The anaerobic ammonia oxidation (Anammox) process is suitable for treating low-carbon high ammonia nitrogen wastewater with low carbon ratio due to the lack of an additional organic carbon source, low sludge yield, low operating cost and high nitrogen removal efficiency. The actual wastewater contains different concentrations and types of organic matter, and it is generally believed that the presence of organic matter will have a negative impact on anaerobic ammonia oxidizing bacteria. In addition, anaerobic ammonia oxidized sludge granulation can retain microbial biomass to a large extent, strengthen the proliferation of functional bacteria, and alleviate the decline in nitrogen removal efficiency caused by environmental changes to a certain extent, which is an effective way to solve this problem. However, how to improve the performance of the anaerobic ammonia oxidation particle sludge itself, it is particularly necessary to improve the resistance of the anaerobic ammonia oxidation system to organic matter interference.
In this regard, the research group of Suzhou University of Science and Technology carried out the following studies: (1) the effects of long-term stress of different concentrations of organic matter on the nitrogen removal efficiency, physical and chemical properties and microbial community structure of anaerobic ammonia oxidized particle sludge; (2) the effect of biochar-mediated organic matter on the nitrogen removal performance, physical and chemical properties and metabolic pathway of anaerobic ammonia oxidized particle sludge. The research results were published in the Journal of Cleaner Production, the Journal of Environmental Science and China Environmental Science to provide a reference for the research and engineering application of anaerobic ammonia oxidized particulate sludge.
Study 1: Effects of long-term stress of organic matter at different concentrations on anaerobic ammonia oxidized particulate sludge
The operating efficiency of the reactor is shown in Figure 1, under the pressure of COD concentrations of 0, 50, 100, 150 and 200 mg/L, the removal rate of ammonia nitrogen showed a downward trend with the increase of COD, which was 97.71%, 97.23%, 83.87%, 68.11% and 46.52%, respectively, while the removal rate of nitrosinous nitrogen was maintained at 96.78-98.62%. The total nitrogen removal rates were 97.20%, 98.00%, 92.12%, 85.06% and 75.02% respectively under each stress concentration, indicating that the low concentration of organic matter (50 mg/L) improved the total nitrogen removal rate by forming a stable synergy between anaerobic ammonia oxidizing bacteria and heterotrophic denitrifying bacteria (see Figure 1). However, the average particle size of the particulate sludge at the organic concentration of 150 mg/L and 200 mg/L showed a tendency to increase first and then decline, and the SVI value of the granular sludge increased, and the sedimentation performance became poor (see Figure 2). Through SEM observation of the microstructure of the granular sludge, it was found that there were obvious cracks on the surface of the granular sludge, and it was speculated that when the concentration of organic matter exceeded 150 mg/L, the disintegration of the granular sludge would be caused by long-term stress. When the concentration of organic matter exceeds 50 mg/L, the dominant gate of anaerobic ammonia oxidized particle sludge changes from Chloroflexi to Proteobacteria. In addition, under long-term organic stress, Candidatus Brocadia replaced Candidatus Kuenenia as the dominant genus of anaerobic ammonia oxidizing bacteria.
Fig. 1 Nitrogen removal performance of anaerobic ammonia oxidized particle sludge at different organic concentrations
Fig. 2 Physicochemical properties of anaerobic ammonia oxidized particle sludge under different organic concentrations
Study 2: Effects of biochar-mediated organic matter on anaerobic ammonia oxidized particulate sludge
The previous research of the research group found that biochar can promote the proliferation of anaerobic ammonia oxidizing bacteria under the condition of presence. In this study, bamboo charcoal was used as the research object to analyze the effect of organic matter on the sludge of anaerobic ammonia oxide particles in the presence of bamboo charcoal. The study found that after 120 days of operation, under the condition of not adding bamboo charcoal, the removal efficiency of ammonia nitrogen gradually decreased as the concentration of COD increased. When COD concentrations were 50, 100 and 150 mg•L-1, the average removal rates of ammonia nitrogen were 89.4%, 77.4%, and 66.2%. However, after the addition of bamboo charcoal, the average ammonia nitrogen removal efficiency increased to 96.2%, 84.5% and 71.5%, respectively. When the COD concentrations were 50, 100 and 150 mg•L-1, the average TN removal efficiency was 85.9%, 82.6% and 81.4%, respectively, and the average TN removal efficiency was 92.3%, 88.9% and 84.6% after the addition of bamboo charcoal, and the removal rate of TN by the reactor adding bamboo charcoal was increased by 3.1 to 6.4% (see Figure 3).
Figure 3 Biochar-mediated effect process of organic matter on nitrogen removal of anaerobic ammonia oxidized particle sludge
The addition of bamboo charcoal also has a significant impact on the physicochemical properties of the anaerobic ammonia oxidized particle sludge, and the study found that with the increase of COD concentration, EPS gradually decreases, excess COD will inhibit the competitive advantage of anaerobic ammonia oxidizing bacteria, which is not conducive to the secretion of anaerobic ammonia oxidized bacteria EPS, and the EPS when adding bamboo charcoal is higher than that of no bamboo charcoal. Due to the secretion of EPS, the average particle size is 0.8 mm without the addition of bamboo charcoal, while the added bamboo charcoal rises to 1.2 mm (see Figure 4). The study also found that the addition of bamboo charcoal can make the surface structure of the granular sludge more dense, and the organic carbon source can be maintained under the pressure. Bamboo charcoal pores are attached to a large amount of sludge, providing a comfortable environment for the hermitage, growth and reproduction of functional microorganisms.
Fig. 4 Effect of biochar-mediated organic matter on the physicochemical properties of anaerobic ammonia oxidized particle sludge
The microbial co-occurrence network diagram was generated by calculating the correlation coefficients of the genera of the first 300 relative abundance of reactor sludge samples using the igraph and Hmisc packets in R (see Figure 5). The anaerobic ammonia oxidizer dominant bacteria Candidatus Brocadia and Candidatus Jettenia were connected to Halomonas, while the relative abundance of Halomonas in the treatment group with bamboo charcoal was higher than that in the control group without charcoal. Halomonas is a moderately salt-loving bacterium that has a denitrifying effect and can produce poly hydroxyalkanoates (PHA), which protects microbial cells from extreme environmental stress and is stored intracellularly as a source of sustained-release carbon. Compared with the general heterotrophic denitrifying bacteria, Halomonas is less susceptible to environmental disturbances and therefore more stable carbon metabolism, which may be the reason why the relative abundance of Candidatus Brocadia and Candidatus Jettenia decreases less under the coexistence of organic matter and bamboo charcoal.
Figure 5 Symbiotic network diagram
Studies have shown that the gene hdh encoding hydrazine dehydrogenase (HDH, EC: 1.7.2.8) and the gene hydrazine synthase (hydrazine synthase, HZS, EC: 1.7.2.7) gene hzsABC are only present in anaerobic ammonia oxides, as shown in the heat map of nitrogen metabolism function gene expression in Figure 6, when C/7/7 Bamboo charcoal at n ratios of 0.28 and 0.83 promoted the expression of hdh and hzsABC genes, but the opposite was true at C/N at 0.56.
Figure 6 Heat map of nitrogen metabolism function gene expression
The glycolytic pathway (EMP) and the tricarboxylic acid cycle (TCA cycle) are sugar catabolic pathways common to most organisms, so further analysis of functional genes in both pathways is necessary. Fig. 7 is the metabolic heat map of EMP and TCA functional genes, it can be seen that there are ten consecutive enzymatic reactions from glucose to pyremonate, of which the three main speed limiting steps are: glucose is catalyzed by glucokinase (glucokinase, EC: 2.7.1.2) to generate glucose-6-phosphate, fructose-6-phosphate is catalyzed by fructose phosphohexokinase (EC: 2.7.1.11) to produce sugar-1,6- Diphosphoric acid and phosphoenol formula pyruvate are catalyzed by pyruvate kinase (EC: 2.7.1.40) to form pyruvate ketoacid, and all three reactions are irreversible reactions. When the C/N ratio was 0.28 and 0.83, the addition of bamboo charcoal significantly promoted the expression of glucokinase gene glk, fructose phosphokinase gene PFK, and pyruvate kinase gene PK, while Glk and PFK were down-regulated under charcoal treatment when C/N was 0.56. In addition, the TCA cycle is also regulated by a series of enzymes, among which the pyremonate dehydrogenase system (glyco1 ketoacid dehydrogenase E1, dihydrothionyl transacetylase E2, dihydrohioleamide dehydrogenase E3, EC: 1.2.4.1, EC: 2.3.1.12, EC: 1.8.1.4) catalyzed pyruvate oxidative decarboxylation to form acetyl-co1-enzyme A is the central link link linking EMP and TCA (irreversible). The pyridoxine ketoacid dehydrogenase system is a multi-enzyme complex located on the membrane of intramitochondrial bodies, involving three functional genes of aceE, DLAT and DLD, and the expression of the three functional genes under different organic concentrations is greater than that of the control group, indicating that bamboo charcoal effectively promotes the connection between the EMP pathway and the TCA cycle.
Fig. 7 Glycolysis pathway functional gene expression heat map and tricarboxylic acid circulation pathway functional gene metabolic heat map
Summary and Outlook
The influence of organic matter on anaerobic ammonia oxidation is a clichéd but timeless research focus, and it is also an unavoidable practical problem in the application of anaerobic ammonia oxidation process engineering. In this study, the effects of different concentrations of organic matter on the nitrogen removal efficiency and microstructural characteristics of sludge of anaerobic ammonia oxidized particles were investigated, and the process characteristics and working mechanism of external mediator materials (such as biochar) on mitigating the inhibition effect of organic matter were explored. The research results will provide a certain reference significance for the engineering application of anaerobic ammonia oxidized particle sludge.