Petroleum refining wastewater is a kind of industrial wastewater with serious pollution and difficult treatment in the field of treatment. It is characterized by high ammonia nitrogen, complex pollutant composition, high concentration, mostly biodegradable toxic and harmful organic compounds, and large fluctuations in water quality and quantity. Compared with physical method and chemical method, biological method has the advantages of removing many kinds of pollutants, high efficiency, strong impact resistance and low treatment cost. At present, for petroleum refining wastewater with poor biodegradability and low biochemical utilization rate, petroleum refining enterprises usually adopt conventional biological denitrification processes such as A2 / O and a / O. however, these technologies have low ammonia nitrogen removal load and large dissolved oxygen consumption. Moreover, due to the long generation cycle of nitrifying bacteria, the above single sludge system operation mode makes ammonia nitrogen nitrification vulnerable to complex high concentration organic matter and unstable operation. New treatment technologies, such as ozone oxidation technology, electrochemical and photochemical methods combined with oxidants (such as H2O2, O3 and Cl2, etc.), although they have certain advantages in sewage treatment and reuse, they have not been widely used in production due to high energy consumption and treatment cost.
The purpose of this experiment is to explore the effects of COD and toxic substances in petroleum refining wastewater on the mixture of anammox denitrifying bacteria in the application of denitrification treatment.
The experimental device consists of raw water tank, upflow moving bed anaerobic ammonia oxidation reactor and reactor inlet pump. The total volume of raw water tank is 20 L. The reactor is made of plexiglass in the form of cylinder, with an inner diameter of 42 mm and a height of 400 mm. The bottom of the reactor is a support layer with a thickness of 70 mm, which is composed of sand and gravel with a particle size of 2 - 20 mm. The supporting layer is filled with 80mm high bacterial mixed culture blocks with anaerobic ammonia oxidizing bacteria and denitrifying bacteria as the core to form an effective biochemical reaction area. The total volume of the reactor is 0 5 L, effective reaction volume 0 11 L, the operation mode adopts upward flow. The outer surface of the reactor is wrapped with black plastic film to prevent the negative impact of light on the mixed culture of bacteria. The reactor inlet pump is a peristaltic pump with a rated flow of 0.2-2.0 L / h. The specific experimental device is shown in Figure 1.
1.2 bacterial source
The mixed culture of anammox denitrifying bacteria used in the reactor is from the anammox biofilter reactor which has been running stably for 6 months. Operating conditions of biofilter: the influent temperature is 33 ℃, the influent substrate concentration is NH4 + - N 200 mg / L (the concentration ratio of NO2 -- N to NH4 + - n is 1.0-1.3), and the TN removal load is 13.5% 5 kg · (M3 · d) - 1, the stoichiometric ratio of three nitrogen is NH4 + - N removal: NO2 -- N removal: NO3 -- N production = 1:1.34:0 20。
1.3 experimental raw water
The experimental raw water is prepared manually and consists of petroleum refining wastewater and basic preparation raw water. The petroleum refining wastewater is taken from the effluent from the air flotation tank of a petrochemical enterprise in Dagang District, Tianjin. The basic components are: NH4 + - N concentration is 80 mg / L, pH is 7.45 and cod concentration is 675 mg / L; The basic prepared raw water is composed of tap water, NH4Cl, NaNO2, KH2PO4, FeCl3 · 6H2O and NaHCO3 with residual chlorine removed.
Preparation method: the composition of experimental raw water prepared in different stages is NH4 + - N concentration 278 28-229 93 mg / L; No2 -- N concentration 201 51 - 319. 55 mg / L (the concentration of NH4 + - N and NO2 -- N is prepared as required according to the experimental requirements, and the proportion is controlled at about 1:1.3); KH2PO4 concentration 10 mg / L; NaHCO3 concentration 200mg / L; FeCl3 · 6H2O concentration 4.0 mg / L; Cod is brought in by petroleum refining wastewater and does not need to be added separately. The pH value of raw water is 2 0 mol / L HCl. In the process of raw water preparation, in order to supplement trace element rope, another volume fraction of 0.5% is added 17% of sterilized domestic sewage. See Table 1 for raw water composition and reactor operating conditions.
1.4 experimental methods
The inlet flow of the experimental device is 0 4 L / h, the water temperature is (29 ± 1) ℃. The concentration ratio of NO2 -- N to NH4 + - n is controlled at 1.5 by controlling the addition ratio of petroleum refining wastewater and continuous water inflow About 30. Through the addition of petroleum refining wastewater in different stages, under the condition of increasing the concentration of refractory COD and toxic substances, the tolerance of bacterial mixture with anaerobic ammonia oxidation denitrification bacteria as the core to cod and toxic substances in the process of denitrification and biochemistry was investigated. At the same time, taking the NH4 + - N removal amount in stage I and the total denitrification nitrogen removal amount in stage II as the activity index and change standard of anammox bacteria and denitrifying bacteria respectively, the effects of COD and toxic substances in petroleum refining wastewater on Anammox denitrifying bacteria mixture were explored.
Considering that the hydraulic retention time of the reaction system is 1 25 h. for this reaction system, the focus is on the impact of petroleum refining wastewater on bacterial mixture rather than denitrification efficiency, so the final effluent index is not controlled in the experiment.
1.5 analysis items and test methods
MPN-PCR technology combines polymerase chain reaction technology with multiple dilution method. Samples with different dilution gradients are selected to do 4 groups of 16 parallel samples with different dilution degrees for PCR amplification. The positive reaction is determined according to the electrophoresis band of the characteristic base sequence of the amplification product. The results are used to calculate the number of positive reactions of each sample, determine the quantitative index, and then find out the corresponding approximate number of bacteria from the MPN statistical calculation table.
1.6 data processing and analysis methods
Number of bacteria in each gram of bacterial block (piece / g) = (value corresponding to the number of bands x dilution multiple of the first dilution in the last three dilutions x total amount of extracted DNA) / mass of bacterial block (2)
2 Result Analysis
2.1 effect of acclimation process on denitrification process
Anammox reaction may be the main energy metabolism pathway of anammox bacteria, so the biochemical activity of the reaction is mainly reflected in the change of NH4 + - N removal concentration. Denitrifying bacteria can use a variety of metabolic pathways to obtain energy in denitrification reaction, so the biochemical activity of the reaction is mainly reflected in the total denitrification amount (NO2 -- N and NO3 -- N). According to the standard established in the experimental method, the effects of COD and toxic substances in petroleum refining wastewater on bacterial mixture were analyzed. The biochemical activities of anammox bacteria and denitrifying bacteria in each stage are shown in Table 3.
2.1.1 phase I and II
Figure 2 shows the denitrification effect of stage I-II. The first stage of the experiment is the adaptation period of the bacterial mixed culture, so the same environment as the original strain (biofilter operation condition) is adopted to shorten the adaptation time and ensure the treatment effect. No petroleum refining wastewater was added to the experimental raw water at this stage. It can be seen from the removal values of NH4 + - N and NO2 -- N that the anaerobic ammonia oxidation reaction is relatively stable, and the TN removal load at this time is 11.5% 978 kg · (M3 · d), while the total nitrogen removal amount of denitrifying bacteria is relatively small, so the biochemical activity of anaerobic ammonia oxidizing bacteria is mainly reflected in the bacterial mixed culture at this stage, while the biochemical activity of denitrifying bacteria exists but is not obvious.
The second stage is the initial stage of domestication experiment. The addition proportion of petroleum refining wastewater is small, and the concentrations of COD and toxic substances in raw water are low. As can be seen from Figure 2, the average removal values of NH4 + - N and NO2 -- N in this stage increased by 1.5% respectively 643 and 7 At 052 mg / L, the average total denitrification increased by 3.5% 56 times. At the same time, 61.5% of COD in raw water can be removed by adsorption and denitrification About 1%, of which the removal proportion of denitrification reaction accounts for about 45%. For the collaborative denitrification system composed of anaerobic ammonia oxidizing bacteria and denitrifying bacteria, the added cod has an obvious promoting effect on the denitrification reaction, but has little effect on the anaerobic ammonia oxidation reaction, indicating that the denitrification system enhances the impact resistance to cod.
It can be seen from figures 3 and 4 that in phase III-V, with the increase of the addition proportion of petroleum refining wastewater, the concentration of COD and toxic substances in raw water gradually increases. During the analysis, the NH4 + - N removal in stage I is taken as the theoretical removal value of NH4 + - N, and the total denitrification in stage II is taken as the theoretical total denitrification, which is used as the activity index and change standard of anaerobic ammonia oxidizing bacteria and denitrifying bacteria respectively.
For anammox bacteria, the average value of NH4 + - N removal decreased and the change gradually increased, and the gap between it and the theoretical removal value of NH4 + - N increased in turn. The NH4 + - N removal value fluctuated in the three stages, but the stability was better and better. It shows that the biochemical activity of anammox bacteria has decreased significantly in this stage, and the decline gradually increases, which is also reflected in the decrease of the overall TN removal load in turn. Because the substrates NH4 + - N and NO2 -- N of anammox reaction in the experimental effluent are surplus, the continuously added COD and toxic substances in petroleum refining wastewater have an obvious adverse impact on anammox bacteria, but with the progress of domestication stage, the bacterial mixture has a certain adaptability to water quality; For denitrifying bacteria, after the reaction with COD in raw water, the biochemical activity of bacteria has increased significantly and the denitrification amount has gradually increased. However, compared with the denitrification theory, the difference of total denitrification amount in stage V is about twice that in stage III. It is speculated that due to the increasing addition proportion of petroleum refining wastewater, the concentration of COD and toxic substances in raw water has increased, while the available NO2 -- N and NO3 -- N in influent are sufficient, Therefore, the increase of COD concentration has a beneficial effect on denitrifying bacteria to a certain extent, but the increase of toxic substance concentration has an obvious negative effect on denitrifying bacteria.
In addition, it can be seen from table 3 that the biochemical activity of anammox bacteria reached the lowest point and that of denitrifying bacteria reached the highest point in the mixed culture of bacteria in stage v. the speculated reasons are: on the one hand, the toxic substances had a significant effect in this stage, and the generation time of anammox bacteria was longer (about 11 days), making the mortality greater than the growth rate, so the anammox activity decreased significantly, And there is competition and interaction between the two bacteria; On the other hand, due to the obvious impact of high concentration cod on anammox bacteria, denitrifying bacteria can use part of COD to maintain a normal reproduction rate, making the growth rate greater than the mortality caused by toxic substances. Therefore, the increase in the number of denitrifying bacteria has no obvious impact on the overall biochemical activity, and denitrifying bacteria have gradually become a dominant strain compared with anammox bacteria in the mixed culture system at this time.
2. 1. 3 phase VI
Stage VI is all composed of petroleum refining wastewater, and the high concentration of COD and toxic substances in the raw water have not been diluted. It can be seen from Figure 5 and table 3 that the average removal value of NH4 + - n increases compared with the previous stage, the removal value of NH4 + - N fluctuates in the stage, and the gap between the removal value of NH4 + - N and the theoretical removal value of NH4 + - N gradually increases; The average total nitrogen removal amount of denitrification decreased compared with the previous stage, and the total nitrogen removal amount of denitrification also fluctuated significantly in the stage. It shows that the high concentration of COD and toxic substances in this stage have a significant adverse impact on the anaerobic ammonia oxidizing bacteria and denitrifying bacteria in the bacterial mixed culture. However, due to the increase in the number of anaerobic ammonia oxidizing bacteria or the strengthening of bacterial adaptability in the long-term operation, the biochemical activity of denitrifying bacteria can be reflected in the average denitrification amount, which is reduced by 25.6%, The biochemical activity of anammox bacteria can be reflected in the average removal value of ammonia nitrogen, which increased by 2.5% 491 mg / L。
Using molecular biology technology (MPN-PCR) to analyze the changes of flora before and after the domestication stage, the biggest advantage of this method is that it can quickly amplify the target sequence in vitro by PCR to replace the isolation and culture of bacteria, which can not only greatly shorten the experimental time, but also count the non culturable bacterial species in the sample, so that the result is closer to the actual number.
Firstly, in the aspect of DNA extraction, three extraction methods were tried: the improved hexadecyl trimethyl ammonium bromide (CTAB) / NaCI chemical lysis method, the improved traditional protease k-dodecyl sulfonate (SDS) - chloroform isoamyl alcohol method (cpsci method) and the improved lysozyme SDS protease K cell lysis method. The extracted DNA was quantitatively determined by UV spectrophotometry, The absorbance values of 260 nm and 280 nm were measured respectively. The integrity of extracted DNA needs to be tested by agarose electrophoresis. Verified by agarose electrophoresis, the length of DNA extracted by the three methods is about 23 KB, the outline of the band is clear, the brightness is appropriate, and there is no obvious dispersion phenomenon, indicating that the extracted bacterial genomic DNA is of high quality and suitable for subsequent experiments. Uv-2550 UV spectrophotometer is used for absorbance measurement, and the measurement results are shown in Table 4. It can be seen from table 4 that the a260 / A280 ratio of the three methods is greater than 1.8, the protein removal efficiency and DNA extraction quality are high, and the three methods can remove organic impurities such as protein well. Although there is a certain degree of RNA interference, it does not affect the operation of subsequent PCR. All three methods do not need purification and can directly obtain PCR amplification products, which shows that these three methods are feasible to extract DNA. From the two aspects of DNA concentration and purity, protease k-sds method was finally determined as the method of extracting DNA from bacterial mixed culture.
The primers used for the amplification of anammox bacteria and denitrifying bacteria in bacterial mixed culture are shown in Table 5, and 25 primers are used for PCR amplification μ L system, each component: PCR buffer 2 five μ L, MgCl2 2 μ L, dNTP 0. five μ 50. 1 upstream and 1 downstream primer μ L, Taq polymerase 0 two μ L (Shanghai Shenggong synthetic), template 1 μ 50. Make up with sterile double distilled water to 25 μ L。
After a series of experimental methods and conditions were tried, explored and optimized, the most suitable combination system of DNA extraction, PCR amplification and MPN counting was obtained. Fig. 6 shows the electrophoretic chart of the count of anammox and denitrifying bacteria in the bacterial mixed culture before and after the domestication experiment.
The number of anammox bacteria and denitrifying bacteria before domestication was 7.7 549 x 10 ^ 14 and 3 523 x 10 ^ 6 / g, and the number after domestication is 8.8 respectively 212 x 10 ^ 8 and 4 693 x 10 ^ 16 / g. Comparing the numbers of the two bacteria before and after domestication, it can be seen that for anammox bacteria, anammox reaction may be the main energy metabolism pathway of anammox bacteria. Due to the long generation time (about 11 days), the increase of denitrifying bacteria and the effect of toxic substances, the number of anammox bacteria and biochemical activity decreased significantly. For denitrifying bacteria, because the reaction can use a variety of metabolic pathways to obtain energy, the effect of matrix on bacteria is relatively small, and the use of COD in petroleum refining wastewater has not been affected in the proliferation of bacteria, but toxic substances have significantly inhibited the biochemical activity of denitrifying bacteria.
3) To a certain extent, the mixed culture denitrification system can effectively resist the negative effects of high concentration COD and high toxic substances on the physiological and biochemical denitrification process of anaerobic ammonia oxidation. The existence of denitrifying bacteria can promote and guarantee the stability of anaerobic ammonia oxidation denitrification system and the reduction of total nitrogen index in effluent.