通過DGGE監(jiān)測活性污泥中的硝化細(xì)菌群落

1、 378 ZIEMBISKA, CIESIELSKI, and MIKSCH Vol. 55 cation of a specic DNA region even in bacteria appearing in small amounts (Boon et al., 2001; Dar et al., 2005; Ward et al., 1997. In this case CTO primers specic for the AOB 16S rRNA gene and 338F and 518R primers for 16S rRNA genes of all the bacteria

2、 were used. It had been established previously that Nitrosomonas was one of the dominant ammonia oxidizing bacteria in the bioreactor. It was also suspected that this bacterium was responsible for efcient nitrication. Interestingly, the sequencing revealed bacteria that previously uncultured were pr

3、esent in abundance in the WWTP activated sludge samples. These clones (clones number 3.13, 4.32, and 5.33, bands c, d and e, respectively, Fig. 2A, identied as the most similar to N. oligotropha, are clustered together in the 16S rRNA gene fragment tree (Fig. 4, which underlines their sequence simil

4、arity. Only clone 1.19 (band a, Fig. 2A, was identied as the closest to N. europea and it was located in the separated cluster of the dendrogram (Fig. 4. This observation proved previous reports that such a group of bacteria might be responsible for effective nitrication (Juretschko et al., 1998; Wa

5、gner et al., 2002. It should be mentioned that the cluster of clones 5.5.15 and 6.31 (band b and f respectively, Fig. 2A was also related to the Nitrosomonas oligotropha cluster, but the differences in the sequences were high enough to locate them in a separate subgroup in the dendrogram. Interestin

6、gly both the Nitrosomonas cluster and the uncultured bacteria cluster were closely related to N. oligotropha, while clone 1.19 (band a, Fig. 2A, also identied as Nitrosomonas, was located closer to Nitrosomonas europea in the dendrogram. This topology underlined the diversity of the Nitrosomonas clo

7、nes in the bioreactor biocenosis. The bacterium identied as the closest to Ferribacterium sp. was present as dominant in all samples (band g, Fig. 2A. This wastewater treatment plant bioreactor is an open system, so it is highly possible that allochtonic bacteria easily drift into the system. Identi

8、cation of Ferribacterium-like bacteria in the WWTP system was probably caused by an ambiguous CTO primer sequence, leading to an amplication of the 16S rRNA gene of bacteria possessing a similar sequence, which belong to the subclass of -Proteobacteria, but are not necessarily ammonia-oxidizers. Suc

9、h a situation can conrm that there are no known primers that amplify only the AOB 16S rRNA gene (Purkhold et al., 2000. The appearance of a Ferribacterium-like clone in acti- vated sludge was conrmed recently by Wittebolle et al. (2008. Nonetheless there has been no research conrming or excluding th

10、e nitrication abilities of these bacteria. A study of the Ferribacterium limneticum strain CdA-1 carried out by Cummings et al. (1999 proved the ability of Fe (III reduction linked with acetate oxidation. We suspect that ammonia can be an electron donor in Fe (III reduction due to the constant prese

11、nce of these bacteria in the WWTP bioreactor. Ferribacterium sp. is an obligate anaerobe, but this fact does not exclude the possibility of ammonia oxidation, because Nitrosomonas sp. is also known to be able to lead the nitrication process in anaerobic conditions (Abeliovich and Vonshak, 1992. Its

12、high volume and the possibility of a niche existence of anaerobic bacteria probably caused anaerobic bacteria identication in an aerated bioreactor. Ammonia oxidation under anaerobic conditions could also suggest the possibility of an Anammox process (anaerobic ammonia oxidation. In this experiment

13、no Anammox research was carried out; therefore it would be difcult to state clearly if we are dealing with Anammox in this system. A phylogenetic analysis of Ferribacterium sp. and Anammox bacteria belonging to Planctomycetes highlighted a large discrepancy between their 16S rRNA gene sequences. All

14、 AmoA gene sequences obtained in the study created one cluster closely related to Nitrosomonas oligotropha (Fig. 5. This group of sequences appeared during the entire length of the experiment and this could suggest that phylogeny and identication based on the AmoA gene sequence could lead to an unde

15、restimation of the AOB taking part in nitrication. This limitation might be avoided in the future by using primers that amplify a longer gene fragment (Norton et al., 2002; Purkhold et al., 2003. AOB biodiversity Shannon diversity index Biological diversity can be mathematically expressed as the Sha

16、nnon diversity index (Shannon and Weaver, 1963. The higher the index value is, the higher the diversity is (Boon et al., 2001. In bacterial biocenosis the species biodiversity estimations are controversial due to problems with bacterial species denitions in microbial ecology (Mes, 2008. The Shannon

17、diversity index of the activated sludge samples in the experiment were at levels of 1.9 2.2 and this seems to be relatively constant (Fig. 3. This could be explained by the stabile experiment condi- 2009 AOB community monitoring using DGGE Acknowledgments 379 tions, such as bioreactor aeration, pH a

18、nd ammonia nitrogen concentration in the inuent. Rapid species changeability was not observed, which might suggest that the gradual and slow ammonia nitrogen concentration did not have a signicant selective inuence on the AOB community in this system. This can conrm the statement that only a high am

19、monia nitrogen concentration in the bioreactor inuent can have a high potential in AOB community modelling (Koops et al., 2003; Webster et al., 2005. Temperature changes also did not have a signicant inuence on AOB biodiversity, nor on the effectiveness of ammonia oxidation (Fig. 1. It was stated pr

20、eviously that temperature has an inuence on the diversity of ammonia oxidizers only when the changes are changeless for longer than 4 weeks (Avrahami and Conrad, 2003; Avrahami et al., 2002, 2003. Additionally, the high bioreactor volume protected the activated sludge from rapid temperature changes.

21、 Conclusions This research was supported by the Polish Ministry of Science and Higher Education, grant no: 2 P04G 086 30. References Abeliovich, A. and Vonshak, A. (1992 Anaerobic metabolism of Nitrosomonas europea. Arch. Microbiol., 158, 267 270. Amann, R. I., Ludwig, W., and Schleifer, K. H. (1995

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