Next Article in Journal
The Aqueducts of Lugdunum
Previous Article in Journal
Evaluation of the Impact of Comprehensive Watershed Management on Carbon Sequestration Capacity of Soil and Water Conservation: A Case Study of the Luodi River Watershed in Changting County, Fujian Province
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Water
Volume 16
Issue 15
10.3390/w16152116
water-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Start-Up and Bacterial Enrichment of an Anammox Reactor with Polyurethane Porous Material: Performance and Microbial Community

by
Zichun Yan
1,2,*,
Weibin Zhang
1,
Zhibin Pei
1 and
Longzhen Jiao
1
1
School of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China
2
Key Laboratory of Yellow River Water Environment of Gansu Province, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
Water 2024, 16(15), 2116; https://doi.org/10.3390/w16152116
Submission received: 14 June 2024 / Revised: 12 July 2024 / Accepted: 24 July 2024 / Published: 26 July 2024
(This article belongs to the Section Wastewater Treatment and Reuse)
Browse Figures
Versions Notes

Abstract

:
To expedite enrichment of anaerobic ammonia-oxidizing bacteria (AnAOB) as a way to reduce the start-up time, leading to a quicker transition into stable operation, the anaerobic ammonia oxidation (anammox) process was initiated by a biofilm reactor with polyurethane porous material. The enrichment of anammox bacteria was studied by progressively increasing the influent substrate concentration while simultaneously decreasing hydraulic retention time. Following a 73 d start-up and subsequent 103 d enrichment phase, the removal rates of ammonia and nitrite reached 97.87% and 99.96%, respectively, and the community was characterized by the development of brick-red anammox biofilms and granules. The predominant bacterial phyla within the reactor were Planctomycetota, Chloroflexi, and Proteobacteria, with relative abundances of 25.25%, 29.41%, and 14.3%, respectively, and the dominant genus was Candidatus brocadia, comprising 20.44% of the microbial community. These findings indicate that the polyurethane porous material biofilm reactor is conducive to the enrichment of AnAOB. After enrichment, the anaerobic microbial community exhibited significant richness and diversity, with anammox bacteria as the primary group.

1. Introduction

Anammox, a promising biological nitrogen-removal technology, boasts several advantages, including low energy consumption, minimal sludge yield, no need for an additional carbon source, efficient nitrogen removal, etc. Notably, it can directly convert ammonia nitrogen and nitrite nitrogen into nitrogen under anaerobic conditions [1]. At present, anammox bacteria are primarily applied to treat industrial wastewater with high ammonia nitrogen and a low carbon−nitrogen ratio, including landfill leachate, aquaculture wastewater, etc. [2]. However, the large-scale application of this process is constrained by the long generation cycle and slow growth of anammox bacteria [3]. For instance, in Rotterdam, the world’s first large-scale anammox reactor required over two years to start up [4]. Therefore, the rapid start-up of anammox reactors and the enrichment of anammox bacteria have emerged as prominent research areas [5].
The key challenges to the successful operation of the anammox process are mainly slow reactor start-up, slow microbial enrichment, difficulties in the formation of granular sludge, and some sludge loss. A review of the literature suggests that providing carriers inside the reactor may be the more effective way to accelerate enrichment on reactor start-up, reduce loss of anammox biomass, and improve the efficiency of anammox treatment [6]. For example, the addition of activated carbon has been shown to accelerate the start-up of the anammox system [7]. Li et al. [8] utilized biocarriers to promote biofilm formation, and that has been shown to be an effective strategy for enrichment and enhancement of microbial biomass. Wang et al. [9] added cylindrical polypropylene suspension carriers to the reactor to provide attachment sites for the formation of biofilms by AnAOB.
To minimize sludge loss and promote the rapid formation of granular sludge, several strategies are commonly employed: (1) adding inorganic salts to induce precipitation in the reactor, promoting the adhesion and growth of microorganisms on the sediment. This approach is particularly applicable to high-salinity wastewater; however, the effluent requires further treatment. Excessive precipitation on particle surfaces can adversely affect mass transfer [10]; (2) introducing carriers to which microorganisms can directly attach, thereby reducing sludge loss and accelerating the formation of granular sludge through hydraulic shear [11]. Both biomass carriers and non-biomass carriers are frequently used [12]. Although the use of biomass filler can expedite the start of anaerobic ammonia oxidation, these fillers have a shorter lifespan and higher cost [13].
In one example of an application of non-biomass carriers, Wu Yang et al. utilized a suspended biofilm carrier to start the anammox reactor, achieving a stable TN-removal rate exceeding 70% after 156 days of operation [14]. Wang XiaoTong et al. demonstrated that porous polyvinyl alcohol (PVA) gel beads could improve the resistance of anammox bacteria to adverse environments [15]. Shelly Verma et al. employed foam materials to regulate the influent NH4+-N/NO2−-N = 1 to start the anammox reactor, achieving an average TN-removal rate of 85% over 150–187 days [16]. These carriers can retain a substantial amount of anammox biomass and improve the adaptability of AnAOB to environmental conditions [17], thereby enhancing the efficiency of reactor start-up and the rate of enrichment of anammox bacteria.
Polyurethane materials possess high porosity, large specific surface area, and high cation-exchange capacity; these features provide efficient biomass retention and substrate uptake and can prolong the retention time of microorganisms [18]. The huge specific surface area and strong hydrophilicity make it a good choice as a biological filler [19]. The structure of tiny interconnected pores gives it great potential to form biofilms, as bacteria can easily adhere to the polyurethane, increasing the density of bacteria and improving the performance of nitrogen removal [20,21].
Based on these excellent properties of polyurethane porous materials, this study applied them to an up-flow anammox reactor to investigate the reactor start-up characteristics, bacterial-enrichment effects, and microbial diversity.

2. Materials and Methods

2.1. Experimental Setup

An up-flow anammox biofilm reactor, depicted in Figure 1, was employed in this experiment. The reactor features an inner diameter of 1000 mm, an effective volume of 6.68 L, and a total column height of 17,000 mm. As shown in Figure 2, the base is supported by a 1000 mm layer of pebbles, while the interior is filled with polyurethane porous material, each piece measuring 15 mm in height, 15 mm in depth, and 25 mm in width. The packing height is 8500 mm. The carrier material is a porous hydrophilic substance with an apparent density of 25.81 mg/cm3 and a bulk density of 14.52 mg/cm3. It is characterized by low price, long service life, resistance to clogging, large specific surface area, and facilitation of biofilm growth. The reactor was maintained within an insulated box at a temperature of 30~32 °C.
The primary focuses of this experiment were the start-up of the reactor and the enrichment of the anammox bacteria. After sludge inoculation, the reactor was allowed to stand for 24 h before continuous water feeding was initiated, maintaining influent NH4+-N and NO2−-N concentrations of 30 and 40 mg/L, respectively, and an HRT of 8 h. anammox bacteria enrichment was achieved after 73 days. During the enrichment period, the influent substrate concentration was gradually increased to 60 mg/L of NH4+-N and 80 mg/L of NO2−-N, while the HRT was progressively reduced to 4 h. Changes in the appearance of the biofilm were documented throughout the enrichment period, and biofilm samples were collected to analyze the structure of the microbial community upon completion of the enrichment.

2.2. Influent and Sludge

Synthetic wastewater served as the influent throughout the study. The influent contained NH4+-N and NO2−-N, provided by NH4Cl and NaNO2, respectively, along with microelements. The microelement solutions included CaCl2 (20 mg/L), KH2PO4 (20 mg/L), MgSO4·7H2O (20 mg/L), trace-element solution I (EDTA 5000 mg/L and FeSO4·7H2O 5000 mg/L), and trace-element solution II (EDTA 15,000 mg/L, H3BO3 14 mg/L, ZnSO4·7H2O 430 mg/L, CoCl2·6H2O 240 mg/L, CuSO4·5H2O 250 mg/L, NiCl2·6H2O 190 mg/L, Na2SeO4·10H2O 210 mg/L, Na2MoO4·2H2O 220 mg/L, MnCl2·4H2O 990 mg/L). The pH of the influent was in the range 7.11~7.54.
The inoculated sludge was sourced from an anammox reactor that had been operating stably in the laboratory for 1 year. The volume of the inoculated sludge was 4 L, and the sludge concentration (MLSS) was 9547 mg/L.

2.3. Analytical Methods

2.3.1. Chemical Methods

The anammox biofilm reactor influent and effluent were sampled and analyzed for total nitrogen (TN), ammonia nitrogen (NH4+-N), nitrate nitrogen (NO3−-N), nitrite nitrogen (NO2−-N), pH, and DO. The water samples were filtered through a 0.45 μm membrane. NH4+-N, NO2−-N and NO3−-N were determined by standard methods [22]. The TN, pH, and DO were directly determined by HACH DR5000, Phs-25, and HACH HQ-10, respectively.

2.3.2. Microbiological Analytical Methods

Upon the achievement of a stable effluent effect and a TN-removal rate exceeding 80% in enrichment phase IV, a biofilm sample from the carrier surface from 16 cm from the bottom of the reactor to the top of the reactor was analyzed. This biofilm sample, designated ANB0_16, was subjected to Illumina MiSeq sequencing to determine the microbial community structure. The sample was sent to Majorbio Company (Shanghai, China). The universal primer pair 338 F (5′-ACTCCTACGGGAGGCAGC AG-3′) and 806 R (5′-GGACTACHVGGGTWTC TAAT-3′) was utilized for amplification and sequencing of the 16 S rRNA gene V3–V4 region. All data analyses were conducted on the I-sanger Cloud Platform (www.i-sanger.com). Sequencing reads were clustered into operational taxonomic units (OTUs) at an identity threshold of 0.97. Functional gene predictions were performed using PICRUSt2 function prediction [23,24]. After subsampling, 30,241 reads were obtained from each, with coverage exceeding 97.5%, indicating robust diversity within the microbial community.

3. Results and Discussion

3.1. Start-Up of the Anammox Reactor

During the start-up stage, the influent−substrate ratio was adjusted to NO2−-N/NH4+-N = 1.32, and the influent NH4+-N, and NO2−-N concentrations were controlled at 30 mg/L and 40 mg/L, respectively. The HRT was set at 8 h, with a hydraulic load of 0.1 m3/(m2∙h). The nitrogen-removal performance of the anammox biofilm reactor was monitored over a continuous 73 d operation period, as illustrated in Figure 3.
During the first 1 to 7 days, a small amount of NO2−-N and NO3−-N was removed in the reactor, while the removal rate of NH4+-N was negative, resulting in the lowest TN removal rate of 27.96%. In the 7 d before start-up, there were microorganisms within the inoculated sludge that were not adapted to the changes in reactor operating conditions, and some microorganisms within the reactor did not adapt to the new environment and died, releasing organic matter, which subsequently provided favorable conditions for the growth of heterotrophic denitrifying bacteria, which use organic matter and nitrite nitrogen and other substances to proliferate rapidly [25]. The substrate of the anaerobic ammonia-oxidizing bacteria was consumed by heterotrophic denitrifying bacteria, which led to a lack of the substrate nitrite nitrogen needed for the growth of anaerobic ammonia-oxidizing bacteria, which thus was inhibited [13]. Because the anaerobic ammonia-oxidizing bacteria are very sensitive to changes in the environment and are highly susceptible to the presence of organic matter, substrate, and other conditions, the activity of the anaerobic ammonia-oxidizing bacteria was limited under the conditions of the presence of organic matter within the system at the beginning of the start-up period and competition for substrate by heterotrophic denitrifying bacteria [26]. During this period, the activity of denitrifying bacteria surpassed that of the anammox bacteria, with NO2−-N and NO3−-N being primarily removed through an anaerobic denitrification process [27]. Later, as the microorganisms not adapted to the environment were gradually eliminated, the remaining microorganisms adapted to the new environment and the removal rate of the reactor gradually returned to normal.
On the 9th day, the removal rate of NH4+-N reached 21.02%, with no significant change observed during the subsequent 32 days; this change was accompanied by NO3−-N accumulation. Between the 17th and 42nd days, the average removal rates of NH4+-N, NO2−-N, and TN were 19.23%, 24.84%, and 15.77%, respectively. Due to the slow growth and long generation cycle of anammox bacteria, their abundance within the system remained low. Consequently, the system exhibited minimal or no anammox activity until a sufficient population of anammox bacteria had been established. During this slow growth phase, the activity of anammox bacteria stagnated, resulting in stable or slightly decreased removal rates of NH4+-N and NO2−-N.
From the 43rd day, the removal rate of NH4+-N began to rise continuously. Notably, between the 51st and 59th days, the NH4+-N-removal rate increased from 14.43% to 74.94%, the NO2−-N-removal rate increased from 49.96% to 86.92%, and NO3−-N accumulation reached 10.1 mg/L. By the 73rd day, the concentrations of NH4+-N, NO2−-N, and TN in the effluent were 2.67, 0.98, and 16.68 mg/L, respectively, and the removal rates reached 90.92%, 97.59%, and 77.20%, respectively. This period marked the lifting stage of the reactor, where anammox activity became prominent. During this stage, anammox bacteria became the dominant strain, with their activity rapidly increasing. These results are comparable to those of the study by Li et al., wherein the UASB reactor successfully started and NH4+-N- and NO2−-N-removal rates exceeded 90% after 91 days [28]. This study demonstrated that using a polyurethane porous carrier in an up-flow anaerobic biofilm reactor, combined with hydraulic shear and the carrier’s effects on sludge loading and retention, resulted in NH4+-N-removal rates of 90% and NO2−-N-removal rates exceeding 95% within 73 days.

3.2. Enrichment of Anammox Bacteria

3.2.1. Changes in the Appearance of the Biofilm during Enrichment

After the 73-day start-up period, the nitrogen-removal effect of the reactor performance stabilized, with a TN-removal rate of 75%. Subsequently, the enrichment of anammox bacteria was conducted by increasing the substrate concentration during phases I and IV and decreasing HRT during phases II and III. The reactor’s operating conditions during the enrichment period are presented in Table 1.
The changes in the appearance of the biofilm on the packing surface within the reactor during enrichment phases II, III, and IV are illustrated in Figure 4. During the second phase, the HRT was reduced to 6 h and light-red particles began to appear on the reactor packing. In the third phase, the HRT was further reduced to 4 h, resulting in the formation of red granules in both the upper and lower sections of the anammox reactor, with some anammox bacteria attached to the lower packing. In phase IV, the concentrations of NH4+-N and NO2−-N were increased to 60 mg/L and 80 mg/L, respectively, leading to the appearance of brick-red biofilms and prominent brick-red granules on the upper and lower carriers. This coloration is likely due to the high amount of cytochrome C in the anammox bacteria [29], which also indicates enhanced bacterial activity. The results demonstrated that the anammox bacteria successfully attached to the polyurethane porous carrier, achieving successful anammox biofilm culture. Additionally, the up-flow reactor, aided by hydraulic shear, facilitated the formation of granular sludge.
In the process of microorganism enrichment, anammox bacteria granules not only grew on the surface of polyurethane porous material, but also accumulated sludge granules inside the carriers and on the inner wall of the reactor. This indicates that the high porosity of the polyurethane material provides ample space for microbial growth and enrichment, while its interception effect minimizes microbial loss. In this study, brick-red sludge granules were observed in both the upper and lower parts of the reactor after 150 days. In contrast, Wu et al. positioned the carrier at the top of the up-flow reactor, and significant brick-red sludge granules did not appear until 200 days had passed [30].

3.2.2. Nitrogen Removal Effect of the Reactor

During the enrichment stage, the concentrations of NH4+-N and NO2−-N were increased from 40 and 53 mg/L to 60 and 80 mg/L, respectively, and the HRT was gradually shortened to 4 h. The changes in the denitrification capacity of the reactor during this process are depicted in Figure 5.
In phase I, the influent NH4+-N, NO2−-N concentrations were increased from 30, 40 mg/L to 40, 53 mg/L. Consequently, the removal rate of TN in the reactor decreased from 77.20% to 45.74%. However, by the 86 th day, the removal rates of NH4+-N, NO2−-N, and TN recovered to 93.59%, 99.89%, and 79.50%. The initial decrease in nitrogen-removal efficiency was attributed to the sudden increase in influent substrate concentration, which adversely impacted the anaerobic ammoxidation bacteria. In enrichment stage II, the removal rates of NH4+-N and NO2−-N decreased to 79.56% and 81.71%, respectively, but recovered to 93.14% and 96.67%, respectively, after 5 days of operation, when the hydraulic retention time was adjusted to 6 h while other conditions were held constant. By the 100th day, the removal rates of NO2−-N and NH4+-N stabilized above 95%, with a minor generation of NO3−-N, indicating an improvement in the reactor’s denitrification performance. This suggested that AnAOB had begun to proliferate and that their activity was increasing. Shortening the HRT increased the nitrogen-loading rate (NLR) of anaerobic ammonia oxidation, and the higher NLR facilitated nutrient removal and microbial activity in the anammox process [31,32]. It has been shown in another publication that a higher nitrogen-removal efficiency (NRE) was obtained by shortening the HRT to achieve the successful initiation of anammox activity [33]. Therefore, it can be concluded that shortening the HRT increased the NLR of the anammox reaction and obtained a higher nitrogen-removal efficiency (NRE), which in turn led to an adequate substrate supply in a high loading environment and an increase in the microbial activity as the AnAOB gradually acclimated to the environment, even though the substrate-contact time was reduced.
After the nitrogen-removal performance stabilized, the hydraulic retention time was further shortened to 4 h, marking the transition to enrichment stage III. Following a 2 day recovery period, the removal rates of NH4+-N, NO2−-N, and TN in the reactor were restored to 90.45%, 90.55%, and 73.99%, respectively, which indicated that shortening the hydraulic retention time enhanced the nitrogen-removal capacity of the anammox reactor [34]. After 34 days of stable operation, the hydraulic retention time remained unchanged for 4 h and the influent substrate concentration was increased to enter enrichment stage IV, with removal rates of NH4+-N, NO2−-N, and TN stabilizing at 95%, 99%, and 85%, respectively.
After 103 days of operation through the above four stages, the average TN-removal rate was 86.69%, and the final concentration of TN in effluent stabilized at 18.70 mg/L. This indicated successful enrichment of AnAOB bacteria and the realization of reactor start-up. The increase in substrate concentration in influent during stage IV had a minimal effect on the denitrification performance of the reactor, indicating that the anammox bacteria in the reactor exhibited a robust denitrification capacity, consistent with the findings from a similar anammox start-up process [15]. This robustness is likely due to the high accumulated biomass and strong anammox activity, which enhance the reactor’s resistance to impact loads, a finding that aligns with that of the study by Wan Yang et al. [35].

3.3. Microbial-Community Analysis after Enrichment

3.3.1. Analysis of Microbial Diversity and Richness

After detection, the library coverage of the sample was approximately 1.00, and the sequencing results could effectively analyze the microbial community in the sample biofilm. Ace and Chao’s values were used to assess the richness of the microbial community in the reactor, and both are positively correlated with the richness of the microbial community [36]. Higher values of these indices indicate a greater number of species in the sample. The Ace and Chao indexes (Table 2) in this study are higher than values of 305 and 299 reported by Luo et.al [37], indicating that the microbial richness in the anammox reactor is relatively high.
It has been shown that the Shannon value is positively correlated with the diversity of bacterial species, while the Simpson value is inversely correlated with the same [38]. Diversity and richness directly affect the stability and functionality of microbial communities. Higher diversity and abundance typically imply more stable reactor performance because they provide a broader range of metabolic pathways and ecological niches, which help to maintain a complex ecological balance and an efficient wastewater-treatment process [39]. When the reactor experiences external influences or load shocks, it can maintain stability without undergoing system collapse. The Shannon value is positively proportional to microbial diversity, and the Simpson value is inversely proportional to diversity [40]. In this study, the Shannon and Simpson values were 4.37 and 0.052, respectively, indicating a high degree of microbial-community diversity. The Ace and Chao indices were 917 and 912, respectively, indicating good community richness. Similar results were obtained in a previous study [41]. The Shannon value in this study was higher than the 2.50 reported by Wang et.al, and the Simpson value was smaller the 0.38 reported in similar studies [42]. These above four indexes collectively indicate good microbial-community diversity in the reactor.

3.3.2. Classification-Level Analysis of Micropyle

The dominant bacteria in the reactor at the gate level include Chloroflexi, Planctomycetes, Proteobacteria, Acidobacteriota, etc. (Figure 6). Chloroflexi emerged as the dominant species, having the highest relative abundance after enrichment. Chloroflexi are known for the function of carbon fixation and can utilize dead cells as organic carbon sources to hydrolyze organic matter under anaerobic conditions, contributing to biofilm formation [43,44]. This finding is consistent with other research results [45]. Planctomycetes accounted for 25.25% of the microbial community, which is higher than the values reported in other anaerobic ammonium oxidation (anammox) start-up studies [46]. Planctomycetes detected in the anaerobic ammonium oxidation reactor were the main functional bacteria capable of removing ammonia nitrogen and nitrite nitrogen from water [47], which is also consistent with the research results of Pía Oyarzúa et al. [48].
The relative abundance value of Proteobacteria was 14.30%, and many microorganisms within Proteobacteria can utilize organic matter for heterotrophic denitrification and nitrogen removal, playing a crucial role in nitrogen removal, biological phosphorus removal, and degradation of various organic compounds [49]. The abundance of Acidobacteriota was 11.89%. Acidobacteriota are capable of simultaneous nitrification and denitrification, utilizing the products of heterotrophic nitrification as the substrate for aerobic denitrification bacteria, and they are sensitive to pH changes in water [50]. Firmicutes had an abundance of 3.67%. Firmicutes, a genus of bacteria common in anaerobic ammoxidation systems, can decompose organic matter and produce volatile fatty acids (VFA) [51]. Planctomycetota and Proteobacteria, the main functional bacteria associated with nitrogen, constituted 39.55% of the total number of bacteria. Therefore, the enrichment of anammox bacteria has been achieved, resulting in effective nitrogen removal. This conclusion is consistent with the conclusion from the efficiency experiment, which demonstrated improved nitrogen-removal performance.

3.3.3. Classification-Level Analysis of Microbe Genera

The relative abundance at the microbial genus level is illustrated in Figure 7. The dominant bacteria within the phylum were Candidatus_Brocadia and Candidatus_Jettenia. The anammox bacteria in the reactor accounted for 22.76% of the total population, with Candidatus_Brocadia at 20.44%. The dominance of anammox bacteria in the reactor significantly enhanced the nitrogen-removal efficiency, which was consistent with the research results of Xiaofei Gong et al. [52]. The abundance of Denitratisoma was 6.56%. These bacteria are known for their ability to degrade organic matter and perform denitrification and nitrogen removal [53]. Additionally, the abundances of Norank_f_Norank_o__sBR1031 and norank_f__A4b were 14.84% and 6.90%, respectively. Both belong to Chloroflexi. JGI_0001001-H03 were 4.6%, it can autotrophic denitrification and electron transfer.

4. Conclusions

(1) After a 73−day start-up, the removal rates of NH4+-N and NO2−-N were 90.92% and 97.59%, respectively. Moreover, after a 103−day enrichment, the removal rates of NH4+-N, NO2−-N, and TN were 97.87%, 99.96%, and 85.87%, respectively.
(2) Planctomycetota, Proteobacteria, Chloroflexi, and Acidobacteriota are the primary nitrogen-removal bacteria. At the genus level, the anammox bacterium Candidatus_Brocadia was the dominant species, accounting for 20.44% of the microbial population. Denitratisoma, which performs denitrification, had an abundance of 6.56%. After successful enrichment, anammox bacteria became the predominant microorganisms.
(3) Porous polyurethane material was added into the reactor as the carrier. The gaps within and between the carriers, along with the inner wall of the reactor, provided ample space for the growth of anammox bacteria. This setup facilitated the formation of large anammox bacterial particles through hydraulic shear and reduced the loss of microorganisms.

Author Contributions

Z.Y.: Methodology, Investigation, Review; W.Z.: Software, Visualization, Writing—original draft, Writing—review & editing; Z.P.: Investigation, Writing—original draft; L.J.: Investigation, Writing—original draft. All authors have read and agreed to the published version of the manuscript.

Funding

The research was financially supported by the National Natural Science Foundation of China [51568034], and the Open Foundation of Key Laboratory of Yellow River Water Environment in Gansu Province [20JR2RA0002].

Data Availability Statement

Data will be made available on request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Li, Y.; Cui, N.; Xuan, K.; Xu, D.; Wang, D.; Li, C.; Li, Z.; Wang, Y. Start-up performance and process kinetics of a UASB-Anammox reactor at low substrate concentration. J. Environ. Chem. Eng. 2021, 9, 106726. [Google Scholar] [CrossRef]
  2. Ren, Z.-Q.; Wang, H.; Zhang, L.-G.; Du, X.-N.; Huang, B.-C.; Jin, R.-C. A review of anammox-based nitrogen removal technology: From microbial diversity to engineering applications. Bioresour. Technol. 2022, 363, 127896. [Google Scholar] [CrossRef] [PubMed]
  3. Liu, J.; Ran, X.; Li, J.; Wang, H.; Xue, G.; Wang, Y. Novel insights into carbon nanomaterials enhancing anammox for nitrogen removal: Effects and mechanisms. Sci. Total Environ. 2023, 905, 167146. [Google Scholar] [CrossRef] [PubMed]
  4. van der Star, W.R.L.; Abma, W.R.; Blommers, D.; Mulder, J.-W.; Tokutomi, T.; Strous, M.; Picioreanu, C.; van Loosdrecht, M.C.M. Startup of reactors for anoxic ammonium oxidation: Experiences from the first full-scale anammox reactor in Rotterdam. Water Res. 2007, 41, 4149–4163. [Google Scholar] [CrossRef] [PubMed]
  5. Lu, Y.; Wang, J.; Feng, Y.; Li, H.; Wang, Z.; Chen, H.; Suo, N.; Yu, Y.; Shoubin, Z. Nitrogen removal performance and rapid start-up of anammox process in an electrolytic sequencing batch reactor (ESBR). Chemosphere 2022, 308, 136293. [Google Scholar] [CrossRef] [PubMed]
  6. Chen, C.-J.; Huang, X.-X.; Lei, C.-X.; Zhu, W.-J.; Chen, Y.-X.; Wu, W.-X. Improving Anammox start-up with bamboo charcoal. Chemosphere 2012, 89, 1224–1229. [Google Scholar] [CrossRef]
  7. Lu, G.; Ma, Y.; Zang, L.; Sun, Y.; Yu, F.; Xue, R. Effects of granular activated carbon and Fe-modified granular activated carbon on anammox process start-up††Electronic supplementary information (ESI) available. RSC Adv. 2021, 11, 10625–10634. [Google Scholar] [CrossRef] [PubMed]
  8. Li, Y.-Y.; Huang, X.-W.; Li, X.-Y. Using anammox biofilms for rapid start-up of partial nitritation-anammox in integrated fixed-film activated sludge for autotrophic nitrogen removal. Sci. Total Environ. 2021, 791, 148314. [Google Scholar] [CrossRef] [PubMed]
  9. Wang, Z.; Zhang, L.; Zhang, F.; Jiang, H.; Ren, S.; Wang, W.; Peng, Y. Enhanced nitrogen removal from nitrate-rich mature leachate via partial denitrification (PD)-anammox under real-time control. Bioresour. Technol. 2019, 289, 121615. [Google Scholar] [CrossRef]
  10. Fernández, I.; Vázquez-Padín, J.R.; Mosquera-Corral, A.; Campos, J.L.; Méndez, R. Biofilm and granular systems to improve Anammox biomass retention. Biochem. Eng. J. 2008, 42, 308–313. [Google Scholar] [CrossRef]
  11. Wang, X.; Yu, H.; Wu, Y.; Lin, S. Enhancement of nitrogen removal and acceleration of anammox start-up with novel gravel contact carriers. Biochem. Eng. J. 2023, 200, 109084. [Google Scholar] [CrossRef]
  12. Yang, C.; Wu, H.; Cai, M.; Zhou, Y.; Guo, C.; Han, Y.; Zhang, L. Valorization of Biomass-Derived Polymers to Functional Biochar Materials for Supercapacitor Applications via Pyrolysis: Advances and Perspectives. Polymers 2023, 15, 2741. [Google Scholar] [CrossRef]
  13. Adams, M.; Xie, J.; Chang, Y.; Kabore, A.W.J.; Chen, C. Start-up of Anammox systems with different biochar amendment: Process characteristics and microbial community. Sci. Total Environ. 2021, 790, 148242. [Google Scholar] [CrossRef]
  14. Wu, Y.; Wang, Y.; De Costa, Y.G.; Tong, Z.; Cheng, J.J.; Zhou, L.; Zhuang, W.-Q.; Yu, K. The co-existence of anammox genera in an expanded granular sludge bed reactor with biomass carriers for nitrogen removal. Appl. Microbiol. Biotechnol. 2019, 103, 1231–1242. [Google Scholar] [CrossRef] [PubMed]
  15. Wang, X.; Yang, H. Nitrogen removal performance of anammox immobilized fillers in response to seasonal temperature variations and different operating modes: Substrate utilization and microbial community analysis. Sci. Total Environ. 2022, 829, 154574. [Google Scholar] [CrossRef]
  16. Verma, S.; Daverey, A.; Lin, J.-G. Successful start-up of anammox process from activated sludge and anaerobic sludge in a sequencing batch reactor using an unconventional strategy. Int. Biodeterior. Biodegrad. 2021, 156, 105132. [Google Scholar] [CrossRef]
  17. Wang, J.; Liang, J.; Sun, L.; Shen, J.; Wang, M. Achieving reliable partial nitrification and anammox process using polyvinyl alcohol gel beads to treat low-strength ammonia wastewater. Bioresour. Technol. 2021, 324, 124669. [Google Scholar] [CrossRef]
  18. Peng, Z.; Lei, Y.; Liu, Y.; Wan, X.; Yang, B.; Pan, X. Fast start-up and reactivation of anammox process using polyurethane sponge. Biochem. Eng. J. 2022, 177, 108249. [Google Scholar] [CrossRef]
  19. Wang, J.; Liang, J.; Ning, D.; Zhang, T.; Wang, M. A review of biomass immobilization in anammox and partial nitrification/anammox systems: Advances, issues, and future perspectives. Sci. Total Environ. 2022, 821, 152792. [Google Scholar] [CrossRef]
  20. Yang, S.; Peng, Y.; Zhang, S.; Han, X.; Li, J.; Zhang, L. Carrier type induces anammox biofilm structure and the nitrogen removal pathway: Demonstration in a full-scale partial nitritation/anammox process. Bioresour. Technol. 2021, 334, 125249. [Google Scholar] [CrossRef]
  21. Yang, S.; Peng, Y.; Zhang, Q.; Li, J.; Zhang, L. Biofilm phenotypes and internal community succession determines distinct growth of anammox bacteria in functional anammox biofilms. Bioresour. Technol. 2022, 349, 126893. [Google Scholar] [CrossRef]
  22. Jenkins, S.H. Standardized methods of water examination. Examination of water for pollution control, a reference handbook: Edited by M. J. Suess. Vol. 1 Sampling, data analysis and laboratory equipment, 360 pp. Vol. 2 Physical, chemical and radiological examination, 555 pp. Vol. 3 Biological, bacteriological and virological examination, 531 pp. published on behalf of the World Health Organization by Pergamon Press, Oxford, 1982. £175.00 or $350.00. Water Res. 1983, 17, 719–720. [Google Scholar] [CrossRef]
  23. Yan, Z.; Shen, L.; Jiao, L.; Tang, R. Effect of Fe (II) on nitrogen removal of anammox under organic matter inhibition. J. Water Process Eng. 2022, 46, 102632. [Google Scholar] [CrossRef]
  24. Ren, Z.; Li, N.; Yu, L.; Zhao, J.; Zhang, H.; Chen, W.; Zhai, Q. An Illumina MiSeq sequencing-based method using the mreB gene for high-throughput discrimination of Pseudomonas species in raw milk. LWT 2022, 163, 113573. [Google Scholar] [CrossRef]
  25. Qian, Y.; Ding, Y.; Ma, H.; Chi, Y.; Yuan, H.; Li, Y.-Y.; Tian, S.; Zhang, B. Startup and performance of a novel single-stage partial nitritation/anammox system for reject water treatment. Bioresour. Technol. 2021, 321, 124432. [Google Scholar] [CrossRef]
  26. Huang, J.; Wang, X.; Qi, Z.; Zhang, M.; Kang, R.; Liu, C.; Li, D. Quantitative effect of adding percentages of anammox granules on the start-up process and microbial community analysis. J. Environ. Manag. 2024, 349, 119361. [Google Scholar] [CrossRef]
  27. Zheng, Z.; Li, Y.; Li, J.; Zhang, Y.; Bian, W.; Wei, J.; Zhao, B.; Yang, J. Effects of carbon sources, COD/NO2−-N ratios and temperature on the nitrogen removal performance of the simultaneous partial nitrification, anammox and denitrification (SNAD) biofilm. Water Sci. Technol. 2017, 75, 1712–1721. [Google Scholar] [CrossRef]
  28. Li, H.; Zhou, S.; Ma, W.; Huang, G.; Xu, B. Fast start-up of ANAMMOX reactor: Operational strategy and some characteristics as indicators of reactor performance. Desalination 2012, 286, 436–441. [Google Scholar] [CrossRef]
  29. Liu, S.; Jiang, J.; Yin, M.; Zheng, Y.; Wang, C.; Yan, L. Promoting performance of Anammox by iron loaded sludge biochar with hydrothermal carbonization (HTC-Fe-BC) addition. Process Saf. Environ. Prot. 2023, 170, 596–607. [Google Scholar] [CrossRef]
  30. Wu, L.; Li, Z.; Huang, S.; Shen, M.; Yan, Z.; Li, J.; Peng, Y. Low energy treatment of landfill leachate using simultaneous partial nitrification and partial denitrification with anaerobic ammonia oxidation. Environ. Int. 2019, 127, 452–461. [Google Scholar] [CrossRef]
  31. Li, H.; Zhang, J.; Zhang, C.; Song, Y.; Han, Y.; Hou, Y.; Zhang, D.; Li, C.; Wang, Y.; Guo, J. Responses of anammox and sulfur/pyrite autotrophic denitrification in one-stage system to high nitrogen load: Performance, metabolic and bacterial community. J. Environ. Manag. 2023, 332, 117427. [Google Scholar] [CrossRef]
  32. Karasuta, C.; Wang, X.; Zheng, X.; Chen, Y.; Chen, Z. Effect of hydraulic retention time on effluent pH in anammox bioreactors: Characteristics of effluent pH and pH as an indicator of reactor performance. J. Environ. Manag. 2021, 280, 111716. [Google Scholar] [CrossRef]
  33. Zhang, M.; Li, B.; Guan, Z.; Fan, Y.; He, L.; Wu, J. Rapid anammox startup in response to nitrogen loading variations: Reactor performance, microbial dynamics and mechanism exploration. J. Water Process Eng. 2024, 59, 104963. [Google Scholar] [CrossRef]
  34. Lu, X.; Wang, Y.; Wang, W.; Li, J.; Li, B.; Huang, X. Characteristics of rapid-biofiltering anammox reactor (RBAR) for low nitrogen wastewater treatment. Bioresour. Technol. 2020, 318, 124066. [Google Scholar] [CrossRef] [PubMed]
  35. Yang, W.; He, S.; Han, M.; Wang, B.; Niu, Q.; Xu, Y.; Chen, Y.; Wang, H. Nitrogen removal performance and microbial community structure in the start-up and substrate inhibition stages of an anammox reactor. J. Biosci. Bioeng. 2018, 126, 88–95. [Google Scholar] [CrossRef] [PubMed]
  36. Ma, J.; Wang, Z.; Yang, Y.; Mei, X.; Wu, Z. Correlating microbial community structure and composition with aeration intensity in submerged membrane bioreactors by 454 high-throughput pyrosequencing. Water Res. 2013, 47, 859–869. [Google Scholar] [CrossRef]
  37. Luo, D.; Qian, J.; Fu, J.-X.; Liu, C.; Zhang, R.-X.; Huang, D.-N.; Zhang, L. Responses of anammox to long-term p-nitrophenol stress: From apparent and microscopic phenomena to mechanism simulation. Bioresour. Technol. 2022, 355, 127265. [Google Scholar] [CrossRef]
  38. Gamoń, F.; Banach-Wiśniewska, A.; Poprawa, I.; Cema, G.; Ziembińska-Buczyńska, A. Insight into the microbial and genetic response of anammox biomass to broad range concentrations of different antibiotics: Linking performance and mechanism. Chem. Eng. J. 2023, 451, 138546. [Google Scholar] [CrossRef]
  39. Huang, T.-H.; Tung, F.-T.; Chen, G.-F.; Chen, W.-H. Variations of N concentrations and microbial community in the start-up of anammox using anaerobic heterotrophic sludge: Influence of a long reaction-phase time and comparison of the efficiencies of attached-versus suspended-growth cultures. Chemosphere 2022, 287, 132151. [Google Scholar] [CrossRef]
  40. Zheng, Y.; Zhang, Y.; An, N.; Shi, L.; Wei, Y.; Ma, B. Microbial community structure and nitrogen conversion rate of size-fractionated granules in partial denitrification and anammox reactor. J. Clean. Prod. 2023, 414, 137714. [Google Scholar] [CrossRef]
  41. Zhang, Y.; Zhang, J.; Li, J.; Wei, P.; Luo, R.; Han, H. Fast start-up of ANAMMOX biofilm processes at low temperatures by economical quorum sensing regulation: The importance of endogenous N-acyl-homoserine lactones from enhanced inoculated sludge. Environ. Res. 2022, 214, 114097. [Google Scholar] [CrossRef]
  42. Wang, B.; Peng, Y.; Guo, Y.; Zhao, M.; Wang, S. Illumina MiSeq sequencing reveals the key microorganisms involved in partial nitritation followed by simultaneous sludge fermentation, denitrification and anammox process. Bioresour. Technol. 2016, 207, 118–125. [Google Scholar] [CrossRef]
  43. Xie, S.; Li, X.; Wang, C.; Kulandaivelu, J.; Jiang, G. Enhanced anaerobic digestion of primary sludge with additives: Performance and mechanisms. Bioresour. Technol. 2020, 316, 123970. [Google Scholar] [CrossRef]
  44. Zhao, Y.; Liu, S.; Jiang, B.; Feng, Y.; Zhu, T.; Tao, H.; Tang, X.; Liu, S. Genome-Centered Metagenomics Analysis Reveals the Symbiotic Organisms Possessing Ability to Cross-Feed with Anammox Bacteria in Anammox Consortia. Environ. Sci. Technol. 2018, 52, 11285–11296. [Google Scholar] [CrossRef]
  45. Ge, C.-H.; Sun, N.; Kang, Q.; Ren, L.-F.; Ahmad, H.A.; Ni, S.-Q.; Wang, Z. Bacterial community evolutions driven by organic matter and powder activated carbon in simultaneous anammox and denitrification (SAD) process. Bioresour. Technol. 2018, 251, 13–21. [Google Scholar] [CrossRef]
  46. Yang, Y.; Zhang, L.; Cheng, J.; Zhang, S.; Li, X.; Peng, Y. Microbial community evolution in partial nitritation/anammox process: From sidestream to mainstream. Bioresour. Technol. 2018, 251, 327–333. [Google Scholar] [CrossRef]
  47. Kong, X.; Yu, S.; Fang, W.; Liu, J.; Li, H. Enhancing syntrophic associations among Clostridium butyricum, Syntrophomonas and two types of methanogen by zero valent iron in an anaerobic assay with a high organic loading. Bioresour. Technol. 2018, 257, 181–191. [Google Scholar] [CrossRef]
  48. Oyarzúa, P.; Bovio-Winkler, P.; Etchebehere, C.; Suárez-Ojeda, M.E. Microbial communities in an anammox reactor treating municipal wastewater at mainstream conditions: Practical implications of different molecular approaches. J. Environ. Chem. Eng. 2021, 9, 106622. [Google Scholar] [CrossRef]
  49. Thu, N.H.T.; Quy, L.V.; Anna, H.A.; Lund, N.J.; Halkjaer, N.P. High diversity and abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated sludge systems. FEMS Microbiol. Ecol. 2011, 76, 256–267. [Google Scholar] [CrossRef]
  50. Costa, M.C.; Carvalho, L.; Leal, C.D.; Dias, M.F.; Martins, K.L.; Garcia, G.B.; Mancuelo, I.D.; Hipólito, T.; Conell, E.F.; Okada, D.; et al. Impact of inocula and operating conditions on the microbial community structure of two anammox reactors. Environ. Technol. 2014, 35, 1811–1822. [Google Scholar] [CrossRef] [PubMed]
  51. Antwi, P.; Li, J.; Boadi, P.O.; Meng, J.; Shi, E.; Chi, X.; Deng, K.; Ayivi, F. Dosing effect of zero valent iron (ZVI) on biomethanation and microbial community distribution as revealed by 16S rRNA high-throughput sequencing. Int. Biodeterior. Biodegrad. 2017, 123, 191–199. [Google Scholar] [CrossRef]
  52. Gong, X.; Zhang, L.; Gong, Q.; Liu, X.; Li, X.; Zhang, Q.; Peng, Y. Rapid cultivation and enrichment of anammox bacteria solely using traditional activated sludge as inoculum and biocarrier in low-strength real sewage treatment. Bioresour. Technol. 2022, 358, 127354. [Google Scholar] [CrossRef]
  53. Qiao, L.; Yuan, Y.; Mei, C.; Yin, W.; Zou, C.; Yin, Y.; Guo, Q.; Chen, T.; Ding, C. Reinforced nitrite supplement by cathode nitrate reduction with a bio-electrochemical system coupled anammox reactor. Environ. Res. 2022, 204, 112051. [Google Scholar] [CrossRef]
Water 16 02116 g001
Figure 1. Anaerobic coupled biofilm reactor.
Figure 1. Anaerobic coupled biofilm reactor.
Water 16 02116 g001
Water 16 02116 g002
Figure 2. Polyurethane porous material.
Figure 2. Polyurethane porous material.
Water 16 02116 g002
Water 16 02116 g003
Figure 3. Performance of the reactor during the start-up period: (a) Effect of ammonia removal; (b) Effect of nitrite removal; (c) Effect of nitrate accumulation; (d) Effect of total nitrogen removal.
Figure 3. Performance of the reactor during the start-up period: (a) Effect of ammonia removal; (b) Effect of nitrite removal; (c) Effect of nitrate accumulation; (d) Effect of total nitrogen removal.
Water 16 02116 g003
Water 16 02116 g004
Figure 4. Changes in the appearance characteristics of biofilms: (a) Phase II (in the upper part of the reactor); (b)Phase III (in the upper part of the reactor); (c) Phase IV (in the upper part of the reactor); (d) Phase II (in the lower part of the reactor); (e) Phase III (in the lower part of the reactor); (f) Phase IV (in the lower part of the reactor).
Figure 4. Changes in the appearance characteristics of biofilms: (a) Phase II (in the upper part of the reactor); (b)Phase III (in the upper part of the reactor); (c) Phase IV (in the upper part of the reactor); (d) Phase II (in the lower part of the reactor); (e) Phase III (in the lower part of the reactor); (f) Phase IV (in the lower part of the reactor).
Water 16 02116 g004
Water 16 02116 g005
Figure 5. Variation in the concentration of nitrogen during the enrichment stage: (a) effect of ammonia removal; (b) effect of nitrite removal; (c) effect of nitrate accumulation; (d) effect of total nitrogen removal.
Figure 5. Variation in the concentration of nitrogen during the enrichment stage: (a) effect of ammonia removal; (b) effect of nitrite removal; (c) effect of nitrate accumulation; (d) effect of total nitrogen removal.
Water 16 02116 g005
Water 16 02116 g006
Figure 6. Percent community abundance on the phylum level.
Figure 6. Percent community abundance on the phylum level.
Water 16 02116 g006
Water 16 02116 g007
Figure 7. Percent community abundance on the genus level.
Figure 7. Percent community abundance on the genus level.
Water 16 02116 g007
Table 1. Operating conditions of the anammox biofilm reactor during enrichment.
Table 1. Operating conditions of the anammox biofilm reactor during enrichment.
PhaseTime/dHRT/hNH4+-N (mg/L)NO2−-N (mg/L)
I74~86840 ± 253 ± 2
II87~110640 ± 253 ± 2
III111~146440 ± 253 ± 2
IV147~176460 ± 280 ± 2
Table 2. Index of biofilm microbial-community richness and diversity.
Table 2. Index of biofilm microbial-community richness and diversity.
Sample NumberSerial NumberShannonSimpsonChaoAceCoverage Rate
1961544.370.0529129170.9972
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Yan, Z.; Zhang, W.; Pei, Z.; Jiao, L. Start-Up and Bacterial Enrichment of an Anammox Reactor with Polyurethane Porous Material: Performance and Microbial Community. Water 2024, 16, 2116. https://doi.org/10.3390/w16152116

AMA Style

Yan Z, Zhang W, Pei Z, Jiao L. Start-Up and Bacterial Enrichment of an Anammox Reactor with Polyurethane Porous Material: Performance and Microbial Community. Water. 2024; 16(15):2116. https://doi.org/10.3390/w16152116

Chicago/Turabian Style

Yan, Zichun, Weibin Zhang, Zhibin Pei, and Longzhen Jiao. 2024. "Start-Up and Bacterial Enrichment of an Anammox Reactor with Polyurethane Porous Material: Performance and Microbial Community" Water 16, no. 15: 2116. https://doi.org/10.3390/w16152116

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop