Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5639
標題: 重金屬污染農地微生物多樣性及其可能之代謝功能探討
Diversity and Possible Function of Microbial Communities in Heavy Metal Polluted Soil
作者: 張飴璇
Jhang, Yi-Shiuan
關鍵字: PCR-DGGE;重金屬污染農地;amoA gene;nifH gene;alkaline phosphatase gene;功能性基因
出版社: 環境工程學系所
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(1994) Functional Diversity of Microbial Communities: A Quantitative Approach Soil Biology and Biochemistry 26: 1101-1108.
摘要: 
受重金屬污染農地經整治後,若能將土壤中重金屬濃度降至管制值之下,農地即可解除列管,但污染農地經整治後常因地力下降導致復耕農地收成不佳,往往需要進行土壤改良以恢復地力。土壤微生物族群之代謝功能與土壤肥力息息相關,重金屬會成為微生物長期的生存壓力,微生物族群之代謝也會因重金屬污染而受到抑制。具特殊功能之微生物(如銨氧化菌、溶磷菌等)族群種類如果因整治而下降則可能會造成農地地力無法於短時間恢復之狀況,因此於整治策略制定時須將整治對微生物多樣性之損害狀況納入考量以降低整治成本並提高復耕農地利用效益。在探討整治對重金屬污染農地微生物多樣性之破壞狀況前,需先瞭解重金屬污染農地微生物與具特殊功能之族群多樣性現況,因此本研究以曾受重金屬污染之農地(標號0119及2023)為目標,利用依照演化基因(rRNA gene)與功能性基因(amoA gene、nifH gene及alkaline phosphatase gene)設計之引子結合PCR-DGGE技術對農地土壤中微生物生態進行調查。
實驗結果顯示,以細菌及真菌演化基因設計的引子依PCR-DGGE技術獲得之圖譜有訊號模糊不清的問題,推測部分原因可能為土壤環境核酸多樣性豐富,因此樣本中多樣的核酸序列其解鏈溫度 ( Tm值,melting temperature)範圍過廣,導致Tm值接近的核酸無法在DGGE圖譜上形成清晰亮帶。利用引子344f/522r對土壤中甲烷古細菌多樣性進行分析,結果顯示有9種及6種甲烷古細菌均勻地分布於農地0119及2023地表下0 ~ 30cm土壤中,甲烷古細菌為絕對厭氧菌生長於土壤團粒中央不含氧之區域,其特殊的生長位置可降低重金屬對其族群多樣性之衝擊。利用引子243f/518r對土壤中放線菌族群進行研究後推測,可能因為放線菌k-strategy之生長策略可減緩低濃度之重金屬對其族群多樣性之衝擊,因此土壤中鉻(Cr)、銅(Cu)、鋅(Zn)及鎳(Ni)濃度須超過法規值兩倍以上於DGGE圖譜上放線菌族群多樣性才會產生變化。
以功能性基因分析所獲得之多樣性資料推測因農地2023受重金屬污染嚴重且土壤pH偏酸,因此於農地2023九個採樣點僅有採樣點I (10 ~ 20cm)土壤中發現兩種銨氧化菌(Ammonia-oxidizing bacteria, AOB)存在。農地0119土壤較接近中性外,九採樣點中,採樣點I (0 ~ 10cm)、M (0 ~ 10cm)、O (0 ~ 10cm)及I (10 ~ 20cm)皆有AOB存在,且其多樣性變化推測與土壤中銨濃度變化有關,故建議銨氧化菌族群多樣性變化可以成為評估環境變化之微生物指標。農地2023受重金屬污染嚴重區域之溶磷菌與固氮菌多樣性與其它採樣點明顯不同,且溶磷菌於污染嚴重之採樣點I (0 ~10cm)與I (10 ~ 20cm)中其菌群種類與樣點M及O不同外,溶磷菌多樣性較採樣點M及O豐富,因此農地雖受重金屬污染但具有將磷酸鹽轉型能力之微生物族群仍可維持一定之多樣性,此外隨污染程度加深,具有磷酸鹽轉型能力之微生物種類會產生改變,故推測高濃度的重金屬確實會對溶磷菌群造成影響,但農地土壤仍具一定之磷酸鹽代謝潛力。

In paddy soil, microbial functions and community diversity are closely involved with the in situ soil fertility. Although performing remediation can reduce the concentration of heavy metal, it also leads to poor soil fertility and low harvest. Therefore, the fertility of the remediation soil will still need to be improved if it will still be used for cultivation. Because of heavy metal cannot be degraded by microorganisms, heavy metal become long-term environmental stressed easily and it will inhibit the activity of soil microorganisms in the polluted site. If the specific microbial diversity decline with remediation process, soil fertility will not recover in short-term. For purpose to develop a reliable method to evaluate the damage from the remediation approaches, which may reduce the value of resumed cultiable land, on microbial diversity, molecular tools were applied in this study. In this study, genes diversity, such as rRNA, amoA, nifH and alkaline phosphatase genes, in the heavy metal polluted soil (marked as land 0119 and 2023) were detected using PCR-DDGE methods.
In soil environment existence of high diversity in nucleic acid might leads to the PCR product have wide range of melting temperature (Tm), this has the possibility to cause the fuzzy bands in DGGE fingerprint that cause the poor resolution of PCR-DGGE fingerprints when applying the eubacteria- and fungal- specific primer sets individually. Applying primer pairs 344f/522r to analyze the methanogens archaea diversity, there were nine and six methanogens archaea species distributed in the soil which was 0 to 30 cm under the surface. Using primer pairs 243f/518r to analyze the Actinobacteria community diversity, the results showed the low Acinobacteria diversity in the paddy land 2023, in which heavy metal concentration exceed than the heavy metal criterions published by Taiwan EPA . Furthermore, it was suggested that k- strategy growth mode of Actinobacteria can reduce the impact of heavy metals. Therefore, it was believed the heavy metal would effects the Actinomycetes diversity when polluted soil it's heavy metal concentration higher than the published criterions.
In Ammonia-oxidizing bacteria(AOB) diversity analysis, there were only five samples amplicons were detected, which were 0119 I (0~10cm), 0119 M (0~10cm), 0119 O (0~10cm), 0119 I (10~20cm)and 2023 I (10~20cm). The AOB diversity analysis demonstrated there were two AOB species exited in the land 2023, the more serious heavy metal pollution and lower pH in the paddy land 2023 might be the reason that reduce AOB diversity. Paddy land 0119 variation of AOB diversity may reflect changes ammonium concentration in soil and the variation of ammonium-oxidizing bacteria diversity was suggested to be applied as the microbial indicators for detecting environmental change. In the high heavy metal polluted soil 2023 the phosphate-solubilizing bacteria diversity in sample site I was higher than it in sites M and O and the high concentrations of heavy metals was demonstrated that to be benefit on phosphate-solubilizing bacterial diversity by the results of DGGE analysis in this study, where the phosphate-solubilizing bacteria have high diversity in sample site 2023 I. It was suspected that heavy metal polluted paddy soil still have potential of phosphate metabolism according to the PCR-DGGE analysis based on amoA, nifH and alkaline phosphatase genes. In summary, inherent specific microbial diversity could be used as an indictor to evaluate the damage in the polluted soil.
URI: http://hdl.handle.net/11455/5639
其他識別: U0005-1008200918191600
Appears in Collections:環境工程學系所

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