陳章波臺灣大學：海洋研究所范嵐楓Fan, Lan- FengLan- FengFan2007-11-272018-06-282007-11-272018-06-282007http://ntur.lib.ntu.edu.tw//handle/246246/56575全世界水域環境污染的情況日益嚴重。以台灣為例，約有三分之二的河川呈現中度 (moderately) 或嚴重 (seriously) 污染，而北部最大河川淡水河由於承受大量生活污水的排放，使得像文蛤Meretrix lusoria這類生存在河口的生物受到嚴重影響。相對在沿岸環境的文蛤人工養殖區同樣也面臨到底質惡化，造成漁獲量降低，以及養殖池壽命縮短的問題。其實，不論河口污染或者是養殖池底泥的惡化，主要都是有機物質蓄積之後，所引發一連串底泥化學、微生物群聚，乃至於底棲動物的變動。 有機物質蓄積是底棲環境的關鍵性議題。水域中有機物質的堆積與否與流體運動及生物地球化學 (biogeochemistry) 代謝兩者息息相關。當流況減弱時，水中懸浮有機物質會沉澱積聚在底泥上，一般水中溶氧會與這些有機物質進行分解作用。當氧氣缺乏時，則以其它替代的化學物質，像是硝酸鹽 (nitrate)、硫酸鹽 (sulfate) 之類與有機物質進行厭氧性的降解 (anaerobic degradation)。這些好氧 (aerobic) 及厭氧代謝的結果都直接反映在底泥化學性質上。為了解有機物質蓄積下對底泥化學性質的影響，本論文測量淡水河河口至中游河段底泥中化學性質的縱向水平空間分布：於2001年採集下游與中游河段之底泥樣品進行硫化物質 (硫化氫, 酸可萃取硫 [acid-volatile sulfide; AVS]，黃鐵礦 [pyrite]) 含量及硫酸還原速率 (sulfate reduction rate; SRR) 的分析；於2002年採集近河口區、下游與中游河段之底泥樣品進行營養鹽含量 (硝酸鹽 [nitrate]、亞硝酸鹽 [nitrate]、磷酸鹽 [phosphate] 及矽酸鹽 [silicate]) 的分析。 綜合底泥化學性質的結果顯示，由間隙水中硝酸鹽、磷酸鹽與硫化氫特性的縱向空間組成分布，可將淡水河系底泥有機物降解的過程界定為三個類型：硝酸鹽富集型 (nitrate-abundance type) 出現在近河口區，間隙水硝酸鹽及磷酸鹽濃度分別為114.24及1.01To understand the integrated effects of organic matter loading on the benthic chemical properties, microbial communities, and benthos in an estuarine system, two field studies and a set of laboratory experiments were conducted. In the Tanshui River, the horizontal characteristics of the estuarine sediments were revealed in three spatial settings from the river mouth to a mid-reach section: a nitrate-abundance type, a phosphate-release type, and a sulfide-generation type; the vertical characteristics in four layers of Guandu sediment were also elucidated. With the exception of the mixed layer, a variety of metabolic processes by denitrifier strains occur in different vertical layers including nitrification-denitrification coupling processes in the nitrate-concentrated layer, denitrification or dissimilatory nitrate reduction to ammonium in the denitrifier-aggregation layer, and fermentation or spore formation in the ammonium-enriched layer. In addition, two laboratory experiments indicated that environmental condition gradually became worse, which leads to decreasing of dissolved oxygen, increasing of ammonium, nitrate and sulfide concentrate. However, high levels of treatment with the photosynthetic bacterium (PSB, Ectothiorhodospira sp.) decreased sulfide and nitrogenous nutrients in both aerated and hypoxic conditions, and then significantly improved the survival rate of the hard clam (Meretrix lusoria). In summary, the degree of organic matter degradation could be defined by the chemical properties in both the horizontal and vertical sediment profiles. A variety of metabolic processes by denitrifiers, which occur in the comparative sediment profile, indirectly correspond to these different stages. The use of the PSB, Ectothiorhodospira sp., actively removed some chemicals and mediated the sediment properties (from polluted to a transitory stage). Benthic hard clams can serve as a good indicator of the shift in benthic chemical processes toxic to the benthos, which are incapable of being detected by environmental measurements.Contents Committee verified document (in Chinese) ………………………………….. i Acknowledgment (in Chinese) ……………………………………………….. iii Abstract in Chinese (中文摘要) ……………………………………………... v Abstract in English …………………………………………………………… xi Contents in English …………………………………………………………... xiii Graph contents ……………………………………………………………….. xviii Table contents ………………………………………………………………… xxii Chapter 1. General introduction ……………………………………………… 1-1 Organic matter loading ………………………………………………….. 1-3 Adverse impacts of organic matter loading …………………………….. 1-4 Mitigating of organic matter loading …………………………………… 1-5 I. Chemical properties of sediments as organic matter is enriched ……... 1-6 II. Roles of microbes (denitrifier) with organic matter loading …………. 1-8 III. Mitigating the impact of organic matter loading using microbial (photosynthetic bacteria; PSB) addition ……………………………... 1-10 Questions to resolve …………………………………………………….. 1-13 Chapter 2. Compositions of nitrate, phosphate, and sulfide in interstitial water as indicators for defining the level of organic matter loading in the benthic environment ………………………………………... 2-1 Abstract …………………………………………………………………. 2-2 Introduction ……………………………………………………………... 2-4 Materials and methods ………………………………………………….. 2-9 Study site ……………………………………………………………… 2-9 Sample collection ……………………………………………………... 2-10 Analysis of chemical parameters in interstitial water ………………… 2-10 Sulfate reduction rate …………………………………………………. 2-11 PyritS and AVS ………………………………………………………... 2-11 Results …………………………………………………………………... 2-13 Distribution of sulfurous compounds …………………………………. 2-13 Distribution of various nutrients ……………………………………… 2-14 Discussion ………………………………………………………………. 2-15 References ………………………………………………………………. 2-21 Chapter 3. Distribution of nitrogenous nutrients and denitrifiers strains in estuarine sediment profiles of the Tanshui River, northern Taiwan . 3-1 Abstract …………………………………………………………………. 3-1 Introduction ……………………………………………………………... 3-1 Materials and methods ………………………………………………….. 3-2 Study site ……………………………………………………………… 3-2 Sample collection ……………………………………………………... 3-3 Analysis of environmental parameters ………………………………... 3-3 Determination of microbial communities …………………………….. 3-3 Isolate characterization ……………………………………………….. 3-4 DNA extraction and PCR amplification of 16S rDNA ……………….. 3-4 Sequence and phylogenic analysis of isolated strains ………………… 3-4 Results …………………………………………………………………... 3-4 Sediment profiles of environmental parameters ……………………… 3-4 Sediment profiles of viable bacteria …………………………………... 3-5 Sediment profiles of microbial strains ………………………………... 3-6 Discussion ………………………………………………………………. 3-7 Distributions of nitrogenous nutrients and denitrifier abundances …… 3-7 Properties of the denitrifying bacteria ………………………………… 3-9 References ………………………………………………………………. 3-10 Chapter 4. The use of photosynthetic bacterium (Ectothiorhodospira sp.) for mitigating the impact of organic matter loading in sediments on the hard clam (Meretrix lusoria) ………………………………….. 4-1 Abstract …………………………………………………………………. 4-2 Introduction ……………………………………………………………... 4-4 Materials and methods ………………………………………………….. 4-7 Experimental design …………………………………………………... 4-7 Sample collection ……………………………………………………... 4-9 Analysis of environmental parameters ……………………………….. 4-10 Data analysis ………………………………………………………….. 4-11 Results …………………………………………………………………... 4-13 Discussion ………………………………………………………………. 4-16 PSB’s effect under different oxic conditions …………………………. 4-17 PSB addition for the removal of sulfide ………………………............. 4-18 PSB addition for the removal of nitrogenous compounds ……………. 4-19 Hard clam as a bioindicator for environmental mitigation …................ 4-20 References ………………………………………………………............. 4-23 Chapter 5. Discussion ………………………………………………………... 5-1 Chemical Properties of Spatial Zonation in the Tanshui River …………. 5-1 Chemicals and Microbial Communities in Sediment Profile in the Estuary of Tanshui River ……………………………………………… 5-3 Microbes, Chemicals and Benthos Interaction in a Mesocosm System … 5-5 Determining the Extent of Organic Matter Loading of the Environment . 5-8 Mitigating the Adverse Effects of an Organic-Matter Accumulated Environment …………………………………………………………... 5-11 References ……………………………………………………………………. r-1 Appendage (in Chinese, 附錄) ………………………………………………. a-1 Publish records (發表記錄) …………………………………………….. a-1 Graph Contents Chapter 1 Figure 1.1. Changes in fauna and sediment structure along a gradient of organic matter loading ……………………………………………………. 1-2 Figure 1.2. Effects of various hydraulic and biogeochemical characteristics on the fate of organic matter in estuarine environments ………………….. 1-3 Figure 1.3. Interactions of chemical properties in a sediment profile receiving organic matter input ………………………………………………………. 1-6 Figure 1.4. Physiological feasibilities of denitrifiers acclimating to the different chemical characteristics of a sediment profile accumulated with organic matter …………………………………………………………….. 1-8 Figure 1.5. Effects of the use of photosynthetic bacteria (PSB: Ectothiorhodospira sp.) to mitigate the adverse impacts of organic matter loading on the hard clam, Meretrix lusoria, as a monitoring indicator in the entire benthic system comprising sediment and water ……………...... 1-10 Figure 1.6. Impacts of organic matter loading on benthic properties, such as chemical properties, microbes, and the resident benthos, and the framework for the three questions addressed in this thesis ......................... 1-14 Chapter 2 Figure 2.1. Location of the Tanshui River in northern Taiwan .……………… 2-25 Figure 2.2. Vertical profiles of environmental factors, sulfide, sulfate, AVS, pyrite and sulfate reducing rate in the sediment of site T2 and T3 (from river mouth to upstream). Notice the scale change for the sulfate reducing rates ……………………………………………………………………….. 2-26 Figure 2.3. Vertical profiles of environmental factors, nitrate, nitrite, phosphate and silicate in the sediment of site T1, T2, and K1 in the Tanshui River system (from river mouth to the upstream) ……………….. 2-27 Figure 2.4. Summary of the spatial distribution of benthos compositions, the correspondent stages of benthic properties, the spatial distribution of chemical properties, nitrate, phosphate, and sulfide, in the Tanshui River, and the concordance of previous sediment profile summarized by author after 90o rotating ………………………………………………………….. 2-28 Chapter 3 Figure 3.1. Location of the Tanshui River Estuary in northern Taiwan ……… 3-2 Figure 3.2. Vertical profiles of environmental factors in the Guandu sediments …………………………………………………………………. 3-5 Figure. 3.3. Phylogenetic (left) and physiological (right) comparisons of denitrifier strains. Underlining indicates a denitrifier strain ……………… 3-7 Figure. 3.4. Summary of the concentrations of nitrate and ammonium, denitrifier abundance profiles, the vertical distribution of denitrifying isolated strains (one bold line for each strain), layer definitions, and corresponding metabolic strategies in the Tanshui River Estuarine sediments …………………………………………………………………. 3-8 Chapter 4 Figure 4.1. Dissolved oxygen, concentration of nitrate, sulfide and ammonium in water, sediment total organic carbon and total nitrogen, and the survival rate of clams with the addition of different levels of photosynthetic bacteria during the first experiment ……………………… 4-26 Figure 4.2. Dissolved oxygen, concentration of ammonium and sulfide in the water with the addition of different levels of photosynthetic, 0 or 0.5% total organic carbon of organic matter loading, and a porous/tight cap in the oxygen-depleting experiment ………………………………………… 4-27 Figure 4.3. A working model of Ectothiorhodospira sp. (PSB) removal of reduced compounds under an organic matter loading condition, through phototrophic sulfide oxidation and nitrogenous nutrient assimilation (ammonium and nitrate) ………………………………………………….. 4-28 Chapter 5 Figure 5.1. Summary of the spatial distribution of benthic compositions, the stages of benthic properties Pearson, the spatial distribution of chemical properties, such as nitrate, phosphate, and sulfide, in the Tanshui River, and the concordance of the previous sediment profile summarized by author after a 90o rotation ………………………………………………… 5-1 Figure 5.2. Summary of the concentrations of nitrate and ammonium, denitrifier abundance profiles, the vertical distribution of isolated denitrifying strains, layer definitions, and corresponding metabolic processes in the estuarine sediments of the Tanshui River ……………….. 5-5 Figure 5.3. A working model of Ectothiorhodospira sp. (PSB) removal of reduced compounds under an organic matter loading condition, through phototrophic sulfide oxidation and nitrogenous nutrient (ammonium and nitrate) assimilation, and which reflects on the survival of the hard clam, Meretrix lusoria …………………………………………………………... 5-7 Figure 5.4. Summary of the effects of fluctuations in benthic properties when undergoing organic matter loading on the chemical composition, the microbial (denitrifier) communities, the resident benthos, and the shift of benthic properties after microbial (photosynthetic bacteria; PSB) addition 5-8 Table Contents Chapter 1 Chapter 2 Table 2.1. Summary of the of the hydraulic and sediment properties of the Tansui River at varying distanced from the river mouth …………………. 2-29 Table 2.2. Summary of the spatial distribution of maximum value of parameters in the sediment profile of the Tansui River at varying distanced from the river mouth …………………………………………… 2-30 Chapter 3 Table 3.1. Correlation analysis (Pearson correlation) of environmental parameters and microbial communities (viable bacteria) in sediments of the estuary of the Tanshui River at Guandu ………………………………. 3-5 Table 3.2. List of physiological tests and character groupings of 13 isolated strains ……………………………………………………………………... 3-6 Table 3.3. 16S rDNA similarity matrix of 13 isolated strains from the Tanshui Estuary and closely related bacteria from the NCBI database …………… 3-6 Chapter 4 Table 4.1. Summary of the two experimental designs ……………………….. 4-29 Table 4.2. Results of a general linear mode analysis of variance for environmental measurements and survival rate of hard clams with the addition of different levels of photosynthetic bacteria, different dates of sampling, and a blocking effect, in the first experiment ………………….. 4-30 Table 4.3. Comparison of regression coefficients of the best-fitting environment measurements to the survival rate of hard clams at each observation dates in different models of backward stepwise regression, including in condition of high and low photosynthetic bacteria, under loading with organic matter ………………………………………………. 4-31 Table 4.4a. Results of an analysis of variance for environmental measurements among the addition of different levels of photosynthetic bacteria, with/without OM loading, and a porously/tightly cap in a full-set design in the oxygen-depleting experiment ………………………………. 4-32 Table 4b. Results of a nonparametric analysis of variance (Kruskal-Wallis test) for environmental measurements among different levels of PSB addition and porous/tight cap in organic material-loading set in the oxygen-depleting experiment …………………………………………….. 4-32 Chapter 5en-US有機物質蓄積底質狀態間隙水化學微生物群聚底棲動物環境改善生物指標organic matter loadingbenthic propertyinterstitial water chemistrymicrobial communitiesbenthosenvironmental mitigationbioindicatorthesis