Efficiency And Mechanism Of Tpyical Ppcps Degradation By Functional Strains Through Co-Metabolism And Different Carbon Sources

Pharmaceuticals and personal care products (PPCPs) are one kind of emerging micropollutant, which are used frequently in daily life and discharged into the environment via sewage and surface runoff. In recent years, many PPCPs were detected in soil, ground water, surface water, etc. In fact, PPCPs can be concentrated through food chains, potentially threatening human health. As a result, various methods have been used to remove PPCPs from different environmental media. The existing studies reported that co-metabolism was one of the most important removal pathways for the persistent organic pollutants and PPCPs as well. However, co-metabolism relied on the additional carbon sources for providing energy to microorganisms as growth substrate to degrade the organic pollutants. Therefore, this research aimed at both the macroscopic and microcosmic influences of different carbon sources on the typical PPCPs co-metabolic processes. Iopromide (IOP), iomeprol (IOM) and bezafibrate (BZF) were chosen as the target PPCPs and two pre-isolated functional strains named Pseudomonas SP.1-24(1-24) and Pseudomonas putida B-31(B-31), were investigated. The effects of different carbon sources on the co-metabolic processes were studied. Also, the enzymology regulatory mechanism was investigated and the enzymatic activity detection methods were established. The optimal reactive conditions and characteristics were discussed. The differential expression of proteome studies revealed inducement of various proteins by different carbon sources. In order to verify the practical applicability of the functional strains and degradation enzymes, biological aerated filters (BAF) were used to compare their performance for the removal of PPCPs and conventional pollution indicators. The results are shown as follows:(1) The investigation of degradation processes of additional carbon sources and target pollutants, as well as the tendency of growth and metabolic activity of functional strains.Methods for the detection of glucose, malt sugar, starch and glycerol were optimized according to the established pathways. The tests of co-metabolism process using different carbon sources indicated that starch and glucose were the most suitable carbon sources for I-24and B-31, respectively. Removal efficiencies of92.70%and38.43%for IOP and IOM by I-24, respectively, were obtained by using starch. While using glucose, B-31degraded BZF by76.98%. However, I-24grew best under glucose condition, indicating that growth condition did not determine degradation efficiency, but it could still exist as one of indicative factors for degradation efficiency. The observation of electron transport system activity (ETSA) of functional strains suggested that malt sugar promoted ETSA mostly. ETSA values of I-24in IOP and IOM, and B-31in BZF were32.12μg/(g·h),100.92μg/(g·h) and215.54μg/(g·h) in the first cultivation day, respectively. From the comparison between non-growth substrate and growth substrate, we guess that the functional strains can hardly get in touch with target pollutants until their surface are activated by carbon electron. As a result, ETSA was useful for evaluating electron releasing efficiency of additional carbon sources in co-metabolism conditon.(2) Establishment of effective and easy detection methods for enzymatic activityThe degradation enzyme excreted by I-24and B-31were defined as IOP enzyme, IOM enzyme and BZF enzyme according to their substrate. The optimum conditions for IOP enzyme and IOM enzyme extraction were:ultrasonic power of150W, running time of20min, working time of3s and resting time of1s. The optimum conditions for BZF enzyme extraction were: ultrasonic power of150W, running time of20min, working time of3s and resting time of2s. The detection methods for enzymatic activity were established and optimized as pH7, reaction temperature of30℃, reaction time of2h and enzyme concentration of80～100mg/L for both IOP and IOM enzymes, and90mg/L for BZF enzyme. The inactivation temperature for IOP and IOM enzymes was80℃, while for BZF enzyme was100℃. Based on the activity tests, the degradation enzymes were found to be intracellular enzyme.(3) Influence of environmental factors on enzyme reactive processesThe characteristics of IOP enzyme, IOM enzyme and BZF enzyme were studied as follows: the pH stability ranges were7～8for IOP enzyme,6～8for IOM enzyme and6～7for BZF enzyme, the temperature stability ranges were10～40℃for IOP enzyme,0～60℃for IOM enzyme and10～40℃for BZF enzyme. Michaelis constant tests indicated that Km of IOP enzyme, IOM enzyme and BZF enzyme were136.70μmol/L,91.08μmol/L and41.85μmol/L, respectively, while the Vm were0.05μmol/(L·min),0.04μmol/(L·min) and0.074μmol/(L·min), respectively. Though IOP, IOM and BZF were not the limiting factors of strain growth, they were still found to be one of inductors of the degradation enzymes. Once there was other carbon source, both of them would induce degradation enzyme in union. However, enzyme inducement was supposed to be strongly restrained in poor energy environment. Enzymatic activities induced by different additional carbon sources verified that starch accelerated the activities of IOP enzyme and IOM enzyme to0.182mU and0.143mU, while glucose accelerated BZF enzyme activity to0.188mU. Due to the accumulation of intermediate products and enzyme aging, their activities decreased. The suitable dosage of starch and glucose were determined as1g/L and3g/L. Excessive starch might result in competitive inhibition with target pollutants and reduced enzyme inducement. Though there was no circumstance suggesting glucose in the concentration between1and3g/L induced inhibition, it did not represent that no inhibition would occur by high concentration of glucose. Double-substrate enzyme reaction method was also established, which showed no IOP, IOM and BZF enzyme activity during2h reaction time, demonstrating the non-specific characteristic of co-metabolic enzymes. Once there was two substrates, the substrate with simple structure would be superior to be degraded.(4) The differential expression of functional strain1-24influenced by verified carbon sourcesIn order to see the influence of verified carbon sources (IOP, IOP+starch and IOP+glucose) on strain, the differential expression of1-24was studied. By means of two-dimensional gel electrophoresis tests, the isoelectric point (pI) of protein in IOP condition was suggested to be between4.5and6.0, while the pIs of the other two proteins were both between4.5and8.5. Nevertheless, the molecular mass ranges were all between25kDa and45kDa. The analysis of protein pages between IOP+starch and IOP+glucose samples pointed out28different spots, of which23spots up regulated and5spots down regulated in IOP+starch sample according to IOP+glucose sample. MALDI-TOF MS analysis of these spots showed that ATP synthesis, protein transcription and transportation were involved in carbon influence. Starch utilized glutamate more efficiency than glucose, therefore, ATP synthesis process was much more outstanding. In the mean time, starch was superior in cell protein metabolism and superoxide metabolism processes.(5) The application of functional strains and degradation enzyme in BAFsThe functional strains and degradation enzymes were applied to two BAF separately to compare the removal efficiencies of conventional pollution indicators and PPCPs under different operating conditions and carbon sources. The results showed that the functional strains and degradation enzymes did not exhibit any acceleration on CODMn, NH3-N and UV254removal, but it showed favorable removal efficiency and load resistance of IOP, IOM and BZF. The BAF exhibited fluctuant removal efficiencies because degradation enzymes could be washed away easily. Compared to complex natural water, simulated micro-polluted water was easier to treat. Carbon sources affected PPCPs removal in accordance with static tests. The lower the hydraulic load was, the contact opportunity the PPCPs would get with biological membrane and filter materials. However, if the DO was too high, the biological membrane might be destroyed and the removal efficiency would be restrained as well. Under the optimum treatment conditions of hydraulic load of0.08m3/m2-h, DO of9.5mg/L and carbon source of starch for IOP and IOM, in simulated micro-polluted water, the IOP and IOM removal rates of three BAF (no functional strain and degradation enzyme, functional strains, degradation enzymes) were97.21%,98.12%and98.68%(IOP),97.15%,96.53%and98.50%(IOM), respectively. Under the optimum treatment conditions of hydraulic load of0.08m3/m2-h, DO of7.5mg/L and carbon source of glucose for BZF, the removal rates in three BAF were90.04%,90.99%and93.08%for simulated micro-polluted water. The removal efficiencies of both conventional pollution indicators and PPCPs in natural water were lower than that in simulated micro-polluted water. However, once the additional carbon sources were added to the natural water, the target pollutants were biodegraded closed to the simulated water. In addition, backwashing parameters were optimized as below:as the simulated micro-polluted water was used as influent, the backwashing period was10d. The mode was air washing of1min with an intensity of9-12L/(m2-s) and the intermittent time was set at min for three cycles. If BAFs run over20days, the backwash mode had to be changed as air and water washing of2min with an air intensity of3-4L/(m2-s) and water intensity of7-8L/(mz·s). Furthermore, when natural water was used as influent, the backwashing period was5d, the mode was air combined with water washing of2min with an air intensity of4-5L/(m2·s) and water intensity of7-8L/(m2·s).