細菌在土壤管柱中移動之研究

Abstract

細菌在土壤中的移動主要受到土壤物理、土壤化學與生物因子的影響，但對於細菌細胞表面特性因子仍不清楚。本研究目的是設計一個土壤管柱系統快速模擬觀察細菌在土壤中移動，並測定不同特性之細菌在管柱內之移動速率，探討多種生物因子與移動之相關性。本研究設計一飽和水土壤管柱系統，將細菌接種至十公分高之土壤管柱後給予固定5 ± 0.1公分高之水壓，分析三小時期間所流出之濾液菌數，並於三小時後測定不同深度土壤之菌數。此外，本研究嘗試以即時定量聚合酶連鎖反應 (real-time PCR) 針對細菌之16S rRNA基因進行偵測。結果顯示乾熱滅菌法具有良好的滅菌效果，滅菌後的土壤土粒乾燥，較能填充均勻的土柱。水流流量會影響Serratia marcescens CC-YM2-1菌株通過土柱的速度，流量較多時，能帶動較多的菌通過土柱。隨著土壤黏粒含量的增加，S. marcescens CC-YM2-1通過土柱的速度變慢，且土壤黏粒含量對S. marcescens CC-YM2-1通過土柱的影響力大於土壤有機質。YEM培養基培養的Rhizobium sp. CC-SB136菌株通過砂質土柱的速度較以NB培養基培養的CC-SB136快，原因可能為生長在YEM培養基的CC-SB136產生較多分泌物質。比較三株Serratia marcescens菌株通過砂質土柱的速度發現，同菌屬菌種的不同菌株，通透土柱的速度會不同，經過鞭毛突變後不具有移動性的F1菌株比具運動性的野生株CH-1略有較多細胞通過土柱。本研究比較八株不同菌屬的細菌通過土柱的速度，結果發現革蘭氏陰性菌通過砂壤土柱的速度較革蘭氏陽性菌快。經比較發現細菌細胞壁組成對細菌通過土柱的影響力大於細菌的運動性。使用3F/4R引子對進行即時定量聚合酶連鎖反應，可以有效增殖DNA片段，並在一小時內即可被偵測到。本研究建議細菌細胞表面特性包括分泌物質與細胞壁組成是影響細菌通過土壤的重要因子，未來應用細菌到環境上進行生物復育及到農業上作為生物肥料的施用時，需要考慮細菌細胞表面特性來選擇合適的菌株。即時定量聚合酶連鎖反應靈敏且快速，未來可應用於偵測細菌在土壤移動的研究上。

Transport of microorganisms in the soil is affected by many factors such as physical, chemical and biological properties of soil. It is still being investigated for a clear understanding of the mechanisms of bacterial transport with an emphasis on the bacterial cell surface. The present study was undertaken to evaluate the transport behavior of bacteria in a specially designed flow-through saturated soil column. Based on the data obtained on transport velocity of bacteria the relationship between many biotic factors and bacterial transport was predicted. The influence of physiological characteristics of bacteria on transport in soil was examined in the soil column system with constant volumetric water flow. Experiment was carried out in a 10 cm long acrylic column packed with three different soil types with different texture. Total cells transported within different depth of column and in the eluants were calculated. Vertical translocation of the introduced bacteria was also visualized after 3 h. In addition, quantification of bacteria was performed by real-time PCR of bacterial 16S rDNA fragment amplified from pure culture. Homogeneous soil particles were packed into column after dry sterilization. Among the tested strains, strain Serratia marcescens (CC-YM2-1) showed better transport behavior in low clay content soil and high flow rate. It is hence, suggested that clay content of the soil plays a significant role in bacterial transport compared to organic matter content in soil. Introduced Rhizobium sp. (CC-SB136) was detected at higher number (10 fold) when YEM compared with NB was used as medium either in different depth of column or in the eluants probably due to the production of secondary metabolites. Significant difference was observed in the transport behavior of three Serratia marcescens strains indicating that even though they represent same species but, display wide physiological diversity. Gram-negative bacteria exhibited higher fractional recovery compared to Gram-positive strains of similar size. All the tested isolates were capable of producing specific pigments, favoring the tracking of bacterial transport behavior easily. The detection limit in real-time PCR amplification was 2 log CFU mL-1 reaction-1 showing higher sensitivity and usefulness of this technique in the detection of bacteria and its further applications in such kind of studies. Transport of Gram-positive strains resulted in a significantly lower number in comparison with Gram-negative strains at constant flow rate. These results hence indicate that cell wall characteristics and cellular secretions are the major biotic factors influencing the transport behavior of bacteria in soils. Hence, further studies are warranted to clearly derive the exact mechanisms of bacterial transport in the natural soil.