Method for strengthening short-cut denitrification in high-salt environment by using glycine betaine

Abstract

The invention discloses a method for strengthening short-cut denitrification in a high-salt environment by using glycine betaine. By exogenously adding glycine betaine, microbe regulation under short-cut denitrification conditions in a high-salt environment is carried out, microbe extracellular polymer secretion is promoted, the inhibition effect of the high-salt environment on the activity of an electron transfer system is relieved, gene and protein expression is regulated, and a cell metabolism pathway is changed; therefore, rapid accumulation of high-level NO2 <->-N is realized at a high NO3 <->-N degradation rate, the application difficulty of short-cut denitrification in a high-salinity environment is overcome, and the method has important application value in the field of high-salinity wastewater treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of industrial wastewater treatment and relates to a method for strengthening short-range denitrification in a high-salt environment by utilizing glycine betaine. Background Art

[0002] Nitrate nitrogen is one of the main causes of eutrophication of water bodies. When discharged with high-salt wastewater, it will not only cause land salinization and surface water pollution, but also the large amount of salt ions contained in the wastewater itself and the ammonia in it will be oxidized into nitrates, which will also cause nitrate or nitrite pollution in water and groundwater. The efficient and clean treatment of high-salt wastewater has always been an important research field in industrial wastewater treatment, which is of great significance for eliminating nitrate pollution, reducing the interactive effects of high salt and high nitrogen, and maintaining the environment and human health. However, traditional treatment methods have problems such as poor economic efficiency and easy secondary pollution. Although traditional biochemical methods have the advantages of relatively high efficiency, good economy, clean and green, they cannot cope with the inhibitory effects of high-salt environments, and the time cost of screening and domesticating halophilic bacteria is high and the scope of application is narrow. Therefore, a method of improving microbial salt tolerance with good universality and rapid action provides a key supplement for the application research of microbial compatible material strategies and the degradation and environmental remediation of other organic pollutants in high-salt water bodies, which is a research direction with far-reaching significance.

[0003] Compared with domesticating non-halophilic and salt-tolerant microorganisms and utilizing limited halophilic organisms, enhancing the osmotic regulation function of cells to improve their tolerance to salinity is an efficient and more economical method in terms of time and cost. Studies have shown that microcells prefer to use the "compatible solute" strategy based on organic osmotic solutes to alleviate osmotic stress, and compatible solutes have the characteristics of low molecular weight, high water solubility, neutral physiological pH, and no interference with cell metabolism. In a high-salt environment, the accumulation of compatible solutes in microbial cells can help maintain the activity of intracellular proteins and enzymes and normal physiological functions. Glycine betaine (GB), as a representative compatible solute, can significantly enhance the degradation performance of organic pollutants in a high-salt environment. As an osmotic pressure protectant, it promotes the secretion of more extracellular polymers, enhances the resistance and regulation ability to the salt environment, thereby enhancing the salt tolerance and activity in a high-salt environment and improving the degradation performance of pollutants. GBs have also been shown to protect enzymes and membranes from salt, heat, and cold stresses, and in addition to their direct protective effects through active effects on enzyme and membrane integrity or as compatible solute strategy substances, GBs may also indirectly protect cells from environmental stresses through their roles in signal transduction.

[0004] Danny et al.'s research shows that under salinity fluctuations, compatible solutes such as GB are conducive to the ion transport of the salt-in strategy, which can synergistically enhance the cell's resistance and promote the dynamic response of the "Salt-in" and "compatible solute" strategies. (Ionescu D, Zoccarato L, Cabello-Yeves PJ, et al. Extreme fluctuations in ambient salinity select for bacteria with a hybrid "salt-in" / "salt-out" osmoregulation strategy [J]. Frontiers in Microbiomes, 2024, 2: 1329925.). Fu et al. compared the two compatible solutes GB and mannitol (MA) at 20 g L -1 Effects of salinity stress on the anaerobic ammonium oxidation (Anammox) process. The results showed that 0.3 mM GB and MA can alleviate salt stress in the anaerobic ammonium oxidation process, but GB has a better effect. The addition of GB restored the denitrification efficiency from 40% to 80% in the experimental group within 13 days. (Fu JJ, Wang Y, Yang JH, et al. Mitigating the detrimental effects of saltstress on anammox process: A comparison between glycine betaine and mannitol [J]. Science of the Total Environment, 2022, 851: 158221.). These studies have shown that the addition of GB can effectively alleviate the effects of high salt stress as an osmotic regulator. However, the methods and possible mechanisms of exogenous addition of GB to regulate short-term denitrification in high-salt environments have not yet been studied. Summary of the invention

[0005] The object of the present invention is to provide a method for strengthening short-range denitrification in a high-salt environment by using glycine betaine. The present invention adds glycine betaine to high-salt wastewater, utilizes the excellent osmotic regulation function of glycine betaine to promote more extracellular polymer secretion, regulates the electron transfer system, further changes the intracellular metabolic pathway through GB, improves the carbon and nitrogen metabolic efficiency, thereby strengthening the salt tolerance and activity of microbial cells in a high-salt environment, maintaining a high nitrate efficiency and achieving rapid accumulation of nitrite, further saving energy, and accumulating intermediate raw materials for anaerobic ammonia oxidation.

[0006] The technical solution for achieving the purpose of the present invention is as follows:

[0007] The method of using glycine betaine to enhance short-range denitrification in a high-salinity environment comprises the following steps:

[0008] Step 1: Add simulated wastewater containing nitrate to an upflow anaerobic bioreactor (UASB) inoculated with activated sludge, re-configure the reactor with simulated wastewater every 2 to 5 days, and gradually increase the influent salt concentration by 0.5 to 1% every 7 to 9 days while keeping other components of the simulated wastewater constant until the salt concentration reaches 30 to 60 g L -1 Then continue acclimation for 10 to 15 days to complete the acclimation;

[0009] Step 2: Add glycine betaine while passing high-salt wastewater containing nitrate into the domesticated UASB, the pH of the high-salt wastewater is 7-9, the carbon source is Na2Ac, the nitrogen source is nitrate, the carbon-nitrogen ratio is 4.5:1-5.5:1, and short-range denitrification is achieved. The concentration of NaCl in the high-salt wastewater is 10 g L -1 above.

[0010] Preferably, in step 1, the concentration of nitrate in the simulated wastewater containing nitrate is 100 mg L -1 In addition to nitrate, the simulated wastewater also contains: 0.76 g L -1 Na2HPO4·12H2O, 0.38 g L -1 KH2PO4, 0.20 g L -1 MgSO4·7H2O, 0.05 g L -1 CaCl2, 0.32 g L -1 Na2Ac. In a specific embodiment of the present invention, NaNO3 is used as a representative example of nitrate.

[0011] Preferably, in step 1, the sludge concentration is 3g VSS L -1 (VSS: Volatile Suspended Solids).

[0012] Preferably, in step 2, the concentration of NaCl in the high-salinity wastewater is 10 to 30 g L -1 , more preferably 20 to 30 g L -1 .

[0013] Preferably, in step 2, the nitrate concentration in the high-salinity wastewater is 50 to 100 mg L -1 .

[0014] Preferably, in step 2, the pH of the high-salt wastewater is 8.95.

[0015] Preferably, in step 2, the nitrogen source is NaNO3, and the carbon-nitrogen ratio is 5.42:1.

[0016] Preferably, in step 2, the concentration of glycine betaine added is 0 to 75 mg L -1 But not 0, more preferably 25 mgL -1 .

[0017] Preferably, in step 1 or 2, the operating temperature of UASB is 25°C to 35°C.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The present invention uses cheap, easily available, and low-cost glycine betaine as an exogenous addition of similar compatible solutes, promotes the secretion of extracellular polymers, changes metabolic pathways, improves the expression and metabolism of salt tolerance-related functional genes in a high-salt environment, and regulates the short-range denitrification process, thereby maintaining a high NO3 - -N degradation rate while achieving high levels of NO2 - -N can be rapidly accumulated, which is suitable for short-range denitrification in high-salinity wastewater containing nitrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Effects of adding GB on nitrate removal efficiency (A) and nitrite conversion rate (B) under different salinities.

[0021] Figure 2 The effects of different GB concentrations on the short-range denitrification treatment performance of saline wastewater, including nitrate removal rate at 1% salinity (A1), nitrite conversion efficiency at 1% salinity (A2), nitrate removal rate at 2% salinity (B1), and nitrite conversion efficiency at 2% salinity (B2).

[0022] Figure 3 25 mg L was added at different salinities -1 Changes in GB secretion of exopolysaccharides (EPS), including soluble microbial products (SMPs, A), loosely bound EPS (LB-EPS, B), and tightly bound EPS (TB-EPS, C), where PN: protein, PS: polysaccharide.

[0023] Figure 4 25 mg L was added at different salinities -1 Effect of GB on electron transport activity ETS.

[0024] Figure 5 Heat map analysis of secondary KEGG metabolic pathways, including C_0 (0% salinity), GB_0 (0% salinity plus GB), C_3 (3% salinity), GB_3 (3% salinity plus GB). DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific embodiments and drawings.

[0026] In biological denitrification technology, short-range denitrification (Partial Denitrification) only converts nitrate (NO3 - ) is reduced to nitrite (NO2 - ), rather than being completely converted into nitrogen (N2), NO2 - It further provides key raw materials for the subsequent anaerobic ammonium oxidation coordinated denitrification, which can significantly improve the denitrification efficiency and reduce the treatment cost, thus helping to overcome the inhibitory effect of high-salinity environment. Therefore, the present invention realizes the short-range denitrification biological denitrification process under different salinities by adding GB on the basis of traditional regulation of carbon-nitrogen ratio (C / N) and pH conditions.

[0027] Example 1

[0028] (1) The activated sludge used was from a chemical wastewater treatment plant. The anaerobic activated sludge was of high concentration and strong activity, and its physical form was black and viscous. The sludge was passed through a 60-mesh sieve and then rinsed three times with deionized water to remove excess ash and physical impurities in the sludge. It was then loaded into a 5-L upflow anaerobic bioreactor (UASB) and the sludge concentration was controlled to be 3 g VSS L -1 Every 3 days, the UASB reactor was re-equipped with simulated wastewater containing nitrate. When other components of the simulated wastewater were fixed, the inlet salt concentration was gradually increased by 0.5% every 8 days until the salt concentration reached 3% (i.e., 30 g L -1 ) and then continued to acclimate for 12 days to complete the acclimatization. The composition of the simulated wastewater containing nitrate is: 100 mg L -1 NaNO3, 0.76 gL -1 Na2HPO4·12H2O, 0.38 g L -1 KH2PO4, 0.20 g L -1 MgSO4·7H2O, 0.05 g L -1 CaCl2, 0.32 g L - 1 Na2Ac.

[0029] (2) Further control the basic conditions of short-range denitrification: in NO3 - -N=51mg L -1 , C / N = 5.42, pH = 8.95, add 25 mg L -1 The effect of exogenous addition of GB on short-range denitrification at salinity of 1% to 3% was investigated. Figure 1 The specific results are analyzed as follows:

[0030] (1) Under 1% salinity, NO3 - -N degradation rate was 51.97%, and the degradation rate reached 97.53% after 6 hours, and NO3 - -N is almost completely degraded. NO2 - The conversion of -N accumulation reached a peak of 17.66% at 4 h.

[0031] (2) Under 2% salinity, NO3 - -N degradation rate was 35.43%, and the degradation rate reached 96.50% after 6 hours. - -N is almost completely degraded. NO2 - The conversion of -N accumulation reached a peak of 31.35% at 6 h.

[0032] (3) Under 3% salinity, NO3 - -N degradation rate was 29.45%, and the degradation rate reached 96.05% after 6 hours. - -N is almost completely degraded. NO2 - The conversion of -N accumulation reached a peak of 37.42% at 6 h.

[0033] In general, when the salinity increased from 1% to 3%, 25 mg L -1 The addition of GB effectively alleviated the high salt stress, making NO3 - The inhibition of -N removal rate was not obvious, and each experimental group could complete the removal of NO3 in about 6 hours. - -N is completely degraded. The addition of GB also shortens the time of NO2 - -N accumulation time, so that the experimental group has a higher NO2 - -N conversion peak.

[0034] Comparative Example 1

[0035] Under the same conditions as in Example 1, the effect of 1% to 3% salinity on short-range denitrification was investigated without adding GB. The results are shown in Table 1. Figure 1 The specific results are analyzed as follows:

[0036] (1) Under 1% salinity, NO3 - -N degradation rate was 49.70%, and the degradation rate reached 99.69% after 6 hours, and NO3 - -N is almost completely degraded. NO2 - The conversion of -N accumulation reached a peak of 13.59% at 4 h.

[0037] Under the same conditions, GB experimental group NO3 was added in Example 1- -N removal rate did not increase significantly, NO2 - The peak value of -N conversion accumulation increased by 29.95%.

[0038] (2) Under 2% salinity, NO3 - -N degradation rate was 28.33%, and the degradation rate reached 90.09% after 6 hours. - -N is almost completely degraded in 8 hours. NO2 - The conversion accumulation of -N reached a peak of 17.56% at 6 h.

[0039] Under the same conditions, the experimental group NO3 in Example 1 with GB added - -N removal efficiency increased by 7.12% and 6.46% at 4h and 6h, respectively, and NO2 - The conversion peak of -N was 31.35%, and the conversion efficiency was increased to 1.81 times the original.

[0040] (3) Under 3% salinity, NO3 - -N degradation rate was 21.46%, and the degradation rate reached 43.91% after 6 hours, and NO3 - -N degradation rate was only 57.74% at 8h, which was not completely degraded. - The conversion accumulation of -N still did not reach the peak at 8h, only 18.90%.

[0041] Under the same conditions, the experimental group in Example 1 with GB added had NO3 - -N removal rate recovered to 96.05%, and the nitrate was completely degraded within 8 hours, NO2 - The conversion peak of -N was 37.42%, and the degradation and conversion efficiencies were increased to 2.19 times and 3.87 times of the original, respectively.

[0042] In general, when the salinity increases from 1% to 3%, NO3 - The -N removal efficiency gradually decreased. Figure 1 (B) NO2 - -N transformation and accumulation showed a consistent pattern, NO2 - The accumulation level of -N gradually increased with the increase of salinity. However, the addition of GB effectively alleviated the high salt stress and increased NO3 - -N removal rate, significantly improving NO2 - -N accumulation level, strengthening the short-range denitrification effect.

[0043] Comparative Example 2

[0044] Under the same conditions as those in Example 1, the effects of short-range denitrification under 0% salinity with GB added and 0% salinity without GB added were compared. The comparison results are shown in Table 1. Figure 1 The specific results are analyzed as follows:

[0045] (1) When GB was not added at 0% salinity, NO3 - -N degradation rate was 78.01%, and the degradation rate reached 100% after 6 hours. - The conversion of -N accumulation reached a peak of 29.75% at 4 h.

[0046] (2) When GB was added at 0% salinity, NO3 - -N degradation rate was 75.08%, and the degradation rate reached 99.06% after 6 hours. - The conversion of -N accumulation reached a peak of 27.00% at 4 h.

[0047] From the comparison results, it can be seen that the effect of adding GB on short-term denitrification under low salinity (0%) is not significant, which excludes the influence of GB's own metabolism on the promotion of short-term denitrification under high-salinity environment.

[0048] Example 2

[0049] Under the same conditions as in Example 1, in a low salt (10 g L -1 NaCl) and high salt (20 g L -1 NaCl) environment, different concentrations of GB were added into the system (GB concentrations were 0, 10, 25, 50, and 75 mg L -1 ) to study the effect of adding different concentrations of GB on the short-range denitrification performance in salt environment. The results are as follows Figure 2 The specific results are analyzed as follows:

[0050] (1) Effect of adding different concentrations of GB on NO3 at 1% salinity - -N degradation was not obvious. Within 4 hours, NO3 - -N removal rate can reach 100%.

[0051] Effect of adding different concentrations of GB on NO2 - -N degradation was not obvious, 10 mg L -1 GB experimental group has the highest effluent NO2 - -N accumulation level was 36.35 mg L -1 , compared to 0 mg L -1 The GB group improved by 51%.

[0052] (2) Effect of adding different concentrations of GB on NO3 at 2% salinity --N degradation played a significant role in promoting the degradation. Adding 25mg L -1 NO3 in GB experimental group - -N degradation is fastest, GB group NO3 is not added at 4h - -N degradation rate was only 56.51±3.60%, while 25 mg L -1 GB experimental group NO3 - The -N removal rate reached 94.94±0.95%, and the complete degradation of nitrate was completed in about 5h.

[0053] Effect of adding different concentrations of GB on NO2 - -N conversion and accumulation played a significant role in promoting the growth of the nutrient solution. -1 ), add 10 mg L -1 GB, 25mg L -1 GB experimental group NO2 - -N conversion accumulation peaks increased from 18.59% to 33.76% and 31.60%, respectively.

[0054] The results show that in low-salinity environments, GB addition has limited promoting effect on short-range denitrification; in high-salinity environments, compared with other addition concentrations, 25 mg L -1 GB has a more significant effect in alleviating high salt stress and regulating osmotic pressure, has better economic efficiency, can better stimulate biological activity, and increase NO3 - -N degradation efficiency and rapid accumulation of NO2 - -N.

[0055] Example 3

[0056] Extracellular polymers (EPS) are secreted by microbial cells and contain different proportions of polysaccharides, proteins, nucleic acids, humus-like substances, etc., which play an important role in enhancing the resistance of microorganisms to toxic substances and maintaining cell vitality. EPS is mainly composed of protein (PN) and polysaccharides (PS), and can be further divided into total EPS (T-EPS), soluble microbial products (SMP), loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) according to the structure.

[0057] This example measured the effect of GB addition on EPS secretion under high salt conditions. The results are as follows: Figure 3As the salinity increases, the contents of SMPs, LB-EPS, and TB-EPS all show an upward trend. As the salinity increases from 0% to 3%, the contents of SMPs, LB-EPS, and TB-EPS increase by 32.44±2.28mg gVSS, respectively. -1 , 8.59±5.47mg gVSS -1 and 21.32±1.96mg gVSS -1 Among them, the contents of SMP and TB-EPS increased more significantly with the increase of salinity. EPS can act as a polymer hydrogel matrix to form a colloidal network structure around microorganisms, promote the sludge granulation process, and thus resist physical shock and abiotic stress.

[0058] At 3% salinity, after adding GB, the contents of SMPs and TB-EPS showed a significant upward trend, reaching 150.97±12.96 mg gVSS, respectively. -1 and 105.99±5.99mg gVSS -1 Among them, SMP is mainly composed of soluble byproducts produced by microbial activities (such as substrate metabolism and cell lysis), while TB-EPS affects microbial aggregation. The significant increase in SMP and TB-EPS content after adding GB indicates that GB promotes microbial metabolism and microbial aggregation activities, thereby protecting cells from osmotic stress.

[0059] EPS can also serve as an electron donor for denitrifying bacteria, and its increase is conducive to the smooth progress of the denitrification process. Compared with the case without GB addition at 3% salinity, the PN content in SMP, LB-EPS and TB-EPS in the experimental group with GB added increased by 20.37±2.47mg gVSS, respectively. -1 、7.95±0.84mg gVSS -1 and 6.08±0.51mg gVSS -1 . Generally speaking, PN behaves as an electrophile due to its positive charge, while the nitrogen atom in nitrate behaves as a nucleophile due to its lone pair of electrons. The addition of GB stimulated more PN secretion, increased the possibility of electrophile-nucleophile interaction, and was more conducive to the denitrification process. The addition of GB also stimulated more PS secretion in TB-EPS, enhanced the complexity and resistance of the cell inner layer structure, and was more conducive to the denitrification process.

[0060] The electron transport system (ETS) refers to the electron transport chain composed of a series of protein complexes inside the cell, also known as the respiratory chain. The degradation efficiency of short-range denitrification depends not only on the growth and metabolism of microorganisms, but also on the denitrification electron transport system (NO3 - →NO2 - →NO→N2O→N2). In order to further explore the electron transfer performance in the denitrification process, the ETS activity was studied. Figure 4 shown.

[0061] With the increase of salinity, the activity of the ETS electron transfer system in denitrifying microorganisms showed a downward trend regardless of whether GB was added or not. The ETS activity of the experimental group with GB added was higher than that of the experimental group without GB. When GB was not added at 2% and 3% salinity, the ETS activity decreased most seriously, decreasing to 64.15% and 38.79%, respectively. The increase in salinity significantly inhibited the activity of ETS. After adding GB, the ETS activity of the 2% and 3% salinity experimental groups recovered to 84.10% and 67.07%, respectively. This observation shows that GB can effectively alleviate the inhibitory effect of salinity on the ETS activity of denitrifying microorganisms, which is consistent with the experimental results of the effect of exogenous addition of GB on the denitrification process under different salt concentrations. It may be because GB participates in the excess electron donor generated as an intermediate.

[0062] Example 4

[0063] Based on the above research, this example studied the further salt tolerance mechanism of GB addition on the short-range denitrification system at the KEGG secondary level. Figure 5 As shown, the addition of GB adopted the strategy of changing metabolic pathways to adapt to the saline-alkali environment.

[0064] At 0% salinity, the addition of GB had a significant effect on 6 key enzymes in nitrogen metabolism (p < 0.05), while at 3% salinity, the number of key enzymes significantly affected increased to 10. This is consistent with previous studies that GB has a more pronounced effect in a high-salinity environment. The relative abundance of nitrate reductase, nitrite reductase, and nitrous oxide reductase was higher than that of the experimental group without GB addition, which resulted in lower NO3 in the denitrification process. - -N concentration, and higher NO2 - -N conversion rate.

[0065] The relative abundance of most enzymes involved in nitrogen metabolism (N metabolism) and carbon metabolism (C metabolism) increased significantly, indicating that GB can resist high-salinity osmotic stress by regulating cellular metabolic processes. GB accelerated intracellular nitrogen metabolism and carbon metabolism to generate more energy to resist high-salinity environments and further improved short-range denitrification efficiency.

Claims

1. A method for enhancing short-range denitrification in a high-salt environment using glycine betaine, characterized in that: The following steps are involved: Step 1: Add simulated wastewater containing nitrate to an upflow anaerobic bioreactor inoculated with activated sludge. Reconfigure the reactor with simulated wastewater every 2 to 5 days. When other components of the simulated wastewater are fixed, gradually increase the salt concentration of the influent by 0.5 to 1% every 7 to 9 days until the salt concentration reaches 30 to 60 g L -1 Then continue acclimation for 10 to 15 days to complete the acclimation; Step 2: Add glycine betaine while introducing nitrate-containing high-salt wastewater into the domesticated upflow anaerobic bioreactor, the pH of the high-salt wastewater is 7-9, the carbon source is Na2Ac, the nitrogen source is nitrate, the carbon-nitrogen ratio is 4.5:1-5.5:1, and short-range denitrification is achieved. The concentration of NaCl in the high-salt wastewater is 10 g L -1 above.

2. The method according to claim 1, characterized in that In step 1, the concentration of nitrate in the simulated wastewater containing nitrate is 100 mg L -1 In addition to nitrate, the simulated wastewater also contains: 0.76 g L -1 Na2HPO4·12H2O, 0.38 gL -1 KH2PO4, 0.20 g L -1 MgSO4·7H2O, 0.05 g L -1 CaCl2, 0.32 g L -1 Na2Ac.

3. The method according to claim 1, characterized in that In step 1, the nitrate in the simulated wastewater containing nitrate is NaNO3.

4. The method according to claim 1, characterized in that In step 1, the sludge concentration was 3 g VSS L -1 .

5. The method according to claim 1, characterized in that In step 2, the concentration of NaCl in the high-salinity wastewater is 10-30 g L -1 The nitrate concentration in high-salinity wastewater is 50-100 mg L -1 .

6. The method according to claim 1, characterized in that In step 2, the concentration of NaCl in the high-salinity wastewater is 20-30 g L -1 .

7. The method according to claim 1, characterized in that In step 2, the pH of the high-salt wastewater is 8.95, the nitrogen source is NaNO3, and the carbon-nitrogen ratio is 5.42:

1.

8. The method according to claim 1, characterized in that In step 2, the addition concentration of glycine betaine is 0~75 mg L -1 But not 0.

9. The method according to claim 1, characterized in that In step 2, the addition concentration of glycine betaine is 25 mg L -1 .

10. The method according to claim 1, characterized in that In step 1 or 2, the operating temperature of the upflow anaerobic bioreactor is 25°C to 35°C.

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