum of freshly synthetized nZVI should remain batch AD experiments using sewage sludge. However, no statistical dif- in the reactor to enhance biogas composition. The loss of the effect ferences were found between nZVI and control group reactors for both could be attributed to changes in nZVI once in contact with the anaero- sets of experiments in this work. bic medium, possibly related to their role as an electron donor (Dong TA remained practically constant, and the IA/PA ratio remained et al., 2019) and possible oxidation. lower than 0.5 for Experiment 1 and 0.3 for Experiment 2 without any significant differences between nZVI and control reactors. IA/PA ratios 3.2.2. Dosing nZVI to sustain the positive effect over a long period of 0.9 were suggested to maintain a total VFA below 2.5 g L−1 during Four doses of 0.27 g L−1 were periodically added to the nZVI reactors the thermophilic AD of sewage sludge (Ferrer et al., 2010). In accor- every 5–7 days to evaluate if the methane content increase could be dance, pH was reasonably constant in Experiment 1 (7.5 ± 0.1 and sustained in long-term operation. Results in Table 1 confirmed the via- 7.4 ± 0.1 for nZVI and control reactors, respectively) and Experiment bility to operate in semi-continuous mode, dosing nZVI every seven 2 (8.0 ± 0.1 and 7.9 ± 0.1). In agreement with the trend followed by al- days to maintain the methane-enriched biogas. During these periods, kalinity and pH, VFA remains low during all the operation for Experi- an increase of 12.6% in methane content on average was observed com- ment 1, being the highest reached value of 0.22 g L−1 (Fig. S3). The pared to control reactors (Fig. 1a). addition of nZVI has been previously related to the promotion of VFA Lastly, after confirming that the effects persisted dosing of 0.27 g L−1 production (Jia et al., 2017). Also, Feng et al. (2014) reported an increase every seven days, the effect of a higher dose (0.81 g L−1) was tested. In of 37.3% of VFA after nZVI supplementation. Both studies were per- this case, methane content was increased from 65.1% to 75.9% (Table 1), formed in batch; however, the trend of VFA observed in this work ap- with a maximum methane content of 78.6%. An averaged increment of peared not to be influenced by dosing nZVI tested in semi-continuous 16.6% for four days was observed with respect to the control reactors. operation. This increment was in accordance with Córdova et al. (2019), who ob- served an increase in methane content from 63.2% to 77.6%, and with 3.2. Effect of nZVI on biogas production and composition Jia et al. (2017), who reported an increase of methane content of 6.93% in a batch test. Methane content in biogas for all Experiment 1 is Unexpectedly, and contrarily to most of the studies on this topic, bio- shown in Fig. 1a. Biogas composition was always analysed before feed- gas production was not statistically different when nZVI were added. ing the reactors. Only methane and CO2 were quantified while H2 was Averaged biogas production rate was 0.25 ± 0.08 for nZVI reactors undetected. and 0.26 ± 0.08 m3 biogas m−3 d−1 for control reactors in Experiment 1, and 0.48 ± 0.06 and 0.45 ± 0.06 m3 biogas m−3 d−1 for Experiment 2 3.2.3. Dose-response effect on methane production (nZVI and control reactors, respectively) (Fig. S4). Volumetric biogas As observed in Experiment 1, increased methane in biogas was de- production was in line with the obtained in Riu Sec WWTP, 0.33 m- tected after the first dose of 0.27 g L−1 for five days in Experiment 2 3 biogas m−3 d−1 (personal communication). As stated above, biogas (Fig. 1b). The next dose (doubling the first one) sustained the effects production improvement has been one of the most reported benefits also for five more days (Table 1). The following doses (each doubling of the use of nZVI. Jia et al. (2017) observed an increment in biogas the previous one) lengthened the impact up to 9 days. Moreover, a 4 R. Barrena, M.C. Vargas-García, G. Capell et al. Science of the Total Environment 777 (2021) 145969 Fig. 1. Methane content in biogas for nZVI and control reactors; a) Experiment 1 and b) Experiment 2. trend can be observed since the higher the dose the higher methane data is skewed to the right for doses 1.08 and 2.17 g L−1. Values below percentage was obtained on average (over the time that the effect the median were less dispersed for these doses, probably because of persists). the higher frequency of high amounts of methane. Instead, high disper- Fig. 2 shows a boxplot with methane values from nZVI reactors for sion above the median (first dose) can be related to the fastest loss of ef- each dose. Only values statistically different (p < 0.05) from control re- fect after the addition. Only the highest dosage tested (4.33 g L−1) could actors were considered in the boxplot. Boxplot lengths showed that sustain the positive effect for more time in high values. Methane data was concentrated in a narrow range of values, being the highest of all doses tested. Accordingly, methane content was sustained for 9 days in the highest values, implying an important improvement in methane yield. Methane yield for this period was 179 ± 24 and 202 ± 13 for con- trol and nZVI reactors respectively, reaching an increment of 12.7% in nZVI reactors. Therefore, the results suggest that higher doses would result in bet- ter performance in long-term operation to achieve reliable improved yields. However, some authors have observed adverse effects when working with lower doses than the ones studied here. For instance, Yang et al. (2013) reported a reduction of 69% in methane production when 1.6 g L−1 nZVI was used in batch experiments. Also, in batch experiments, Wang et al. (2018) established the optimum dosage at 1.0 g L−1 because further addition could cause inhibition of methanogenesis step. In this sense, the gradual acclimation and adapta- tion of the microbiota after punctual doses raises as a hypothesis for the better performance of the process, as well as differences in the morphol- ogy and purity of the employed materials. As observed in Table 1, the same dose (0.27 g L−1) did not show the same effect at day 3 when sig- nificant differences were found between them. In this sense, it should be Fig. 2. Significantly improved methane values from nZVI reactors for each dose in interesting to study the effect of higher doses in a continuous operation Experiment 2. Dotted lines in boxplots indicate mean values. mode using an acclimated microbial consortium having in mind the 5 R. Barrena, M.C. Vargas-García, G. Capell et al. Science of the Total Environment 777 (2021) 145969 possible changes that higher doses can have on microbial communities. Fig. 3e an image of a control reactor is shown, where sludge flocs Indeed, Dong et al. (2019), using 25 g L−1 of nZVI reported the promo- displayed a rigid and compact structure, hallmark of a NPs free sludge. tion of homoacetogenesis, with acetic acid production instead of con- The size of the NPs obtained is similar to that used by Su et al. (2013) version of CO2 to CH4, and inhibition of the methanogenesis, which is who used the 1 g L−1 of commercial NPs (size 20 nm) and obtained a dose significantly higher than the employed in this work. 13.2% increase in the methane content of biogas. Jia et al. (2017), stud- The magnetic retention of nZVI in the reactors allowed showing the ied the effect of 50–70 nm NPs reaching an increase of 6.9% in the meth- loss of its effect on methane content over time. This could be an impor- ane content of biogas using the same concentration of 1 g L−1. As tant knowledge to operate in semi-continuous mode, avoiding the con- mentioned previously, in our study, the methane content was increased tinuous nZVI addition and working efficiently without adding more 16.6 and 11.6%, in Experiments 1 and 2, respectively. It has been re- nZVI than necessary. In this sense, the study of NPs properties once ported different factors affecting nZVI effect on AD related to both added to the reactors can help to understand the loss of efficiency nZVI particle characteristics (particle size, surface properties, dosage, over time and the mechanisms involved in the enhancement of AD. etc.) but also microbial structure in the system (Wang et al., 2018). Spe- cial attention should be paid to phosphate concentration in the medium 3.3. Physical and chemical nZVI properties due to its high affinity with nZVI and other iron species. Phosphate can be easily adsorbed onto iron surface, which makes of it a good candidate 3.3.1. NPs morphology and size distribution after the anaerobic digestion for phosphate removal from aqueous solutions. One of the reported process mechanisms for this affinity is that hydroxyl species are formed on Fig. 3a shows SEM images of nZVI before their addition to the anaer- iron surfaces, and this group is easily replaced by phosphate (Wen obic digesters. It can be observed how nZVI of sizes ranging from 10 to et al., 2014). Thus, this interaction can affect the process in two main 30 nm tend to agglomerate. It has been previously reported that these ways: 1) modifying the surface structure and properties of nZVI, since NPs have a strong tendency to form microscale aggregates, likely due adsorbed phosphate could combine with iron ions to form stable com- to the weak surface charges (Sun et al., 2006; Zhou et al., 2020). This plexes, among which vivianite is one of the most common (Wen et al., phenomenon could partially explain their loss of efficiency over time 2014); and 2) affecting methanogenic activity since the aforementioned in the present study, as the contact surface is reduced as aggregation complexes could hinder the phosphorus uptake by methanogenic mi- increases. croorganisms (Zhu et al., 2021). Indeed, phosphate analysis should be Regarding nZVI extracted from the digester at the end of the process necessary to really understand the efficiently of nZVI dosage in view (Experiment 1, day 99), in some areas of the sample, it is noted how the to further experiments regarding dose optimization. surface of nZVI can become scraggy and rough (Fig. 3b). Zhen et al. (2015) reported that many “flower-shaped” crystals made by prismatic 3.3.2. Oxidation state after the anaerobic digestion process iron (hydr)oxides clusters (e.g., FeCO3. Fe3O4. Fe(OH)3 and FeOOH) are TEM-EELS analysis was used to evaluate the oxidation state of iron at found on the nZVI surface. Fig. 3c, shows a representative image of a the beginning and the end of the studied processes. The most common sample extracted from a reactor towards the end of the study (Experi- procedure to assess the oxidation state of a metal is based on the L2,3 ment 1, day 99). It shows the presence of nZVI in the anaerobic digester, white-line ratio, which has a value of 2.99 for zero-valent iron, 3.99 where these can be clearly identified by its spherical shape and for Fe2+ and 4.55 for Fe3+ (Tan et al., 2012). In the present case, nZVI re- scattered surface (Fig. 3d). An EDX analysis of this area revealed an vealed a 3.70 ratio at the beginning of the process, a value that increased iron composition of 78% in weight. It can be observed that nZVI are up to 5.60 at the end. Thus, there is clear oxidation of the nZVI during mixed with sludge, and they have not been washed off the reactor. In the AD, confirming the electron donor capacity of nZVI (Jia et al., 2017). A B E 5 µm C D 100 µm 10 µm Fig. 3. SEM images of A) nZVI particles before being used for AD; B) and C) sample from nZVI reactors; D) Zoom picture of the indicated area of picture C; E) sample from control reactors. 6 R. Barrena, M.C. Vargas-García, G. Capell et al. Science of the Total Environment 777 (2021) 145969 Zhen et al. (2015) observed that the use of zero-valent scrap iron nZVI reactors, from 4.36% to 6.01%, might enhance the activity of could offer electrons and promote H2 as a source of electron donors hydrogenotrophic methanogens. for the AD of sewage sludge, accelerating different AD steps. Karri Among the numerous OTUs without direct assignation stood out a et al. (2005) indicated that Fe0 could serve as a significant slow- group of five sequences related to no cultivated microorganisms affili- release electron donor, noticing that Fe could directly be used for reduc- ated to the division-level “candidate” phylogenetic group WS6 ing CO2 into CH4 through autotrophic methanogenesis. So, the study of (Table S1, supporting material). Such species are mostly associated microbial community changes can also help understand how the oxida- with anaerobic environments including AD (Qiao et al., 2013) and tion of nZVI could be exploited for AD performance, understanding have been described as capable of producing acetate, lactate and/or hy- some of the involved mechanisms. drogen (Wrighton et al., 2016). Moreover, some members of this clade carry genes encoding RuBisCO forms specific to Archaea (Hernsdorf 3.4. Effect of nZVI on microbial community et al., 2017). These characteristics emphasize their importance in me- thanogenic processes and explain their high relative abundance, the It is widely accepted that nZVI supplementation affects the structure third most dominant group, in both reactors. Data about the influence of the indigenous microbial communities (Zhang et al., 2020). In some of nZVI on representatives of WS6 group is practically non-existent, al- cases, such affectation has been described as negative to a greater or lesser though the limited information available regarding anaerobic processes extent (Xiu et al., 2010). On the contrary, methanogenic populations suggests a slight positive effect (Zhang et al., 2018; Xiang et al., 2019). seem to respond positively to them (Antwi et al., 2017). At first sight, The most dominant phylum, both in the absence and in the presence our results confirm the existence of alterations in the microbial commu- of nZVI, was Firmicutes. All of the dominant OTUs assigned to this phylum nity structure because of the application of nZVI. According to α- belong to Clostridia class, which is expected since members of this class biodiversity indices (Table 2), both processes can be considered as very are close associates to AD and methanogenesis because of their ability rich and heterogeneous from a prokaryotic point of view, which corre- to produce different substrates that promote methanogenic activity sponds well to the fact pointed out by other authors studying AD supple- (Dos Reis et al., 2015). The relative abundance of Firmicutes decrease in mented with Fe nanoparticles (Ambuchi, 2018). However, β-biodiversity nZVI reactors, 20.59%, in comparison to control, 30.76%. Results are con- Sørensen-Dice index revealed differences derived from the presence of tradictory concerning this phylum, since some studies report a positive ef- nZVI, not associated to the identity of species but because of the relative fect of nZVI supplementation (Antwi et al., 2017; He et al., 2017), but abundance (>0.5%) of dominant bacteria. Fig. 4a shows the composition many others describe a decrease in the quantitative presence of their rep- of the microbial community (phylum taxonomic level) at the end of the resentatives (Pan et al., 2017; Zhang et al., 2018). Iron can play a dual role processes and Fig. 4b presents the percentage changes for each phylum regarding viability of microbial cells, a positive one associated to its nutri- in nZVI and control reactors. Most of the prokaryotic groups showed in- tional function, and a negative one derived from the production of reac- creased relative abundances when nZVI particles were added, especially tive oxygen species that strongly affect cell membranes (Daraei et al., Aminicenantes, Verrucomicrobia and Proteobacteria. Aminicenantes and 2019), and promote disruption of cell integrity (Yang et al., 2013). Indeed, Verrucomicrobia are not considered as common members of usual oxidation of nZVI is responsible for both, to act as co-factor (co-reducer) microbiome of AD and their presence, if so, can be termed as marginal to promote the conversion of CO2 into CH4, or, by the same mechanisms, (Calusinska et al., 2018). Considering the methanotrophic potential of the generation of toxic free radicals. Consequently, nZVI need to be care- some species assigned to Verrucomicrobia phylum (van Teeseling et al., fully introduced into the AD processes. Besides, the prevalence of one or 2014), their relatively low significance has a positive character. On the another effect and, in consequence, the response of specific microorgan- contrary, Proteobacteria are usually common members of the AD isms, will be mostly depending on environment (Kong et al., 2018). microbiome, and their stimulation on account of nZVI supplementation A more detailed analysis of the direction of the changes taking has already been reported in similar conditions (Wang et al., 2018). This place in Firmicutes at species level (Fig. 5) revealed decreases in can be assumed as positive since they are strongly related to the Sedimentibacter and Syntrophomonas representatives, as well as acidification process, which is essential for methanogens activity. A simi- OTU 6892, affiliated to Peptococcaceae family (Accession number lar role can be assigned to the members of phyla Bacteroidetes and LT625357.1), and an increase in OTU 18225, also identified as a member Cloacimonetes, for which the stimulating effect of nZVI was not as marked of the Clostridia group. Clostridia are considered one of the most signif- as in the case of Proteobacteria, although their relative abundances, be- icant communities integrating the so-called core microbiome in AD (De tween 13% and 20%, were far outstripping than those observed for Vrieze et al., 2016). Species belonging to Clostridium, Sedimentibacter, Proteobacteria, never above 3%. Both microbial groups are significant mi- and Syntrophomonas have been recognized as prevalent OTUs within crobiota members responsible for the hydrolysis of carbon compounds this core microbiome. However, the dominance of one or other is condi- and further transformation into molecules that support methanogenic ac- tioned by both nutritional and environmental parameters (Rui et al., tivity (Chojnacka et al., 2015). This is also the case of representatives of 2015). In this sense, the point is the maintenance of the community Thermotogae, a phylum integrated by microorganisms showing a close overall functionality to provide the substrates needed for the methano- relation with members of Archaea, which are recognized H2 producers genic activity (Treu et al., 2016). The balance between the main types of (Bhandari and Gupta, 2014). The increase in their relative abundance in substrates, in turn, determines which methanogenic community, acetotrophic or hydrogenotrophic, will lead the process. The methanogenic community remained stable in both reactors Table 2 (Fig. 6), with the only exception of species assigned to Methanothrix α- and β-biodiversity estimated according to Shannon-Wiener, Chao 1 and Simpson and Sørensen-Dice qualitative and quantitative indices, respectively. The last one was calcu- genus, which increased their relative abundance from 0.2% in control up lated for both the whole community and the dominant community (relative abundance to 0.57% in nZVI reactors. Members of this genus are recognized as spe- >0.5%). Indices were calculated at species taxonomic level. cialist methanogenic bacteria that only use acetate for their growth (Stams et al., 2019), a substrate for which they have greater affinity Index Control nZVI than other acetoclastic species (Smith and Ingram-Smith, 2007). This Shannon-Wiener richness index 4.43 4.48 greater affinity could explain its marked rise, as it has been suggested Chao 1 estimator of total richness 1891 1712 Simpson dominance index 0.04 0.04 by other studies (Röske et al., 2014), since it would be reasonable to ex- Sørensen-dice qualitative index Absolute >0.5% pect that acetate concentration in nZVI reactors is lower because of the re- nZVI 0.64 0.97 duction in the relative abundances of acetate-producer bacteria, such as Sørensen-dice quantitative index Sedimentibacter, or Syntrophomonas (Breitenstein et al., 2002; Kong nZVI 0.71 0.74 et al., 2018). The higher relative abundance of other acetate- 7 R. Barrena, M.C. Vargas-García, G. Capell et al. Science of the Total Environment 777 (2021) 145969 a) 16000 Aminicenantes Bacteroidetes 14000 31% Chloroflexi Number of reads 12000 Calsiderica 10000 Candidate division WS6 21% 8000 Cloacimonetes Firmicutes 6000 Proteobacteria 4000 Spirochaetae 2000 Synergistetes Thermotogae 0 R_Control R_nZVI Verrucomicrobia b) Verrucomicrobia Thermotogae Calsiderica Synergistetes Spirochaetae Proteobacteria Firmicutes Candidate división WS6 Cloacimonetes Chloroflexi Bacteroidetes Aminicenantes -60 -40 -20 0 20 40 60 80 Fig. 4. a) Number of reads and relative importance of the prevalent OTUs (relative abundance >0.5%) in both samples at phylum taxonomic level. Relative importance within the sample is expressed as bubble size (diameter). Data for maximum abundances are included in respective bubbles as a reference. b) Percentage changes in phyla relative abundances among prevalent OTUs representing both microbiomes. Green color means higher abundance in nZVI reactors and red color in control reactors. producer species, like those assigned to Cloacimonetes taxon or 4. Conclusions Porphyromonadaceae family (OTU 7658) (Esquivel-Elizondo et al., 2016; Hahnke et al., 2015) appears not to be sufficient to maintain the Dossing a few g L−1 zero-valent iron nanoparticles (nZVI) every same level as in control reactors but enough to assure the prevalence of 5–7 days could be a successful strategy in the semi-continuous anaero- acetotrophic pathway over hydrogenotrophic methanogenesis. The in- bic digestion of sewage sludge to enhance methane content in biogas creased presence of some H2-producers, as in the case of OTU 18825 (16.6%) and sustain it over time, avoiding the continuous addition of assigned to Hydrogenospora genus (Azizi et al., 2016), have been previ- nZVI. The acclimatization of the microbial community seems to be an ously reported in AD supplemented of nZVI (Huang et al., 2019). This re- important step to the efficient use of nZVI. According to the structure sult might suggest a larger contribution of hydrogenotrophic processes of prokaryotic communities, acetotrophic methanogenesis seems to be for the production of methane. However, it has been recently reported the prevalent via in both reactors, although the supplementation with that its activity could be associated to H2 consumption rather than H2 nZVI altered the balance among acetogenic eubacteria and stimulated production (Zhu et al., 2019), which would be consistent with results the presence of apparently H2-producer species. Methanothrix, an here described. On the other side, a recent study reveals the isolation acetoclastic archaea showing high acetate affinity, was strongly encour- of a methanogen that clusters with Methanothrix and shows aged by nZVI, which sustain the dominance of acetotrophic processes. In hydrogenotrophic activity (Blesy et al., 2019). Further and more ex- any case, further research is needed the study the effects of nZVI tensive studies will be necessary to establish the existence of one or accumulation in the reactors in a long-term operation once their another option, or even the co-existence of both of them. oxidation occurs and contemplating recovery and reuse options. Dose 8 R. Barrena, M.C. Vargas-García, G. Capell et al. Science of the Total Environment 777 (2021) 145969 R_Control R_nZVI Rectinema cohabitans Fervidobacterium riparium OTU 11490 OTU 11571 OTU 1953 OTU 63 OTU 27895 OTU 13083 OTU 13571 OTU 13373 OTU 13779 OTU 16575 OTU 20711 Levilinea saccharolytica OTU 11961 Cloacibacillus porcorum OTU 75 Peptoclostridium acidaminophilum Sedimentibacter bydroxybenzoicus Sedimentibacter saalensis OTU 2411 OTU 18225 OTU 6892 Syntrophomonas bryantii Caloramator proteoclasticus Smithella propionica Ottowia shaguanensis OTU 140 OTU 70 OTU 6804 OTU 6874 OTU 8004 OTU 8578 OTU 7658 Petrimonas sulphuriphila Proteiniphilum acetatigenes OTU 7268 OTU 8038 OTU 8689 Spirochaetae Thermototogae Verrucomicrobia Candidatus Parcubacteria 0% 9% Candidate división WS6 Candidate división WW3 Chloroflexi Aminicenantes Synergistetes Caldiserica Firmicutes Proteobacteria Cloacimonetes Bacteroidetes Fig. 5. Evolutionary history, inferred from rRNA 16S gene sequences associated to prevalent OTUs (relative abundance >0.5%) in at least one of the samples, and corresponding heatmap. The tree was constructed using the Neighbor-Joining method and the p-distance method for computing the evolutionary distances. 9 R. Barrena, M.C. Vargas-García, G. Capell et al. Science of the Total Environment 777 (2021) 145969 R_nZVI R_Control Syntrophomonas Sedimentibacter OTU 7658 OTU 8004 OTU 6804 OTU 70 OTU 6892 OTU 18225 OTU 13779 OTU 13571 OTU 13373 OTU 13083 Methanothrix Methanothrix Methanospirillum Methanospirillum Methanosphaerula Methanosphaerula Methanosarcina Methanosarcina Methanosaeta Methanosaeta Methanomassiliicoccus Methanoculleus Methanomassiliicoccus Methanobrevibacter Methanoculleus Methanobacterium Methanobrevibacter 0 0,2 0,4 0,6 0,8 1 Methanobacterium Re lative abundance (%) 0 2 4 6 8 10 Relative abundance (%) Fig. 6. Relative abundance of methanogens and more significant eubacteria to methanogenic activity. optimization should also be tackled to operate efficiently in a semi- Appendix A. Supplementary data continuous mode without adding more nZVI than necessary and consid- ering phosphate interactions with iron ions. Further consideration Supplementary data to this article can be found online at https://doi. should be given to the quality of digestate in order to avoid any problem org/10.1016/j.scitotenv.2021.145969. related to its valorisation. An enrichment methane streams of biogas in sewage sludge anaerobic digestion could be an interesting alternative References facilitating the upgrading of biogas. Ambuchi, J.J., 2018. Hematite and multi-walled carbon nanotubes stimulate a faster CRediT authorship contribution statement syntrophic pathway during methanogenic beet sugar industrial wastewater degrada- tion. Appl. Microbiol. 102, 7147. 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