Effect of salinity changes on extracellular polymers in anaerobic sludge

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　1 Introduction The rapid development of food processing, leather manufacturing, petroleum and other industries has resulted in an increasing discharge of salty organic wastewater. In addition to high salinity, this type of wastewater usually contains high concentrations of organic matter. Therefore, anaerobic biological treatment technology is used to treat such salty organic wastewater. Wastewater is more practical. Anaerobic biological treatment technologies such as upflow anaerobic sludge fluidized bed (UASB), anaerobic sequencing batch biofilm reactor (ASBBR) and expanded granular sludge bed (EGSB) have been proven to effectively treat saline organic wastewater. Extracellular polymers (EPS) are macromolecular viscous organic polymers formed by microorganisms through metabolism and cell autolysis and are attached to the outside of the cell wall. They have a rheological double-layer structure on the surface of the cell wall. The inner layer is tightly bound EPS (TB-EPS), which is closely combined with the cell wall, has a certain shape, and is relatively stably attached to the outside of the cell wall; the outer layer is loosely bound EPS (LB-EPS), which can diffuse to the surrounding environment. , is a mucus layer without obvious edges. EPS is mainly composed of protein (PN) and polysaccharide (PS), and contains a small amount of humic acid, nucleic acid, etc. This composition of EPS is conducive to the adhesion and aggregation between microbial cells, and affects the physical and chemical properties of sludge floc, such as sedimentation, flocculation, dehydration, surface chargeability, etc. Therefore, studying the role of extracellular polymers in wastewater biological treatment systems is necessary to maintain the stable operation of the process. Changes in EPS content and composition are related to nutrient concentration, matrix type, dissolved oxygen content, concentration of heavy metal ions and other toxic and harmful substances in wastewater, and salinity. In recent years, the impact of changes in salinity in wastewater on the content and composition of sludge EPS has attracted widespread attention from scholars. Abbasi et al. found that an increase in incoming water salinity can stimulate microorganisms to secrete more extracellular polysaccharides to reduce the damage to cells caused by an increase in osmotic pressure. Ismail et al. found that the PN content in anaerobic sludge EPS increased when the incoming water Na+ concentration was 10 g· There is no significant difference between L-1 and 20 g · L-1, while the Na+ concentration is 10 g · When L-1, the PS content in EPS is higher. Wang et al. studied the impact of salinity changes on aerobic granular sludge EPS and found that a gradual increase in inlet water salinity from 0 to 8% will lead to an increase in the PN and PS content in aerobic sludge LB-EPS and TB-EPS, and a decrease in the PN/PS ratio. The content of LB-EPS and TB-EPS is related to the volume index of granular sludge. (SVI) is positively correlated. A lot of research has been done on the impact of changes in salinity in the incoming water on the EPS content and composition of anaerobic and aerobic sludge. However, there are few reports on the impact of increasing salinity on the content and composition of anaerobic sludge LB-EPS and TB-EPS, and there are no reports on the relationship between the physical and chemical properties of anaerobic sludge and extracellular polymers under different salinities. In response to the above problems, this article intends to study the changing patterns of PN and PS contents in UASB anaerobic sludge LB-EPS and TB-EPS as the incoming water salinity increases, and use three-dimensional fluorescence (3D-EEM) and Fourier transform infrared (FTIR) technology to analyze The composition and structure of LB-EPS and TB-EPS under different salinities were discussed, and the relationship between the changes in the composition and content of PN and PS in LB-EPS and TB-EPS and the settleability of sludge were discussed, in order to provide useful reference for the research on anaerobic treatment of saline organic wastewater. 2 Materials and methods 2.1 Experimental device The UASB reactor is shown in Figure 1. The reactor is made of organic glass columns. The reactor body is divided into upper and lower parts. The upper part is the precipitation zone and the lower part is the reaction zone. The inner diameter of the reaction zone is 8 cm, the height is 80 cm, and the effective volume is 4 L. The inner diameter of the precipitation zone is 15 cm, the effective height is 15 cm, and the effective volume is 2 L. The total height of the reactor is 100 cm, and the total effective volume is 6 L. Use a peristaltic pump to feed water into the UASB reactor, with a flow rate of 0.42 L·h-1, a hydraulic retention time of 24 h, and a circulation pump outside the reactor to fully contact the wastewater and sludge. The UASB reactor operates at normal temperature. http://www.dowater.com/jishu/UploadFiles_9416/201610/20161026142552591.jpg 　　Figure 1 Schematic diagram of UASB reactor 2.2 The main components of experimental water and inoculated sludge simulated saline organic wastewater are as follows (mg · L-1): Glucose 1536, NaNO3 364, KH2PO4 79, salinity and trace elements are provided by sea crystal. At a salinity of 3% (containing 30 g sea crystal per liter), the composition of sea crystal is as follows (mg · L-1): Na+ 9880, Mg2+ 950, Cl- 18025, SO42- 2500, K+ 360, Ca2+ 300, Zn2+ 0.015, Mn2+ 0.013, Fe2+ 0.13, Co2+ 3×10-4, Mo6+ 3×10-3, Sr+ 7.5×10-3, I- 0.07, Se6+ 3.5×10-4, Cu2+ 0.05, active phosphate 0.045, nitrite 0.01, nitrate 0.3. The inlet water pH is controlled at around 7.5. The digested sludge of Qingdao Licunhe Wastewater Treatment Plant is used as the inoculated sludge in the UASB reactor, and the sludge concentration (MLSS) is 4.23 g·L-1. 2.3 Analysis method 2.3.1 Extraction and determination of extracellular polymers Extract LB-EPS and TB-EPS according to the method of Li et al. with appropriate modifications. Take 40 mL of sludge sample from the reactor, centrifuge at 6000 r·min-1 for 5 min, discard the supernatant, and obtain concentrated sludge; dilute to 40 mL with NaCl solution preheated to 70 °C (same salinity as the sludge sample), cover tightly and shake quickly 1 min; centrifuge the extracted mixture at 6000 r·min-1 for 10 min, collect the supernatant, which is LB-EPS. Dilute to 40 mL with NaCl solution (same salinity as the sludge sample), incubate in 60°C water bath for 30 min, and then centrifuge at 6000 r·min-1 for 15 min, collect the supernatant, which is TB-EPS. The supernatant is passed through 0.45 After filtration with μm cellulose acetate membrane, analyze the PN and PS content in LB-EPS and TB-EPS. The PN content is determined by the Folin phenol method, and the PS content is determined by the anthracene colorimetric method. The total amount of LB-EPS (or TB-EPS) is expressed as the sum of the PN and PS contents in LB-EPS (or TB-EPS). 2.3.2 Three-dimensional fluorescence spectrum analysis The three-dimensional fluorescence spectra of LB-EPS and TB-EPS were measured using a fluorescence spectrophotometer (F-4600, Hitachi, Japan). The excitation wavelength (λEx) range was 200~400 nm, and the scanning interval was 5 nm; the emission wavelength (λEm) range was 200~500 nm, and the scanning interval was 5 nm; the slits of the excitation light and the emission light were both 10 nm, and the scanning speed was 1200 nm·min-1. Origin was used. 8.1 Use software to draw spectra. 2.3.3 Fourier transform infrared spectroscopy analysis Fourier transform infrared spectrometer (Tensor 27, Bruker Optics, Germany) was used to measure the infrared spectra of LB-EPS and TB-EPS. After freeze-drying, the LB-EPS and TB-EPS extracts were ground and mixed with spectrally pure KBr according to a mass ratio of 1:100, and kept under a certain pressure for several minutes to make translucent flakes. Scan within the cm-1 wave number range, and the detector resolution is 4 cm-1. 2.3.4 Conventional analysis and determination methods COD, NO3--N, NO2--N, MLSS, MLVSS and SVI are all used * * The pH is measured according to the prescribed standard method, and the pH is measured using a portable pH meter. 3 Results and analysis (Results and discussion) 3.1 The effect of salinity changes on the denitrification performance of the UASB reactor. The changes in COD, NO3--N and NO2--N in the inlet and outlet water at different salinities are shown in Figure 2. The increase in salinity in the wastewater will increase the extracellular osmotic pressure, which may cause plasmolysis, dehydration and activity reduction of the cells, or even disintegration and death. Each time In the early stage of increasing the salinity of the incoming water, because the denitrifying bacteria are impacted by the increase in osmotic pressure, the metabolic activity of the cells is inhibited, resulting in higher concentrations of COD and NO3--N in the effluent. As the ability of the denitrifying bacteria to withstand high osmotic pressure continues to increase, the concentrations of COD and NO3--N in the effluent also gradually decrease. Inlet water salt The degree gradually increased from 0 to 8%, the average COD removal rate dropped from 90% to 44%, and the average NO3--N removal rate dropped from 95% to 45%. The increase in salinity will cause the denitrifying bacteria to grow slowly and reduce their metabolic activity, which will cause the COD and NO3--N removal rates to decrease with the increase in incoming water salinity. In addition, salt The increase in concentration will also stimulate microorganisms to secrete more extracellular polymers, and excess extracellular polymers will block the channels for substrate transmission, thereby reducing the mass transfer rate of the substrate. This may also cause the removal rate of COD and NO3--N to decrease with the increase in inlet water salinity. There is no significant accumulation of NO2--N in the effluent at different salinities. http://www.dowater.com/jishu/UploadFiles_9416/201610/20161026142553986.jpg 　　Figure 2 Changes in COD, NO3--N and NO2--N in the inlet and outlet water under different salinities (a. COD,b.NO3--N,c.NO2--N) 3.2 Changes in PN and PS contents in anaerobic sludge LB-EPS and TB-EPS under different salinities. After the effluent quality of the UASB reactor stabilized, the anaerobic sludge was taken for analysis of the changes in PN and PS contents in LB-EPS and TB-EPS. The results are shown in Figure 3. The inlet water salinity gradually increased from 0 to 8%, and the PN and PS contents in LB-EPS increased from 3.75 and 1.19 mg respectively. g-1 (based on VSS) increased to 26.82 and 14.65 mg · g-1 (based on VSS), and the PN and PS contents in TB-EPS increased from 9.70 and 2.55 mg · g-1 (based on VSS) to 30.11 and 12.92 mg · respectively. g-1 (based on VSS). Under different salinities, the PN content in LB-EPS and TB-EPS is always higher than the PS content, which is related to the presence of a large number of extracellular enzymes in EPS. The increase in salinity in the incoming water leads to an increase in the difference in ion concentration inside and outside the cell. In order to adjust this difference, cells under the stress of increased osmotic pressure enhance active transport and diffusion by secreting a large number of enzymes and other auxiliary substances to adapt to environmental changes, thereby maintaining normal metabolism, which may be the cause of EPS The reason why the PN and PS content in EPS increases as the incoming water salinity increases. In addition, the disintegration of cells that cannot adapt to the increase in salinity in the environment will also release macromolecules such as intracellular proteins and polysaccharides, which will also lead to an increase in the PN and PS content in EPS. Li et al. reported similar results. A gradual increase in the incoming water salinity from 0.4% to 2% will lead to an increase in the PN and PS content in anaerobic sludge EPS. However, Ismail et al. found that the PN content in anaerobic sludge EPS increased when the incoming water Na+ concentration was 10 There is no significant difference between g · L-1 and 20 g · L-1, while the Na+ concentration is 10 g · When L-1, the PS content in EPS is higher. Scholars' reports on the impact of changes in incoming water salinity on the PN and PS content in anaerobic sludge EPS are inconsistent, which may be related to the differences in experimental conditions. The incoming water salinity gradually increased from 0 to 8%, LB-EPS and TB-EP The PN/PS ratio in S decreased from 3.2 and 3.8 to 1.8 and 2.3 respectively, indicating that the increase in salinity is more likely to cause changes in the PS content in EPS. PS molecules contain a large number of polar groups, which have strong binding ability to water molecules, so PS can limit the flow of water. Under high osmotic pressure, the increase in PS content in EPS can reduce the water loss of cells, which can be regarded as a way for cells to resist the increase in osmotic pressure in the environment. Abbasi et al. found that the increase in extracellular salinity stimulates bacteria to secrete more extracellular PS to reduce the impact of increased osmotic pressure on cells. Cell destruction. Zou et al. also reported similar results. Increased salinity led to an increase in extracellular PS content. When the incoming water salinity was lower than 4%, the PS content in LB-EPS was lower than that in TB-EPS, while when the incoming water salinity was higher than 4%, the PS content in LB-EPS was higher. http://www.dowater.com/jishu/UploadFiles_9416/201610/20161026142554459.jpg 　　Figure 3 Changes of PN and PS in LB-EPS and TB-EPS under different salinities (a.LB-EPS, b.TB-EPS, c.PN/PS ratio) 3.3 The three-dimensional fluorescence spectra of anaerobic sludge LB-EPS and TB-EPS under different salinities. The three-dimensional fluorescence spectra of anaerobic sludge LB-EPS and TB-EPS under different salinities are shown in Figure 4. The X-axis and Y-axis represent the emission spectrum (λEx) and the excitation spectrum (λEm) respectively, and the contour lines represent the fluorescence intensity. Fluorescence peaks A (275~280 nm /335~345 nm) and B (220~225 nm /335~350 nm) is protein-like fluorescence, which is related to tryptophan protein substances and aromatic ring protein substances respectively; fluorescence peak C (325~340 nm/435~440 nm) is humic acid-like fluorescence, which is related to humic acid substances. Under different salinities, fluorescence peak A and fluorescence peak B were detected in LB-EPS and TB-EPS, indicating that the proteins in the anaerobic sludge extracellular polymers are composed of tryptophan protein substances and aromatic ring protein substances. At salinities of 4% and 8%, fluorescence peak C was detected in TB-EPS, indicating the existence of humic acid substances, which is related to dead cells and macromolecular substances (such as proteins and polysaccharides). ) is related to the decomposition of ), reflecting the toxic effect of increasing salinity on cells. No humic acid-like fluorescence was detected in the 3D-EEM spectrum of LB-EPS, which may be related to the low content of humic acid-like substances. Under different inlet water salinities, the fluorescence peak positions and peak intensities are shown in Table 1. Compared with the fluorescence peak positions of LB-EPS and TB-EPS when the inlet water salinity is 0, when the inlet water salinity is 8%, the fluorescence peak A in LB-EPS is red-shifted by 5 along the direction of the metabolism spectrum and the excitation spectrum. nm, the fluorescence peak A in TB-EPS is red-shifted by 5 nm along the direction of the excitation spectrum. The red-shift of the fluorescence peak indicates the increase in carbonyl, carboxyl, hydroxyl and amine groups in the fluorescent group. When the inlet water salinity is 8%, the fluorescence peak B in LB-EPS is red-shifted by 5 nm along the direction of the emission spectrum. When the salinity is 4% and 8%, the fluorescence peak B is blue-shifted by 5 nm and 10 nm respectively along the direction of the excitation spectrum. nm, the blue shift of the fluorescence peak indicates that the aromatic ring in the fluorescent group is reduced. The shift of the fluorescence peak reflects that the increase in salinity causes changes in the content of tryptophan protein substances and aromatic ring protein substances in the extracellular polymer of anaerobic sludge. The mechanism causing this change and the law of change need to be further studied. http://www.dowater.com/jishu/UploadFiles_9416/201610/20161026142555586.jpg 　　Figure 4 Three-dimensional fluorescence spectra of LB-EPS and TB-EPS under different salinities (a. LB-EPS(0),b.TB-EPS(0),c.LB-EPS(4%),d. TB-EPS(4%),e.LB-EPS(8%),f. LB-EPS(8%)) Table 1 Characteristics of fluorescence peaks in anaerobic sludge LB-EPS and TB-EPS at different salinities http://www.dowater.com/jishu/UploadFiles_9416/201610/2016102514084304.jpg 　　3.4 Infrared spectra of anaerobic sludge LB-EPS and TB-EPS at different salinities. The infrared spectra of LB-EPS and TB-EPS at different salinities are shown in Figure 5. The broad absorption peak at 3440~3454 cm-1 is caused by the stretching vibration of hydroxyl groups (from polysaccharide compounds) and amino groups (from proteins). Due to the complexity of the composition of EPS, it is difficult to accurately determine which group’s stretching vibration causes the broad absorption peak near 3450 cm-1; 1630 The absorption peak near cm-1 is caused by the CO stretching vibration of β-sheets in the protein secondary structure; the absorption peak near 1400 cm-1 is caused by the vibration of the C-H bond in the methyl group; the absorption peak near 1100 cm-1 is caused by the stretching vibration of the C-O bond of polysaccharides; less than 1000 cm-1 is the fingerprint area; 600~900 The absorption peak of cm-1 indicates the presence of unsaturated bonds in the sample. LB-EPS and TB-EPS are at 3450, 1600 and 1100 There are obvious absorption peaks at cm-1, indicating the presence of proteins and polysaccharides in the LB-EPS and TB-EPS samples. Under different inlet water salinities, the peak positions of the infrared spectra of anaerobic sludge LB-EPS and TB-EPS are similar, but the relative intensity of the peaks shows different changes with the increase in inlet water salinity. 1100 in LB-EPS and TB-EPS The relative intensity of the absorption peak near cm-1 increased significantly as the salinity increased, indicating that the increase in incoming water salinity led to an increase in the relative content of the C-O component of the polysaccharides in the anaerobic sludge LB-EPS and TB-EPS. http://www.dowater.com/jishu/UploadFiles_9416/201610/20161026142555428.jpg 　　Figure 5 Infrared spectra of LB-EPS and TB-EPS at different salinities (a.LB-EPS, b.TB-EPS) 3.5 The relationship between LB-EPS and TB-EPS and anaerobic sludge settleability under different salinities. When the inlet water salinity is 0, 1%, 2%, 3%, 4%, 6% and 8%, after the UASB reactor outlet water quality is stabilized, the results are at 78, 94, 115, 136, 155, 190 and 240 respectively. d. Anaerobic sludge was taken for LB-EPS, TB-EPS and SVI analysis. The results are shown in Figure 6. The inlet water salinity gradually increased from 0 to 8%, and the anaerobic sludge LB-EPS and TB-EPS contents increased from 4.94 and 12.25 mg·g-1 (based on VSS) to 41.47 and 43.04 mg· respectively. g-1 (based on VSS). The changes in LB-EPS and TB-EPS have an obvious linear relationship with the increase in salinity. R2 is 0.9003 (LB-EPS) and 0.8851 (TB-EPS) respectively. It can be seen that the change in LB-EPS is more related to the increase in influent salinity than TB-EPS. Tighter. TB-EPS is more closely combined with the cell wall and has a stable shape, while LB-EPS is a mucus layer without obvious edges and can diffuse to the surrounding environment. This may cause the content of LB-EPS to be more susceptible to changes in environmental factors. As the incoming water salinity gradually increases from 0 to 8%, the anaerobic sludge SVI increases from 88 mL · g-1 increased to 140 mL · g-1, SVI and salinity show a good linear relationship, R2 is 0.9391. The increase in SVI indicates that the increase in incoming water salinity leads to the deterioration of anaerobic sludge sedimentation. Some scholars have found that an increase in salinity will cause microorganisms to secrete more extracellular polymers to resist the damage to cells caused by increased osmotic pressure. Extracellular polymers are combined with a large number of water molecules and have a loose structure. Therefore, an increase in their content leads to pollution The compression performance of the mud flocs deteriorates, which in turn causes the deterioration of the sludge settling performance. In order to have a deeper understanding of the impact of the increase in incoming water salinity on the SVI of anaerobic sludge, the relationships between LB-EPS and TB-EPS and SVI under different salinities were analyzed separately (Figure 7). SVI is positively correlated with LB-EPS and TB-EPS, and the correlation coefficients r are 0.915 and 0.911 respectively (p