Screening With High Capability P-Accumulating, Cloning Of Ppk Gene And Application

Eutrophication of water bodies is one of the ten global environmental problems. Nitrogen and phosphate, especially phosphate is the key contributor to eutrophication of water bodies. Discharge of untreated domestic sewage is one of the major sources of the phosphorus pollutants in the water bodies. In order to eliminate the hazard of phosphorus pollution, it is essential to treat the sewage to reduce its P concentration before it is discharged into rivers. Currently most large-scaled wastewater treatment plants use enhanced biological phosphorus removal(EBPR) process characterized by less sludge generation, freedom of chemicals and economical operation, which is extensively used in countries all over the world. So far, people have separated only a few PABs, such as Microlunatus phosphorus, Lampropedia spp., and Pseudomonas spp., which have few anaerobic characteristics of typical PABs in EBPR system, and little has been reported about engineering application of PABs. What's more, microbiologists and engineers donot unite tightly, system EBPR has its characteristic of faintness, and microbiological and molecular mechanisms are not known, so it is difficult to control the stabilization of running EBPR and development of the whole biological P-accumulating study is very slow. Therefore, it is quite necessary to improve strain isolating methods, intensify screening of the strains, investigate characteristics, dominating gene and engineering application of PABs.The masterstroke of this text was the study on mechanism of PABs. Commencing with the issue of how to screen high-efficiency PABs, P-accumulating features of PABs, P-accumulation-related ppk gene, colonization of GM6 and its capability of enhanced wastewater biological phosphorus removal were investigated by the numbers. The foreground of GM6's engineering application was explored. The technique platform of biological P-removal investigation in the country was constructed. And many rich harvests about basic research of sewage enhanced bilogical P-removal and application field in the country at present were obtained.I. Screening of PABs and its following characteristicsThe methods of screening PABs with high capability was innovated. Four strains with high capability of accumulating poly-P which capability was much higher than the one reported by other country were screened. Based on the high flux principle, the methods of plate count, colony-dyeing, and blue- and white-colored screening, were used to screen PABs. A total of 25 strains were obtained. After culturing in aerobic and anaerobic conditions, 4 strains named GM1,GM6,GM18,GM21 were confirmed as PABs strains. GM1 and GM6 were chosen as objects of the study. GM1 was primarily identified as Citrobacter freundii and GM6 as Pseudomonas putida according to its physiological & biochemical characteristics and the homology analysis of its 16S rDNA sequence. GM1 could grow in synthetic wastewater with optimal initial pH of 7.0 and optimal temperature of 30℃. When pH was above 8.5 or below 5.5, or temperature went above 33℃or below 5℃, it would grow slower. And GM6 could grow in synthetic wastewater with optimal initial pH of 6.5 and optimal original temperature of 27℃. When pH was above 8.0 or below 5.0, or temperature rose above 37℃or dropped below 5℃, it would also grow slower. But volume of the liquid had little influence on growth of GM1 and GM6.Ⅱ. Characterization of P accumulation of GM1 and GM6Strains GM1 and GM6 exhibited characteristics of typical PABs under pure culture conditions which offered invaluable materials for biological P-removal study. When GMI and GM6 were cultured in the synthetic wastewater, the optimal initial pH for the strains to remove phosphorus was 6.5 and 7.0, respectively. When pH was above 7.5 or below 5.5, the P-removing capability of both strains would come down. The optimal initial temperature for the strains to remove phosphorus was 20℃and 27℃, respectively. But for GM1, when temperature went up above 30℃or down below 10℃, its P-removing efficiency would decline significantly and for GM6 when temperature was above 33℃or below 5℃something similar would occur. When GM1 and GM6 grew in the synthetic wastewater, MOPS, LB, and YG media under aerobic conditions, their P removing rate was 60％-89.6％and 63％-96.6％, respectively (as a control the P-removing rate of Strain E. coli was 13.6％-30.8％). GM6 had a higher P takeup and release capability. When cultured in acetic acid under aerobic conditions, GM1 and GM6 could accumulate PHB. Ascending of PHB Concentration in the strains was negatively correlated with descending of acetic acid concentration,. While GM1 and GM6 grew under anaerobic conditions, descending of acetic acid concentration in the supematant was negatively correlated with ascending of PHB and P concentration. Within the first hour, the acetic acid absorbing rate and the P release rate were quite high, but with the absorption of acetic acid and decomposition of polyphosphate, the acetic acid concentration in the solution would descend while the P concentration therein would ascend, forming a negative correlation.Ⅲ. Cloning and expression of the polyphosphate kinase (ppk) gene from GM6 (Pseudomonas putida)Ppk gene of PABs was cloned and expressed for the first time in the country, and the expressed strain had a relatively high efficiency of P-removal. In this research a primer was designed in light of the conserved region of the ppk gene. a fragment, about 528bp, of ppk gene was augmented into the total DNA of GM6. Then a complete ppk gene (GenBank accession number DQ0133537 ) and its upstream and downstream sequences was cloned by SEFA-PCR, a new PCR technology used to amplified relatively long flanking sequences of known regions. Sequences analysis with OMIGA program revealed the whole size of the ppk gene was 2220bp, and its upstream gene was hemB gene with the likely size of 1119bp. The nucleotide sequence analysis showed that their homology between ppk gene and Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas syringae, Klebsiella aerogenes, Neisseria meningitidis, Acinetobacter sp. ADP1 and Escherichia coli K12-MG1655 was 82％, 79％, 84％, 45％, 63％, 53％, and 22％, respectively. And the nucleotide sequence analysis also showed that their homology in amino acid sequence between PPK protein and Pseudomonas putida KT2440, Acinetobacter.sp.ADP1, Escherichia coli K12-MG1655, Neisseria meningitidis MC58 and Pseudomonas aeruginosa PAO1 was 89％, 58％, 33％, 50％, and 80％, respectively.When the constitutive expression strain E.coli BL21(DE3)/ pET29a-ppk was indued by IPTG for 3 hours, the expression product with the molecular weight of about 81 kDa was obtained. The P-removing rate of expression strain E.coli BL21(DE3)/pET29a-ppk reached 80％while the control strain was only 18％after being cultured for 12h, which was far more than 40％reported before. It suggested that excessive expression of ppk gene in E. coli lead to accumulation of a great deal of polyphosphate in E. coli, thus removing the phosphate from the media.Ⅳ. Colonization of GM6 in activated sludge and application of the technology of intensified biological P-removalStrain GM6 was applicated in engineering for the first time which gained the capability of enhanced wastewater biological phosphorus removal rapidly. GM6 was labelled by tri-parents conjugation, and strain GMTR with gfp gene was obtained. The test indicated that, strain GMTR should be added in the original of anaerobic phase. GMTR could not have the dominant in the sludge when the inoculation was below 1％. The optimal GMTR application rate was 2％, when sewage was under anaerobic treatment. After it was added, the sewage should be kept under anaerobic conditions for at least 2 hours. Colonization of GMTR in Device R2 was that, Strain GMTR accounted for 1.5％-3％of the whole count of bacteria in activated sludge in the initial period after GMTR was added. After 21d to 60d, the proportion of GMTR rose to 6.5％-7.6％, suggesting that GMTR colonized well after 21d. The P-removing rate began to increase gradually on D6, and up to 96％on D21. The concentration of phosphorus in the drainage was stabilized around 0.2 mg L^-1 from D 28 and on, meeting criteria for wastewater discharge. After the apparatus ran for 30 days, it was shifted into anaerobic conditions for one hour, the strain would quickly release phosphorus to remove organic substances, and when it was put back under aerobic conditions for 2 hours, it would again remove phosphorus efficiently. Consequently, additon of Strain GM6 could enhance biological phosphorus removal and quickly restore the biological P-removing ability of deteriorated strains. Some sewage treatment plants that were low in P-removing efficiency and could only deal with 30 tons of sewage per day were selected as pioneer plants for engineering application of the technology. GM6 was applied at a rate of 2％, P concentration in the drainage began to decline gradually on D6 and dropped down to or below 0.5mg/L on D15, and further down to or below 0.3mg/L from D18 and on. The results show that GM6 could enhance biological P-removing efficiency notably, which could serve as scientific basic for further engineering application on a large scale.Ⅴ. Main factors affecting biological P-removal in the EBPR systemAccording to the research findings, concentration of acetic acid in the influent, anaerobic conditions, pH of influent, concentration of DO, sludge age, and so on are key factors affecting P-removing efficiency of the EBPR system. Keeping anaerobic for 45 minutes made P release rate rise with the concentration of acetic acid in the influent. The P-releasing rate varied with the environment. For example, it was only 2.25mg/l when glucose was used as the exclusive carbon source, was 16.69mg/l when the concentration of acetic acid was 15％, and remained unchanged if the concentration of acetic acid was above 30％. Under anaerobic condition for 120min, concentration of acetic acid had little influence on P-releasing rate. If the churning speed was too high mixing under anaerobic conditions, the P-releasing rate would descend. As far as P-releasing of the system is concerned, the optimal pH was 6.5. When pH was above 7.0 or below 6.0 in the influent, the P-releasing rate declined. And when pH was above 7.5, the P-release rate dropped drastically. As far as the P-removing rate of the system is concerned, the optimal pH was also 6.5. When pH was above 7.0 in influent, the P-removing efficiency fell sharply. And when pH was 8.0, the P-removing efficient was only 39.7％and the system lost its capability of enhanced biological phosphorus removal. Aeration intensity did not have much influence on concentration of COD, but great influence on concentration of phosphorus in the drainage. When the aeration intensity led DO to 2.10mg/l, concentration of phosphorus in the drainage declined down to 0.26mg/l. In addition, sludge age influenced P-removing efficiency greatly. When the age was 4d, concentration of phosphorus and COD_cr was 0.25 mg L^-1 and 25mg L^-1, respectively, MLVSS 2.97mg L^-1; P-releasing rate 33.8mg L^-1 under anaerobic conditions, and concentration of phophorus in the sludge 6.36％. Then the EBPR system was running in a good condition.