| It is common that high ammonium can be detected in the surface water in China. In 2006,the new Standard for Drinking Water Quality was issued, in which the ammonium standard was more strict than before and should be lower than 0.5 mg/L. In most region of China, the water temperature is low and about 1-6°C in winter. Especially in Heilongjiang Province, the water temperature is 1-2°C in winter. Although break point chlorination can remove ammonium in water by increase addition of chlorine, the high amount of chlorine in water raise the cost of drinking water treatment and the risk of producing chlorination by-products. It would greatly influence the safety of drinking water. The traditional nitrification methods have the virtues of low cost and easy operation, which can be used in drinking water treatment for removing ammonium. However, the nitrifying bacteria are usually influenced by low temperature. They show low activity and ammonium removal efficiency when temperature is lower than 10°C. It has been proved that heterotrophic nitrifying bacteria can remove ammonium and grow at low temperature. Thus, it is possible to realize ammonium removal at low temperature by heterotrophic nitrification, which will benefit for innovating novel technology of drinking water treatment.In this study, 24 bacteria were isolated from Songhua River in winter. 5 bacteria were heterotrophic nitrifying bacteria that had the ability of ammonium removal. When the initial ammonium concentration was 5 mg/L, HITLi 7T showed the highest ammonium removal efficiency. The characteristics of the strain HITLi 7T were studied. It was aerobic, Gram-negative, non-motile, pleomorphic coccobacillus(0.7-1.3μm×0.8-1.4μm).Colonies were milk-white, circular, opaque, and slightly convex; colonies were 0.2-0.6 mm in diameter. Physiological and biochemical characteristics, metabolic properties and substrate utilization of strain HITLi 7Twere tested. No growth occurred at 37°C and above in 7 days. The strain HILi 7T is specializedby the inability to hydrolyze gelatin and utilize adipate and benzoate, assimilation of β-alanine and malonate. The strain HITLi 7T showed an unusual growth at 2°C. The major fatty acids of HITLi 7T were C18:1ω9c(41.92%)and C16:1ω7c(32.97%). Strain HITLi 7T especially had a small amount of C10:0(2.98%), C12:0(3.37%), C12:0 3OH(3.90%), C16:0(7.11%) C18:0(2.41%) and C18:1ω7c(4.82%). The G+C content of strain HITLi 7Twas 45.5 mol%. The similarity values of 16 S r DNA gene sequences of the strain HITLi 7Twere 93.5%-98.1% with other related species in the genus of Acinetobacter.rpo B and gyr B gene sequences showed low similarity with other type strains of recognized species and genomic species of the genus(lower than 85%). The DNA-DNA hybridization value of HITLi 7Twith Acinetobacter lwoffii DSM 2403 Tand Genomic species 6 ATCC 17979 was 36.2% and 25.5%, respectively. The genotypic and phenotypiccharacteristics clearly distinguishedthe strain HITLi 7Tfrom other species within the genus Acinetobacter. Based on the above results and the name rule, a new species Acinetobacter harbinensiswas proposed.The type strain was HITLi 7T and submitted to China General Microbiological Culture Collection Center(CGMCC) and Korean Collection of Type Culture(KCTC) with the accession number CGMCC 1.12528 and KCTC 32411.amo A, hao and nap A gene sequences were amplified by PCR from the strain HITLi 7T. The activity of NAP was detected by nitrate reduction method. The AMO and HAO were purified from by the anion-exchange chromatography and gel filtration chromatography. The AMO of the strain HITLi 7T was 31 k Da monosome, which kept high activity at 4-15°C. The HAO of the strain HITLi 7T was 61 k Da monosome. The highest HAO activities were detected at 15 °C and p H 7.5. Only potassium ferricyanide could be the electron acceptor of HAO.The amino acid sequences of the peptides hydrolyzed from HAO were LPSGLYQFR, VTVPHEDFIAK and SEFGPLPDQSMHEK. AMO and HAO played key role on ammonium removal by the novel heterotrophic nitrifying bacterium HITLi 7T.1550mg/kg of HITLi 7T that was 1000 times of amount exposure to human were supplied to the mice ICR by intraperitoneal injection. The mice ICR was 6 weeks old and clean. After 15 days of injection, the weigh, growth, organ and blood condition of the mice were in the normal values. The results showed that the strain HITLi 7T was not the infectious bacterium without biotoxicity. 550mg/kg·d of HITLi 7T were supplied to mice ICR as food. After 90 days, the weigh, growth, organ and blood condition of the mice were in the normal values.Two generations were bred by the mice. The baby mice grew well without malformation. The Ames test of the cell of the strain HITLi 7Tand it?s production showed that there was no mutagenesis risk. The results proved that the strain HITLi 7T was safe.The novel strain HITLi 7T was inoculated on the surface of filtration materials for constructing bio-enhanced filter process. The process was used in ground water treatment polluted by high ammonium. The water flow was 10000 t/d. The influent water temperature was 6-8°C. The ammonium concentration in the influent was 0.8-3.6 mg/L. The ammonium removal efficiency was stable and higher than 90%. The highest ammonium removal efficiency reached to 99.1%. The biofilms were observed on the surface of the anthracites and manganese sands by Scanning Electron Microscopy. The dehydrogenase activities of bio-enhanced filters were detected after 234 days of running. The activities of biomass on the anthracites and manganese sands were 167.66 mg/L/g and 105.61 mg/L/g in the first filter, and 96.94 mg/L/g and 87.59 mg/L/g in the second filter. PCR-DGGE results showed that the novel strain HITLi 7T was competed and correlated with the indigenous bacteria in the filter. The stable bacterial community structure was developed and played key role in ammonium removal.Through the study, the novel heterotrophic nitrifying bacterium HITLi 7T was isolated. AMO and HAO with high activity at low temperature were purified from the novel strain HITLi 7T. The novel strain HITLi 7T was used in treating ground water with high ammonium concentration. The ammonium removal efficiency was stable and high. The results in the study would be important theoretical and technical references for ammonium removal from micro-pollutant raw water under low temperature. |
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