Li Yanzhe; Gai Jianing; Zhang Xiaofei; Zhao dongfeng; Guo Yadong;Yu Gengxing; Zhao Chaocheng; Liu Fang; Zhao Ruiyu; Liu Chunshuang

(1. College of Chemical Engineering, China University of Petroleum, Qingdao 266580;2. Research Institute of Safety and Environmental Technology, Beijing 102206;3. Harbin Jianshi Technology Co., Ltd, Harbin 150040)

Abstract: In this study, the nitrogen removal performance of partial denitrificaiton/anammox (PDA) process was investigated by using an UASB reactor. High total nitrogen (TN) removal efficiency (91.97%) was achieved at an influent nitrogen loading rate of 0.64 kg/(m3·d). Anammox bacteria did execute the function of converting nitrate to nitrite in PDA system according to 15N isotope labeling experiments and the contribution was approximately 36.3%. Candidatus_Brocadia,Candidatus_Kuenenia and Thauera were functional strains for anammox and denitrification process, respectively. Thauera and Candidatus_Brocadia were more important for TN removal at high loading rates (0.64 kg/(m3·d)). This result can provide a theoretical and technical foundation for the application of the PDA process.

Key words: partial-denitrification; anammox; 15N isotope labeling experiments; biological nitrogen removal

1 Introduction

Generally, petroleum refining and chemical industries produce wastewater with high levels of nitrogen compounds. As an example, the coal chemical wastewater usually contains more than 5 000 mg/L of chemical oxygen demand (COD) and 200 mg/L of ammonium nitrogen (NH4+-N)[1]. Nitrogen is supposed to be removed through the nitrification and denitrification system.However, this process is energy-intensive with 50% or more of the energy consumption in wastewater treatment plants being used to supply air for ammonium oxidation during nitrification. By contrast, biological nitrogen removal via anammox can reduce oxygen requirements,as a fraction of ammonium ions is oxidized under anoxic conditions without oxygen consumption[2].

The partial-denitrification/anammox (PDA) process is regarded as a promising way to apply anammox in wastewater treatment plants[3]. Compared to the conventional nitrification/denitrification process, the PDA process can reduce oxygen consumption by 45% and organic matter requirements by 79%[4]. The PDA process includes partial-denitrification reaction and anammox reaction, with the reaction stoichiometry shown in Equations (1)-(3)[5～7]:

Ordinarily, partial-denitrification reaction is generally considered to be implemented by denitrifying bacteria andThauerawas the dominant genus responsible for nitrite accumulation in the DPA process[8]. However,many studies have shown that anammox bacteria also can carry out partial denitrification in the presence of a small amount of volatile fatty acids (VFAs)[9]. It is unknown whether anammox bacteria also play an important role in nitrite accumulation in PDA system. In this study,the nitrogen removal performance of PDA process was investigated by using the UASB reactor. Isotope labeling method was firstly adopted to answer the question regarding whether anammox bacteria were involved in partial denitrification metabolism in the DPA system.Moreover, the changes of community structure at different stages were also investigated. The results will present a reference for practical application.

2 Materials and Methods

2.1 Reactor set-up

An UASB reactor with a dimension of 5 cm (diameter)×75 cm (height) and a working volume of 1.25 L operating at 30±1 °C was set up. An internal reflux peristaltic pump drove an internal up-flow at a velocity of 2.0 m/s. The reactor was covered with a tin foil to protect anammox bacteria from the influence of light. Synthetic wastewater was prepared by mixing prescribed quantities of sodium acetate, potassium nitrate and ammonia chloride[10].The reactor was operated in 3 stages, with the operating details presented in Table 1. Other material compositions in synthetic wastewater were prepared according to Liu’s report[11]. The inoculated sludge was a mixture of anammox sludge and denitrifying sludge (at a volume ratio of 2:1), and the initial volatile suspended solid (VSS)concentration of the inoculated sludge was 1.35 g/L.

Table 1 Operating parameters for the DPA system

2.2 15N tracer batch incubation experiments

An isotope experiment was conducted for an incubation time of Day 80 - 85, when the reactor was operated in stable state. Isotope experiment was conducted in several serum bottles. 1 mg of sodium sulfite was added into the serum bottles for oxygen depletion and all bottles were sealed with rubber stoppers ensuring the elimination of all bubbles, which were considered to be airtight in other studies[12]. Subsequently, N2-purged stock solutions of14NH4++15NO3-and13CH3COOH were injected to maintain the final concentrations at the set values for each treatment. All isotope labeled bottles were incubated in a shaker at a rate of 250 r/min at 30 °C and were sampled at the end of incubated period by transferring 200 μL of supernatant to the He-flushed, 12.6-mL glass vials. The product29N2was derived from anammox pathway by combining one molecule of15NO2-and one molecule of NH4+, whereas30N2was generated from two molecules of15NO2-via denitrification.29N2and30N2were measured by isotope-ratio mass spectrometry (Thermo Delta V Advantage, Germany). The potential contribution of anammox and denitrification to N2formation was calculated[12].

2.3 Microbial community

Total genomic DNA of sludge samples was extracted from 0.15-0.20 g of dried sludge using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA,USA) following the manufacturer’s instructions. The extracted DNA amount was detected via electrophoresis with 1% (w/v) of agarose gel, and its concentration was measured with a UV-Vis spectrophotometer NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).The bacterial V3-V4 region of the 16S rRNA gene was amplified using the forward primers 338F (5′ - ACT CCT ACG GGA GGC AGC AG - 3′) and the reverse primer 806R (5′- GGA CTA CHV GGG TWT CTA AT - 3′). The detailed PCR mixture and reaction procedure can be found in the papers of Liu, et al.[13], and Shi, et al.[14]The PCR product was purified by a GeneJet PCR purification kit (Thermo Scientific) before sequencing on the Illumina Miseq PE300 platform (Illimina, USA).

2.4 Chemical analysis

After being filtered through a 0.22-μm millipore filter,the NO3--N, NO2--N, and NH4+-N values in the samples of continuous experiments were measured by an ion chromatography instrument equipped with a thermal type conductivity detector and an Ionpac column (ICS-3000;Dionex, USA). The pH value of the liquid samples, the suspended solids (SS), and the volatile suspended solids of the sludge were measured by standard methods[15].

3 Results and Discussion

3.1 Nitrogen removal performance of the PDA reactor

The reactor was operated for 85 days, including 3 stages (Stage I to Stage III) according to the operation parameters (Table 1). The nitrogen removal performance is illustrated in Figure 1. At Stage I, the averaged effluent concentration of nitrate and ammonium ions was 14.19 mgN/L and 4.36 mgN/L, respectively, and the corresponding removal efficiency was 64.52%and 85.46%, respectively. The averaged TN removal efficiency was only 64.52%. The low TN removal rate is probably due to the insufficient concentration of organic carbon in PDA system. Thus, the influent acetate was enhanced from 100 mgCOD/L to 120 mgCOD/L at the flowing stage (Stage II) to maintain a COD to nitrate ratio of 3.0. Meanwhile the influent concentration of ammonium ions was increased from 30 mgN/L to 40 mgN/L. The efficiency for removal of nitrate and ammonium ions significant increased to 75.77%and 90.72%, respectively at this stage. The averaged TN removal efficiency increased to 82.74% and the effluent TN concentration was merely 7.84 mg/L. This phenomenon indicated the efficient nitrogen removal performance of the PDA reactor.

On day 59, the hydraulic retention time (HRT) of the reactor was decreased from 4 h to 3 h (Stage III). The change trend toward removal of nitrate and ammonium ions was similar to that measured at Stage II. The efficiency for removal of nitrate and ammonium ions was further increased to 87.95% and 94.53%, respectively.The corresponding TN removal rate was as high as 91.97% towards the end of the experiment. The above results suggest that the PDA process can maintain a relatively efficient nitrogen removal performance at a nitrogen loading rate of 0.64 kg/m3d.

3.2 Isotope labeling experiments for revealing the contribution of anammox process to nitrite production in PDA system

Figure 1 Performance of PDA reactor at different stages

Batch incubation experiments with15NO3-,14NH4+and13CH3COOH were conducted to investigate the nitrogen conversion pathways (Figure 2). The29N2gas was mainly produced as a product of14NH4+and15NO2-ions, and15NO2-ions were mainly generated from reduction of15NO3-. The30N2gas was mainly produced due to the reduction of15NO3-via15NO2-. To determine if anammox bacteria are responsible for reducing nitrate to nitrite,the penicillin G (0.5 mmol/L), a compound that was not active against anammox but could inhibit specifically the activity of most heterotrophs, was added to the vials. Interestingly, there was a large amount of29N2gas produced during the incubation with penicillin G,indicating that anammox bacteria did execute the function of converting nitrate to nitrite in the PDA system. In contrast, during the batch incubation with acetylene(0.1 mmol/L), a compound that could selectively inhibit anammox bacteria, the30N2gas was also produced, further supporting the contribution of heterotrophic denitrifiers to nitrite accumulation. Therefore, the proportion of anammox pathway to nitrite production in PDA system was approximately 36.3%, and the nitrogen removal rate by anammox bacteria reached 0.518 kg/m3d.

Figure 2 Production of 29N2 and 30N2 in batch tests with 15N labeled on nitrate separately

3.3 Microbial communities

To further confirm the important role of anammox bacteria responsible for reducing nitrate to nitrite in the PDA system, high-throughput sequencing approach was used to establish the changes of microbial communities under different conditions.

The parameters related to the alpha diversity of microbial community for each sample at an increment of 0.03 are shown in Table 2. The species richness for bacteria in the reactor varied significantly during the 85-day operation, which was revealed by OTUs and Chao 1.This is confirmed by the coverage values of four samples,indicating that almost all OTUs in the reactor were detected in this study.

Table 2 Richness and diversity of the four samples based on 0.03 distance

In total, nine known bacterial phyla were detected in the three samples, includingProteobacteria, Actinobacteriota,Chloroflexi, Acidobacteria, Bacteroidetes, Ignavibacteriae, Firmicutes, ArmatimonadetesandPlanctomycetes(Figure 3).Proteobacteria, ActinobacteriaandChloroflexipredominated in all communities.

Figure 3 Taxonomic classification of the bacterial communities at phylum level(Note: Phylum making up less than 0.5% of total composition in the sample was classified as “others”.)

The phylaProteobacteria,Actinobacteriota,Chloroflexi,Acidobacteria,BacteroidetesandIgnavibacteriaewere identified as dominanting phyla at Stage I. The abundance ofAcidobacteria,ArmatimonadetesandBacteroidetesgradually decreased from 8.51%, 1.77% and 8.93% (Stage I) to 4.36%, 0.33% and 3.33% (Stage III), respectively.On the contrary, the corresponding abundance ofChloroflexigradually increased from 13.47% (Stage I)to 20.33% (Stage III). The abundance ofProteobacteria,Acidobacteria,Ignavibacteriae,FirmicutesandPlanctomyceteshad little change and remained at around 35.49%, 18.27%, 3.62%, 2.13% and 1.53%, respectively,during the whole process.

A wide range of bacteria genera were identified as dominant ones during the whole process, in which three genera, includingThauera,Candidatus_BrocadiaandCandidatus_Kueneniaare the typical functional bacteria in the PDA system (Figure 4 and Table 3).Thauerais a typical heterotrophic denitrifier, which could utilize acetate as the electron donor to reduce nitrate into nitrite or nitrogen gas[16～18].Candidatus_BrocadiaandCandidatus_Kueneniaare typical anammox bacteria,which have been detected in many anaerobic ammonium oxidation reactors[19-20].

Figure 4 Taxonomic classification of the bacterial communities at genus level(Note: Phylum making up less than 0.5% of total composition in the sample was classified as “others”.)

The TN removal maintained as high as 64.52% at stage I and the predominant phylum is simplyCandidatus_Kuenenia(＞1%). The efficient TN removal at this stage is probably owing to initial reduction of nitrate to nitrite and then the N2production emanated from the nitrite ions previously generated and the ammonium ions fulfiled via the anammox bacteria. On the other hand, the abundance ofThaueraandCandidatus_Brocadiagradually increased from 0.04% and 0.01% (Stage I) to 3.24% and 1.25%(Stage III), respectively. On the contrary, the abundance value ofCandidatus_Kueneniashowed obvious decrease from 1.37% (Stage I) to 0.28% (Stage III). The identified TN removal efficiency has significantly increased from 64.52% (Stage I) to 91.97% (Stage III). This phenomenon has indicated thatThaueraandCandidatus_Brocadiaare essential for TN removal at Stage III. In all, both anammox bacteria and heterotrophic denitrifiers are probably capable of reducing nitrate to nitrite in the PDA system, while heterotrophic denitrifiers are more important under high loading rates (Stage III).

Table 3 The abundance of functional bacteria at genus level

4 Conclusions

High TN removal efficiency (＞91.97%) was achieved in the PDA system at an influent nitrogen loading rate of 0.64 kg/m3d.Candidatus_Brocadia,Candidatus_KueneniaandThauerawere functional strains for anammox and denitrification process, respectively. Anammox bacteria did execute the function of converting nitrate to nitrite in the PDA system and the contribution was approximately 36.30%. Heterotrophic denitrifiers are more important for nitrite production at high loading rates (0.64 kg/m3·d ).

Acknowledgements: This research was supported by the Natural Science Foundation of Shandong Province(ZR2019MEE038), the Fundamental Research Funds for the Central Universities (19CX02038A), and the Key R&D Program of Shandong Province (Major Scientific and Technological Innovation Project 2019JZZY020502).