微污染水源扬水曝气强化原位生物脱氮特性与试验研究
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摘要
由于污废水大量排放及农业长期大量施用化肥,饮用水源氮源污染日益严重,已引起世界各国的高度重视。三十多年来人们采用各种方法对受污染水源水进行脱氮处理,其中生物脱氮法是目前去除水体氮源污染物最经济、有效的方法。尽管国内外对微污染水体生物脱氮进行了大量研究,但现有地表水源生物脱氮技术主要以异位生物修复为主,其水力停留时间受到限制,对总氮去除效果不理想,且大多需投加电子供体,增加了水处理成本,同时有些基质或其氧化的中间产物对生物有毒害作用。
本文将扬水曝气与原位生物净化两种技术有机结合,针对微污染水体原位生物脱氮存在的低温、贫营养及好氧问题,采用贫营养脱氮功能菌群及低温贫营养脱氮功能菌群制成微生态菌剂,形成能有效去除水中氮源及有机污染物的原位生物投菌技术和原位生物膜技术,并结合扬水曝气技术,揭示了该组合技术用于微污染水源水生物脱氮的特性及效能。论文成果主要包括以下几方面:
(1)贫营养好氧反硝化细菌的驯化、筛分及脱氮特性研究。在低营养条件下驯化、筛分出具有较好脱氮性能的贫营养好氧反硝化细菌PY8、DA15和DF7,结合菌株形态、生理生化试验及16S rDNA序列分析,可基本确定菌株PY8属于Rhizobium sp.,菌株DA15属于Arthrobacter sp.,菌株DF7属于Bacillus sp.。在前期已筛分出的高效脱氮菌种FY3、FY5和FY7的基础之上,采用生态位分离策略构建了贫营养脱氮功能菌群Z4(PY8+FY3+FY7),并对其中各功能菌的生长曲线、脱氮特性进行了系统的研究。结果表明,各菌株生长速率较慢,pH、温度、C/N、NaNO3浓度及接种量对各贫营养好氧反硝化细菌的脱氮性能影响有差异,且PY8异养硝化性能明显。周质硝酸盐还原酶亚基基因(napA)PCR扩增结果证实这3株菌具有可在好氧环境中还原硝酸盐的系统,好氧反硝化作用的确是由周质硝酸盐还原酶来催化的。
(2)低温好氧反硝化细菌的驯化、筛分及脱氮特性研究。通过逐渐降低温度条件驯化、筛分出耐冷脱氮细菌DW3、DW4、D3和D4,经过生理生化鉴定及16S rDNA序列分析,基本确定菌株DW3属于Pseudomonas sp.,菌株DW4、D3和D4均属于Acinetobacter sp.。各耐冷脱氮菌株生长曲线及脱氮特性试验表明,除D3菌株外,其他菌株在培养过程中都存在明显的衰亡期,各菌株的反硝化过程都发生在对数生长期,pH、温度、C/N及接种量对各菌株的脱氮性能有影响,各菌株具有明显的异养硝化性能,且在10℃温度条件下仍能保持一定的脱氢酶活性。选择菌株DW3和D3进行周质硝酸盐还原酶亚基基因(napA)PCR扩增,证实了这2株菌具有周质硝酸盐还原酶,可实现好氧反硝化。采用自适应及菌源生态重组策略构建出在极端环境中具有较好反硝化效果的低温贫营养脱氮功能菌群L1(PY8+DW3+D3)和L2(PY8+DW3+D4)。
(3)微污染水体原位生物投菌技术脱氮特性与效能研究。小试研究以贫营养脱氮功能菌群Z4作为菌源制成微生态菌剂,采用不同菌剂投量直接投加到水库原水中,菌投量为0.1mg/L时净化效果最好,整个运行期间(32d),硝氮、总氮、CODMn及TOC去除率分别达到72.3%、71.3%、32.3%和34.8%,稳定期的脱氮效果可满足地表水环境Ⅲ类水体的质量标准要求;同时溶解氧、水温、pH及C/N会影响生物菌剂的脱氮性能。将低温贫营养脱氮功能菌群L1用于微污染水原位生物投菌技术中,在低温环境条件下运行36d,该菌剂对原水硝氮及总氮去除率最大可达到46%和53%。
(4)微污染水体原位生物膜技术脱氮特性与效能研究。选择脱氮性能较稳定的硬性悬浮填料,同时研制出适合于微污染水体原位修复、且可创造适宜微生物生长的不同溶解氧环境的新型多孔悬浮填料作为生物载体,以贫营养脱氮功能菌群Z4作为菌源对载体进行人工强化挂膜,小试研究结果表明,试验运行46d,贫营养原位生物膜技术对原水硝氮及总氮去除率均可达到75%以上,CODMn去除率在25%以上,稳定时期脱氮效果可满足地表水环境Ⅲ类水体的质量标准要求,且温度、填料填充率及C/N比对原位生物膜系统脱氮效果有一定影响。贫营养细菌以较低速率进行生长时,对外界环境具有较大的抗性,是微生物适应贫营养环境的结果,在贫营养条件下,吸附和生物膜形成是微生物的一种生存策略。
(5)原位生物投菌系统对混合充氧技术的条件要求。DO浓度对贫营养好氧反硝化菌群的脱氮性能有一定影响。当DO浓度大于7mg/L时,反硝化速率很慢,始终保持在较低的水平;当DO浓度低于7mg/L时,反硝化速率随DO值的降低而较大幅度地升高,说明贫营养好氧反硝化菌群的阈值较高,对氧气有较高的耐受能力。DO浓度为3~4mg/L、5~6mg/L和7~8mg/L时,系统运行期间对原水总氮去除率分别为91%、84%和31%。好氧反硝化细菌的反硝化酶系和有氧呼吸系统同时存在,氧不再是抑制反硝化酶活性和反硝化酶生成的直接因素,周质硝酸盐还原酶亚基基因的扩增进一步解释了好氧反硝化菌可以在DO浓度相对较高的条件下进行反硝化作用。
(6)扬水曝气—原位生物净化组合技术中试研究。在扬水曝气强化混合条件下,进行原位生物净化系统中试研究。以贫营养脱氮功能菌群Z4为菌源,分别构成扬水曝气—贫营养生物膜和扬水曝气—贫营养生物投菌组合技术,在溶解氧浓度为5.0~7.0mg/L,水温范围为10℃~ 23℃、CODMn/TN≈1.56的贫营养条件下,扬水曝气—贫营养生物膜组合系统稳定运行时的氨氮、硝氮、总氮和TOC去除率范围分别为82%~100%、62%~79%、70%~80%和72%~80%,BOD5/CODMn比值可由初始0.476降至0.054,且三维荧光光谱图显示运行期间原水生源性DOM变化较大;扬水曝气—贫营养生物投菌组合系统稳定运行时的氨氮、硝氮、总氮、TOC和CODMn去除率分别为82%~100%、43%~58%、63%~70%、65%~73%和51%~57%。PCR-DGGE图谱分析表明,生物膜上种群演替进程缓慢,α-proteobacterium纲是生物膜中最优势的细菌类群,固定的菌株PY8和FY3成为生物膜上的优势菌群;生物投菌系统中FY3和FY7成为系统中的优势菌群。消毒剂灭活试验、群落结构多样性及小鼠急性经口毒性试验表明,生物菌剂不会对原生态系统产生较大的破坏,且不会对饮用水水质构成威胁。采用低温贫营养脱氮功能菌群L1与扬水曝气技术有机结合,在初期水温不足15℃,CODMn/TN≈1.0的低温、贫营养条件下,稳定运行阶段的硝氮及总氮去除率分别在50%和60%以上,最大硝氮及总氮去除率分别为81%和84%,TOC、CODMn及BOD5月平均去除率分别为63%、49%和62%。上述结果表明,该组合技术用于净化微污染原水是可行的,稳定运行期的脱氮效果均可满足地表水环境Ⅲ类水体的质量标准要求。
Nitrate often contaminated surface water resources due to excessive use of fertilizers and uncontrolled on-land discharges of raw and treated wastewater and can therefore limit the direct use of surface water for drinking water purposes. Many researches on the denitrification of source water had been reported in the last three decades with the increasing nitrogen pollution concerns; biological treatment was the most attractive process to remove nitrogen from surface water sources due to its low cost and high efficient. However, there are still some problems on denitrification of drinking water resources. Firstly, biological nitrogen removal of ectopic widely used in surface water sources has a restriction of biological contact time——an important limiting factor for influencing the removal efficiency of total nitrogen. Secondly, electron donor used to improve the denitrification efficiency can increase water treatment costs and some substrate or its intermediate products of biological oxidation may produce toxic effects on life.
In this research, a multifunctional device for water lifting and aeration (WLA) was combined with in-situ biological purification technology (ISBP) for the denitrification of surface water. The main objective of this research was to investigate the key issues of nitrate removal of micro-polluted raw water with the combined system under low temperature, oligotrophic and aerobic condition. Oligotrophic denitrifiers and cold-resistant denitrifiers were cultured for microbial agents, which was the important source of bacteria for in-situ biological inoculation or in-situ bio-contact oxidation system. Both ISBPs were combined with WLA in order to obtain a satisfactory performance on nitrogen and organic matter removal. Properties and experiments of enhanced in-situ biological nitrogen removal of micro-polluted raw water were discussed in this paper. The main results and conclusions of this study are as follows:
(1) Domestication, isolation and denitrification properties of oligotrophic aerobic denitrifiers. Oligotrophic denitrifiers strain PY8, DA15 and DF7 was isolated after the acclimatization with low-nutrient medium in order to remove nitrogen desirably under oligotrophic conditions. According to their physiological, biochemical methods and the sequence analysis of 16S rDNA, strain PY8, DA15, DF7 was identified as Rhizobium sp., Arthrobacter sp. and Bacillus sp., respectively. On the base of the isolated strain FY3, FY5 and FY7, oligotrophic denitrifying functional bacteria populations Z4 (PY8, FY3 and FY7) was formulated by niche separation, and all the functional bacteria were studied for nitrogen removal performance. The results showed that all the strains grew slowly, and pH value, temperature, C/N ratio, NaNO3 concentration and inoculum had different effects on denitrification performance of strain PY8, FY3 and FY7. Also, the strain PY8 had a significantly heterotrophic nitrification performance. It was confirmed that the strain PY8, FY3 and FY7 had the capacity of express periplasm nitrate reductase through the PCR reaction, and periplasmic nitrate reductase played an important role in aerobic gentrification.
(2) Domestication, isolation and denitrification properties of low-temperature- resistant aerobic denitrifiers. Low-temperature-resistant denitrifying bacteria were isolated by gradually reducing temperature condition, and the strain DW3 was identified as Pseudomonas sp., the strains of DW4, D3 and D4 as Acinetobacter sp., based on biochemical index analysis and 16S rDNA sequence homology analysis. Growth curve and nitrogen removal capacity was investigated for the four strains. The results indicated that in addition to D3 strain, other strains were present a decline phase, and the strains of the denitrification took place in the logarithmic phase. Also, pH value, temperature, C/N ratio and inoculum could influence denitrification performance of low-temperature- resistant denitrifiers. A desirable ammonia removal rate was obtained for the four strains in nitrification medium. D3 and D4 still maintained a certain dehydrogenase activity at 10℃. The results also proved that DW3 and D3 strains had periplasmic nitrate reductase and could realize aerobic denitrification. Low-temperature-resistant and oligotrophic denitrifying functional bacteria groups L1 (PY8, DW3 and D3) and L2 (PY8, DW3 and D4) could were constructed by self-adjustment and eco-recombination for the purpose of denitrification under oligotrophic and low temperature conditions.
(3) Properties and efficiency of in-situ biological inoculation nitrogen removal of micro-polluted raw water. Oligotrophic denitrifying functional bacteria populations Z4 as bacteria source was used for in situ biological inoculation technology in small scale tests. The showed that a preferable purification could be presented under 0.1 mg/L inoculating dose, and the removal rate of nitrate, total nitrogen, CODMn and TOC during the entire operation (32 days) could reach 72.3%, 71.3%, 32.3% and 34.8%, respectively. Also, nitrogen removal effects in steady running period could meet the requirements of classⅢof surface water quality according to GB3838-2002. DO, temperature, pH and C/N ratio was important influencing factors for denitrification performance of oligotrophic dominant agents. The cold-resistant and oligotrophic denitrifying functional groups L1 showed a high efficiency for nitrogen removal in low temperature, and the optimum removal rates of nitrate and total nitrogen reached 46% and 53% during 36 days operation.
(4) Properties and efficiency of in-situ biological biofilm nitrogen removal of micro-polluted raw water. A rigid suspended packing and a novel suspended packing chosen as biological carriers was applied to in-situ biofilm system. The novel suspended packing was developed for in-situ remediation and could create an appropriate environment for the growth of aerobic, anaerobic and facultative bacteria. Artificially hanged membrane was formed on both carriers with oligotrophic denitrifiers functional groups Z4. The small test results showed that the removal rates of nitrate, total nitrogen and CODMn could reach over 75%, 75% and 25%, respectively, and nitrogen removal effects in steady running period could meet the requirements of classⅢof surface water quality according to GB3838-2002. Temperature, filling ratio of suspended packing and C/N had a certain influence on nitrogen removal efficiency of in-situ biological biofilm system. Oligotrophic bacteria grown at a lower rate and had a greater resistance to external environment, and these properties could provide a better adaptability to poor nutrition environment for them. The adsorption and microbial biofilm formation was a survival strategy in oligotrophic environment.
(5) Requirements of mixed aeration technology for in-situ biological inoculation system. Dissolved oxygen concentration could affect denitrification performance of oligotrophic denitrifying functional groups. When the DO concentration greater than 7 mg/L, the denitrification rate was very slow and always maintained a low level. The nitrogen removal rate would increase significantly with the decrease of DO value under condition of DO concentration less than 7 mg/L. These results indicated that aerobic denitrifiers with a higher threshold had a desirable oxygen tolerance. The removal rates of total nitrogen were 91%, 84% and 31% respectively under conditions of DO concentration 3~4 mg/L, 5~6 mg/L and 7~8 mg/L. Both denitrification enzyme and aerobic respiratory system could exist in aerobic denitrifiers, and oxygen was no longer inhibited denitrification enzyme activity and denitrification enzyme generation directly. Periplasmic nitrate reductase subunit gene amplification further explained that aerobic denitrification could occur under high DO concentration condition.
(6) Pilot research on combined process of WLA and ISBP. Under the condition of enhanced mixing of water-lifting aeration, a pilot research on ISBP was made for the purpose of pretreatment of micro-polluted raw water. Oligotrophic denitrifying functional groups Z4 as bacterial source constituted combined process of WLA and in-situ biofilm and combined process of WLA and in-situ bio-inoculation. For the combined technology of WLA and in oligotrophic biofilm system, the results showed that removal rates of ammonia, nitrate, total nitrogen and TOC in steady running period ranged from 82%~100%, 62%~79%, 70%~80% and 72%~80%, respectively, under conditions of dissolved oxygen 5~7 mg/L, temperature 10℃~23℃and C/N ratio 1.56 for source water, also the ratio of BOD5 to CODMn was changed from 0.476 to 0.054. Three-dimensional excitation-emission matrix (3DEEM) fluorescence spectroscopy showed that the biogenic dissolved organic matter of raw water had experienced an obvious change during the operation period. As for the combined technology of WLA and oligotrophic bio-inoculation system, removal rates of ammonia, nitrate, total nitrogen, TOC and CODMn in steady running period ranged from 82%~100%, 43%~58%, 63%~70%, 65%~73% and 51%~57% respectively under the same conditions. The PCR-DGGE profiles showed that the microbial populations on the biofilm evolved slowly andα-proteobacterium was the largest fraction. In addition, sequences revealed that strains PY8 and FY3 became the dominant bacteria in the combined system WLA and oligotrophic biofilm, and FY3 and FY7 strain became the dominant bacteria in the combined system WLA and oligotrophic bio-inoculation system. Disinfection tests, biological community structure diversity and acute oral toxicity tests in mice showed that biological agents would not pose a threat to original ecosystems and drinking water. Under conditions of initial temperature of less than 15℃and CODMn/TN of 1.0, cold-resistant and oligotrophic denitrifying functional groups L1 combined with WLA could obtain a desirable denitrification effects during the stable period for more than 50% of nitrate removal rate and 60% of total nitrogen removal rate, and the optimum removal rate for nitrate and total nitrogen was 81% and 84%. Also, the monthly average removal efficiency of TOC、CODMn and BOD5 could reach 63%, 49% and 62%. The experiment results showed that the combined technique of WLA and ISBP was feasible for the denitrification of raw water, and nitrogen removal effects in steady running period could meet the requirements of classⅢof surface water quality according to GB3838-2002.
本文将扬水曝气与原位生物净化两种技术有机结合,针对微污染水体原位生物脱氮存在的低温、贫营养及好氧问题,采用贫营养脱氮功能菌群及低温贫营养脱氮功能菌群制成微生态菌剂,形成能有效去除水中氮源及有机污染物的原位生物投菌技术和原位生物膜技术,并结合扬水曝气技术,揭示了该组合技术用于微污染水源水生物脱氮的特性及效能。论文成果主要包括以下几方面:
(1)贫营养好氧反硝化细菌的驯化、筛分及脱氮特性研究。在低营养条件下驯化、筛分出具有较好脱氮性能的贫营养好氧反硝化细菌PY8、DA15和DF7,结合菌株形态、生理生化试验及16S rDNA序列分析,可基本确定菌株PY8属于Rhizobium sp.,菌株DA15属于Arthrobacter sp.,菌株DF7属于Bacillus sp.。在前期已筛分出的高效脱氮菌种FY3、FY5和FY7的基础之上,采用生态位分离策略构建了贫营养脱氮功能菌群Z4(PY8+FY3+FY7),并对其中各功能菌的生长曲线、脱氮特性进行了系统的研究。结果表明,各菌株生长速率较慢,pH、温度、C/N、NaNO3浓度及接种量对各贫营养好氧反硝化细菌的脱氮性能影响有差异,且PY8异养硝化性能明显。周质硝酸盐还原酶亚基基因(napA)PCR扩增结果证实这3株菌具有可在好氧环境中还原硝酸盐的系统,好氧反硝化作用的确是由周质硝酸盐还原酶来催化的。
(2)低温好氧反硝化细菌的驯化、筛分及脱氮特性研究。通过逐渐降低温度条件驯化、筛分出耐冷脱氮细菌DW3、DW4、D3和D4,经过生理生化鉴定及16S rDNA序列分析,基本确定菌株DW3属于Pseudomonas sp.,菌株DW4、D3和D4均属于Acinetobacter sp.。各耐冷脱氮菌株生长曲线及脱氮特性试验表明,除D3菌株外,其他菌株在培养过程中都存在明显的衰亡期,各菌株的反硝化过程都发生在对数生长期,pH、温度、C/N及接种量对各菌株的脱氮性能有影响,各菌株具有明显的异养硝化性能,且在10℃温度条件下仍能保持一定的脱氢酶活性。选择菌株DW3和D3进行周质硝酸盐还原酶亚基基因(napA)PCR扩增,证实了这2株菌具有周质硝酸盐还原酶,可实现好氧反硝化。采用自适应及菌源生态重组策略构建出在极端环境中具有较好反硝化效果的低温贫营养脱氮功能菌群L1(PY8+DW3+D3)和L2(PY8+DW3+D4)。
(3)微污染水体原位生物投菌技术脱氮特性与效能研究。小试研究以贫营养脱氮功能菌群Z4作为菌源制成微生态菌剂,采用不同菌剂投量直接投加到水库原水中,菌投量为0.1mg/L时净化效果最好,整个运行期间(32d),硝氮、总氮、CODMn及TOC去除率分别达到72.3%、71.3%、32.3%和34.8%,稳定期的脱氮效果可满足地表水环境Ⅲ类水体的质量标准要求;同时溶解氧、水温、pH及C/N会影响生物菌剂的脱氮性能。将低温贫营养脱氮功能菌群L1用于微污染水原位生物投菌技术中,在低温环境条件下运行36d,该菌剂对原水硝氮及总氮去除率最大可达到46%和53%。
(4)微污染水体原位生物膜技术脱氮特性与效能研究。选择脱氮性能较稳定的硬性悬浮填料,同时研制出适合于微污染水体原位修复、且可创造适宜微生物生长的不同溶解氧环境的新型多孔悬浮填料作为生物载体,以贫营养脱氮功能菌群Z4作为菌源对载体进行人工强化挂膜,小试研究结果表明,试验运行46d,贫营养原位生物膜技术对原水硝氮及总氮去除率均可达到75%以上,CODMn去除率在25%以上,稳定时期脱氮效果可满足地表水环境Ⅲ类水体的质量标准要求,且温度、填料填充率及C/N比对原位生物膜系统脱氮效果有一定影响。贫营养细菌以较低速率进行生长时,对外界环境具有较大的抗性,是微生物适应贫营养环境的结果,在贫营养条件下,吸附和生物膜形成是微生物的一种生存策略。
(5)原位生物投菌系统对混合充氧技术的条件要求。DO浓度对贫营养好氧反硝化菌群的脱氮性能有一定影响。当DO浓度大于7mg/L时,反硝化速率很慢,始终保持在较低的水平;当DO浓度低于7mg/L时,反硝化速率随DO值的降低而较大幅度地升高,说明贫营养好氧反硝化菌群的阈值较高,对氧气有较高的耐受能力。DO浓度为3~4mg/L、5~6mg/L和7~8mg/L时,系统运行期间对原水总氮去除率分别为91%、84%和31%。好氧反硝化细菌的反硝化酶系和有氧呼吸系统同时存在,氧不再是抑制反硝化酶活性和反硝化酶生成的直接因素,周质硝酸盐还原酶亚基基因的扩增进一步解释了好氧反硝化菌可以在DO浓度相对较高的条件下进行反硝化作用。
(6)扬水曝气—原位生物净化组合技术中试研究。在扬水曝气强化混合条件下,进行原位生物净化系统中试研究。以贫营养脱氮功能菌群Z4为菌源,分别构成扬水曝气—贫营养生物膜和扬水曝气—贫营养生物投菌组合技术,在溶解氧浓度为5.0~7.0mg/L,水温范围为10℃~ 23℃、CODMn/TN≈1.56的贫营养条件下,扬水曝气—贫营养生物膜组合系统稳定运行时的氨氮、硝氮、总氮和TOC去除率范围分别为82%~100%、62%~79%、70%~80%和72%~80%,BOD5/CODMn比值可由初始0.476降至0.054,且三维荧光光谱图显示运行期间原水生源性DOM变化较大;扬水曝气—贫营养生物投菌组合系统稳定运行时的氨氮、硝氮、总氮、TOC和CODMn去除率分别为82%~100%、43%~58%、63%~70%、65%~73%和51%~57%。PCR-DGGE图谱分析表明,生物膜上种群演替进程缓慢,α-proteobacterium纲是生物膜中最优势的细菌类群,固定的菌株PY8和FY3成为生物膜上的优势菌群;生物投菌系统中FY3和FY7成为系统中的优势菌群。消毒剂灭活试验、群落结构多样性及小鼠急性经口毒性试验表明,生物菌剂不会对原生态系统产生较大的破坏,且不会对饮用水水质构成威胁。采用低温贫营养脱氮功能菌群L1与扬水曝气技术有机结合,在初期水温不足15℃,CODMn/TN≈1.0的低温、贫营养条件下,稳定运行阶段的硝氮及总氮去除率分别在50%和60%以上,最大硝氮及总氮去除率分别为81%和84%,TOC、CODMn及BOD5月平均去除率分别为63%、49%和62%。上述结果表明,该组合技术用于净化微污染原水是可行的,稳定运行期的脱氮效果均可满足地表水环境Ⅲ类水体的质量标准要求。
Nitrate often contaminated surface water resources due to excessive use of fertilizers and uncontrolled on-land discharges of raw and treated wastewater and can therefore limit the direct use of surface water for drinking water purposes. Many researches on the denitrification of source water had been reported in the last three decades with the increasing nitrogen pollution concerns; biological treatment was the most attractive process to remove nitrogen from surface water sources due to its low cost and high efficient. However, there are still some problems on denitrification of drinking water resources. Firstly, biological nitrogen removal of ectopic widely used in surface water sources has a restriction of biological contact time——an important limiting factor for influencing the removal efficiency of total nitrogen. Secondly, electron donor used to improve the denitrification efficiency can increase water treatment costs and some substrate or its intermediate products of biological oxidation may produce toxic effects on life.
In this research, a multifunctional device for water lifting and aeration (WLA) was combined with in-situ biological purification technology (ISBP) for the denitrification of surface water. The main objective of this research was to investigate the key issues of nitrate removal of micro-polluted raw water with the combined system under low temperature, oligotrophic and aerobic condition. Oligotrophic denitrifiers and cold-resistant denitrifiers were cultured for microbial agents, which was the important source of bacteria for in-situ biological inoculation or in-situ bio-contact oxidation system. Both ISBPs were combined with WLA in order to obtain a satisfactory performance on nitrogen and organic matter removal. Properties and experiments of enhanced in-situ biological nitrogen removal of micro-polluted raw water were discussed in this paper. The main results and conclusions of this study are as follows:
(1) Domestication, isolation and denitrification properties of oligotrophic aerobic denitrifiers. Oligotrophic denitrifiers strain PY8, DA15 and DF7 was isolated after the acclimatization with low-nutrient medium in order to remove nitrogen desirably under oligotrophic conditions. According to their physiological, biochemical methods and the sequence analysis of 16S rDNA, strain PY8, DA15, DF7 was identified as Rhizobium sp., Arthrobacter sp. and Bacillus sp., respectively. On the base of the isolated strain FY3, FY5 and FY7, oligotrophic denitrifying functional bacteria populations Z4 (PY8, FY3 and FY7) was formulated by niche separation, and all the functional bacteria were studied for nitrogen removal performance. The results showed that all the strains grew slowly, and pH value, temperature, C/N ratio, NaNO3 concentration and inoculum had different effects on denitrification performance of strain PY8, FY3 and FY7. Also, the strain PY8 had a significantly heterotrophic nitrification performance. It was confirmed that the strain PY8, FY3 and FY7 had the capacity of express periplasm nitrate reductase through the PCR reaction, and periplasmic nitrate reductase played an important role in aerobic gentrification.
(2) Domestication, isolation and denitrification properties of low-temperature- resistant aerobic denitrifiers. Low-temperature-resistant denitrifying bacteria were isolated by gradually reducing temperature condition, and the strain DW3 was identified as Pseudomonas sp., the strains of DW4, D3 and D4 as Acinetobacter sp., based on biochemical index analysis and 16S rDNA sequence homology analysis. Growth curve and nitrogen removal capacity was investigated for the four strains. The results indicated that in addition to D3 strain, other strains were present a decline phase, and the strains of the denitrification took place in the logarithmic phase. Also, pH value, temperature, C/N ratio and inoculum could influence denitrification performance of low-temperature- resistant denitrifiers. A desirable ammonia removal rate was obtained for the four strains in nitrification medium. D3 and D4 still maintained a certain dehydrogenase activity at 10℃. The results also proved that DW3 and D3 strains had periplasmic nitrate reductase and could realize aerobic denitrification. Low-temperature-resistant and oligotrophic denitrifying functional bacteria groups L1 (PY8, DW3 and D3) and L2 (PY8, DW3 and D4) could were constructed by self-adjustment and eco-recombination for the purpose of denitrification under oligotrophic and low temperature conditions.
(3) Properties and efficiency of in-situ biological inoculation nitrogen removal of micro-polluted raw water. Oligotrophic denitrifying functional bacteria populations Z4 as bacteria source was used for in situ biological inoculation technology in small scale tests. The showed that a preferable purification could be presented under 0.1 mg/L inoculating dose, and the removal rate of nitrate, total nitrogen, CODMn and TOC during the entire operation (32 days) could reach 72.3%, 71.3%, 32.3% and 34.8%, respectively. Also, nitrogen removal effects in steady running period could meet the requirements of classⅢof surface water quality according to GB3838-2002. DO, temperature, pH and C/N ratio was important influencing factors for denitrification performance of oligotrophic dominant agents. The cold-resistant and oligotrophic denitrifying functional groups L1 showed a high efficiency for nitrogen removal in low temperature, and the optimum removal rates of nitrate and total nitrogen reached 46% and 53% during 36 days operation.
(4) Properties and efficiency of in-situ biological biofilm nitrogen removal of micro-polluted raw water. A rigid suspended packing and a novel suspended packing chosen as biological carriers was applied to in-situ biofilm system. The novel suspended packing was developed for in-situ remediation and could create an appropriate environment for the growth of aerobic, anaerobic and facultative bacteria. Artificially hanged membrane was formed on both carriers with oligotrophic denitrifiers functional groups Z4. The small test results showed that the removal rates of nitrate, total nitrogen and CODMn could reach over 75%, 75% and 25%, respectively, and nitrogen removal effects in steady running period could meet the requirements of classⅢof surface water quality according to GB3838-2002. Temperature, filling ratio of suspended packing and C/N had a certain influence on nitrogen removal efficiency of in-situ biological biofilm system. Oligotrophic bacteria grown at a lower rate and had a greater resistance to external environment, and these properties could provide a better adaptability to poor nutrition environment for them. The adsorption and microbial biofilm formation was a survival strategy in oligotrophic environment.
(5) Requirements of mixed aeration technology for in-situ biological inoculation system. Dissolved oxygen concentration could affect denitrification performance of oligotrophic denitrifying functional groups. When the DO concentration greater than 7 mg/L, the denitrification rate was very slow and always maintained a low level. The nitrogen removal rate would increase significantly with the decrease of DO value under condition of DO concentration less than 7 mg/L. These results indicated that aerobic denitrifiers with a higher threshold had a desirable oxygen tolerance. The removal rates of total nitrogen were 91%, 84% and 31% respectively under conditions of DO concentration 3~4 mg/L, 5~6 mg/L and 7~8 mg/L. Both denitrification enzyme and aerobic respiratory system could exist in aerobic denitrifiers, and oxygen was no longer inhibited denitrification enzyme activity and denitrification enzyme generation directly. Periplasmic nitrate reductase subunit gene amplification further explained that aerobic denitrification could occur under high DO concentration condition.
(6) Pilot research on combined process of WLA and ISBP. Under the condition of enhanced mixing of water-lifting aeration, a pilot research on ISBP was made for the purpose of pretreatment of micro-polluted raw water. Oligotrophic denitrifying functional groups Z4 as bacterial source constituted combined process of WLA and in-situ biofilm and combined process of WLA and in-situ bio-inoculation. For the combined technology of WLA and in oligotrophic biofilm system, the results showed that removal rates of ammonia, nitrate, total nitrogen and TOC in steady running period ranged from 82%~100%, 62%~79%, 70%~80% and 72%~80%, respectively, under conditions of dissolved oxygen 5~7 mg/L, temperature 10℃~23℃and C/N ratio 1.56 for source water, also the ratio of BOD5 to CODMn was changed from 0.476 to 0.054. Three-dimensional excitation-emission matrix (3DEEM) fluorescence spectroscopy showed that the biogenic dissolved organic matter of raw water had experienced an obvious change during the operation period. As for the combined technology of WLA and oligotrophic bio-inoculation system, removal rates of ammonia, nitrate, total nitrogen, TOC and CODMn in steady running period ranged from 82%~100%, 43%~58%, 63%~70%, 65%~73% and 51%~57% respectively under the same conditions. The PCR-DGGE profiles showed that the microbial populations on the biofilm evolved slowly andα-proteobacterium was the largest fraction. In addition, sequences revealed that strains PY8 and FY3 became the dominant bacteria in the combined system WLA and oligotrophic biofilm, and FY3 and FY7 strain became the dominant bacteria in the combined system WLA and oligotrophic bio-inoculation system. Disinfection tests, biological community structure diversity and acute oral toxicity tests in mice showed that biological agents would not pose a threat to original ecosystems and drinking water. Under conditions of initial temperature of less than 15℃and CODMn/TN of 1.0, cold-resistant and oligotrophic denitrifying functional groups L1 combined with WLA could obtain a desirable denitrification effects during the stable period for more than 50% of nitrate removal rate and 60% of total nitrogen removal rate, and the optimum removal rate for nitrate and total nitrogen was 81% and 84%. Also, the monthly average removal efficiency of TOC、CODMn and BOD5 could reach 63%, 49% and 62%. The experiment results showed that the combined technique of WLA and ISBP was feasible for the denitrification of raw water, and nitrogen removal effects in steady running period could meet the requirements of classⅢof surface water quality according to GB3838-2002.
引文
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