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Bioaugmentation of Anammox Activated Sludge with a Nitrifying Bacterial Community as a Way to Increase the Nitrogen Removal Efficiency

Bioaugmentation of Anammox Activated Sludge with a Nitrifying Bacterial Community as a Way to... ISSN 0026-2617, Microbiology, 2022, Vol. 91, No. 2, pp. 133–142. © The Author(s), 2022. This article is an open access publication. Russian Text © The Author(s), 2022, published in Mikrobiologiya, 2022, Vol. 91, No. 2, pp. 160–170. EXPERIMENTAL ARTICLES Bioaugmentation of Anammox Activated Sludge with a Nitrifying Bacterial Community as a Way to Increase the Nitrogen Removal Eff iciency a a, a a a a N. V. Pimenov , Yu. A. Nikolaev *, A. G. Dorofeev , V. A. Grachev , A. Yu. Kallistova , V. V. Mironov , a a a b b A. V. Vanteeva , N. V. Grigor’eva , Yu. Yu. Berestovskaya , E. V. Gruzdev , Sh. A. Begmatov , b b N. V. Ravin , and A. V. Mardanov Winogradsky Institute of Microbiology, Research Centre of Biotechnology, Russian Academy of Sciences, Moscow, 119071 Russia Skryabin Institute of Bioengineering, Research Centre of Biotechnology, Russian Academy of Sciences, Moscow, 119071 Russia *e-mail: nikolaevya@mail.ru Received November 23, 2021; revised November 26, 2021; accepted November 27, 2021 Abstract—Bioaugmentation, i.e., increasing the abundance of certain microorganisms in the community by adding appropriate cells or establishing the conditions promoting their growth, is widely used in environmen- tal technologies. Its application for launching of the anammox reactors is usually limited to introduction of anammox bacteria. We expected addition of nitrif iers during anammox bioreactor launching to stimulate the anammox process due to rapid production of nitrite, which anammox bacteria use for ammonium oxidation. The present work investigated the effect of introduction of a nitrifying community on the composition and activity of the microbial community in an anammox reactor. At the time of inoculation of a laboratory SBR reactor, an active nitrifying community (5 days old) (ASB) (bioaugmenting activated sludge, ASB) containing group I nitrifiers, primarily Nitrosospira, was added (1 : 100 by biomass) to anammox activated sludge (ASA) stored for 1 month at 4°C and exhibiting low metabolic activity. The use of ASB resulted in increased effi- ciency of nitrogen removal. While noticeable nitrogen removal in the control (7%) was observed since day 11 of incubation, nitrogen removal in the experimental reactor began on day 4 at the level of 20%. Nitrogen removal after 30 days of incubation was ~60% in the experiment and 20% in the control. The rate of ammo- nium oxidation in the presence of ASB increased due to activity of nitrifying bacteria (during the f irst 10 days of operation) and anammox bacteria of the genus Brоcadia, which were already present in ASA (throughout all period of operation). Activity of group II nitrifiers (genera Nitrobacter and Nitrococcus), which were pres- ent in ASB, prevented accumulation of nitrite, which in high concentrations is toxic to both nitrifiers and anammox bacteria. High activity of the Nitrosospira nitrifiers introduced with ASB probably provided the anammox bacteria with one of the substrates (nitrite), promoting their rapid growth. During subsequent operation of the reactor, nitrifiers of the genus Nitrosomonas from the initial ASA community were mainly responsible for growth of the anammox bacteria. Thus, ASA bioaugmentation at the loading of the anammox reactor by active nitrifiers resulted in significantly improved efficiency of ammonium removal via the anam- mox process and accelerated transition of the reactor to the working mode. Keywords: anammox, deammonification, bioaugmentation, nitrifying community, nitritation, wastewater treatment, increasing the efficiency of Anammox, community composition DOI: 10.1134/S0026261722020102 Biotechnological processes using the nitrita- requires relatively strict regulation of the physico- tion/anammox scheme (deammonification) have chemical conditions to maintain the activity and com- been actively used in recent years, since they are eco- petitiveness of AOB and AnB (Kallistova et al., 2016; nomically and ecologically more attractive than tradi- Cho et al., 2020). This is especially the case for pro- tional processes based on the nitrif ication/denitrif ica- cessing of concentrated return f lows from facilities for tion scheme (Agraval et al., 2018; Kevbrina et al., processing wastewater sludge (methane fermentation) 2019). Deammonification involves two stages: (Zhang et al., 2018; Izadi et al., 2021; Ochs et al., (1) approximately half of ammonium is oxidized to 2021; Pedrouso et al., 2021). The main task for nitrite by ammonium-oxidizing bacteria (AOB) and research work in this area is to increase the activity and (2) anammox bacteria (AnB) oxidize ammonium with stress resistance of the major groups involved in deam- nitrite to dinitrogen. The deammonification process monification, AOB and AnB, and to improve resis- 133 134 PIMENOV et al. tance of this process to unfavorable factors (Shourjeh era Nitrosomonas and Nitrosospira predominate in the et al., 2021). activated sludge. Addition of these bacteria was planned in the study. One of the ways to achieve higher efficiency, The goal of the present work was to investigate the quicker startup process, and stability of the biochemi- possibility of acceleration of the anammox process at cal parameters of wastewater treatment is bioaugmen- launching the reactor by adding the physiologically tation by adding to the reactor individual strains or active group I nitrifying bacteria. mixed cultures in order to accelerate the transforma- tion of pollutants (Herrero and Stuckey, 2015; Raper et al., 2018). Bioaugmentation with nitrifiers (Tang MATERIALS AND METHODS and Chen, 2015; Stenström and la Cour Jansen, 2017) or anammox bacteria (Jin et al., 2014; Zhang et al., Cultivation of the activated sludge (anammox bac- 2018) was shown to enhance the eff iciency of nitrif ica- terial community) was carried out in the tion or the anammox process, respectively, as well as reactor described previously (Kallistova et al., 2020). their stability under unfavorable conditions. Addition The 4.5-L reactor consisted of two coaxial polymethyl of AOB-enriched activated sludge to the bioreactor methacrylate cylinders. The hermetic space between accelerated the onset of nitritation and established the the cylinders was used for temperature control. Acti- conditions favoring the subsequent AnB development vated sludge was immobilized on a cylindrical carrier (Zhang et al., 2012; Ma et al., 2013). Nitrifiers are of fibrous plastic, a mixture of polyethylene and poly- most often used for bioaugmentation of traditional propylene (Polyvom, ETEK, Russia) with an internal nitrification/denitrification processes. The works on diameter 85 mm, height 200 mm, and total surface addition of nitrifiers to enhance the anammox process area 11.6 dm . Air was supplied from the bottom of the are few. Bioaugmentation with activated sludge reactor by means of a SCHEGO SW2 compressor enriched with AOB and AnB facilitated stabilization of (Germany). When aeration was not applied, mixing the deammonification process and provided for more was carried out using an IKA C-MAG MS7 magnetic rapid commissioning of the bioreactors (Wett et al., stirrer (Germany) at 120‒150 rpm. The medium was 2013; Miao et al., 2017; Pedrouso et al., 2021). Ma supplied at the bottom of the reactor by Masterflex et al. (2013) did not carry out AOB identif ication; acti- L/S economy drive peristaltic pumps (United States), vated sludge was added daily. Miao et al. (2017) and processed water was displaced through the upper achieved enrichment with the biomass of anammox fitting. The temperature mode was adjusted using an bacteria by its monthly addition to the reactor. ELMI TV 2.03 water bath (Latvia) equipped with a circulation pump for the outer contour. The parame- The f irst stage (ammonium nitritation) is known to ters were maintained with a Siemens LOGO 6ED1 be the vulnerable point of the anammox process (Tro- programmed timer (Germany). The reactor was oper- janowicz et al., 2021), since nitrifiers are sensitive to ated in the sequencing batch mode (SBR), which toxic compounds and rapidly die off in the course of storage or starvation (Salem et al., 2006). Anammox included the stages of settling, medium supply with the simultaneous removal of treated water, and aera- bacteria are considerably more robust, recover rapidly tion. The cycle duration was 6 h, and the average after periods of exposure to toxic agents, and die off hydraulic time for the medium in the reactor was 27 h. slowly, not more than 1‒2% a day (Wang et al., 2018), The reactor operated at 30°C and oxygen concentra- compared to up to 20% a day for the nitrifiers (Salem et al., 2006). tions of 0.4‒0.8 mg/L, with alternating phases of aer- ation and its absence (20 min each), and air f low of Planning of the experiment was based on two pre- 20 L/h. Two identical reactors, the experimental and requisites. Of two major bioaugmentation types, exter- control ones, were used in the work. Oxygen concen- nal (when the target biological agent is introduced into tration was determined with a WTW INOLAB 7310 the system) and internal (when conditions are estab- meter equipped with a Sellox sensor (Germany). lished for enrichment of the system with desired bac- The incoming medium contained the following teria, i.e., selection), the former was chosen as the only (g/L): (NH ) SO , 0.942; NaCH COO·3H O, 0.04; one possible at launching of new reactors and during 4 2 4 3 2 KH PO , 0.044; NaHCO , 2.1; pH 8.3 (Boeije et al., activity recovery in the reactor after poisoning of the 2 4 3 activated sludge. Moreover, internal bioaugmentation 1999). The medium was obtained by diluting the con- is well-studied and is widely used, though often not centrate (prepared on distilled water) with tap water at termed bioaugmentation. This is the principle the time of injection into the reactor. The anammox involved in the operation of skimmers enriching the activated sludge (ASA) obtained from a previous run activated sludge with phosphate-accumulating bacte- of the same reactor and stored for 30 days at 4°C was ria (Lema and Suarez, 2017) and partial nitrification used as an inoculum. To launch the reactor, 1 L of the reactors, enriching the sludge with ammonium-oxi- inoculum containing 2 g/L suspended matter was dizing bacteria (Tchobanoglous et al., 2014). Group I added to 3.5 L of the medium. The experimental reac- nitrifiers are required to support the activity of the tor was supplemented with the association of nitrifying anammox process, among which members of the gen- bacteria obtained as described below and containing MICROBIOLOGY Vol. 91 No. 2 2022 BIOAUGMENTATION OF ANAMMOX ACTIVATED SLUDGE 135 20 mg bacterial biomass; this corresponded to the (NH ) SO , 2; K HPO ·3H O, 1; MgSO ·7H O, 0.5; 4 2 4 2 4 2 4 2 100 : 1 mass ratio of ASA and the nitrifying/bioaug- NaCl, 2; FeSO ·7H O, 0.05; CaCO , 5; pH 7. The 4 2 3 menting activated sludge (ASB). The concentrations suspension was mixed thoroughly for 10 min on a Vor- of ammonium, nitrate, and nitrite ions were deter- tex V-1 plus laboratory shaker (Biosan, Latvia). The mined weekly in processed water using the standard enrichment culture was obtained by adding 5 mL of techniques (Rice and Bridgewater, 2012). The amount the compost suspension to 250 mL of the medium. of removed nitrogen (dN mg/L) was calculated as the The enrichment was cultured for 7 days at 30°C on a difference between the concentration of ammonium Biosan OS-20 incubator shaker (Biosan, Latvia) at nitrogen (N-NH ) in the inf lowing medium and the 4 140 rpm. The culture was used as the inoculum (20%) total concentration of mineral nitrogen species in pro- to obtain the bioaugmenting culture (2 L), which was cessed water (N-NH , N-NO , N-NO ). The effi- grown for 5 days under the same conditions and used 4 2 3 ciency of nitrogen removal was calculated as the share as the bioaugmenting activated sludge (ASB) added to (%) of removed nitrogen to its concentration in sup- the anammox bioreactor. Cell titer in ASB (2 × plied water. 10 cells/mL) was determined using an Axio Imager M2 epif luorescence microscope (Carl Zeiss Micros- Activity of ammonium-oxidizing bacteria (AOB), copy, Germany). anammox bacteria (AnB), and nitrite-oxidizing bacte- ria (NOB) was calculated using the amount of The composition of the activated sludge microbial removed nitrogen (dN) and the stoichiometry of the communities (in the inoculum, bioaugmenting ASB, anammox process (Lotti et al., 2014): and those immobilized on the carrier) was analyzed by high-throughput sequencing of the 16S rRNA gene +− − + NH++ 1.146NO 0.071НCO+ 0.57H 42 3 fragments. Activated sludge was samples at the time of inoculation and after 25, 45, and 60 days of incuba- →+ 0.986N 0.161NO tion. ++ 0.071CH O N 2.002H O. 1.74 0.31 0.20 2 Metagenomic DNA from the samples was isolated Specific activity of AnB = dN/T, mg/L/h; using the DNeasy PowerSoil Kit (Qiagen, Germany) + + according to the manufacturer’s protocols. The specific activity of AOB = ((N-NH – N-NH ) – 40 4 V3‒V4 variable region of the 16S rRNA gene was (dN/1.99))/T, mg/L/h; amplif ied using the universal primers 341F CCTAYG- GGDBGCWSCAG and 806R GGACTACNVGG- specif ic activity of NOB = (N-NO – 0.08dN)/T, 3r GTHTCTAAT (Frey et al., 2016). The amplicon mg/L/h, libraries with barcodes were prepared from the where T is hydraulic time of occurrence in the biore- obtained PCR fragments using the Nextera XT Index actor, h; Kit v2 (Illumina, United States) according to the + + manufacturer’s protocols. PCR fragments were N- and N- are concentrations of NH NH 40 4 sequenced using Illumina MiSeq in the 2 × 300 nt for- ammonium nitrogen in the inf lowing and processed mat. medium, respectively, mg N/L, Paired reads were combined with FLASH v. 1.2.11 N-NO is the concentration of nitrate nitrogen in (Magoc and Salzberg, 2011). Low-quality reads, sin- 3r the eff luent processed water. gletons, and chimeras were excluded at the next stage of analysis. The contribution of denitrification was not taken into account, since this process could be responsible The remaining reads were clustered into opera- for not more than 5% of nitrogen removal. Contribu- tional taxonomic units (OTUs) with at least 97% tion to assimilation for the AnB growth was below 1% sequence identity. To determine the share of an OTU and was therefore also disregarded. The coefficients in each sample, original reads (including the low- were taken from the work by Lotti et al. (2014). quality and singleton ones) were overlaid over the rep- resentative OTU sequences using the USEARCH v. 11 Bioaumenting activated sludge (ASB) was a nitrify- software package (Edgar, 2010). Taxonomic identifi- ing enrichment culture obtained from composted cation of the OTUs was carried out using the mixture of excessive sludge from a processing plant of VSEARCH v. 2.14.1 algorithm and the Silva v. 138 a dairy industry waste water and vegetable and wood database (Rognes et al., 2016). waste in the 3 : 3 : 4 ratio (vol/vol). Compost samples were collected during the cooling stage at the tempera- The interaction between microbial groups was ture of 45°C from a 950-m industrial pile with the characterized using network analysis. Analysis of membranous opaque coverage and active aeration simultaneous OTU presence or of their mutual exclu- (Grunt Eko, Russia). Dry matter content in the com- sion was performed based on the Spearman correla- post (by mass) was 27.5 ± 1.3%. Water suspension of tional matrix (Langfelder et al., 2012) using only the the compost was prepared by mixing 1 part of the com- significant correlation values (Barberan et al., 2014). post with 9 parts (by mass) of the sterile nitrifier The threshold value accepted for the correlation coef- medium containing the following (g/L tap water): ficients was 0.6, and the one for the corrected p values MICROBIOLOGY Vol. 91 No. 2 2022 136 PIMENOV et al. (а) 140 60 40 1 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Days Days (b) Fig. 2. Amounts of mineral nitrogen removed in the course of operation of the control (1) and experimental (2) biore- actors. were carried out. The arithmetical mean and the mean absolute deviation were calculated. This value corre- sponded to the experimental dispersion and did not exceed 3%. The values are presented on the graphs as average ± mean deviation. 0 5 10 15 20 25 30 35 Days RESULTS AND DISCUSSION (c) Parameters of bioreactor operation. Addition of ASB resulted in signif icantly more eff icient removal of both ammonium and total mineral nitrogen from the bioreactor (Figs. 1, 2). On day 4, the concentrations of ammonium nitrogen in processed water decreased compared to the incoming water (with N-NH con- centration 200 mg/L) by 40 and 14% in the bioreactors with and without (control) ASB bioaugmentation. Noticeable removal of total nitrogen (7%) in the control reactor was observed from day 11 on; in the 0 5 10 15 20 25 30 35 experimental reactor the process commenced on day 4 Days (20%). By the end of the experiment, ~60 and 20% of nitrogen was removed in the experimental and control Fig. 1. Dynamics of the concentrations of ammonium (a), reactors, respectively. During the first 10 days, nitrite nitrite (b), and nitrate (c) in processed water in the course concentrations in the experimental and control reac- of operation of the control anammox bioreactor (1) and tors did not differ considerably; subsequently, how- the bioreactor supplemented with ASB (2). ever, the concentration increased to 70‒75 mg/L in the control and decreased to 10‒15 mg/L in the exper- was 0.001 (Luo and Bhattacharya, 2006). Only the iment. Decreased concentrations of ammonium and OTUs with the relative abundance of at least 2.0% of nitrites, together with low concentrations of nitrate the 16S rRNA gene sequences in at least one sample (and the absence of conditions for denitrification) were included in the analysis. The networks were visu- indicated the key role of stage I nitrification and ana- alized using Cytoscape v. 3.8.2 (Shannon et al., 2003; mmox reaction in the nitrogen balance. Faust et al., 2016). The calculated values of specific activity for anam- The 16S rRNA gene sequences obtained in the mox bacteria, AOB, and NOB are shown on Fig. 3. work were deposited to the NCBI database and are Bioaugmentation resulted in increased nitrification available at BioProject PRJNA556270. activity at the beginning of the experiment. On day 4, Statistical processing of the data. In the experi- AOB specific activity in the experimental variant was ments on cultivation of the anammox community and 1.8 times higher than in the control. At this time, the assessment of activity of various bacterial physiologi- activity of anammox bacteria in the variant with bio- cal groups, three measurements of each parameter augmentation was 25 times higher than in the control. (concentrations of oxygen and of nitrogen species) While AnB activity subsequently increased, even on MICROBIOLOGY Vol. 91 No. 2 2022 [N(NO )], mg/L [N(NO )], mg/L [N(NO )], mg/L 3 2 4 d[N], mg/L BIOAUGMENTATION OF ANAMMOX ACTIVATED SLUDGE 137 day 32 specific activity of anammox bacteria in the (а) 5.0 experimental bioreactor was 2.7 times higher than in 4.5 the control one. In both reactors activity of group II 4.0 nitrif iers (NOB) was lower than the AOB activity by an 3.5 order of magnitude. NOB activity in the presence of 3.0 ASB was 5 times higher than in the control on day 4; 2.5 during the next 2.5 weeks it rapidly decreased almost 2.0 to zero. In the control activity of this group peaked on 1.5 day 11 and then decreased to zero by day 18. 1.0 0.5 Composition of microbial communities. In order to determine the composition of the activated sludge 0 5 10 15 20 25 30 35 microbial communities, 153698 sequences of the Days V3‒V4 variable regions of the 16S rRNA genes were determined. Clusterization resulted in 1383 OTUs (b) 4.5 (Table S1). Alpha-diversity indices indicated high 4.0 diversity of the communities in the original activated 3.5 sludge samples (Table 1). In the course of operation, biodiversity in both the experimental and control bio- 3.0 reactors decreased and became similar (Table 1), 2.5 which indicated selection of the microbial communi- 2.0 ties. 1.5 1.0 The OTUs found in the communities belonged to 22 bacterial phyla identified using GTDB (genome 0.5 taxonomy database; Parks et al., 2018). Only seven of 0 5 10 15 20 25 30 35 them were dominant and represented at least 1% of the Days 16S rRNA gene sequences in at least one sample (Fig. 4). Predominant groups belonged to the phyla (c) Proteobacteria, Chloroflexi, Bacteroidota, Actinobacte- 0.6 riota, Planctomycetota, Verrucomicrobiota, and Spiro- chaetota; their total abundance in microbial commu- 0.5 nities exceeded 92%. Members of Acidobacteriota, 0.4 Armatimonadota, Bdellovibrionota, Cyanobacteria, Deinococcota, Dependentiae, Desulfobacterota, Firmic- 0.3 utes, Gemmatimonadota, Hydrogenedentes, Myxococ- cota, Patescibacteria, Sumerlaeota, Synergistota, and 0.2 WPS-2 were present as minor components, with their abundance not changing significantly in the course of 1 0.1 community development. Archaea, belonging to the phylum Halobacterota, were found only in ASB, where m m 0 5 10 15 20 25 30 35 their share was below 0.01%. Days ASB differed signif icantly from other samples, with predomination of Proteobacteria (the group to which Fig. 3. Specific activities of anammox bacteria (AnB) (a), nitrifiers belong), while the shares of Chloroflexi and ammonium-oxidizing bacteria (AOB) (b), and nitrite-oxi- Planctomycetota were low. Other activated sludge sam- dizing bacteria (NOB) (c) in an anammox bioreactor: con- ples exhibited similar composition of microbial com- trol (1) and experiment (2). munities at the phylum level (Fig. 4). Microorganism involved in nitrogen removal. The Nitrosospira (Otu2) in the activated sludge decreased analyzed samples were found to contain nitrifiers of 100-fold (to 0.15%) and remained almost stable during the genera Nitrosomonas (Otu8, Otu25, Otu359, subsequent cultivation (Table S1). Otu31, etc.) and Nitrosospira (mainly Otu2) belonging to the phylum Proteobacteria. In the course of cultiva- Detected anammox bacteria belonged to the gen- tion, abundance of Nitrosomonas in the control AAS era Ca. Brocadia (Otu48 and Otu67) and Ca. Jettenia samples decreased from 7 to 4.5%, while in the ASB- (Otu371). The share of Ca. Brocadia was significantly supplemented bioreactor it increased from 5 to 10%. higher (7.8%); it decreased to 1.7% during operation Interestingly, Nitrosospira were predominant nitrifiers of the bioreactor not supplemented with nitrifiers. In in ASB (15.4%), while in the original AAS they were the bioreactor to which nitrif iers were added, the share not present (Fig. 5). After addition of ASB (1 : 100 by of Ca. Brocadia remained almost stable (~6.5%). Rel- biomass) to the experimental bioreactor, the share of ative abundance of Ca. Jettenia in all activated sludge MICROBIOLOGY Vol. 91 No. 2 2022 mg N/(L h) mg N/(L h) mg N/(L h) 138 PIMENOV et al. Table 1. Diversity indices for microbial communities of the analyzed activated sludge samples Diversity indices Cultivation Samples duration, days Chao1 Shannon_e AAS 346 3.48 ASB 839 4.49 Control 180.5 3.32 Experiment 213.6 3.43 Control 147.3 3.27 Experiment 194.3 3.25 Control 141.6 3.34 Experiment 168.4 3.34 samples decreased from 0.37 to 0.1%. Thus, nitrogen bacteria. The analysis was carried out for the OTUs removal via the anammox reaction was probably car- comprising over 2% in at least one sample. The results ried out by Ca. Brocadia (Otu67); its relative abun- are presented on Fig. 6. It may be seen that Otu2 (Nitrospira) occurred together with Otu17 (Pedo- dance in the bioreactor supplemented with nitrifiers increased to 5.5%. Otu67 belonged to Ca. Brocadia bacter) and Otu5 (uncultured Micavibrionales), which were dominant in ASB. However, no positive relations fulgida (98.93% identity of the 16S rRNA gene were found between Otu2 and the microorganisms sequences), which are common in anammox bioreac- of AAS. tors. The second Ca. Brocadia phylotype, Otu48, belonged to another species, Ca. Brocadia caroliensis; It should be noted that the presence of anammox its share decreased in the course of bioreactors opera- bacteria of Otu48 (Ca. Brocadia) correlated with the tion. presence of nitrifiers Nitrosomonas (Otu8) and planc- tomycetes of the family Phycisphaeraceae (Otu50). OTU simultaneous presence and mutual exclusion. Their relative abundance decreased in the course of The functional characteristics of microorganisms are operation of the anammox bioreactor. closely related to the properties of their habitats. Anal- ysis of the presence of the major microbial groups in Thus, ASB introduced into the microbial commu- different microbial communities makes it possible to nity had a significant effect on the efficiency of the reveal the patterns of interaction between different anammox process, while the nitrifiers present in ASB microbial groups. We carried out network analysis of could not adapt to the bioreactor conditions. Bacterial the studied microbial communities to reveal the satellites introduced with ASB probably acted as the groups most affected by the introduction of nitrifying stimulators of the anammox process. Proteobacteria Relative Chloroflexi abundance, % Bacteroidota 1 Actinobacteriota Planctomycetota Verrucomicrobiota Spirochaetota Other phyla Fig. 4. Relative abundance of bacterial phyla in the studied activated sludge samples: activated sludge of the control anammox bioreactor (ASA); activated sludge containing nitrifiers (ASB); and activated sludge from the experimental bioreactor supple- mented with ASB (AB). The numerals after the sample designation indicate operational time in days. MICROBIOLOGY Vol. 91 No. 2 2022 ASA _0 ASB_0 ASA _25 АB_25 ASA _45 АB_45 ASA _60 АB_60 BIOAUGMENTATION OF ANAMMOX ACTIVATED SLUDGE 139 Ca. Jettenia Ca. Brocadia Nitrosospira Nitrosomonas ASA ASB ASA АB ASA АB ASA АB 025 45 60 Fig. 5. Relative abundance of nitrifiers and anammox bacteria in activated sludge samples: activated sludge from the anammox bioreactor (ASA); bioaugmented activated sludge with nitrifiers (ASB); and activated sludge from the anammox reactor supple- mented with the bioaugmentinf sludge (AB). The numerals on the X axis indicate days of cultivation. Otu17 Otu8 Otu28 Otu2 Otu48 Otu5 Otu50 Otu112 Bactroidota Chloroflexi Otu415 Otu4 Otu11 Planctomycetota Proteobacteria Verrumicrobiota Possible presence Otu9 Otu135 Otu3 Mutual exclusion Fig. 6. Network analysis of microbial interactions according to correlation analysis. Positive correlation is marked by green lines; mutual exclusion of the OTUs is marked by red lines. Increased eff iciency of nitrogen removal correlated dance of Comamonas, Kapabacteria, Desulfuromona- with increased abundance of members of the phyla dia, Sorangiineae, Bdellovibrionia, Hyphomicrobium, Chloroflexi, Bacteroidetes, Leptospiraceae and Plancto- and Chitinophagales was revealed. The reason for the mycetes (Table S1). Negative correlation with abun- negative correlation may be explained only for MICROBIOLOGY Vol. 91 No. 2 2022 140 PIMENOV et al. Bdellovibrionia, predatory bacteria capable of lysing We performed bioaugmentation using the commu- other microbial cells. No functional relationships nity enriched with Nitrosospira, bacteria less wide- could be determined for other bacteria. spread in waste treatment plants, and obtained unex- pected results. Although the nitrifying and anammox Another microbial group, addition of which could activities increased rapidly after bioaugmentation, in stimulate ammonium removal are the ASB bacteria, the course of subsequent operation Nitrosomonas that preserved or increased their abundance after (Otu25 and Otu359), belonging to the original com- introduction into the bioreactor. In the initial AAS munity, grew more actively than the introduced Nitro- these organisms were not found or their share was sospira. This was probably due to the initially high minimal. These organisms belonged to Otu31 (Nitro- activity of the introduced Nitrosospira nitrif iers, which somonas), Otu125 (Lacunisphaera), Otu118 (uncul- provided the substrate (nitrite) for anammox bacteria tured planctomycete), Otu959 (Castellaniella_hirudi- and promoted a rapid increase in anammox activity. nis), Otu66 (Castellaniella_ginsengisoli), Otu247 Nitrosospira was subsequently replaced by more com- (Pedomicrobium), Otu383 (Sphingopyxis_sp.), Otu80 petitive autochthonous nitrifiers. (Bordetella petrii), Otu1147 (Rhizobiales), Otu732 (Lacunisphaera), and Otu810 (Micavibrionales). It should be noted that the share of stage II nitrifi- FUNDING ers (nitrite oxidizers) was low in all activated sludge samples. Thus, ASB contained 0.01‒0.04% of this The work was supported by the Russian Foundation for nitrifier type: Otu611 and Otu623 (Nitrobacter alkali- Basic Research, project no. 18-29-08008 (operation of the cus) and Otu853 (Nitrococcus sp.). This finding was in anammox bioreactor), Russian Science Foundation, proj- agreement with their low activity (Fig. 3). Thus, both ect no. 21-64-00019 (molecular analysis of microbial com- analysis of mineral nitrogen balance in the bioreactors munities), and State Assignment for the Research Centre of and molecular biological investigation suggest the Biotechnology, Russian Academy of Sciences (obtaining conclusion that the transformation of nitrogen com- the nitrifying bacteria). pounds in the studied microbial communities fol- lowed mainly the “stage I nitrification‒anammox process” pathway. Both stage II nitrification and het- COMPLIANCE WITH ETHICAL STANDARDS erotrophic denitrification were insignificant. The authors declare that they have no conf licts of inter- ASB bioaugmentation with the Nitrosospira- est. enriched microbial community resulted in a rapid (in several hours‒days) increase of nitrification activity This article does not contain any studies involving ani- and anammox activity, with the latter subsequently mals or human participants performed by any of the maintained at the level several times higher than the authors. anammox activity in the control bioreactor (without bioaugmentation). Increasing anammox activity cor- related with increasing relative abundance of Ca. Bro- OPEN ACCESS cadia fulgida (Otu67). In general, our results conf irmed the positive effect This article is licensed under a Creative Commons Attri- of bioaugmentation with nitrifiers, which has been bution 4.0 International License, which permits use, shar- used worldwide. However, bioaugmentation of waste- ing, adaptation, distribution and reproduction in any water treatment bioreactors is usually carried out with medium or format, as long as you give appropriate credit to the material obtained on the spot and enriched with the original author(s) and the source, provide a link to the the microorganisms common in the activated sludge Creative Commons licence, and indicate if changes were of a given waste treatment plant. Thus, Nitrosomonas made. The images or other third party material in this article predominates among stage I nitrifiers in activated are included in the article’s Creative Commons licence, sludge of industrial facilities (Agraval et al., 2018; Kev- unless indicated otherwise in a credit line to the material. If brina et al., 2019), since it oxidizes ammonium more material is not included in the article’s Creative Commons actively than Nitrosospira, the second most abundant licence and your intended use is not permitted by statutory group (Dytczak et al., 2007). Bioaugmentation with regulation or exceeds the permitted use, you will need to the material obtained on the spot is presently used at obtain permission directly from the copyright holder. To several large-scale plants. Bioaugmentation with nitri- view a copy of this licence, visit http://creativecommons. fying microorganisms from activated sludge is used in org/licenses/by/4.0/. the BABE process, increasing the rate of nitrification in ammonium-enriched wastewater (Kallistova et al., 2016); a full-scale deammonification process was SUPPLEMENTARY INFORMATION implemented in the main flow of the Strass Waste Water Treatment Plant (Strass, Austria) (Cho et al., The online version contains supplementary material 2020). available at https://doi.org/10.1134/S0026261722020102. MICROBIOLOGY Vol. 91 No. 2 2022 BIOAUGMENTATION OF ANAMMOX ACTIVATED SLUDGE 141 from wastewater, Microbiology (Moscow), 2016, vol. 85, REFERENCES pp. 140‒156. 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Bioaugmentation of Anammox Activated Sludge with a Nitrifying Bacterial Community as a Way to Increase the Nitrogen Removal Efficiency

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Copyright © The Author(s), 2022. ISSN 0026-2617, Microbiology, 2022, Vol. 91, No. 2, pp. 133–142. © The Author(s), 2022. This article is an open access publication. Russian Text © The Author(s), 2022, published in Mikrobiologiya, 2022, Vol. 91, No. 2, pp. 160–170.
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ISSN 0026-2617, Microbiology, 2022, Vol. 91, No. 2, pp. 133–142. © The Author(s), 2022. This article is an open access publication. Russian Text © The Author(s), 2022, published in Mikrobiologiya, 2022, Vol. 91, No. 2, pp. 160–170. EXPERIMENTAL ARTICLES Bioaugmentation of Anammox Activated Sludge with a Nitrifying Bacterial Community as a Way to Increase the Nitrogen Removal Eff iciency a a, a a a a N. V. Pimenov , Yu. A. Nikolaev *, A. G. Dorofeev , V. A. Grachev , A. Yu. Kallistova , V. V. Mironov , a a a b b A. V. Vanteeva , N. V. Grigor’eva , Yu. Yu. Berestovskaya , E. V. Gruzdev , Sh. A. Begmatov , b b N. V. Ravin , and A. V. Mardanov Winogradsky Institute of Microbiology, Research Centre of Biotechnology, Russian Academy of Sciences, Moscow, 119071 Russia Skryabin Institute of Bioengineering, Research Centre of Biotechnology, Russian Academy of Sciences, Moscow, 119071 Russia *e-mail: nikolaevya@mail.ru Received November 23, 2021; revised November 26, 2021; accepted November 27, 2021 Abstract—Bioaugmentation, i.e., increasing the abundance of certain microorganisms in the community by adding appropriate cells or establishing the conditions promoting their growth, is widely used in environmen- tal technologies. Its application for launching of the anammox reactors is usually limited to introduction of anammox bacteria. We expected addition of nitrif iers during anammox bioreactor launching to stimulate the anammox process due to rapid production of nitrite, which anammox bacteria use for ammonium oxidation. The present work investigated the effect of introduction of a nitrifying community on the composition and activity of the microbial community in an anammox reactor. At the time of inoculation of a laboratory SBR reactor, an active nitrifying community (5 days old) (ASB) (bioaugmenting activated sludge, ASB) containing group I nitrifiers, primarily Nitrosospira, was added (1 : 100 by biomass) to anammox activated sludge (ASA) stored for 1 month at 4°C and exhibiting low metabolic activity. The use of ASB resulted in increased effi- ciency of nitrogen removal. While noticeable nitrogen removal in the control (7%) was observed since day 11 of incubation, nitrogen removal in the experimental reactor began on day 4 at the level of 20%. Nitrogen removal after 30 days of incubation was ~60% in the experiment and 20% in the control. The rate of ammo- nium oxidation in the presence of ASB increased due to activity of nitrifying bacteria (during the f irst 10 days of operation) and anammox bacteria of the genus Brоcadia, which were already present in ASA (throughout all period of operation). Activity of group II nitrifiers (genera Nitrobacter and Nitrococcus), which were pres- ent in ASB, prevented accumulation of nitrite, which in high concentrations is toxic to both nitrifiers and anammox bacteria. High activity of the Nitrosospira nitrifiers introduced with ASB probably provided the anammox bacteria with one of the substrates (nitrite), promoting their rapid growth. During subsequent operation of the reactor, nitrifiers of the genus Nitrosomonas from the initial ASA community were mainly responsible for growth of the anammox bacteria. Thus, ASA bioaugmentation at the loading of the anammox reactor by active nitrifiers resulted in significantly improved efficiency of ammonium removal via the anam- mox process and accelerated transition of the reactor to the working mode. Keywords: anammox, deammonification, bioaugmentation, nitrifying community, nitritation, wastewater treatment, increasing the efficiency of Anammox, community composition DOI: 10.1134/S0026261722020102 Biotechnological processes using the nitrita- requires relatively strict regulation of the physico- tion/anammox scheme (deammonification) have chemical conditions to maintain the activity and com- been actively used in recent years, since they are eco- petitiveness of AOB and AnB (Kallistova et al., 2016; nomically and ecologically more attractive than tradi- Cho et al., 2020). This is especially the case for pro- tional processes based on the nitrif ication/denitrif ica- cessing of concentrated return f lows from facilities for tion scheme (Agraval et al., 2018; Kevbrina et al., processing wastewater sludge (methane fermentation) 2019). Deammonification involves two stages: (Zhang et al., 2018; Izadi et al., 2021; Ochs et al., (1) approximately half of ammonium is oxidized to 2021; Pedrouso et al., 2021). The main task for nitrite by ammonium-oxidizing bacteria (AOB) and research work in this area is to increase the activity and (2) anammox bacteria (AnB) oxidize ammonium with stress resistance of the major groups involved in deam- nitrite to dinitrogen. The deammonification process monification, AOB and AnB, and to improve resis- 133 134 PIMENOV et al. tance of this process to unfavorable factors (Shourjeh era Nitrosomonas and Nitrosospira predominate in the et al., 2021). activated sludge. Addition of these bacteria was planned in the study. One of the ways to achieve higher efficiency, The goal of the present work was to investigate the quicker startup process, and stability of the biochemi- possibility of acceleration of the anammox process at cal parameters of wastewater treatment is bioaugmen- launching the reactor by adding the physiologically tation by adding to the reactor individual strains or active group I nitrifying bacteria. mixed cultures in order to accelerate the transforma- tion of pollutants (Herrero and Stuckey, 2015; Raper et al., 2018). Bioaugmentation with nitrifiers (Tang MATERIALS AND METHODS and Chen, 2015; Stenström and la Cour Jansen, 2017) or anammox bacteria (Jin et al., 2014; Zhang et al., Cultivation of the activated sludge (anammox bac- 2018) was shown to enhance the eff iciency of nitrif ica- terial community) was carried out in the tion or the anammox process, respectively, as well as reactor described previously (Kallistova et al., 2020). their stability under unfavorable conditions. Addition The 4.5-L reactor consisted of two coaxial polymethyl of AOB-enriched activated sludge to the bioreactor methacrylate cylinders. The hermetic space between accelerated the onset of nitritation and established the the cylinders was used for temperature control. Acti- conditions favoring the subsequent AnB development vated sludge was immobilized on a cylindrical carrier (Zhang et al., 2012; Ma et al., 2013). Nitrifiers are of fibrous plastic, a mixture of polyethylene and poly- most often used for bioaugmentation of traditional propylene (Polyvom, ETEK, Russia) with an internal nitrification/denitrification processes. The works on diameter 85 mm, height 200 mm, and total surface addition of nitrifiers to enhance the anammox process area 11.6 dm . Air was supplied from the bottom of the are few. Bioaugmentation with activated sludge reactor by means of a SCHEGO SW2 compressor enriched with AOB and AnB facilitated stabilization of (Germany). When aeration was not applied, mixing the deammonification process and provided for more was carried out using an IKA C-MAG MS7 magnetic rapid commissioning of the bioreactors (Wett et al., stirrer (Germany) at 120‒150 rpm. The medium was 2013; Miao et al., 2017; Pedrouso et al., 2021). Ma supplied at the bottom of the reactor by Masterflex et al. (2013) did not carry out AOB identif ication; acti- L/S economy drive peristaltic pumps (United States), vated sludge was added daily. Miao et al. (2017) and processed water was displaced through the upper achieved enrichment with the biomass of anammox fitting. The temperature mode was adjusted using an bacteria by its monthly addition to the reactor. ELMI TV 2.03 water bath (Latvia) equipped with a circulation pump for the outer contour. The parame- The f irst stage (ammonium nitritation) is known to ters were maintained with a Siemens LOGO 6ED1 be the vulnerable point of the anammox process (Tro- programmed timer (Germany). The reactor was oper- janowicz et al., 2021), since nitrifiers are sensitive to ated in the sequencing batch mode (SBR), which toxic compounds and rapidly die off in the course of storage or starvation (Salem et al., 2006). Anammox included the stages of settling, medium supply with the simultaneous removal of treated water, and aera- bacteria are considerably more robust, recover rapidly tion. The cycle duration was 6 h, and the average after periods of exposure to toxic agents, and die off hydraulic time for the medium in the reactor was 27 h. slowly, not more than 1‒2% a day (Wang et al., 2018), The reactor operated at 30°C and oxygen concentra- compared to up to 20% a day for the nitrifiers (Salem et al., 2006). tions of 0.4‒0.8 mg/L, with alternating phases of aer- ation and its absence (20 min each), and air f low of Planning of the experiment was based on two pre- 20 L/h. Two identical reactors, the experimental and requisites. Of two major bioaugmentation types, exter- control ones, were used in the work. Oxygen concen- nal (when the target biological agent is introduced into tration was determined with a WTW INOLAB 7310 the system) and internal (when conditions are estab- meter equipped with a Sellox sensor (Germany). lished for enrichment of the system with desired bac- The incoming medium contained the following teria, i.e., selection), the former was chosen as the only (g/L): (NH ) SO , 0.942; NaCH COO·3H O, 0.04; one possible at launching of new reactors and during 4 2 4 3 2 KH PO , 0.044; NaHCO , 2.1; pH 8.3 (Boeije et al., activity recovery in the reactor after poisoning of the 2 4 3 activated sludge. Moreover, internal bioaugmentation 1999). The medium was obtained by diluting the con- is well-studied and is widely used, though often not centrate (prepared on distilled water) with tap water at termed bioaugmentation. This is the principle the time of injection into the reactor. The anammox involved in the operation of skimmers enriching the activated sludge (ASA) obtained from a previous run activated sludge with phosphate-accumulating bacte- of the same reactor and stored for 30 days at 4°C was ria (Lema and Suarez, 2017) and partial nitrification used as an inoculum. To launch the reactor, 1 L of the reactors, enriching the sludge with ammonium-oxi- inoculum containing 2 g/L suspended matter was dizing bacteria (Tchobanoglous et al., 2014). Group I added to 3.5 L of the medium. The experimental reac- nitrifiers are required to support the activity of the tor was supplemented with the association of nitrifying anammox process, among which members of the gen- bacteria obtained as described below and containing MICROBIOLOGY Vol. 91 No. 2 2022 BIOAUGMENTATION OF ANAMMOX ACTIVATED SLUDGE 135 20 mg bacterial biomass; this corresponded to the (NH ) SO , 2; K HPO ·3H O, 1; MgSO ·7H O, 0.5; 4 2 4 2 4 2 4 2 100 : 1 mass ratio of ASA and the nitrifying/bioaug- NaCl, 2; FeSO ·7H O, 0.05; CaCO , 5; pH 7. The 4 2 3 menting activated sludge (ASB). The concentrations suspension was mixed thoroughly for 10 min on a Vor- of ammonium, nitrate, and nitrite ions were deter- tex V-1 plus laboratory shaker (Biosan, Latvia). The mined weekly in processed water using the standard enrichment culture was obtained by adding 5 mL of techniques (Rice and Bridgewater, 2012). The amount the compost suspension to 250 mL of the medium. of removed nitrogen (dN mg/L) was calculated as the The enrichment was cultured for 7 days at 30°C on a difference between the concentration of ammonium Biosan OS-20 incubator shaker (Biosan, Latvia) at nitrogen (N-NH ) in the inf lowing medium and the 4 140 rpm. The culture was used as the inoculum (20%) total concentration of mineral nitrogen species in pro- to obtain the bioaugmenting culture (2 L), which was cessed water (N-NH , N-NO , N-NO ). The effi- grown for 5 days under the same conditions and used 4 2 3 ciency of nitrogen removal was calculated as the share as the bioaugmenting activated sludge (ASB) added to (%) of removed nitrogen to its concentration in sup- the anammox bioreactor. Cell titer in ASB (2 × plied water. 10 cells/mL) was determined using an Axio Imager M2 epif luorescence microscope (Carl Zeiss Micros- Activity of ammonium-oxidizing bacteria (AOB), copy, Germany). anammox bacteria (AnB), and nitrite-oxidizing bacte- ria (NOB) was calculated using the amount of The composition of the activated sludge microbial removed nitrogen (dN) and the stoichiometry of the communities (in the inoculum, bioaugmenting ASB, anammox process (Lotti et al., 2014): and those immobilized on the carrier) was analyzed by high-throughput sequencing of the 16S rRNA gene +− − + NH++ 1.146NO 0.071НCO+ 0.57H 42 3 fragments. Activated sludge was samples at the time of inoculation and after 25, 45, and 60 days of incuba- →+ 0.986N 0.161NO tion. ++ 0.071CH O N 2.002H O. 1.74 0.31 0.20 2 Metagenomic DNA from the samples was isolated Specific activity of AnB = dN/T, mg/L/h; using the DNeasy PowerSoil Kit (Qiagen, Germany) + + according to the manufacturer’s protocols. The specific activity of AOB = ((N-NH – N-NH ) – 40 4 V3‒V4 variable region of the 16S rRNA gene was (dN/1.99))/T, mg/L/h; amplif ied using the universal primers 341F CCTAYG- GGDBGCWSCAG and 806R GGACTACNVGG- specif ic activity of NOB = (N-NO – 0.08dN)/T, 3r GTHTCTAAT (Frey et al., 2016). The amplicon mg/L/h, libraries with barcodes were prepared from the where T is hydraulic time of occurrence in the biore- obtained PCR fragments using the Nextera XT Index actor, h; Kit v2 (Illumina, United States) according to the + + manufacturer’s protocols. PCR fragments were N- and N- are concentrations of NH NH 40 4 sequenced using Illumina MiSeq in the 2 × 300 nt for- ammonium nitrogen in the inf lowing and processed mat. medium, respectively, mg N/L, Paired reads were combined with FLASH v. 1.2.11 N-NO is the concentration of nitrate nitrogen in (Magoc and Salzberg, 2011). Low-quality reads, sin- 3r the eff luent processed water. gletons, and chimeras were excluded at the next stage of analysis. The contribution of denitrification was not taken into account, since this process could be responsible The remaining reads were clustered into opera- for not more than 5% of nitrogen removal. Contribu- tional taxonomic units (OTUs) with at least 97% tion to assimilation for the AnB growth was below 1% sequence identity. To determine the share of an OTU and was therefore also disregarded. The coefficients in each sample, original reads (including the low- were taken from the work by Lotti et al. (2014). quality and singleton ones) were overlaid over the rep- resentative OTU sequences using the USEARCH v. 11 Bioaumenting activated sludge (ASB) was a nitrify- software package (Edgar, 2010). Taxonomic identifi- ing enrichment culture obtained from composted cation of the OTUs was carried out using the mixture of excessive sludge from a processing plant of VSEARCH v. 2.14.1 algorithm and the Silva v. 138 a dairy industry waste water and vegetable and wood database (Rognes et al., 2016). waste in the 3 : 3 : 4 ratio (vol/vol). Compost samples were collected during the cooling stage at the tempera- The interaction between microbial groups was ture of 45°C from a 950-m industrial pile with the characterized using network analysis. Analysis of membranous opaque coverage and active aeration simultaneous OTU presence or of their mutual exclu- (Grunt Eko, Russia). Dry matter content in the com- sion was performed based on the Spearman correla- post (by mass) was 27.5 ± 1.3%. Water suspension of tional matrix (Langfelder et al., 2012) using only the the compost was prepared by mixing 1 part of the com- significant correlation values (Barberan et al., 2014). post with 9 parts (by mass) of the sterile nitrifier The threshold value accepted for the correlation coef- medium containing the following (g/L tap water): ficients was 0.6, and the one for the corrected p values MICROBIOLOGY Vol. 91 No. 2 2022 136 PIMENOV et al. (а) 140 60 40 1 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Days Days (b) Fig. 2. Amounts of mineral nitrogen removed in the course of operation of the control (1) and experimental (2) biore- actors. were carried out. The arithmetical mean and the mean absolute deviation were calculated. This value corre- sponded to the experimental dispersion and did not exceed 3%. The values are presented on the graphs as average ± mean deviation. 0 5 10 15 20 25 30 35 Days RESULTS AND DISCUSSION (c) Parameters of bioreactor operation. Addition of ASB resulted in signif icantly more eff icient removal of both ammonium and total mineral nitrogen from the bioreactor (Figs. 1, 2). On day 4, the concentrations of ammonium nitrogen in processed water decreased compared to the incoming water (with N-NH con- centration 200 mg/L) by 40 and 14% in the bioreactors with and without (control) ASB bioaugmentation. Noticeable removal of total nitrogen (7%) in the control reactor was observed from day 11 on; in the 0 5 10 15 20 25 30 35 experimental reactor the process commenced on day 4 Days (20%). By the end of the experiment, ~60 and 20% of nitrogen was removed in the experimental and control Fig. 1. Dynamics of the concentrations of ammonium (a), reactors, respectively. During the first 10 days, nitrite nitrite (b), and nitrate (c) in processed water in the course concentrations in the experimental and control reac- of operation of the control anammox bioreactor (1) and tors did not differ considerably; subsequently, how- the bioreactor supplemented with ASB (2). ever, the concentration increased to 70‒75 mg/L in the control and decreased to 10‒15 mg/L in the exper- was 0.001 (Luo and Bhattacharya, 2006). Only the iment. Decreased concentrations of ammonium and OTUs with the relative abundance of at least 2.0% of nitrites, together with low concentrations of nitrate the 16S rRNA gene sequences in at least one sample (and the absence of conditions for denitrification) were included in the analysis. The networks were visu- indicated the key role of stage I nitrification and ana- alized using Cytoscape v. 3.8.2 (Shannon et al., 2003; mmox reaction in the nitrogen balance. Faust et al., 2016). The calculated values of specific activity for anam- The 16S rRNA gene sequences obtained in the mox bacteria, AOB, and NOB are shown on Fig. 3. work were deposited to the NCBI database and are Bioaugmentation resulted in increased nitrification available at BioProject PRJNA556270. activity at the beginning of the experiment. On day 4, Statistical processing of the data. In the experi- AOB specific activity in the experimental variant was ments on cultivation of the anammox community and 1.8 times higher than in the control. At this time, the assessment of activity of various bacterial physiologi- activity of anammox bacteria in the variant with bio- cal groups, three measurements of each parameter augmentation was 25 times higher than in the control. (concentrations of oxygen and of nitrogen species) While AnB activity subsequently increased, even on MICROBIOLOGY Vol. 91 No. 2 2022 [N(NO )], mg/L [N(NO )], mg/L [N(NO )], mg/L 3 2 4 d[N], mg/L BIOAUGMENTATION OF ANAMMOX ACTIVATED SLUDGE 137 day 32 specific activity of anammox bacteria in the (а) 5.0 experimental bioreactor was 2.7 times higher than in 4.5 the control one. In both reactors activity of group II 4.0 nitrif iers (NOB) was lower than the AOB activity by an 3.5 order of magnitude. NOB activity in the presence of 3.0 ASB was 5 times higher than in the control on day 4; 2.5 during the next 2.5 weeks it rapidly decreased almost 2.0 to zero. In the control activity of this group peaked on 1.5 day 11 and then decreased to zero by day 18. 1.0 0.5 Composition of microbial communities. In order to determine the composition of the activated sludge 0 5 10 15 20 25 30 35 microbial communities, 153698 sequences of the Days V3‒V4 variable regions of the 16S rRNA genes were determined. Clusterization resulted in 1383 OTUs (b) 4.5 (Table S1). Alpha-diversity indices indicated high 4.0 diversity of the communities in the original activated 3.5 sludge samples (Table 1). In the course of operation, biodiversity in both the experimental and control bio- 3.0 reactors decreased and became similar (Table 1), 2.5 which indicated selection of the microbial communi- 2.0 ties. 1.5 1.0 The OTUs found in the communities belonged to 22 bacterial phyla identified using GTDB (genome 0.5 taxonomy database; Parks et al., 2018). Only seven of 0 5 10 15 20 25 30 35 them were dominant and represented at least 1% of the Days 16S rRNA gene sequences in at least one sample (Fig. 4). Predominant groups belonged to the phyla (c) Proteobacteria, Chloroflexi, Bacteroidota, Actinobacte- 0.6 riota, Planctomycetota, Verrucomicrobiota, and Spiro- chaetota; their total abundance in microbial commu- 0.5 nities exceeded 92%. Members of Acidobacteriota, 0.4 Armatimonadota, Bdellovibrionota, Cyanobacteria, Deinococcota, Dependentiae, Desulfobacterota, Firmic- 0.3 utes, Gemmatimonadota, Hydrogenedentes, Myxococ- cota, Patescibacteria, Sumerlaeota, Synergistota, and 0.2 WPS-2 were present as minor components, with their abundance not changing significantly in the course of 1 0.1 community development. Archaea, belonging to the phylum Halobacterota, were found only in ASB, where m m 0 5 10 15 20 25 30 35 their share was below 0.01%. Days ASB differed signif icantly from other samples, with predomination of Proteobacteria (the group to which Fig. 3. Specific activities of anammox bacteria (AnB) (a), nitrifiers belong), while the shares of Chloroflexi and ammonium-oxidizing bacteria (AOB) (b), and nitrite-oxi- Planctomycetota were low. Other activated sludge sam- dizing bacteria (NOB) (c) in an anammox bioreactor: con- ples exhibited similar composition of microbial com- trol (1) and experiment (2). munities at the phylum level (Fig. 4). Microorganism involved in nitrogen removal. The Nitrosospira (Otu2) in the activated sludge decreased analyzed samples were found to contain nitrifiers of 100-fold (to 0.15%) and remained almost stable during the genera Nitrosomonas (Otu8, Otu25, Otu359, subsequent cultivation (Table S1). Otu31, etc.) and Nitrosospira (mainly Otu2) belonging to the phylum Proteobacteria. In the course of cultiva- Detected anammox bacteria belonged to the gen- tion, abundance of Nitrosomonas in the control AAS era Ca. Brocadia (Otu48 and Otu67) and Ca. Jettenia samples decreased from 7 to 4.5%, while in the ASB- (Otu371). The share of Ca. Brocadia was significantly supplemented bioreactor it increased from 5 to 10%. higher (7.8%); it decreased to 1.7% during operation Interestingly, Nitrosospira were predominant nitrifiers of the bioreactor not supplemented with nitrifiers. In in ASB (15.4%), while in the original AAS they were the bioreactor to which nitrif iers were added, the share not present (Fig. 5). After addition of ASB (1 : 100 by of Ca. Brocadia remained almost stable (~6.5%). Rel- biomass) to the experimental bioreactor, the share of ative abundance of Ca. Jettenia in all activated sludge MICROBIOLOGY Vol. 91 No. 2 2022 mg N/(L h) mg N/(L h) mg N/(L h) 138 PIMENOV et al. Table 1. Diversity indices for microbial communities of the analyzed activated sludge samples Diversity indices Cultivation Samples duration, days Chao1 Shannon_e AAS 346 3.48 ASB 839 4.49 Control 180.5 3.32 Experiment 213.6 3.43 Control 147.3 3.27 Experiment 194.3 3.25 Control 141.6 3.34 Experiment 168.4 3.34 samples decreased from 0.37 to 0.1%. Thus, nitrogen bacteria. The analysis was carried out for the OTUs removal via the anammox reaction was probably car- comprising over 2% in at least one sample. The results ried out by Ca. Brocadia (Otu67); its relative abun- are presented on Fig. 6. It may be seen that Otu2 (Nitrospira) occurred together with Otu17 (Pedo- dance in the bioreactor supplemented with nitrifiers increased to 5.5%. Otu67 belonged to Ca. Brocadia bacter) and Otu5 (uncultured Micavibrionales), which were dominant in ASB. However, no positive relations fulgida (98.93% identity of the 16S rRNA gene were found between Otu2 and the microorganisms sequences), which are common in anammox bioreac- of AAS. tors. The second Ca. Brocadia phylotype, Otu48, belonged to another species, Ca. Brocadia caroliensis; It should be noted that the presence of anammox its share decreased in the course of bioreactors opera- bacteria of Otu48 (Ca. Brocadia) correlated with the tion. presence of nitrifiers Nitrosomonas (Otu8) and planc- tomycetes of the family Phycisphaeraceae (Otu50). OTU simultaneous presence and mutual exclusion. Their relative abundance decreased in the course of The functional characteristics of microorganisms are operation of the anammox bioreactor. closely related to the properties of their habitats. Anal- ysis of the presence of the major microbial groups in Thus, ASB introduced into the microbial commu- different microbial communities makes it possible to nity had a significant effect on the efficiency of the reveal the patterns of interaction between different anammox process, while the nitrifiers present in ASB microbial groups. We carried out network analysis of could not adapt to the bioreactor conditions. Bacterial the studied microbial communities to reveal the satellites introduced with ASB probably acted as the groups most affected by the introduction of nitrifying stimulators of the anammox process. Proteobacteria Relative Chloroflexi abundance, % Bacteroidota 1 Actinobacteriota Planctomycetota Verrucomicrobiota Spirochaetota Other phyla Fig. 4. Relative abundance of bacterial phyla in the studied activated sludge samples: activated sludge of the control anammox bioreactor (ASA); activated sludge containing nitrifiers (ASB); and activated sludge from the experimental bioreactor supple- mented with ASB (AB). The numerals after the sample designation indicate operational time in days. MICROBIOLOGY Vol. 91 No. 2 2022 ASA _0 ASB_0 ASA _25 АB_25 ASA _45 АB_45 ASA _60 АB_60 BIOAUGMENTATION OF ANAMMOX ACTIVATED SLUDGE 139 Ca. Jettenia Ca. Brocadia Nitrosospira Nitrosomonas ASA ASB ASA АB ASA АB ASA АB 025 45 60 Fig. 5. Relative abundance of nitrifiers and anammox bacteria in activated sludge samples: activated sludge from the anammox bioreactor (ASA); bioaugmented activated sludge with nitrifiers (ASB); and activated sludge from the anammox reactor supple- mented with the bioaugmentinf sludge (AB). The numerals on the X axis indicate days of cultivation. Otu17 Otu8 Otu28 Otu2 Otu48 Otu5 Otu50 Otu112 Bactroidota Chloroflexi Otu415 Otu4 Otu11 Planctomycetota Proteobacteria Verrumicrobiota Possible presence Otu9 Otu135 Otu3 Mutual exclusion Fig. 6. Network analysis of microbial interactions according to correlation analysis. Positive correlation is marked by green lines; mutual exclusion of the OTUs is marked by red lines. Increased eff iciency of nitrogen removal correlated dance of Comamonas, Kapabacteria, Desulfuromona- with increased abundance of members of the phyla dia, Sorangiineae, Bdellovibrionia, Hyphomicrobium, Chloroflexi, Bacteroidetes, Leptospiraceae and Plancto- and Chitinophagales was revealed. The reason for the mycetes (Table S1). Negative correlation with abun- negative correlation may be explained only for MICROBIOLOGY Vol. 91 No. 2 2022 140 PIMENOV et al. Bdellovibrionia, predatory bacteria capable of lysing We performed bioaugmentation using the commu- other microbial cells. No functional relationships nity enriched with Nitrosospira, bacteria less wide- could be determined for other bacteria. spread in waste treatment plants, and obtained unex- pected results. Although the nitrifying and anammox Another microbial group, addition of which could activities increased rapidly after bioaugmentation, in stimulate ammonium removal are the ASB bacteria, the course of subsequent operation Nitrosomonas that preserved or increased their abundance after (Otu25 and Otu359), belonging to the original com- introduction into the bioreactor. In the initial AAS munity, grew more actively than the introduced Nitro- these organisms were not found or their share was sospira. This was probably due to the initially high minimal. These organisms belonged to Otu31 (Nitro- activity of the introduced Nitrosospira nitrif iers, which somonas), Otu125 (Lacunisphaera), Otu118 (uncul- provided the substrate (nitrite) for anammox bacteria tured planctomycete), Otu959 (Castellaniella_hirudi- and promoted a rapid increase in anammox activity. nis), Otu66 (Castellaniella_ginsengisoli), Otu247 Nitrosospira was subsequently replaced by more com- (Pedomicrobium), Otu383 (Sphingopyxis_sp.), Otu80 petitive autochthonous nitrifiers. (Bordetella petrii), Otu1147 (Rhizobiales), Otu732 (Lacunisphaera), and Otu810 (Micavibrionales). It should be noted that the share of stage II nitrifi- FUNDING ers (nitrite oxidizers) was low in all activated sludge samples. Thus, ASB contained 0.01‒0.04% of this The work was supported by the Russian Foundation for nitrifier type: Otu611 and Otu623 (Nitrobacter alkali- Basic Research, project no. 18-29-08008 (operation of the cus) and Otu853 (Nitrococcus sp.). This finding was in anammox bioreactor), Russian Science Foundation, proj- agreement with their low activity (Fig. 3). Thus, both ect no. 21-64-00019 (molecular analysis of microbial com- analysis of mineral nitrogen balance in the bioreactors munities), and State Assignment for the Research Centre of and molecular biological investigation suggest the Biotechnology, Russian Academy of Sciences (obtaining conclusion that the transformation of nitrogen com- the nitrifying bacteria). pounds in the studied microbial communities fol- lowed mainly the “stage I nitrification‒anammox process” pathway. Both stage II nitrification and het- COMPLIANCE WITH ETHICAL STANDARDS erotrophic denitrification were insignificant. The authors declare that they have no conf licts of inter- ASB bioaugmentation with the Nitrosospira- est. enriched microbial community resulted in a rapid (in several hours‒days) increase of nitrification activity This article does not contain any studies involving ani- and anammox activity, with the latter subsequently mals or human participants performed by any of the maintained at the level several times higher than the authors. anammox activity in the control bioreactor (without bioaugmentation). Increasing anammox activity cor- related with increasing relative abundance of Ca. Bro- OPEN ACCESS cadia fulgida (Otu67). In general, our results conf irmed the positive effect This article is licensed under a Creative Commons Attri- of bioaugmentation with nitrifiers, which has been bution 4.0 International License, which permits use, shar- used worldwide. However, bioaugmentation of waste- ing, adaptation, distribution and reproduction in any water treatment bioreactors is usually carried out with medium or format, as long as you give appropriate credit to the material obtained on the spot and enriched with the original author(s) and the source, provide a link to the the microorganisms common in the activated sludge Creative Commons licence, and indicate if changes were of a given waste treatment plant. Thus, Nitrosomonas made. The images or other third party material in this article predominates among stage I nitrifiers in activated are included in the article’s Creative Commons licence, sludge of industrial facilities (Agraval et al., 2018; Kev- unless indicated otherwise in a credit line to the material. If brina et al., 2019), since it oxidizes ammonium more material is not included in the article’s Creative Commons actively than Nitrosospira, the second most abundant licence and your intended use is not permitted by statutory group (Dytczak et al., 2007). Bioaugmentation with regulation or exceeds the permitted use, you will need to the material obtained on the spot is presently used at obtain permission directly from the copyright holder. To several large-scale plants. Bioaugmentation with nitri- view a copy of this licence, visit http://creativecommons. fying microorganisms from activated sludge is used in org/licenses/by/4.0/. the BABE process, increasing the rate of nitrification in ammonium-enriched wastewater (Kallistova et al., 2016); a full-scale deammonification process was SUPPLEMENTARY INFORMATION implemented in the main flow of the Strass Waste Water Treatment Plant (Strass, Austria) (Cho et al., The online version contains supplementary material 2020). available at https://doi.org/10.1134/S0026261722020102. MICROBIOLOGY Vol. 91 No. 2 2022 BIOAUGMENTATION OF ANAMMOX ACTIVATED SLUDGE 141 from wastewater, Microbiology (Moscow), 2016, vol. 85, REFERENCES pp. 140‒156. 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Journal

Microbiology – Springer Journals

Published: Apr 1, 2022

Keywords: anammox; deammonification; bioaugmentation; nitrifying community; nitritation; wastewater treatment; increasing the efficiency of Anammox; community composition

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