臺灣博碩士論文加值系統

近年來，隨著高技術產業發展，都市廢水越來越複雜。去除含氮污染物的方法有很多，其中厭氧氨氧化（Anammox）在不添加有機碳源的情況下，不僅減少一氧化二氮氣體逸出而且節能和省成本，但廢水的特性不同，容易造成Anammox優勢族群變遷，影響除氮能力，藉由改變菌種繁殖環境或尋找新品種可以解決此問題。
台灣位於適合紅樹林生長的亞熱帶，綜合紅樹林的缺氧環境與出海口之具有氨氮物質環境，再加上新豐紅樹林的上游是新竹工業區，推估紅樹林具有高機會取得Anammox之樣本，目前已有許多文獻證實在紅樹林中發現anammox菌。
本研究針對固定採樣點進行每月的水質之PH值、溫度、氧化還原電位等環境因子偵測，再利用分子生物技術對紅樹林沉積物中氮循環相關細菌做定量分析，其中分析的細菌包含ammonia-oxidizing bacteria (AOB)、nitrite-oxidizing bacteria (NOB)、total anammox bacteria (TA)，比較環境因子與菌相的動態變化關係，並針對Anammox菌中的Kuenenia stuttgartiensis (KS)、Brocadia anammoxidans (BA)和Scalindua spp.做研究。
結果表明，紅樹林表層的總菌量比裡層總菌量多10倍，而且anammox菌在紅樹林是氮循環菌中的優勢種。調查發現ORP值的影響比營養鹽對anammox菌更劇烈。基於16S rDNA，anammox菌在金屬元素多時呈現數量少，但不同種anammox菌卻有不一樣的現象，因此可推斷各種anammox菌對金屬元素的接受度不同，未來可以透過基因轉錄組來判斷。除了上述已知菌屬，紅樹林中必定存在其他菌種，若能純化培養，將能突破處理低溫、低氨氮污水的瓶頸。

In recent years, high-tech industries develop and the compositions of municipal wastewater are increasingly complex. There are several methods for removing nitrogen-containing pollutants. Anaerobic ammonium oxidation (Anammox) is a new process which is energy-saving and economical in wastewater treatment without adding organic carbon source and reducing nitrous oxide gas escaping. However, the characteristics of wastewater are complex and it is easy to cause down the nitrogen removal efficiency of anammox bacteria. In order to solve this problem, exploring for new strain is a good way.
Hsinfeng mangroves forests locate in the north of Taiwan. Hsinchu Industrial Park is located in the upstream of the mangrove forests. The rich organic matter from Hsinchu Industrial Park pile to mangrove forests. Summary of environmental parameters, there is an opportunity of obtaining new species of anammox bacteria from mangrove sediments. To date, there are many literatures confirmed that anammox bacteria exactly exist in the mangrove sediments.
In this study, the pH value, temperature, and redox potential of the mangrove forest were measured at the sampling site every month. The nitrogen cycle related bacteria, which includes ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and total anammox bacteria (TA), in the mangrove sediments were quantitatively analyzed by molecular biology techniques. This study is investigation of the environmental factors with the dynamic variation of microflora and the distribution of Kuenenia stuttgartiensis (KS), Brocadia anammoxidans (BA) and Scalindua spp..
The results showed that the total bacteria of the surface soil in the mangrove sediments was 10 times higher than layer soil and anammox bacteria was dominant species in the mangrove sediments. The effects of ORP value were more sensitive than nutrients on anammox bacteria. Base on the 16S rDNA, the copies of anammox bacteria was less when the concentration of metal elements was high. An amazing finding was that different kinds of anammox bacteria had different inhibition phenomena, and it can be inferred that a variety of anammox bacteria on the acceptance of metal elements. In the future, it can be judged by gene transcription. Besides the known genus, there is definitely other species of anammox bacteria which exist in the mangrove sediments. By the dominant species from mangrove sediments, It will be able to break the bottleneck which apply in the wastewater treatment with lower temperature and lower ammonia wastewater.

Content
摘要 I
Abstract III
Content V
List of Figures VII
List of Tables VIII
Chapter 1 Introduction 1
1.1 The characteristics of ammonia wastewater 1
1.1.1 Hazard of ammonia wastewater 1
1.1.2 The methods of ammonia wastewater treatment 2
1.2 The characteristics of mangrove forests 5
1.3 Nitrogen removal bacteria 6
1.3.1 Nitrogen cycle 6
1.3.2 The types and characteristics of nitrifying bacteria 7
1.3.3 The types and characteristics of denitrifying bacteria 8
1.4 Anammox bacteria 9
1.4.1 The basic position of molecular biology 11
1.4.2 Effect of heavy metals on anammox bacteria 14
1.4.3 The application and trend in ANAMMOX process 15
Chapter 2 Experimental Strategy 17
Chapter 3 Materials and methods 19
3.1 Site description, Sampling and collection 19
3.2 Analytical Methods 21
3.2.1 Water quality 21
3.2.2 Metal elements 21
3.2.3 Microbiology community 23
Chapter 4 Results and Discussions 29
4.1 Characteristics of sampling sites 29
4.2 Variation of bacteria 33
4.2.1 Seasonal variation with nitrogen cycle related bacteria 33
4.2.2 Seasonal variation with anammox bacteria 33
4.2.3 Environmental factors with anammox bacteria 34
4.2.4 Metal elements with anammox bacteria 37
Chapter 5 Conclusion and Suggestions 41
Reference 42
 
List of Figures
Figure 1 Global distribution of mangrove forest in the world 5
Figure 2 Microorganisms play important roles in the nitrogen cycle 7
Figure 3 The anammox cell envelope 12
Figure 4 The structures of ladderane lipids 12
Figure 5 The steps of anammox metabolism 13
Figure 6 Overview of the anammox catabolism 14
Figure 7 The publications in anammox research during the year 1995 to 2016 16
Figure 8 The hot topics in anammox research 16
Figure 9 Conceptual framework 17
Figure 10 Location map for sampling sites in Hsinfeng mangroves forests 20
Figure 11 Distribution of Scalindua spp. 34
Figure 12 The environmental factors of water sample of site B with anammox bacteria. 35
Figure 13 The environmental factors of water sample of site D with anammox bacteria. 35
Figure 14 Variation of nitrogen cycle related bacteria with sample site and season. 39
Figure 15 Variation of anammox bacteria with sample site and season. 40
 
List of Tables
Table 1 Comparison of advantages and disadvantages of common methods for ammonia nitrogen removal in wastewater treatment. 4
Table 2 Anammox bacteria species[23-43]. 10
Table 3 Location information of sample site 20
Table 4 Method of water quality analysis 21
Table 5 Overview of qPCR Primers set 27
Table 6 Average of environmental parameters of the water 31
Table 7 Metal ions of the water and soil samples (Unit: g/m3) 32
Table 8 Correlation coefficients among bacteria and environmental factors. 36
Table 9 The metal elements and anammox bacteria in surface soil. 38
Table 10 The metal elements and anammox bacteria in layer soil. 38

1. Van De Graaf, A.A., et al., Metabolic pathway of anaerobic ammonium oxidation on the basis of 15N studies in a fluidized bed reactor. Microbiology, 1997. 143(7): p. 2415-2421.
2.van Niftrik, L., et al., Linking ultrastructure and function in four genera of anaerobic ammonium-oxidizing bacteria: cell plan, glycogen storage, and localization of cytochrome C proteins. Journal of bacteriology, 2008. 190(2): p. 708-717.
3.Wang, C.-C., et al., Simultaneous partial nitrification, anaerobic ammonium oxidation and denitrification (SNAD) in a full-scale landfill-leachate treatment plant. Journal of hazardous materials, 2010. 175(1): p. 622-628.
4.Giri, C., et al., Status and distribution of mangrove forests of the world using earth observation satellite data. Global Ecology and Biogeography, 2011. 20(1): p. 154-159.
5.Kathiresan, K. and B.L. Bingham, Biology of mangroves and mangrove ecosystems. Advances in marine biology, 2001. 40: p. 81-251.
6.Nagelkerken, I., et al., The habitat function of mangroves for terrestrial and marine fauna: A review. Aquatic Botany, 2008. 89(2): p. 155-185.
7.Ho, C.-y., Estimation of carbon sequestration for a mangrove wetland in southern Taiwan. CHNA University of Pharmacy and Science, department of environmental engineering and science., 2012.
8.Siao, M.-F., Uptake and accumulation of heavy metals in sediment by mangroves. Fooyin University, department of environmental engineering and science., 2011.
9.Chiang, C.-A., The Effects of Mangroves on Pollutant Removal Efficiencies in Salty Water Types of Constructed Wetlands. 2013.
10.Capone, D.G., et al., Follow the nitrogen. Atmosphere, 2006. 1: p. 2.5.
11.Burrell, P.C., C.M. Phalen, and T.A. Hovanec, Identification of bacteria responsible for ammonia oxidation in freshwater aquaria. Applied and environmental microbiology, 2001. 67(12): p. 5791-5800.
12.Schramm, A., et al., Identification and activities in situ of nitrosospiraand Nitrospira spp. as dominant populations in a nitrifying fluidized bed reactor. Applied and environmental microbiology, 1998. 64(9): p. 3480-3485.
13.Teske, A., et al., Evolutionary relationships among ammonia-and nitrite-oxidizing bacteria. Journal of bacteriology, 1994. 176(21): p. 6623-6630.
14.Ward, B., Nitrification and ammonification in aquatic systems. Life support & biosphere science: international journal of earth space, 1995. 3(1-2): p. 25-29.
15.Schmidt, I., et al., Anaerobic ammonia oxidation in the presence of nitrogen oxides (NOx) by two different lithotrophs. Applied and environmental microbiology, 2002. 68(11): p. 5351-5357.
16.Chen, S.-S., Investigation of the Distribution of Ammonia-Oxidation Bacteria in Landfill-leachate Treatment Plant. Department of Civil Engineering, Chung Yuan Christian University, 2012.
17.Purkhold, U., et al., Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys. Applied and Environmental Microbiology, 2000. 66(12): p. 5368-5382.
18.Prosser, J., Autotrophic nitrification in bacteria. Adv. Microbiol. Physiol, 1989. 30: p. 125-181.
19.Lu, H., K. Chandran, and D. Stensel, Microbial ecology of denitrification in biological wastewater treatment. Water research, 2014. 64: p. 237-254.
20.Tsai, R.-l., Investigation of the characteristics of ammonia-oxidation bacteria and novel nitrogen removal technologies. Department of Biological Sciences, National Sun Yat-sen University, 2009.
21.Huang, X., et al., Effects of ferrous and manganese ions on anammox process in sequencing batch biofilm reactors. Journal of Environmental Sciences, 2014. 26(5): p. 1034-1039.
22.Devol, A.H., Nitrogen cycle: solution to a marine mystery. Nature, 2003. 422(6932): p. 575-576.
23.Khramenkov, S., et al., A novel bacterium carrying out anaerobic ammonium oxidation in a reactor for biological treatment of the filtrate of wastewater fermented sludge. Microbiology, 2013. 82(5): p. 628-636.
24.Jetten, M.S., et al., Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process. Current opinion in biotechnology, 2001. 12(3): p. 283-288.
25.Nikolaev, A., et al., [Candidatus "Jettenia moscovienalis" sp. nov., a New Species of Bacteria Carrying out Anaerobic Ammonium Oxidation]. Mikrobiologiia, 2015. 84(2): p. 236-43.
26.Ali, M., et al., Physiological characterization of anaerobic ammonium oxidizing bacterium ''Candidatus Jettenia caeni''. Environ Microbiol, 2015. 17(6): p. 2172-89.
27.Quan, Z.X., et al., Diversity of ammonium-oxidizing bacteria in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor. Environ Microbiol, 2008. 10(11): p. 3130-9.
28.Ali, M., et al., The increasing interest of ANAMMOX research in China: bacteria, process development, and application. Biomed Res Int, 2013. 2013: p. 134914.
29.Kartal, B., et al., Candidatus "Anammoxoglobus propionicus" a new propionate oxidizing species of anaerobic ammonium oxidizing bacteria. Syst Appl Microbiol, 2007. 30(1): p. 39-49.
30.Brandsma, J., et al., A multi-proxy study of anaerobic ammonium oxidation in marine sediments of the Gullmar Fjord, Sweden. Environ Microbiol Rep, 2011. 3(3): p. 360-6.
31.Hong, Y.G., et al., Residence of habitat-specific anammox bacteria in the deep-sea subsurface sediments of the South China Sea: analyses of marker gene abundance with physical chemical parameters. Microb Ecol, 2011. 62(1): p. 36-47.
32.Li, H., et al., Molecular detection of anaerobic ammonium-oxidizing (anammox) bacteria in high-temperature petroleum reservoirs. Microb Ecol, 2010. 60(4): p. 771-83.
33.van de Vossenberg, J., et al., The metagenome of the marine anammox bacterium ''Candidatus Scalindua profunda'' illustrates the versatility of this globally important nitrogen cycle bacterium. Environ Microbiol, 2013. 15(5): p. 1275-89.
34.Dang, H., et al., Molecular detection of Candidatus Scalindua pacifica and environmental responses of sediment anammox bacterial community in the Bohai Sea, China. PLoS One, 2013. 8(4): p. e61330.
35.Schmid, M., et al., Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria. Syst Appl Microbiol, 2003. 26(4): p. 529-38.
36.Fuchsman, C.A., et al., Free-living and aggregate-associated Planctomycetes in the Black Sea. FEMS Microbiol Ecol, 2012. 80(2): p. 402-16.
37.Kuypers, M.M., et al., Anaerobic ammonium oxidation by anammox bacteria in the Black Sea. Nature, 2003. 422(6932): p. 608-11.
38.Woebken, D., et al., A microdiversity study of anammox bacteria reveals a novel Candidatus Scalindua phylotype in marine oxygen minimum zones. Environ Microbiol, 2008. 10(11): p. 3106-19.
39.Schmid, M., et al., Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation. Syst Appl Microbiol, 2000. 23(1): p. 93-106.
40.Araujo, J.C., et al., Anammox bacteria enrichment and characterization from municipal activated sludge. Water Sci Technol, 2011. 64(7): p. 1428-34.
41.Rothrock, M.J., Jr., et al., Long-term preservation of anammox bacteria. Appl Microbiol Biotechnol, 2011. 92(1): p. 147-57.
42.Oshiki, M., et al., Physiological characteristics of the anaerobic ammonium-oxidizing bacterium ''Candidatus Brocadia sinica''. Microbiology, 2011. 157(Pt 6): p. 1706-13.
43.Kartal, B., et al., Candidatus ''Brocadia fulgida'': an autofluorescent anaerobic ammonium oxidizing bacterium. FEMS Microbiol Ecol, 2008. 63(1): p. 46-55.
44.Egli, K., et al., Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate. Archives of Microbiology, 2001. 175(3): p. 198-207.
45.Schmidt, I., et al., New concepts of microbial treatment processes for the nitrogen removal in wastewater. FEMS microbiology reviews, 2003. 27(4): p. 481-492.
46.Dosta, J., et al., Short-and long-term effects of temperature on the Anammox process. Journal of Hazardous Materials, 2008. 154(1): p. 688-693.
47.Strous, M., et al., Missing lithotroph identified as new planctomycete. Nature, 1999. 400(6743): p. 446-449.
48.Kartal, B., et al., Anammox—Growth physiology, cell biology, and metabolism. Advances in microbial physiology, 2012. 60: p. 212.
49.van Teeseling, M.C., et al., Anammox Planctomycetes have a peptidoglycan cell wall. Nature communications, 2015. 6.
50.van Niftrik, L. and M.S. Jetten, Anaerobic ammonium-oxidizing bacteria: unique microorganisms with exceptional properties. Microbiology and Molecular Biology Reviews, 2012. 76(3): p. 585-596.
51.van Niftrik, L.A., et al., The anammoxosome: an intracytoplasmic compartment in anammox bacteria. FEMS Microbiology Letters, 2004. 233(1): p. 7-13.
52.Jetten, M.S., et al., Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria. Applied Microbiology and Biotechnology, 2003. 63(2): p. 107-114.
53.Li, M., et al., Cytochrome cd1-containing nitrite reductase encoding gene nirS as a new functional biomarker for detection of anaerobic ammonium oxidizing (Anammox) bacteria. Environmental science & technology, 2011. 45(8): p. 3547-3553.
54.Strous, M., et al., Deciphering the evolution and metabolism of an anammox bacterium from a community genome. Nature, 2006. 440(7085): p. 790-794.
55.van de Vossenberg, J., et al., The metagenome of the marine anammox bacterium ‘Candidatus Scalindua profunda’illustrates the versatility of this globally important nitrogen cycle bacterium. Environmental microbiology, 2013. 15(5): p. 1275-1289.
56.de Almeida, N.M., et al., Immunogold localization of key metabolic enzymes in the anammoxosome and on the tubule-like structures of Kuenenia stuttgartiensis. Journal of bacteriology, 2015. 197(14): p. 2432-2441.
57.Li, G., et al., Inhibition of anaerobic ammonium oxidation by heavy metals. Journal of Chemical Technology and Biotechnology, 2015. 90(5): p. 830-837.
58.Lotti, T., et al., Inhibition effect of swine wastewater heavy metals and antibiotics on anammox activity. Water Science and Technology, 2012. 66(7): p. 1519-1526.
59.Daverey, A., et al., Effect of zinc on anammox activity and performance of simultaneous partial nitrification, anammox and denitrification (SNAD) process. Bioresource technology, 2014. 165: p. 105-110.
60.Zhang, Z. and S. Liu, Hot topics and application trends of the anammox biotechnology: a review by bibliometric analysis. SpringerPlus, 2014. 3(1): p. 220.
61.Kallistova, A.Y., et al., Role of anammox bacteria in removal of nitrogen compounds from wastewater. Microbiology, 2016. 85(2): p. 140-156.
62.Han, P., et al., A comparison of two 16S rRNA gene-based PCR primer sets in unraveling anammox bacteria from different environmental samples. Applied microbiology and biotechnology, 2013. 97(24): p. 10521-10529.