不同碳氮比值下亚硝酸盐氧化菌和异养菌混合体系的微生物多样性

应用与环境生物学报 2009，15 ( 3 ): 351~355Chin J Appl Environ Biol=ISSN 1006-687X2009-06-25DOI: 10.3724/SP.J.1145.2009.00351Biological nitri f i c ation process has been widely applied to remove nitrogen pollutants (ammonia and nitrite) in municipal wastewater treatment plants (WWTPs). The process of nitrification, including ammonia-oxidizing process and nitrite-oxidizing process, was performed using two phylogenetically unrelated groups of obligately chemolithotrophic bacteria. Ammonia-oxidizing bacteria (AOB) first oxidize ammonia to nitrite, and subsequently nitrite-oxidizing bacteria (NOB) oxidizenitrite to nitrate. Various parameters influence the nitrification process. Major factors of the process include dissolved oxygen (DO), temperature, pH, ammonia and nitrite concentrations, organic loading, and hydraulic loading rate [1]. The effect of soluble organic matter on nitrification was extensively investigated by many researchers [2~4]. Their results indicated that high C/N ratios induced interspecific competition for oxygen between nitrifying bacteria and heterotrophic bacteria, which resulted in nitri f i cation retardance.However, so far the effects of organic carbon on nitri f i cation were studied with respect either to reactor performance or不同C/N值下亚硝酸盐氧化菌和异养菌混合体系的微生物多样性*胡 杰 李大平** 陶 勇 张金莲 钟 琦 何晓红 王晓梅(中国科学院成都生物所应用与环境微生物中心 成都 610041)Microbial Diversity of Nitrite-oxidizing and Heterotrophic Bacterial Communities under Different C/N Ratios *HU Jie, LI Daping **, TAO Yong, ZHANG Jinlian, ZHONG Qi, HE Xiaohong & WANG Xiaomei(Center for Applied and Environmental Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences , Chengdu 610041, China)Abstract The microbial diversity of nitrite-oxidizing and heterotrophic bacterial communities at three C/N ratios of feed solutions was experimentally investigated using PCR- restriction fragment length polymorphism (RFLP) technique and correlated with nitrification performances of the mixture. At C/N = 0, the dominant populations were mainly autotrophic and oligotrophic bacteria (85.1%), including nitrite-oxidizing bacteria (NOB), and some strains of Bacteroidetes, Alphaproteobacteria, Actinobacteria, and green nonsulfur bacteria. At C/N = 0.44, autotrophic bacteria reduced, while heterotrophic bacteria (members of Gammaproteobacteria) increased. At C/N =8.82, members of Gammaproteobacteria dominated (93.8%), especially denitrifiers, i.e., Pseudomonas sp. And on the other side, a striking shift of nitrification performance occurred from a normal nitrite oxidation process to a simultaneous nitri fi c ation and denitri f i c ation process along with increasing C/N. The nitri fi c ation performances at the three C/N ratios could be well explained by the obtained microbial diversities. This study demonstrated the linkage between the functional performance (nitrification performance) and the microbial diversity, suggesting that PCR- RFLP technique together with functional parameter analysis has potential as a tool for relating functional variety to bacterial community shifts. Fig 3, Tab 2, Ref 13Keywords microbial diversity; PCR-restriction fragment length polymorphism (RFLP); nitrite-oxidizing bacteria (NOB);C/N ratioCLC Q938.15摘 要 采用PCR-RFLP 技术研究了不同C/N 比下亚硝酸盐氧化菌及异养菌混合体系的微生物多样性，并探讨了微生物菌群结构与其功能(硝化性能)的关系. C/N=0时，混合体系主要由自养菌和寡营养菌(85.1%)组成，包括亚硝酸盐氧化菌(NOB)、拟杆菌门、α-变形菌纲、浮霉菌门和绿色非硫细菌中的一些菌株. C/N=0.44时，混合体系中的自养菌减少，异养菌(主要是γ-变形菌纲的成员)大量出现. C/N=8.82时，γ-变形菌纲的菌株尤其是反硝化菌Pseudomonas sp.占主导(93.8%). 与此同时，随着C/N 升高，该混合体系的硝化性能也由专一的亚硝酸盐氧化过程转变为同时硝化反硝化过程. 微生物菌群结构的转变较好地解释了其硝化性能的改变. 本研究揭示了微生物菌群结构与其功能的内在联系，同时表明PCR-RFLP 技术与化学分析相结合是研究微生物菌群结构与功能的有力工具. 图3 表2 参13关键词 微生物多样性；PCR-限制性片段长度多态性(RFLP)；亚硝酸盐氧化菌(NOB)；C/N 比CLC Q938.15Received: 2008-03-27 Accepted: 2009-05-06∗Supported by the National High-tech Research and Development Program (“863” Program) of China (Nos. 20062AA05Z103,2007AA06Z324)**Corresponding author (E-mail: lidp@)35215 卷应 用 与 环 境 生 物 学 报 Chin J Appl Environ Biol to microbial spatial distribution, but hardly to the microbial community shifts. Understanding of the biodiversity and dominant species of the microbial community is greatly important in studying contaminant degradation pathways, optimizing treatment processes and improving removal efficiencies. The use of molecular techniques allows a more complete understanding of the diversity and distribution of bacteria than simply using cultivation-based methods. PCR- restriction fragment length polymorphism (RFLP) provides information about sequence variation in a mixture of PCR fragments of almost full-length 16S rDNA, and it can be used conveniently to infer the differences in microbial community compositions [5, 6]. PCR-RFLP allowed the detection of specific groups of bacteria and permitted their presence to be correlated with key environmental factors. Hence, in this study we chose PCR-RFLP technique to investigate microbial population shifts when supplied with organic carbon.This study focused on the linkage between nitrification performance and the microbial diversity. Furthermore, unlike other studies focusing on the ammonia-oxidizing process, and the community shifts of AOB and heterotrophic bacteria, we examined the nitrite-oxidizing process and the community shifts of NOB and heterotrophic bacteria.1 Material & methods1.1 Bacteria preparation and experimental designThe nitrite-oxidizing and heterotrophic bacteria were separated and enriched from Sanwayao Wastewater Treatment Plant (WWTP) in Sichuan Province (China) and were further studied under laboratory conditions. Three batch reactors (with 100 mL working volume) were used to culture the mixture under agitation (150 r/min) at 30 °C. The nutrient medium contained K 2HPO 4·H 2O 2 g/L, KH 2PO 4 1.5 g/L, MgSO 4·7H 2O 0.1 g/L, nitrogen source (NaNO 2) 1g/L and sodium acetate 0 g/L, 0.5 g/L and 10g/L (for C/N ratios of 0, 0.44, and 8.82, respectively). The changes in effluent water quality, that is, (1) nitrite-N, (2) nitrate-N, (3) total nitrogen and (4) chemical oxygen demand (COD), were measured after three days. When the nitrite was absolutely removed, the bacterial community was centrifuged and analyzed for microbial diversity. The experiment was repeated twice.1.2 PCRThe genomic DNA was extracted by using TIANamp bacterial genomic DNA extraction kit (TIANGEN, China). PCRs targeting total bacteria were performed with primers (27F [5’-AGAGTTTGATCCTGGCTCAG-3’] and 1492R [5’- GGTTACCTTGTTACGACTT-3’]) [7]. PCRs were carried out in a Peltier thermal cycler with 50 μL reaction volume (Bio-Rad, Hercules, CA, USA). The reaction mixture contained 10 ng of template DNA, 0.2 μmol/L of each primer, 1×PCR buffer, 200 μmol/L of each deoxynucleoside triphosphate, 2 mmol/L MgCl 2 and 1 U of Taq DNA polymerase (TaKaRa). The PCR conditionswere: at 95 °C for 3 min, 33 cycles at 95 °C for 1 min, at 51°C for 1 min and at 72 °C for 90 s, and final extension at 72 °C for 3 min. Amplification products were firstly examined by standard agarose gel electrophoresis (1.2% agarose, 0.5×TAE) with ethidium bromide staining to confirm the product size and then purified with TIANgel midi purification kit (TIANGEN, China).1.3 Restriction fragment length polymorphism(RFLP)The purified PCR products were ligated into the TaKaRa pMD18-T vector in Escherichia coli JM109 transformants. A total of 140 clones were randomly selected and were analyzed. The cloned inserts of 1 500 bp were grouped by RFLP to form operational taxonomic units (OTUs) using the type II restriction enzymes Hha I [GCG’C] and Msp I [C’CGG] simultaneously. After restriction digestion at 37 °C for 4 h, the banding patterns were compared by electrophoresis, and the clones showing the same RFLP pattern were grouped into a single OTU. The cloned inserts were sequenced in the Bioasia Company Shanghai Ltd. The OTUs with chimeric 16S rRNA gene sequences were removed by CHECK_CHIMERA program of Ribosomal Database Project II.1.4 Phylogenetic analysisAll sequences were submitted to GenBank database (Accession numbers: EU305563- EU305581 and EU305596- EU305601) and were searched for homology using the Ribosomal Database Project (RDP-II, ). The sequences were aligned with Clustal X, and phylogenetic tree was constructed with MEGA software (version 4.0) [8].1.6 Diversity indicesThe Shannon index (H’) [9] was calculated applying theformula H’ =, where P i is the proportion of theindividuals of OTUs pattern, and S relative to the total number of OTUs patterns. The Shannon evenness index (E ) [9] was calculated using the formula E = H’/ln S , where S is the total number of OTUs patterns. Coverage was calculated using the equation C = 1－n /N , where n is the number of clones that occurred only once and N is the total number of the examined clones.2 Results2.1 Nitrification performanceThe initial conditions used in the reactors and nitrification performance at different C/N ratios are listed in Table 1. The nitrite-N could be absolutely removed under all the conditions. The nitrate-N concentration was decreased as the C/N ratios increased. Total nitrogen (TN) removal rates were 0% and 70% at C/N = 0.44 and 8.82, respectively. The COD removal was around 87% with supplement of organic matter.2.2 Microbial diversityAfter excision of 16 non-target sequences and 6 chimeric sequences, a total of 25 different phylotypes were obtained. Three3533 期HU Jie, et al .：Microbial Diversity of Nitrite-oxidizing and Heterotrophic Bacterial Community under Different C/N Ratiosfull-length 16S rDNA clone libraries (reflecting the total bacterial community structure under three C/N ratios) were generated by PCR-RFLP (Fig. 1). The coverage was about 87.2%~93.8%, indicating that a majority of microbial community was covered in the three full-length 16S rDNA clone libraries. Diversity analysisof the three 16S rDNA clone libraries is shown in Table 2. The Shannon diversity indices (H ) were calculated to be 1.76, 1.25 and 1.08, and the Shannon evenness indices (E ) were found to be 0.71, 0.64 and 0.60. It was demonstrated that the three clone libraries had low diversity and evenness of the distribution of OTUs.The sequences obtained from the study were subjected to phylogenetic analysis. The sequenced clones were affiliated withTable 1 Initial conditions used in the reactor and nitrification performance at different C/N ratiosDifferent C/N ratios00.448.82Initial conditionsNitrite-N (ρ/mg L -1)100±3100±3100±3Nitrate-N (ρ/mg L -1)000Nitrification performance Nitrite-N (ρ/mg L -1)000Nitrate-N (ρ/mg L -1)101±490±30TN removal (r /%)02±0.570.0±1.8COD removal (r /%)88.5±3.086.4±1.5Fig. 1 Patterns after restriction digestion of the cloned inserts in different16S rDNA clone librariesa. C/N = 0;b. C/N = 0.44;c. C/N = 8.82Table 2 Diversity analysis of the three 16S rDNA clonelibraries at different C/N ratiosDifferent C/N ratios 00.448.82No. of clones 473932No. of OTUs 1276Coverage (r /%)87.292.393.8H 1.76 1.25 1.08E0.710.640.60Fig. 2 Patterns and their proportions of the three 16S rDNA clone libraries atdifferent C/N ratiosa. C/N = 0,b. C/N = 0.44,c. C/N = 8.8235415 卷应 用 与 环 境 生 物 学 报 Chin J Appl Environ Biolsequences from five major lineages of the bacteria, namely the Bacteroidetes, Alpha-, Beta- and Gamma-subdivisions of the Proteobacteria and Actinobacteria (Fig. 2). Phylogenetic tree was derived from full-length 16S rDNA by the neighbor-joining algorithm (Fig. 3).For C/N = 0, the sequences were obtained mainly from Bacteroidetes, Alpha- and Gamma-subdivisions of Proteobacteria and Actinobacteria. Phylogenetic analysis showed that 24 clones (53.2% of total clones) were members of Bacteroidetes and were mainly grouped with unclassified Sphingobacteriales. Eight clones (17.0%) were found in Alphaproteobacteria and those of Nitrobacter accounted for 37.5% of the total of Alphaproteobacteria. Seven clones were found within Gammaproteobacteria and those of Pseudomonas accounted for 28.6% of the total of Gammaproteobacteria. Six clones (12.8%) belonged to green nonsulfur bacteria (GNS), and the rest one to Actinobacteria.Fo r C /N =0.44, t h e m e m b e r s of B a c t e r oid e t e s , Alphaproteobacteria, Actinobacteria and GNS decreased, whilethese of Gammaproteobacteria increased to 87.2% and were found mainly belonging to Pseudomonas sp. (64.1%). The OTU 2-A and OTU 2-B were 99.9% and 95.2% similar to Pseudomonas stutzeri (88.0% of Pseudomonas sp.) and Pseudomonas nitroreducens (12.0% of Pseudomonas sp.), respectively. One clone was classified to Betaproteobacteria and its similarity to AB186827 was 99.7%.For C/N=8.82, the members of Gammaproteobacteria increased to 93.8%, and the rests belonged to Alphaproteobacteria (6.3%). The OTU 3-A and OTU 3-D were 99.9% and 99.6% similar to Pseudomonas stutzeri (80.0% of Pseudomonas sp.) and Pseudomonas nitroreducens (20.0% of Pseudomonas sp.), respectively. The main strains of Alphaproteobacteria were similar to Mesorhizobium sp. S1-8 and their similarity was 99.4%.3 DiscussionThe study on microbial diversity using PCR-RFLP technique and nitrification performance provides a moreFig. 3 Neighbor-joining phylogenetic tree based on the three 16S rDNA clone libraries at different C/N ratiosFrom 1-A to 1-L, C/N = 0; from 2-A to 2-G, C/N = 0.44; from 3-A to 3-F, C/N = 8.82. GenBank accession numbers were indicated in the following brackets respectively. Bootstrap values (1 000 replicates) are shown at the nodes. GNS: Green nonsulfur bacteria355 3 期HU Jie, et al.：Microbial Diversity of Nitrite-oxidizing and Heterotrophic Bacterial Community under Different C/N Ratioscomplete understanding of the functional parameters and community diversity shift of nitrite-oxidizing and heterotrophic bacterial communities with treatment of organic matter. At C/N =0, the culture was under autotrophic condition, nitrite-N was converted to equal quantity of nitrate-N, and the dominant populations were found belonging to the strains of Bacteroidetes, Alphaproteobacteria, Actinobacteria and green nonsulfur bacteria (mainly autotrophic and oligotrophic bacteria). At C/N = 0.44, the culture was under mixotrophic condition, the increase in nitrate-N was not equal to the decrease in nitrite-N, indicating a partial loss of nitrogen, and the strains dominating under the autotrophic condition decreased, but the members of Gammaproteobacteria (mainly Pseudomonas sp.) increased. At C/N = 8.82, the culture was also under mixotrophic condition, the concentration of nitrate-N, as well as that of nitrite-N, was zero, suggesting a obvious loss of nitrogen, and the dominated bacteria were Pseudomonas stutzeri and Pseudomonas nitroreducens. In the course of C/N ratios increasing, TN removal increased from 0% to 70%, and the dominant populations shifted from autotrophic bacteria to mainly Pseudomonas sp. The denitrification ability of the strains of Pseudomonas stutzeri, P. pseudoalcaligenes and P. nitroreducens had been proved by many articles [10~12]. TN removal is obviously performed by denitrifying bacteria; and it increased along with the increasing populations of denitrifying bacteria. On the other hand, the denitrifiers could also consume nitrite (besides nitrate) by denitrification [10, 13], and it was so presumed that nitrite was mainly removed by Pseudomonas sp. (not the decreasing NOB) at high C/N ratios.This study showed that the mixture treated with different C/N ratios had clearly distinctive communities, which had a linkage with nitrification performances. The shift of nitrification performance could be sufficiently explained by the obtained microbial diversities, suggesting that PCR- RFLP technique together with functional parameter analysis has potential as a tool for relating functional variety to bacterial community shifts. This study, therefore, demonstrates the usefulness of monitoring the performance of the nitrification process through a combined analysis of both nitrification parameters and microbial diversities.References1 Sharma B, Ahlert RC. Nitrification and nitrogen removal. Water Res,1977, 11: 897~9252 Ohashi A, de Silva DG, Mobarry B, Manem JA, Stahl DA, Rittmann BE.Influence of substrate C/N ratio on the structure of multi-species biofilms consisting of nitrifiers and heterotrophs. Wat Sci Tech, 1995, 32: 75~843 Okabe S, Hiratia K, Ozawa Y, Watanabe Y. Spatial microbialdistributions of nitrifiers and heterotrophs in mixed-population biofilms.Biotechnol Bioeng, 1996, 50: 24~354 Michaud L, Blancheton JP, Brunl V, Piedrahita R. Effect of particulateorganic carbon on heterotrophic bacterial populations and nitrification efficiency in biological filters. Aquacult Eng, 2006, 34: 224~2335 Urakawa H, Kita-Tsukamoto K, Ohwada K. 16S rDNA genotyping usingPCR/RFLP (restriction fragment length polymorphism) analysis among the family Vibrionaceae. FEMS Microbiol Lett, 1997, 152: 125~1326 Ramirez-Moreno S, Martinez-Alonso MR, Mendez-Alvarez S, Esteve I,Gaju N. Seasonal population changes in the restriction fragment length polymorphism (RFLP) patterns from PCR-amplified 16S rRNA genes of predominant ribotypes in microbial mat samples from the Ebro Delta (Spain). Curr Microbiol, 2003, 46: 190~1987 Miyoshi T, Iwatsuki T, Naganuma T. Phylogenetic characterization of16S rRNA gene clones from deep-groundwater microorganisms that pass through 0.2-micrometer-pore-size filters. Appl Environ Microbiol, 2005, 71: 1084~10888 Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular EvolutionaryGenetics Analysis (MEGA) software version 4.0. Mol Biol & Evol, 2007, 24: 1596~15999 Hill TCJ, Walsh KA, Harris JA, Moffett BF. Using ecological diversitymeasures with bacterial communities. FEMS Microbiol Ecol, 2003, 43: 1~1110 Blaszczyk M. Effect of various sources of organic carbon and highnitrite and nitrate concentrations on the selection of denitrifying bacteria. II. Continuous cultures in packed bed reactors. Acta Microbiol Pol, 1983, 32: 65~7111 Ping L, De-Li L, Nahimana L, Chen SL, Yang X, Zhao L. High nitrogenremoval from wastewater with several new aerobic bacteria isolated from diverse ecosystems. J Environ Sci (China), 2006, 18: 525~52912 Song L, Wang H, Jiang J, Gao JS, Shi HC. Isolation, idetification of1,2,4-trichlorobenzene-degrading strain Pseudomonas nitroreducens J5-1 and cloning of chlorocatechol 1,2-dioxygenase gene. Environ Sci, 2007, 28: 1878~188113 Braker G, Zhou J, Wu L, Devol AH, Tiedje JM. Nitrite reductasegenes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities.Appl Environ Microbiol, 2000, 66: 2096~2104。