200909590 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種不但可以有效地提昇復育速率、且能 夠顯著地縮短用以處理油污染土壤及/或地下水、或者將污 染物轉換成無害成分所需之生物復育期程，並且具有簡便' 經濟、快速等特點之油污染土壤及/或地下水之生物復育方 法。 更詳細地來說，本發明是關於一種藉由在油污染土壤及 /或地下水中投入油生物分解促進劑、微生物成長所需營養 源、以及視情況需要地添加油分解優勢菌種；並予以充分混 合均勻來分解清除油污染成分之一種油污染土壤及/或地下 水之生物復育方法。 【先前技術】 由於人類大量而廣泛地使用石油系石化產品的結果，已 對於自然環境造成莫大的影響與破壞，尤其是因老舊加油 站、油槽、輸油管等滲漏所引起的土壤及/或地下水污染、 環境破壞等之問題日趨嚴重。例如，依據民國九十年的「地 下水潛在污染源調查計畫」執行報告指出：台灣目前至少有 十九座加油站及六座大型石化儲槽附近之土壤及地下水處 於高污染潛勢狀態，同時由於污染源所引起的環境破壞污染 紛爭案例與其他意外事件亦層出不窮。因此，對於地狹人稠 的台灣而言，毫無疑問地，解決加油站、儲油槽附近土壤及 地下水受到污染的問題，日益迫切且刻不容緩。 對於該等環境污染之防治與環境復育，在可資利用的科 200909590 學手段中，最爲經濟且有效的方法即是一種運用環境分子生 物科技、應用生物處理技術來解決土壤、地下水等環境之污 染問題的方法，近年來已引起科學界廣泛地硏究，並已大量 地應用於環境整治與環境復育領域上。 又，已知道於土壤及/或地下水中所存在的一部分微生 物，具有分解或降解前述之汽油、柴油等石油系污染成分的 能力（Hess, et al·，1996 ; Boopathy, 2003 ； Widrig and Manning, Jr.， 1995 ； Richard and Vogel, 1999 ； Rahman et al., 2002 ; Margesin and Schinner, 2001)。因此，近年來有一部分專家學者建議利用該 等微生物可將石油系污染物分解成無害的化合物的特性，來 解決由於原油、汽油、柴油、重油等所引起的土壤及/或地 下水中石油系化學品污染之問題（Balba，et al, 1998 ; Banat, 1995; Banat，2000; Rahman, etal·, 2001 )。然而，很不幸地， 由於石化油品類污染成分多半是脂肪族或芳香族等疏水性 化合物，因此有限種類與數量之微生物很難將它分解成無害 化合物，縱使存在於自然界中的部分原生菌可以降解石油系 污染成分，它的降解速度也是非常的緩慢，因而不適合實際 使用受污染的土壤及/或地下水之復育工作上。 爲了解決土壤及/或地下水等環境污染問題’近年來各 國專家學者均建議應用生物科技來協助解決，即藉由所謂環 境生物科技技術來實施生物復育，例如’藉由採用植種外來 優勢的微生物之生物優植法（bioaugmentation)或添加促進微生 物活性之助劑之生物促進法（biostimulation)等方案，在生物處理 的過程中提供微生物最適的環境條件，以保持微生物的活性 200909590 並加速微物物分解污染成分速率，來達成生物復育與恢復環 境景觀之目標。 舉例來說，例如，在美國第5436 1 60號專利中已建議 一種利用受烴類化合物污染的土壤中之原生菌，藉由添加界 面活性劑並提供最適的微生物生長環境條件，來提昇原生菌 分解該受污染土壤的烴類化合物之速率，以及促進受污染土 壤之生物復育效能之方法。然而，在該方法卻未記載該受污 染土壤中原生菌群之形態及分析方法，由於未能掌握存在於 該烴類化合物污染土壤中原生菌族群的動態，且無後續的追 蹤與監測，因而利用該方法來進行土壤之復育工作，會有不 能徹底復原污染環境的問題。 如以上所述，由於在土壤及/或地下水之生物復育技術 領域上，雖然已提議添加界面活性劑之利用原生菌進行土壤 之生物復育的方法，但由於未分析原生菌群分布形態且無後 續的追蹤與監測的緣故，生物復育速率提昇程度不理想，其 應用效能因而受到限制，所以強烈期望進行改良。也就是 說，目前的現況是：尙未開發出一種可以精確地提昇復育速 率具有簡便、經濟、快速等特點之土壤及/或地下水之生物 復育方法；所以，亟需開發出一種不但可以有效地解決土壤 及/或地下水中所存在的原生菌之分解速率緩慢之問題，而 且可以大幅地提昇污染土壤及/或地下水之復育速率之簡 便、經濟、快速的土壤及/或地下水之生物復育方法。 【發明內容】 【發明所欲解決的課題】 200909590 因此，有鑒於上述此種情況，則本發明之目的即在於解 決土壤及/或地下水之生物復育速率不理想之問題點，提供 一種可以有效地提昇復育速率具有簡便、經濟、快速、精確 等特點之土壤及/或地下水之生物復育方法。更詳細地來 說，本發明提供不但可以有效地提昇復育速率、且能夠顯著 地縮短用以處理油污染土壤及/或地下水或者將污染物轉換 成無害成分所需之生物復育期程，並且具有簡便、經濟、快 速等特點之油污染土壤及/或地下水之生物復育方法。確保 污染土壤及/或地下水之環境品質淨化及再利用之可行性。 【用以解決課題之手段】 爲了達成上述之目的，本發明人等刻意進行檢討的結果 ，開發出一種利用系統化環境分子生物復育技術，而且發現 一種不但可以有效地提昇復育速率、且能夠顯著地縮短用以 處理油污染土壤及/或地下水、或者將污染物轉換成無害成 分所需之生物復育期程，並且具有簡便、經濟、快速等特點 之油污染土壤及/或地下水之生物復育方法。 也就是說，上述之課題可藉由本發明之下述觀點與構成 手段加以解決。首先，本發明之觀點係藉由模廠試驗評估生 物菌劑與生物界面活性劑於整治油污染場址之成效評估，建 立生物復育之技術策略來實施生物復育。本發明之另一觀點 爲組合生物優植法及/或生物促進法的一種系統化生物復育 之方法，並藉由利用微生物晶片精確檢測生物菌種，以監控 整治成效，縮短污染者處理污染所需之時間與費用，促使其 提早使用該場址繼續其事業發展。 200909590 又，本發明之再一觀點係依照污染場址之微生物社會群 族結構可精確檢測而從生物優植法中生物促進法中選用任 何適用之方法或兩者倂用之方法，以達成節省整治經費、快 速降低污染物之目標。 另外，本發明之其他觀點爲開發設計一種可定性定量之 微矩陣生物晶片與即時聚合酵素鏈鎖反應所需要的引子 (primer)與基因探針（probe)，並調整至最適用於油污染分解 菌分析、監測之適化條件，建立可應用在油污染場址生物復 育技術的生物晶片監測方法，藉以監測多樣化目標微生物並 達成整治效果之目的。 基於上述觀點，本發明至少提供一種結合生物優植法與 生物促進法處理油類污染土壤及/或地下水之系統化之生物 復育技術及多樣菌種微矩陣生物晶片檢測技術；以及提供一 種高效率、節省處理成本及可縮短1倍以上整治時間之優異 的處理油類污染土壤及/或地下水之系統化生物復育技術。 亦即，提供一種先用分生技術變性凝膠梯度電泳分析土壤及 /或地下水微生物之社會族群結構，決定實施生物優植法或 生物促進法或結合兩法之策略，以微生物晶片之分生檢测技 術監測目標微生物之多樣菌相種類，以生物計數法估算油分 解微生物菌數，利用GC-FID分析目標污染物濃度降解效果 之處理油類污染土壤及/或地下水之系統化環境分子生物復 育方法。 從而’本發明提供一種生物復育方法，其係利用系統化 環境分子生物技術處理油類污染土壤及/或地下水、或者將 -10- 200909590 油類污染物轉換成無害成分之方法，包括：分析受污染土壤 及/或地下水中微生物之社會族群結構，決定生物復育策 略’監測目標微生物之菌相種類’及分析目標污染物之降解 效果。 又’本發明還提供一種生物復育方法，其係用於處理油 類污染土壤及/或地下水、或者將油類污染物轉換成無害成 分之方法’其係包括：於油污染土壤及/或地下水中投入油 生物分解促進劑，其中油生物分解促進劑係由鼠李糖脂構 成；及視需要添加微生物成長所需營養源。 另外’本發明亦提供一種生物復育方法，其係用於處理 油類污染土壤及/或地下水、或者將油類污染物轉換成無害 成分，包括：於油污染土壤及/或地下水中投入油分解優勢 菌種，及視需要添加微生物成長所需營養源。 【發明效果】 依照本發明的話，如以下之說明所述，即可以一種具有 簡便、經濟、快速、精確等特點之土壤及/或地下水之生物 復育方法，配合污染場址之微生物社會群族結構形態及基因 特性，藉由添加具有優異效能的油分解促進劑及/或油分解 菌於油污染土壤及/或地下水中，而有效地提昇利用生物來 進行土壤及/或地下水之生物復育速率而得到令人滿意之復 育功效。 【實施方式】 【用以實施發明的最佳形態】 以下，就本發明更進一步詳細地說明之。另外，在本發 -11- 200909590 明說明書中’所謂「（數値1 )〜（數値2)」之記載方式，係代 表包括在「（數値1)或以上〜（數値2)或以下」的範圍內所有 數値之意。「油污染」係泛指受到石油系化合物，例如，包 括柴油（C10〜C28)、較難分解且不具揮發性之由20個碳以 上的烴類混和物、重油（fuel heavy oil) (C28〜C40)等之油品污 染之意。 爲了方便說明起見，以下特以受到柴油污染的土壤及/ 或地下水做爲生物復育對象，來詳細地說明本發明之油污染 土壤之生物復育方法；然而，熟習本項技術者當能輕易明 白，本發明方法非僅限定使用於此種柴油污染土壤及/或地 下水而已，其也可以將本發明說明書中所描述者應用於受到 柴油以外之可生物降解之其他成分所污染的土壤及/或地下 水之生物復育方面。 首先，本發明中所謂的系統化環境分子生物復育技術之 流程主要包括監測、分析、診斷及控制等程序；其中監測程 序係採用微矩陣生物晶片（microarray biochip)及分生技術變 性凝膠梯度電泳分析（DGGE)進行微生物族群調查，而分析程 序則在於判定於復育環境中是否已有油分解微生物之存在 ，又，診斷程序爲利用分析之結果以做爲復育方案之決策標 準，而控制程序則可區分成生物添加與生物促進等二種方式 。於復育期間則採用分子生物技術進行監控微生物消長變動 與污染物濃度變化。 又，依照本發明，在進行生物復育之前，較宜是先進行 土壤環境原生菌群分析試驗，更宜是以多樣性微矩陣生物晶 -12- 200909590 片進行原生菌群分析試驗，然而也可以在沒有油污染土壤及 /或地下水中原生菌群分析試驗之情況下，利用本發明之方 法來進行土壤及/或地下水生物復育。又，按照本發明之第 一觀點，本發明之油污染土壤之·生物復育方法，一般是利用 原先存於油污染土壤中之原生菌來進行生物復育，通常主要 包括於油污染土壤中添加油生物分解促進劑及微生物成長 所需營養源，較宜是充分混合均勻。另外，也可以視需要以 多樣性微矩陣生物晶片進行原生菌群分析試驗以外，特佳者 €) 爲進一步地進行各種土壤特性分析，包括污染成分及污染 量、碳氮比、溫度、含水率、pH、氧及二氧化碳等氣體含量 等。 按照本發明之第一觀點，雖然沒有要求必須進行油污染 土壤中原生菌群分析試驗，然而較佳爲在決定使用本發明之 分子生物復育方法以前先進行原生菌群分析試驗，更宜是以 多樣性生物晶片進行原生菌群分析試驗。關於使用於本發明 的原生菌群分析試驗之方法係可以採用公知或習用之方 lj 法，並沒有特別地限定。舉例來說，例如，可以使用於中華 民國發明專利申請案第931 1 0828號所揭示之分生技術變性 凝膠梯度電泳分析方法（DGGE法），來分析存在於受污染環 境中微生物之社會族群結構。又，由於中華民國發明專利申 請案第931 1 08 28之開示全部皆可供參照，因而該專利文獻 內容亦納入本說明書中。 其次，本發明中所使用之油生物分解促進劑的種類、成 分並沒有特別地限定，只要是可以促進生物分解污染成分的 200909590 速度及效率之物質即可，較宜是一種對生物無毒害性、 可生物分解性且不會造成環境之生物累積的化合物或 物。在本發明中，舉例來說，例如可以使用含以鼠李糖 主成分之油生物分解促進劑。因爲鼠李糖脂包含1或2 碳糖與一長鍊脂肪的化學結構之化合物，且係屬糖脂類 物界面活性劑，並對於油類具有高溶解性因而有利於油 菌之利用。所以，從生物復育成效之觀來看，本發明較 採用含有鼠李糖脂之生物界面活性劑。 Γ- 其次，鼠李糖脂之來源並沒有特別地限定，可以是 傳統化學合成方法製造而得的化合物，也可以是藉由於 民國發明專利公告第丨270534號之生物方法，利用銅 單胞菌（Psewctomonas_ aenvg/nosa)於適當發酵條件下所產生， 離心菌體與一連串之粹取純化而得。由於中華民國發明 第I 270 534之開示全部皆可供參照，因而該專利文獻 亦納入本說明書中。 另外，在本發明之生物復育方法所使用的油生物分 進劑中，除了可以含有做爲主成分的鼠李糖脂以外，尙 含有其他的副成分。舉例來說，例如，本發明之油生物 促進劑中可以含有葡萄糖等之其他成分。再者，關於油 分解促進劑之添加量，油生物分解促進劑之添加量通常 公斤乾土壤重及/或地下水施用10〜50毫克，較宜是每 公尺添加20〜100毫克之範圍，更宜是每立方公尺添) 〜2 00毫克之範圍，特佳爲每公斤乾土壤重添加100 -毫克。 具有 組成 脂爲 個五 之生 分解 宜是 藉由 中華 綠假 再經 專利 內容 解促 可以 分解 生物 是每 立方 J口 5 0 〆250 -14- 200909590 再者，按照本發明第一觀點之油污染土壤及/或地下水 之生物復育方法，其中油生物分解促進劑與營養源之添加方 式並沒有特別地限定，可以從公知技術中視需要選取任何一 種添加方法來使用，舉例來說，例如，其可以使用連續加入 或分批加入油生物分解促進劑與營養源，也可以是在充分混 合油污染土壤與油生物分解促進劑之後，再加入營養源。 另外，油生物分解促進劑之形態並沒有特別地限定，例 如，依照本發明，可以將溶液、乳化液、固態顆粒、粉末、 膠囊、錠劑等各種形態之油生物分解促進劑添加於生物復育 對象之土壤及/或地下水之中。又，在溶液、乳化液等之液 態的情況下，也可以更進一步地含有界面活性劑、乳化劑、 標示劑等之其他添加劑。 此外，本發明之微生物成長所需營養源主要爲氮營養 源、磷營養源及碳源等；可以使用來做爲本發明之微生物成 長所需營養源並沒有特別地限定，舉例來說，例如，其可以 是尿素、硝酸鉀、硝酸銨、磷酸銨、磷酸鉀、磷酸氫鉀、磷 酸氫鈉、磷酸鈉等等。又，一般而言，微生物成長所需營養 源之添加量，通常係控制在使得土壤中之C : N : P之比例 界成爲100: 1: 0.1至100: 10: 5之範圍。 其次’按照本發明之第二觀點，本發明係提供於油污染土壤 中投入油分解優勢菌種，添加微生物成長所需營養源；及視 情況需要而添加以鼠李糖脂爲主成分之油生物分解促進 劑；並予以充分混合均勻之一種油污染土壤及/或地下水之 生物復育方法。又，可以使用於本發明之油分解優勢菌種， 200909590 只要是具有或可以促進生物分解污染成分的速度及效率的 微生物即可，並沒有特別地限定，舉例來說，例如，可以使 用由叢毛單胞菌株（Cowa/wonai1 iesiosierowe)(以下簡稱爲 ct)、紅呈球菌（Λ/ζοί/ococcMS er少 i/iro/7〇/z’5·，以下簡稱爲 Re)、 戈登氏菌（<^〇"^〇”/0!〇/^：(3«&0〃<3«1?)(以下簡稱爲63)、戈登氏 去硫菌（Gori/ow/α cans)(以下簡稱爲Gd)、糞產驗桿 菌（J/ca/igewes ?;?.)(以下簡稱爲 As)、土地戈登氏菌 (Gorc/odo! ierrae)(以下簡稱爲 Gt)、銅綠假單胞菌 (: aerwgiwosa)(以下簡稱爲Pa)、ί貪氏不動桿菌 (jc/neioftacier _/wn/Z)(以下簡稱爲 Aj)、金橙黃微小桿菌 {Exiguob acter ium aur ant i a cum) {\>X T M IS M Ea)中之任一種 構成之單一菌株油分解優勢菌種；也可以是由上述群組中所 選出的至少一種以上之菌種，依一定比例組合而成的混合油 分解優勢菌種。依照本發明之生物復育方法，從降低微生物 取得' 培養成本來看，油分解優勢菌較宜使用只有一種微生 物所構成之單一菌種；然而，從加速生物復育速率減低復育 I ’ 成本的觀點來看，則較宜是使用一種以上微生物所組合而成 之混合菌種。另一方面，從所欲處理污染成分之複雜程度來 看，更宜是由二種以上菌株構成之多菌株混合油分解優勢菌 種。舉例來說，當以受到油品污染土壤及/或地下水做爲生 物復育對象時，可以使用單一菌株之油分解優勢菌種；然而 在考慮油污成分之複雜程度的情況下，則可以使用一種以上 菌株構成之二菌株混合油分解優勢菌種混、三或以上菌株構 成的多菌株合油分解優勢菌種。 200909590 又’適用於本發明生物復育方法之二菌株混合油分解優 勢園種之組合’舉例來說’例如’其可以是叢毛單胞菌株（ct) 與紅呈球困（Re)之組合、戈登氏菌（Ga)與戈登氏去硫菌（Gd) 之組合、叢毛單胞菌株（Ct)和戈登氏菌（Ga)之組合、紅呈球 囷（Re)和戈登氏去硫菌（Gd)之組合等。再者，適用於本發明 生物復育方法之三菌株混合油分解優勢菌種之組合，舉例來 說，例如，其可以是叢毛單胞菌株（ct)與紅呈球菌（Re)和戈 登氏菌（Ga)之組合、叢毛單胞菌株（ct)與紅呈球菌（Re)和戈 登氏去硫囷（Gd)之組合、紅呈球菌（Re)與戈登氏菌（Ga)和戈 登氏去硫菌（Gd>之組合、叢毛單胞菌株（ct)與戈登氏菌（Ga) 和銅綠假單胞菌（Pa)之組合等；更具體爲叢毛單胞菌株（ct) 與戈登氏菌（Ga)和銅綠假單胞菌cc_rsi之 組合等。另外，適用於本發明生物復育方法之四菌株混合油 分解優勢菌種之組合’舉例來說，例如，其可以是叢毛單胞 菌株（Ct)與紅呈球菌（Re)和戈登氏菌（Qa)及戈登氏去硫菌 (Gd)之組合、紅呈球菌（Re)與戈登氏菌（〇8)和寝氏不動桿菌 (A j)及金橙黃微小桿菌（E a)之組合等。 又’油分解優勢菌種之取得來源並沒有特別地限定，例 如’可以使用由歐、美、日等各國生物寄存中心或我國食品 科學工業硏究所生物菌種寄存中心分讓或購得之菌種，或者 可以由市售購得’也可以自行利用生物方法分離純化而得之 菌種。另外’上述多菌株混合油分解優勢菌種之各菌株的構 成比例也沒有特別地限定，舉例來說，例如，可以按照生物 復育期限、污染成分種類、濃度等而適當選定。 200909590 例如，在由叢毛單胞菌株（Ct)、戈登氏菌（Ga)、金橙黃 微小桿菌（Ea)組合而成的三菌株油分解優勢菌種之情況 下，各菌株之構成比例’以菌落數比計通常是控制在C t : Ga: Ed = 1〜1000: 1〜1000: 1〜1000之範圍，較宜是控 制在1〜100 : 1〜1〇〇 : 1〜100之範圍，更宜是控制在1〜 10: 1〜10: 1〜10之範圍。 又，當使用由叢毛單胞菌株（ct)、紅呈球菌（Re)、戈登 氏菌（Ga)、戈登氏去硫菌（Gd)組合而成的四菌株油分解優勢 菌種的情況，各菌株之構成比例，以菌落數比計通常是控制 在 Ct: Re: Ga: Gd = 1 〜1000: 1 〜1000: 1 〜1000: 1 〜1000 之範圍，較宜是控制在1〜1 00 : 1〜100 : 1〜1〇〇:1〜1〇〇 之範圍，更宜是控制在1〜1〇: 1〜1〇: 1〜10: 1〜1〇之範 圍。 又，在使用由紅呈球菌（Re)、戈登氏菌（Ga)、瓊氏不動 桿菌（Aj)、金橙黃微小桿菌（Ea)組合而成的四菌株油分解優 勢菌種之情況下，則各菌株之構成比例’以菌落數比計通常 是控制在 Re: Ga: Aj: Ea = 1 〜1000: 1 〜1〇00: 1 〜1〇〇〇: 1〜1000之範圍，較宜是控制在1〜100: 1〜100: 1〜100: 1〜100之範圍’更宜是控制在1〜10: 1〜1(): 1〜10: 1 〜1 0之範圍。 再者，油分解優勢菌種之添加濃度通常是界於108〜 1 012 CFU/毫升菌液之範圍，較宜是104〜108 CFU/克乾土重’ 更宜是105〜108CFU/克乾土重，特佳爲1〇6〜1〇9 CFU/克乾 土重。又，油生物分解促進劑之添加量通常是每公斤乾土壤 -18- 200909590 重及/或地下水施用10〜50毫克，較宜是每立方公尺添加 20〜100毫克之範圍，更宜是每立方公尺添加50〜200毫克 之範圍，特佳爲每公斤乾土壤重添加100〜250毫克。 另外，按照本發明第二觀點之油污染土壤及/或地下水 之生物復育方法，其中油分解優勢菌與營養源之添加方式並 沒有特別地限定，可以從公知技術中視需要選取任何一種添 加方法來使用，舉例來說，例如，其可以使用連續加入或分 批加入油分解優勢菌與營養源，也可以是在充分混合油污染 V 土壤與油分解優勢菌之後，再加入營養源。又，按照本發明 第二觀點而視情況需要而添加的油生物分解促進劑之添加 方式，沒有特別地限定，可以從公知技術中視需要選取任何 一種添加方法來使用，舉例來說，例如，其可以將油生物分 解促進劑和油生物分解促進劑與營養源一起添加，也可以在 加入油生物分解促進劑與營養源之後，再添加油生物分解促 進劑。又且，也可以分開添加油生物分解促進劑、油生物分 解促進劑與營養源。 ί 另外，按照本發明之其他觀點，本發明又提供一種可以 有效地提昇復育速率、並具有簡便、經濟、快速定性定量分 析等特點之土壤及/或地下水之生物復育方法。此生物復方 法包括導入系統化環境分子生物復育技術（SEMBT)，以偵測 原生菌群社會結構並藉以決定整治油污染土壤及/或地下水 之最佳解決方案；以及在生物復育期間，藉由實施快速且精 確定性定量之分子生物技術來檢測各種油類分解菌種之消 長程度。 -19- 200909590 更具體而言，本發明係提供一種油類污染土壤及/或地 下水之生物復育方法，其係包括分析土壤及/或地下水微生 物之社會族群結構，決定實施生物優植法或生物促進法或結 合兩法之生物復育策略，以多樣性微生物晶片之分生檢测技 術監測目標微生物之菌相種類，及以生物計數法估算油分解 微生物，利用GC-FID分析目標污染物濃度降解效果。 關於土壤及/或地下水微生物之社會族群結構之分析方 法，雖然並沒有特別地限定，然而，如前述，通常是採用分 f 生技術變性凝膠梯度電泳分析法（D G G E法），以展示土壤地 下水中現存多樣性菌種族群結構，較宜是採用微矩陣生物晶 片（Microarray Biochip法），以快速鑑定油分解菌分解優勢 菌種學名，更宜是採用末端限制酵素片段長度多型性檢測法 (TRFLP),以鑑定追蹤生物復育進行期程中各時程當時土壤 與地下水中既存多樣性菌種之消長現況及菌數增減槪況，最 宜是採用選殖基因定序資料庫分析法（Cloneing sequence lib ra ly)，已總括分類統計土壤與地下水中既存多樣性菌群 ί 之豐富度及分類相似度。 舉例來說，例如，可以採用於中華民國發明專利申請案 第931 1 0828號所揭示之分生技術變性凝膠梯度電泳分析方 法。其次，關於油類污染土壤及/或地下水之微生物監測， 較宜是採用可監測3 0種以上之目標微生物的微生物晶片。 又，微生晶片之製作方法，舉例來說，例如，可以參照於中 華民國發明專利公開第200636073號所揭示之方法製作而 得。更詳細地來說，例如，可以設計具有油分解優勢菌特異 -20 - 200909590 性的寡核苷酸探針，利用細菌16S - 23S核糖體核酸基 內轉錄區（intergenic space ’丨T S)序列，分析存在菌種間高 留的專一性序列，將數段寡核苷酸點漬於基材上製成生 片’利用D N A雜合反應來檢測土壤及/或地下水中是否 生分解優勢菌組之存在。又，由於中華民國發明專利公 931 1 0828號及1 997年應用與環境微生物期刊發表尾 制酶片段長度多樣圖譜（T-RFLP)方法之開示全部皆可 照’因而該專利文獻內容亦納入本說明書中。 另外’關於在本說明上所記載的全部文獻、專利申 技術規格，雖然各個文獻、專利申請及技術規格因参照 入’然而與具體且個別記載的情況程度上相同者，亦因 而納入本說明書中。 以下’例示本發明之實施例，但是本發明不僅限定 等實施例而已。 【實施例A1〜A3】 於實施例A 1〜A 3中，按照以下之作法分別對於已 燃料油污染土壤進行生物復育。 又，在進行生物復育前土壤中油污染成分含量，經 GC-FID分析法，依照環保署公告之NIEAS703.60B標 法，測定結果其中所含之柴油污染成分的含量爲2801 (mg/kg soil)。 又，該受油污染土地之質地係爲壤質性砂土，其基 理化物性質詳如下表所示。 因間 度保 物晶 有油 開第 端限 供參 請及 而納 参照 於此 受到 利用 準方 TPH 本物 200909590 分析項目 測量値 備註 有機質（％) 2.6 濕化法 質地 壤質性沙土 砂粒 S a n d (%) 90.4 玢粒 Silt(%) 9.6 粘粒 C I a y (% ) 0 陽離子交換能力（c mol/kg) 4.1 全氮N ( % ) 0.052 鐵 Fe(mg/kg) 723 0.1 N HCI Olsen 磷（P mg/kg) 5.7 交換性鈣 C a (m g / k g) 2941 交換性鎂 M g (m g / k g) 47 ~~ — 其次，使用參照中華民國發明專利公開第200636073 號所揭示之方法製作微生物晶片’設計如表彳所示之具有油 分解優勢菌特異性的寡核苷酸探針，利用細菌1 6 S - 2 3 S核 糖體核酸基因間內轉錄區（intergenic space，ITS)序列，分析存 在菌種間高度保留的專一性序列，將數段寡核苷酸點漬於基 材上製成生物晶片，利用DNA雜合反應來檢測土壤及/或地 下水中是否有油生分解優勢菌組之存在，結果詳如下表所 示0 -22 - 200909590 表1 菌株名稱 探針名稱 Comamonas testosteroni Ctesl Ctes2 Ctes3 Ctes4 Ctes5 Gordonia alkanivorans Galk2-1 GalkJG-1 GalkJG-1 Gordonia desulfuricans Gdes4 Gdesl3 Gdesl5 Gordonia nitida Gnitl-1 Gnitl-2 Gnit3-1 Pseudomonas sp. PI P2 P3 Pseudomonas aeruginosa Paer2 Paer6 Pseudomonas fluorescens Pflul-l Pflul-2 Pflu2-1 Pflu2-2 Pseudomonas mendocina Pmenl Pseudomonas putida Pputl-l Pput2-2 Pput3-1 Pput3-2 Pput3-3 Pput4-1 Pseudomonas stutzeri Pstul-2 Pstu2-1 Pstu2-2 Pstu2-3 Pstu3-2 Pstu3-3 Rhodococcus erythropolis Reryl3 Reryl4 -23 - 200909590 生物復育土壤原生油分解菌群： •1 2200909590 IX. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for not only effectively improving the rate of re-cultivation, but also significantly shortening the treatment of oil-contaminated soil and/or groundwater, or converting pollutants into harmless The bioremediation period required for the ingredients, and the biological remediation method of oil-contaminated soil and/or groundwater with simple 'economical and rapid characteristics'. More specifically, the present invention relates to an oily biodegradation accelerator for oil-contaminated soil and/or groundwater, a nutrient source for microbial growth, and, if necessary, an oil-decomposing dominant species; A method of biological remediation of an oil-contaminated soil and/or groundwater that is thoroughly mixed to decompose and remove oil-contaminated components. [Prior Art] As a result of the extensive use of petroleum-based petrochemicals by humans, it has caused great impact and damage to the natural environment, especially soils caused by leakage from old gas stations, oil tanks, oil pipelines, etc. Problems such as groundwater pollution and environmental damage are becoming more serious. For example, according to the implementation report of the “Groundwater Potential Pollution Source Survey Project” in the 1990s, the soil and groundwater near at least 19 gas stations and six large petrochemical storage tanks in Taiwan are currently in a high pollution potential. Cases of environmental damage caused by pollution sources and other accidents are also emerging. Therefore, for Taiwan, which has a narrow population, there is no doubt that it is increasingly urgent and urgent to solve the problem of pollution of soil and groundwater near gas stations and oil storage tanks. For the prevention and environmental remediation of such environmental pollution, the most economical and effective method in the available 200909590 method is to use environmental molecular biotechnology and applied biological treatment technology to solve the environment such as soil and groundwater. The method of pollution has been widely studied in the scientific community in recent years and has been widely applied in the fields of environmental remediation and environmental remediation. Further, it is known that a part of microorganisms present in soil and/or groundwater have the ability to decompose or degrade petroleum-like pollutants such as gasoline and diesel oil (Hess, et al., 1996; Boopathy, 2003; Widrig and Manning, Jr., 1995; Richard and Vogel, 1999; Rahman et al., 2002; Margesin and Schinner, 2001). Therefore, in recent years, some experts and scholars have suggested that these microorganisms can be used to decompose petroleum-based pollutants into harmless compounds to solve petroleum-based chemicals in soil and/or groundwater caused by crude oil, gasoline, diesel oil, heavy oil, etc. The problem of pollution (Balba, et al, 1998; Banat, 1995; Banat, 2000; Rahman, et al, 2001). However, unfortunately, since most of the polluting components of petrochemical oils are hydrophobic compounds such as aliphatic or aromatic, it is difficult for a limited number and quantity of microorganisms to decompose them into harmless compounds, even though some of the protobacteria existing in nature can Degradation of petroleum-based pollutants, its degradation rate is also very slow, so it is not suitable for the actual use of contaminated soil and / or groundwater rejuvenation work. In order to solve environmental pollution problems such as soil and/or groundwater, in recent years, experts and scholars in various countries have recommended the application of biotechnology to solve the problem, that is, through the so-called environmental biotechnology technology to carry out biological re-education, such as 'by using planting external advantages Microbial bioaugmentation or biostimulation with additives to promote microbial activity, providing optimal environmental conditions for microbes during biological treatment to maintain microbial activity 200909590 and accelerate micro-organisms The substance decomposes the rate of contaminated components to achieve the goal of biological rehabilitation and restoration of the environmental landscape. For example, in U.S. Patent No. 5,436,160, it is proposed to use a protobacteria in a soil contaminated with a hydrocarbon compound to enhance the protobacteria by adding a surfactant and providing optimum microbial growth environmental conditions. The rate at which the hydrocarbon compounds of the contaminated soil are decomposed, and the method of promoting the biological remediation efficiency of the contaminated soil. However, the method does not describe the morphology and analytical methods of the native flora in the contaminated soil. Because of the failure to grasp the dynamics of the protozoa population present in the contaminated soil of the hydrocarbon compound, and without follow-up tracking and monitoring, The use of this method for the re-cultivation of soil will have the problem of not completely recovering the polluted environment. As described above, since in the field of bioremediation technology of soil and/or groundwater, although the method of adding a surfactant to the biological re-cultivation of the soil using the protobacteria has been proposed, since the distribution pattern of the native flora is not analyzed and Without follow-up tracking and monitoring, the rate of bioremediation is not ideal, and its application efficiency is limited. Therefore, improvement is strongly expected. That is to say, the current situation is: 尙 has not developed a biological remediation method that can accurately improve the re-education rate with simple, economical, and rapid characteristics of soil and/or groundwater; therefore, it is urgent to develop a Effectively solve the problem of slow decomposition rate of native bacteria present in soil and/or groundwater, and can easily improve the remediation rate of contaminated soil and/or groundwater, simple, economical, and rapid soil and/or groundwater organisms. Rehabilitation method. SUMMARY OF THE INVENTION [Problems to be Solved by the Invention] 200909590 Therefore, in view of the above circumstances, the object of the present invention is to solve the problem that the rate of biological re-cultivation of soil and/or groundwater is not ideal, and to provide an effective one. The method of biological re-cultivation of soil and/or groundwater with simple, economical, rapid and accurate characteristics for improving the re-cultivation rate. In more detail, the present invention provides a bioremediation schedule that not only can effectively increase the rate of re-cultivation, but also can significantly shorten the process for treating oil-contaminated soil and/or groundwater or converting pollutants into harmless components. And it is a simple, economical, rapid and other biological remediation method for oil contaminated soil and/or groundwater. Ensure the feasibility of environmental quality purification and reuse of contaminated soil and/or groundwater. [Means for Solving the Problem] In order to achieve the above-mentioned object, the inventors of the present invention deliberately conducted a review and developed a technique for utilizing a systematic environmental molecular biological remediation technique, and found that not only can the reproductive rate be effectively improved, but It can significantly shorten the biological re-feeding period required to treat oil-contaminated soil and/or groundwater, or convert pollutants into harmless components, and has oily soil and/or groundwater that is simple, economical, and fast. Biological rehabilitation methods. That is to say, the above-mentioned problems can be solved by the following viewpoints and constituent means of the present invention. First, the idea of the present invention is to evaluate the effectiveness of biobacteria and biosurfactant in remediating oil contaminated sites by a mold factory test, and to establish a bioremediation technical strategy to implement bioremediation. Another aspect of the present invention is a systematic bioremediation method combining bio-implantation and/or bio-promoting methods, and by using microbial wafers to accurately detect biological strains, to monitor the remediation effect and shorten the pollution treatment of polluters. The time and cost required will enable it to use the site early to continue its career development. 200909590 In addition, another aspect of the present invention is to select any suitable method or both methods from the biological promotion method of the bio-optimal method according to the precise detection of the microbial social group structure of the contaminated site to achieve savings. Remediation of funds and the goal of rapidly reducing pollutants. In addition, the other aspects of the present invention are to develop a primer and a gene probe which are required for qualitative and quantitative micro-matrix biochips and real-time polymerase chain reaction, and are adjusted to be most suitable for oil pollution decomposition. The conditions for the analysis and monitoring of the bacteria are established, and a biochip monitoring method that can be applied to the bioremediation technology of the oil pollution site is established, thereby monitoring the diversified target microorganisms and achieving the purpose of remediation. Based on the above, the present invention provides at least one systemic bioremediation technique for combining oil-contaminated soil and/or groundwater in combination with bio-implantation and bio-promoting methods, and micro-matrix bio-wafer detection technology for various strains; and providing a high Systematic bioremediation technology for the treatment of oil-contaminated soils and/or groundwater with excellent efficiency, cost savings and excellent refining time. That is, providing a social group structure for analyzing soil and/or groundwater microorganisms by using a sub-technical denaturing gel gradient electrophoresis, and deciding to implement a bio-optimal or bio-promoting method or a combination of two methods to separate the microbial wafers The detection technology monitors the diverse microbial species of the target microorganism, estimates the number of microbial bacteria in the oil by biometric method, and analyzes the degradation effect of the target pollutant concentration by GC-FID. The systemic environmental molecular biology of the oil contaminated soil and/or groundwater is treated by GC-FID. Rehabilitation method. Thus, the present invention provides a biological re-cultivation method which utilizes systematic environmental molecular biotechnology to treat oil-contaminated soil and/or groundwater, or converts -10-200909590 oil pollutants into harmless components, including: analysis The social group structure of microorganisms in contaminated soil and/or groundwater determines the biological remediation strategy 'monitoring the species type of target microorganisms' and analyzing the degradation effects of target pollutants. Further, the present invention also provides a biological re-cultivation method for treating oil-contaminated soil and/or groundwater or converting oil contaminants into harmless components, which includes: oil contaminated soil and/or The oil biodegradation accelerator is put into the groundwater, wherein the oil biodegradation accelerator is composed of rhamnolipid; and the nutrient source required for the growth of the microorganism is added as needed. In addition, the present invention also provides a biological re-cultivation method for treating oil-contaminated soil and/or groundwater, or converting oil pollutants into harmless components, including: putting oil into oil-contaminated soil and/or groundwater. Decompose dominant species and add nutrients needed for microbial growth as needed. [Effect of the Invention] According to the present invention, as described in the following description, a biological and/or groundwater biological remediation method having characteristics of simplicity, economy, rapidity, and accuracy can be used, and a microbial social group of contaminated sites can be used. Structural morphology and genetic characteristics, by adding oil-decomposing accelerators and/or oil-decomposing bacteria with excellent efficacy to oil-contaminated soil and/or groundwater, effectively improving the use of organisms for soil and/or groundwater bioremediation A satisfactory rejuvenation effect is obtained at a rate. [Embodiment] [Best Mode for Carrying Out the Invention] Hereinafter, the present invention will be described in further detail. In addition, in the description of the present specification -11-200909590, the description of "so-called" (number 値 1) ~ (number 値 2)" is included in "(number 値 1) or above ~ (number 値 2) or All of the following are within the scope of the following. "Oil contamination" refers to petroleum-based compounds, such as diesel (C10~C28), hydrocarbons containing more than 20 carbons that are difficult to decompose and non-volatile, and heavy fuel (C28~ C40) The meaning of oil pollution. For the convenience of description, the following is a biological re-cultivation method for oil-contaminated soil according to the present invention, which is specifically treated with diesel-contaminated soil and/or groundwater; however, those skilled in the art can It will be readily understood that the method of the present invention is not limited to use in such diesel contaminated soil and/or groundwater, and that the methods described in the specification of the present invention can also be applied to soil contaminated with other biodegradable components other than diesel. / or the biological rehabilitation of groundwater. First, the process of the so-called systemized environmental molecular biological remediation technology in the present invention mainly includes monitoring, analysis, diagnosis and control procedures; wherein the monitoring program uses a microarray biochip and a sub-technical denaturing gel gradient. Electrophoretic analysis (DGGE) is used to conduct microbial population surveys, and the analysis procedure is to determine whether there is an oil-decomposing microorganism in the re-cultivation environment. Moreover, the diagnostic procedure is to use the results of the analysis as a decision criterion for the re-education program. The control program can be divided into two ways: biological addition and biological promotion. During the re-education period, molecular biotechnology was used to monitor changes in microbial growth and changes in pollutant concentrations. Moreover, according to the present invention, before the biological re-cultivation, it is preferred to carry out the soil environment primary flora analysis test, and it is more suitable to carry out the primary flora analysis test with the diversity micro-matrix biocrystal-12-200909590 piece, however Soil and/or groundwater bioremediation can be carried out using the method of the present invention in the absence of oil contaminated soil and/or native flora analysis in groundwater. Moreover, according to the first aspect of the present invention, the biological remediation method of the oil-contaminated soil of the present invention generally utilizes the original bacteria originally stored in the oil-contaminated soil for biological re-cultivation, and is usually mainly included in the oil-contaminated soil. The oil biodegradation accelerator and the nutrient source required for the growth of the microorganism are added, and it is preferable to mix well. In addition, it is also possible to carry out primary flora analysis experiments with a variety of micro-matrix biochips as needed, especially for the analysis of various soil properties, including pollution components and pollution, carbon to nitrogen ratio, temperature, moisture content. , gas content such as pH, oxygen and carbon dioxide. According to the first aspect of the present invention, although it is not required to carry out a virgin flora analysis test in oil-contaminated soil, it is preferred to carry out a primordial flora analysis test before deciding to use the molecular biological re-cultivation method of the present invention, preferably Primary flora analysis experiments were performed on a variety of biochips. The method for the virgin flora analysis test used in the present invention may be a well-known or conventional method, and is not particularly limited. For example, the sub-technical denaturing gel gradient electrophoresis analysis method (DGGE method) disclosed in the Republic of China Patent Application No. 931 1 0828 can be used to analyze the social groups of microorganisms present in a polluted environment. structure. Further, since the disclosure of the Patent Application No. 931 1 08 28 of the Republic of China is for reference, the contents of the patent document are also incorporated in the present specification. In addition, the type and component of the oil biodegradation accelerator used in the present invention are not particularly limited as long as it is a substance which can promote the biodegradation of the contaminated component at the speed and efficiency of 200909590, and is preferably a non-toxic substance. A compound or substance that is biodegradable and does not cause accumulation of environmental organisms. In the present invention, for example, an oil biodegradation accelerator containing a main component of rhamnose can be used. Since rhamnolipids contain a compound of a chemical structure of 1 or 2 carbon sugars and a long-chain fat, and are a glycolipid surfactant, and have high solubility for oils, they are advantageous for the utilization of oil bacteria. Therefore, from the viewpoint of the effectiveness of biological rejuvenation, the present invention uses a biosurfactant containing rhamnolipid. Γ - Secondly, the source of rhamnolipid is not particularly limited, and may be a compound produced by a conventional chemical synthesis method, or may be a biological method by the method of the Republic of China Patent Publication No. 270534, using Xanthomonas oryzae (Psewctomonas_ aenvg/nosa) is produced under appropriate fermentation conditions, and the cells are centrifuged and purified. Since the disclosure of the invention of the Republic of China No. I 270 534 is hereby incorporated by reference in its entirety, it is hereby incorporated by reference. Further, in the oil bio-ingredients used in the biological re-cultivation method of the present invention, in addition to the rhamnolipid which is a main component, 尙 contains other sub-components. For example, the oil biopromoting agent of the present invention may contain other components such as glucose. Further, regarding the addition amount of the oil decomposition accelerator, the addition amount of the oil biodegradation accelerator is usually 5,000 kg of dry soil weight and/or groundwater application, preferably 20 to 100 mg per metre, and more It should be added in a range of ~2 00 mg per cubic meter. It is especially good to add 100-mg per kg of dry soil. The decomposition of the constituents with a composition of five is preferably achieved by the Chinese green leave and then by the patent content. The decomposition of the organism is 5 K 每 250 -14 - 200909590 per cubic J. Furthermore, the oil pollution according to the first aspect of the present invention The method for bioremediation of soil and/or groundwater, wherein the manner in which the oil biodegradation accelerator and the nutrient source are added is not particularly limited, and any one of the addition methods may be selected from the known art, for example, for example, The oil biodegradation accelerator and the nutrient source may be added continuously or in batches, or after the oil contaminated soil and the oil biodegradation accelerator are sufficiently mixed, the nutrient source may be added. Further, the form of the oil biodegradation accelerator is not particularly limited. For example, according to the present invention, various forms of oil biodegradation accelerators such as a solution, an emulsion, a solid granule, a powder, a capsule, and a tablet can be added to the biological complex. Among the soil and/or groundwater of the breeding object. Further, in the case of a liquid state such as a solution or an emulsion, other additives such as a surfactant, an emulsifier, and a labeling agent may be further contained. In addition, the nutrient source required for the growth of the microorganism of the present invention is mainly a nitrogen nutrient source, a phosphorus nutrient source, a carbon source, etc.; and the nutrient source which can be used for the growth of the microorganism of the present invention is not particularly limited, for example, for example, It may be urea, potassium nitrate, ammonium nitrate, ammonium phosphate, potassium phosphate, potassium hydrogen phosphate, sodium hydrogen phosphate, sodium phosphate, and the like. Further, in general, the amount of the nutrient source required for the growth of microorganisms is usually controlled so that the ratio of the ratio of C:N:P in the soil becomes 100: 1: 0.1 to 100: 10: 5. Secondly, according to the second aspect of the present invention, the present invention provides an oil-decomposing dominant species in an oil-contaminated soil, and adds a nutrient source for the growth of the microorganism; and, if necessary, an oil containing rhamnolipid as a main component A biodegradation accelerator; and a method of biological remediation in which oil is contaminated with soil and/or groundwater. Further, it can be used in the oil-decomposing dominant species of the present invention, and 200909590 is not particularly limited as long as it has or can promote the speed and efficiency of biodegrading the contaminating component, and for example, it can be used, for example, Monochacilis strain (Cowa/wonai1 iesiosierowe) (hereinafter referred to as ct), Rhodococcus erythropolis (Λ/ζοί/ococcMS er less i/iro/7〇/z'5·, hereinafter referred to as Re), Gordonia (<^〇"^〇"/0!〇/^:(3«&0〃<3«1?) (hereinafter referred to as 63), Gordon's desulfurization bacteria (Gori/ow/α Cans) (hereinafter referred to as Gd), human bacillus (J/ca/igewes?;?.) (hereinafter referred to as As), and Gord/odo! ierrae (hereinafter referred to as Gt), Pseudomonas aeruginosa (: aerwgiwosa) (hereinafter referred to as Pa), Acinetobacter baumannii (jc/neioftacier _/wn/Z) (hereinafter referred to as Aj), P. triticum {Exiguob acter ium aur ant ia cum a single strain of oil decomposition dominant species consisting of any one of {\>XTM IS M Ea); or at least one species selected from the above group, according to a certain ratio The combined mixed oil decomposes the dominant species. According to the biological re-cultivation method of the present invention, from the viewpoint of reducing the microorganism's 'cultivation cost, the oil-decomposing dominant bacteria preferably uses a single species composed of only one microorganism; however, From the standpoint of speeding up the rate of re-remediation to reduce the cost of re-cultivation, it is better to use a mixture of more than one kind of microorganisms. On the other hand, from the complexity of the contaminated components to be treated, It is suitable to use a mixture of two or more strains to isolate the dominant strains of oil. For example, when oil-contaminated soil and/or groundwater is used as a biological re-cultivation object, a single strain of oil-decomposing dominant bacteria can be used. However, in consideration of the complexity of the oil component, it is possible to use the two strains composed of one or more strains to mix the oil to decompose the dominant species, and the three strains or more of the strains to isolate the dominant strains of the oil. 200909590 A combination of two strains of mixed oil decomposition dominant gardens suitable for use in the biological remediation method of the present invention, for example, Combination of Pleurotus sinensis strain (ct) with red ball (Re), combination of Gordon's bacteria (Ga) with Gordon's desulfurization bacteria (Gd), C. sylvestris (Ct) and Gordon a combination of bacteria (Ga), a combination of red globules (Re) and Gordon's desulfurization bacteria (Gd), etc. Further, the three strains suitable for the biological remediation method of the present invention are mixed oil-decomposing dominant species. Combinations, for example, may be, for example, a combination of C. pluvialis (ct) with Rhodococcus erythropolis (Re) and Gordonia (Ga), Pleurotus tuberculosis (ct) and Rhodococcus erythropolis ( Combination of Re) and Gordon's desulfurization (Gd), combination of Rhodococcus erythropolis (Re) with Gordonia (Ga) and Gordon's desulfurization (Gd), Pleurotus tuberculosis (ct) Combination with Gordonia (Ga) and Pseudomonas aeruginosa (Pa); more specifically, the combination of C. sylvestris (ct) with Gordonia (Ga) and Pseudomonas aeruginosa cc_rsi . Further, a combination of the four strains of the mixed oil-decomposing dominant species suitable for use in the biological re-cultivation method of the present invention is exemplified, for example, as C. sylvestris (Ct) and Rhodococcus erythropolis (Re) and Gordon's Combination of bacteria (Qa) and Gordon's desulfurization bacteria (Gd), Rhodococcus erythropolis (Re) and Gordonia (〇8) and Acinetobacter baumannii (A j) and P. oxysporum (E a) Combinations, etc. Moreover, the source of the 'oil decomposition dominant strains' is not particularly limited. For example, 'can be used by the bio-storage centers of Europe, the United States, Japan, etc. or the bio-species storage center of the China Food Science and Industry Research Institute. The strain is either commercially available or can be isolated and purified by biological methods. Further, the ratio of the respective strains of the above-mentioned multi-strain mixed oil-dissolving dominant strains is not particularly limited, and for example, it can be appropriately selected according to the biological reproduction period, the type of the contaminated component, the concentration, and the like. 200909590 For example, in the case of the three strains of oil-decomposing dominant strains composed of the combination of C. sylvestris (Ct), Gordonia (Ga), and P. trichoenum (Ea), the composition ratio of each strain' The colony ratio is usually controlled in the range of C t : Ga: Ed = 1~1000: 1~1000: 1~1000, preferably controlled in the range of 1~100: 1~1〇〇: 1~100 It is more suitable to control the range of 1~10: 1~10: 1~10. In addition, when four strains of oil are used to decompose dominant species from the combination of C. pluvialis (ct), Rhodococcus erythropolis (Re), Gordonia (Ga), and Gordon's desulfurization (Gd) In the case, the proportion of each strain, in terms of colony ratio is usually controlled in Ct: Re: Ga: Gd = 1 ~ 1000: 1 ~ 1000: 1 ~ 1000: 1 ~ 1000 range, preferably controlled at 1 ~ 1 00 : 1~100 : 1~1〇〇: 1~1〇〇 range, more preferably controlled in 1~1〇: 1~1〇: 1~10: 1~1〇 range. Further, in the case of using four strains of oil-decomposing dominant species composed of a combination of Rhodococcus erythropolis (Re), Gordonia (Ga), Acinetobacter junii (Aj), and Polygonum aeruginosa (Ea), Then the composition ratio of each strain 'in terms of colony ratio is usually controlled in Re: Ga: Aj: Ea = 1 ~ 1000: 1 ~ 1〇00: 1 ~ 1〇〇〇: 1~1000 range, preferably Control in the range of 1~100: 1~100: 1~100: 1~100' is more suitable to control in the range of 1~10: 1~1(): 1~10: 1~1 0. Furthermore, the concentration of the oil-decomposing dominant species is usually in the range of 108 to 1 012 CFU/ml of bacterial liquid, preferably 104 to 108 CFU/g dry soil weight. More preferably 105 to 108 CFU/gram dry soil. Heavy, especially good for 1〇6~1〇9 CFU/g dry weight. Moreover, the amount of the oil biodegradation accelerator is usually from -18 to 200909590 per kg of dry soil and/or from 10 to 50 mg of groundwater, preferably 20 to 100 mg per cubic meter, more preferably per kg. Add a range of 50 to 200 mg for the cubic meter, and add 100 to 250 mg for each kilogram of dry soil. In addition, according to the second aspect of the present invention, the method for bioremediation of oil-contaminated soil and/or groundwater, wherein the manner in which the oil-decomposing dominant bacteria and the nutrient source are added is not particularly limited, and any one of the addition methods may be selected from known techniques. For example, it may be used by continuously adding or adding oil to decompose the dominant bacteria and the nutrient source in batches, or after fully mixing the oil-contaminated V soil and the oil-decomposing dominant bacteria, and then adding the nutrient source. Further, the manner of adding the oil biodegradation accelerator added as the case requires, according to the second aspect of the present invention, is not particularly limited, and any one of the addition methods may be selected from the known art, for example, for example, The oil biodegradation accelerator and the oil biodegradation accelerator may be added together with the nutrient source, or an oil biodegradation accelerator may be added after the oil biodegradation accelerator and the nutrient source are added. Further, an oil biodegradation accelerator, an oil biodegradation accelerator, and a nutrient source may be separately added. In addition, according to other aspects of the present invention, the present invention further provides a biological re-cultivation method for soil and/or groundwater which can effectively improve the re-cultivation rate and has the characteristics of simple, economical, rapid qualitative and quantitative analysis. The bioreplication method includes introduction of a systematic environmental molecular biological remediation technique (SEMBT) to detect the social structure of the native flora and thereby determine the optimal solution for remediation of oil contaminated soil and/or groundwater; and during biological rehabilitation, The degree of growth and decline of various oil-decomposing strains is detected by performing rapid and precise quantitative molecular biotechnology. -19- 200909590 More specifically, the present invention provides a method for biological remediation of oil-contaminated soil and/or groundwater, which comprises analyzing the social group structure of soil and/or groundwater microorganisms, and determining the implementation of bio-optimal methods or The bio-promoting method or the bioremediation strategy combining the two methods, the species of the target microorganism is monitored by the multi-division detection technology of the microbial wafer, and the oil-decomposing microorganism is estimated by the biometric method, and the target pollutant is analyzed by GC-FID. Concentration degradation effect. Although the analysis method of the social group structure of soil and/or groundwater microorganisms is not particularly limited, as described above, the degraded gel gradient electrophoresis analysis (DGGE method) is usually used to display soil groundwater. The ethnic group structure of the existing diversity bacteria is preferably a microarray biochip (Microarray Biochip method) to quickly identify the dominant species of the oil-decomposing bacteria decomposition, and it is more suitable to use the end-limiting enzyme fragment length polymorphism detection method ( TRFLP), in order to identify the current state of growth and the number of bacteria in the soil and groundwater at the time of the bioremediation process, and the number of bacteria is increased or decreased. (Cloneing sequence lib ra ly), the classification and statistics of the richness and classification similarity of the existing diversity flora in soil and groundwater have been summarized. For example, a method of degenerative gel gradient gel electrophoresis analysis disclosed in the Republic of China Patent Application No. 931 1 0828 can be used. Secondly, for microbial monitoring of oil-contaminated soils and/or groundwater, it is preferred to use microbial wafers that can monitor more than 30 target microorganisms. Further, a method of producing a micro-battery can be produced, for example, by the method disclosed in the Chinese Patent Publication No. 200636073. In more detail, for example, an oligonucleotide probe having an oil-decomposing dominant bacteria specific -20 - 200909590 can be designed, using a bacterial 16S-23S ribosomal nucleic acid transcribed region (intergenic space '丨TS) sequence, Analyze the sequence of high specificity between strains, and spot several segments of oligonucleotides on the substrate to make green slices. Use DNA hybridization to detect whether the dominant bacteria in the soil and/or groundwater are biodegradable. presence. Moreover, since the Republic of China Invention Patent No. 931 1 0828 and the Journal of Applied and Environmental Microbiology published the publication of the T-RFLP method for the length of the tail enzyme fragment, all the patents are also included. In the manual. In addition, as regards all the documents and patent application specifications described in the present specification, each document, patent application, and technical specification are incorporated in the present specification by reference to the same extent as the specific and individual case. . The following examples illustrate the invention, but the invention is not limited to the embodiments. [Examples A1 to A3] In Examples A 1 to A 3, biological re-cultivation of fuel-contaminated soil was carried out in accordance with the following procedures. In addition, the content of oil-contaminated components in the soil before bioremediation is determined by GC-FID analysis according to the NIEAS703.60B standard published by the Environmental Protection Agency. The content of diesel pollution components contained in the measurement results is 2801 (mg/kg soil). ). Moreover, the texture of the oil-contaminated land is loamy sand, and the basic properties of the soil are shown in the following table. Because of the inter-preservation of the crystal, the first end of the oil is available for reference and the reference is used here. It is measured by the use of the quasi-party TPH. 200909590 Analytical project 値Remarks organic matter (%) 2.6 Wet-processed loamy sand S and %) 90.4 bismuth Silt (%) 9.6 Cosmid CI ay (%) 0 cation exchange capacity (c mol / kg) 4.1 total nitrogen N (%) 0.052 iron Fe (mg / kg) 723 0.1 N HCI Olsen phosphorus (P Mg/kg) 5.7 Exchangeable calcium C a (mg / kg) 2941 Exchangeable magnesium M g (mg / kg) 47 ~~ - Next, the microbial wafer is produced by the method disclosed in the Chinese Patent Publication No. 200636073. Design an oligonucleotide probe with the specificity of the oil-decomposing dominant bacteria as shown in Table ,, and analyze the existing bacteria by using the intergenic space (ITS) sequence of the bacterial 1 6 S - 2 3 S ribosomal nucleic acid gene. A highly-retained sequence of specificity in which a plurality of oligonucleotides are spotted on a substrate to form a biochip, and DNA heterozygous reaction is used to detect the presence of a dominant group of oil-decomposing bacteria in the soil and/or groundwater. The results are detailed in the following table. 0 -22 - 200909590 Table 1 Strain name Probe name Comamonas testosteroni Ctesl Ctes2 Ctes3 Ctes4 Ctes5 Gordonia alkanivorans Galk2-1 GalkJG-1 GalkJG-1 Gordonia desulfuricans Gdes4 Gdesl3 Gdesl5 Gordonia nitida Gnitl-1 Gnitl-2 Gnit3-1 Pseudomonas sp. PI P2 P3 Pseudomonas aeruginosa Paer2 Paer6 Pseudomonas fluorescens Pflul-l Pflul-2 Pflu2-1 Pflu2-2 Pseudomonas mendocina Pmenl Pseudomonas putida Pputl-l Pput2-2 Pput3-1 Pput3-2 Pput3-3 Pput4-1 Pseudomonas stutzeri Pstul-2 Pstu2- 1 Pstu2-2 Pstu2-3 Pstu3-2 Pstu3-3 Rhodococcus erythropolis Reryl3 Reryl4 -23 - 200909590 Bioremediation of soil primary oil decomposition bacteria: • 1 2
參》. Φ 7 春參.纛黪參_参》. Φ 7 Chunshen. 纛黪参_
9wm pc # 1. Acinetobacter sp. 2. A. junii 3. Godonia alkanivorans 4. G. desulfuricans 5. Pseudomonas sp. 6. P. aeruginosa 7. P. putida 8. Ralstonia picketti 9. R. erythropolis 接著，使用 MgSO4-7H2O(0_5 g/L)、K2HP〇4(5 g/L)、 KH2P04(5 g/L)、NH4N03(1.3 g/L)、CaCI2(0_2 g/L)、洋菜 粉（13 g/L)及柴油（2% v/v)組成之培養基培養分解柴油微 生物菌落數（Diesel aerobic bacteria);使用濃度爲 8 g/L 之營養（Nutrient Broth，其組成參照 Marshall ed_, 1 993)與 濃度爲13 g/L之合成媒介（synthetic medium，其組成參照 Gallego et a丨.，200 1 )培養總異營菌菌落數原生油分解菌 數。然後，在1〇 g之待生物復育土壤中加入2〇 g之 0.8%NaCI溶液及一滴Tween 80，震盪5分鐘沉澱10分鐘 後取上層液進行序列稀釋，取〇 . 1 mI稀釋液以塗抹棒塗碟 (ΝΙΕΑ E203.54B)。將培養皿至於30°C恆溫箱避光培養72 小時，以菌數塗碟法（Plate count)測定待生物復育土壤中原 生菌數。經計數後，待生物復育土壤中含有約1.〇x1〇6CFU/ 克土壤之土壤原生菌含量。 另外，在實施例A1中不添加油生物分解促進劑，而於 實施例A2、A3之待生物復育土壤中，另外加入以鼠李糖脂 -24 - 200909590 表2 rd '''''---- 3污染成分含量((TPH(mg/kg soil)) 進行生物復育初期 生物復育14天後 生物復育49天後 生物復育84¾ 實施例A1 2,801 1.914 1,885 1,816 實施例A2 2,801 1,035 980 ------ 632 實施例A3 2,801 2,209 1,954 ———_ 1,790 【實施例B1〜B3】 於實施例B 1〜B 3中，按照以下之作法分別對於已受到 柴油污染土壤進行生物復育。又，在進行生物復育前土壤中 油污染成分含量，經利用GC-FID分析法，依照環保署公告 之NIEAS703.60B標準方法，測定結果其中所含之柴油污染 成分的含量爲 32 76 TPH (mg/kg soil)。 又，該受油污染土地之質地係爲砂質性土壤，其基本物 理化物性質詳如下表所示。9wm pc # 1. Acinetobacter sp. 2. A. junii 3. Godonia alkanivorans 4. G. desulfuricans 5. Pseudomonas sp. 6. P. aeruginosa 7. P. putida 8. Ralstonia picketti 9. R. erythropolis Next, use MgSO4 -7H2O (0_5 g/L), K2HP〇4 (5 g/L), KH2P04 (5 g/L), NH4N03 (1.3 g/L), CaCI2 (0_2 g/L), acacia powder (13 g/ L) and diesel (2% v/v) medium cultured to disintegrate diesel microbial colonies (Diesel aerobic bacteria); use a concentration of 8 g / L of nutrients (Nutrient Broth, its composition with reference to Marshall ed_, 1 993) and concentration The number of primary oil-decomposing bacteria was determined by the total number of colonies of 13% of the synthetic medium (synthetic medium, the composition of which is referred to Gallego et a., 2001). Then, add 1〇g of 0.8% NaCI solution and 1 drop of Tween 80 to 1〇g of the biologically replenished soil, shake for 5 minutes and precipitate for 10 minutes, then take the supernatant to serially dilute and take 〇. 1 mI dilution to smear. Bar coated dish (ΝΙΕΑ E203.54B). The culture dishes were incubated in a 30 ° C incubator for 72 hours in the dark, and the number of native bacteria in the soil to be reconstituted was determined by Plate Count. After counting, the soil to be re-fed contains about 1.〇x1〇6 CFU/g soil soil native bacteria content. In addition, in the example A1, no oil biodegradation accelerator was added, and in the soil to be reconstituted in Examples A2 and A3, rhamnolipid-24 - 200909590 was additionally added. Table 2 rd '''''- --- 3 Contaminant content ((TPH(mg/kg soil)) Bioremediation after 14 days of bioremediation, bioremediation after 49 days, bioremediation 843⁄4 Example A1 2,801 1.914 1,885 1,816 Example A2 2,801 1,035 980 ------ 632 Example A3 2,801 2,209 1,954 ——— _ 1,790 [Examples B1 to B3] In Examples B 1 to B 3, biological reconstitution of soil contaminated with diesel oil was respectively carried out according to the following method. In addition, the content of oil-contaminated components in the soil before bioremediation is determined by GC-FID analysis according to the NIEAS703.60B standard method announced by the Environmental Protection Agency. The content of diesel pollutants contained in the results is 32 76 TPH. (mg/kg soil). The texture of the oil-contaminated land is sandy soil, and its basic physical properties are shown in the following table.
分析項目 測値 備註 質地 沙質壤土 砂粒 S a n d (% ) 64 玢粒 Silt(%) 22 粘粒 C I a y ( %) 14 陽離子交換能力（c mol/kg) 5.4 全氮N(%) 0.04 鐵 Fe(mg/kg) 723 0.1 N HCI Olsen 磷 P (mg/kg) 5.7 交換性鈣 Ca (mg/kg) 1686 交換性鎂 Mg (mg/kg) 76 -26 - 200909590 其次，使用和實施例A1〜A3同樣之微生物晶片’測定 待生物復育土壤中原生菌，結果詳如下表所示。 l.Acinetobacter sp. 2.Pseudomonas sp. 1 m / ™ • ·.. · ·. . ·. / 應. ·. . ^ ； .，· " 'μ·· 接著，和實施例A1〜A3同樣的作法’以菌數塗碟法 (Plate count)測定待生物復育土壤中原生菌數。經計數後’ 待生物復育土壤中含有約1.〇x1〇6 CFU/克土壤之土壤原生 菌含量。 另外，在實施例B1中不添加油生物分解促進劑，而於 實施例B2、B3之待生物復育土壤中，另外加入以鼠李糖脂 爲主成分之油生物分解促進劑，藉由土壤中所存在之原生菌 進行生物復育。其中，實施例B2、B3之油生物分解促進劑 的添加量分別爲10、80(mg/kg dry soil)。 然後，再利用 GC-FID分析法，依照環保署公告之 NIEAS703.60B標準方法，分別對於實施例B1〜B3測定經 進行生物復育第21天、第56天與第84天於土壤中所殘留 的油污染成分含量測定，結果如表3所示。 -27 - 200909590 表3 油 污染成分含量((TPH-d(mg/kg soil) )__ 進行生物復育初期 生物復育21天後 生物復育56天後 生物復育84天後 實施例B1 3,276 2,891 1,820 1.780 實施例B2 3,276 2,168 348 173 實施例B3 3,276 2,436 552 247 【實施例C1〜C4】 於實施例C 1〜C4中，按照以下之作法分別對於已嚴重 受到柴油污染土壤（實施例C1〜C2)、燃料油污染土壤（實施 例C3〜C4)進行生物復育。又，在進行生物復育前土壤中油 污染成分含量，經利用GC-FID分析法，依照環保署公告之 NIEAS703.60B標準方法，測定結果其中所含之油污染成分 的含量，實施例C1〜C4分別爲16962、16385、17820、 1 0826TPH (mg/kg soil)。 又，該受油污染土地之質地係爲壤質性砂土，其基本物 理化物性質詳如下表所示。 分析項目 測値 備註 質地 壤質沙土 砂粒 S a n d (%) 95.2 玢粒 S i 11 (%) 4.8 粘粒 Clay(%) 0 陽離子交換能力（c mol/kg) 3.7 全氮N(%) 0.03 鐵 Fe(mg/kg) 61 0 0.1 N HCI Olsen 磷 P (%) 0.046 交換性鈣 C a (m g / k g > 251 0 交換性鎂 Mg (mg/kg) 45 -28 - 200909590 其次，使用和實施例A 1〜A3同樣之微生物晶片，測定 待生物復育土壤中原生菌，結果詳如下表所示。 1. Acinetobacter sp. 2. A. junii 3. G alkanivorans 4. G desulfuricans 5. Pseudomonas sp. 6. R aeruginosa 7. Ralstonia picketti 8· R· erythropolis •1 5 2 6 3 8 pc 參 f 接著，和實施例A1〜A3同樣的作法，以菌數塗碟法 (Plate count)測定待生物復育土壤中原生菌數。經計數後， 待生物復育土壤中含有約1.0x106 CFU/克土壤之土壤原生 菌含量。 另外，在實施例C1、C3中不添加油分解優勢菌，而於實施 例C2、C4之待生物復育土壤中則加入106 CFU/g soil之油分 解優勢菌，藉由土壤中所存在之原生菌及油分解優勢菌進行 生物復育。其中，所添加的油分解優勢菌種係由紅呈球菌 (Re)、戈登氏菌（Ga)、瓊氏不動桿菌（Aj)、金橙黃微小桿菌 (Ea)組合而成，以菌落數比計算，各菌株之構成比例爲Re : Ga:Aj:Ea = 1: 1:1:1。 然後，再利用 G C - F丨D分析法，依照環保署公告之 NIEAS703.60B標準方法，分別對於實施例B1〜B3測定經 進行生物復育第7天 '第42天與第69天於土壤中所殘留的 油污染成分含量測定，結果表4所示。 -29- 200909590 表4 油污染成分含量((TPH-d(mg/kg soil)) 進行生物復育初期 生物復育7天後 生物復育42天後 生物復育69天後 實施例C1 16,962 11,061 3,923 3,864 實施例C2 16,385 9,133 3,365 3,346 油污染成分含量((TPH(mg/kg soil)) 實施例C3 17,820 13,693 6,431 6,930 實施例C4, 10,826 12,902 5,648 7,998 【實施例D1〜D3】 於實施例D1〜D3中，使用於不同高度（10cm、30cm、 5〇cm)處預留1吋孔徑之土壤氣體採集管、且於槽體底部設 ^ 有排水口以利收集滲出水之約1 m長x1 m寬x50 cm的生物 復育槽體中，分別於按照以下所說明之作法，分別在已受到 某輸送柴油管線所洩漏的柴油污染之酸鹼値爲6.9、陽離子 交換能力（CEC)爲5.4 cmol/kg、總體積爲約〇.5m3的土壤進 行生物復育。 又，在進行生物復育前土壤中油污染成分含量，經利用 GC-FID分析法，依照環保署公告之NIEAS703.60B標準方 法，測定結果其中所含之柴油污染成分的含量，實施例D1 〜D3 分另!J-爲 6557、4549、4770 TPH-d (mg/kg soil)。 又，該受油污染土地之質地係爲砂質性土壤，其基本物 理化物性質詳如下表所示。 -30- 200909590 分析項目 測値 備註 質地 沙質壤土 ------- 砂粒 S a n d (%) 64 __---- 玢粒 Silt(%) 22 _______ 粘粒 Clay(%) 14 _______ 陽離子交換能力（c mol/kg) 5.4 ----- 全氮N(%) 0.04 — 鐵 Fe(mg/kg) 723 0.1 N HCI — Olsen 憐 P (mg/kg) 5.7 交換性鈣 C a ( m g / k g) 1686 ----- 交換性鎂 M g ( m g / k g ) 76 -—-1 其次，使用和實施例A 1〜A3同樣之微生物晶片’測定 待生物復育土壤中原生菌。接著，和實施例A1〜A3同樣的 作法，以菌數塗碟法（Plate count)測定待生物復育土壤中原 生菌數。經計數後，待生物復育土壤中含有約1 .0x1 〇6 〇FU/ 克土壤之土壤原生菌含量。 另外，在實施例D1中不添加油生物分解促進劑及油分 解優勢菌種，而於實施例D 2中僅添加油分解優勢菌種，於 實施例D3則只加入以鼠李糖脂爲主成分之油生物分解促進 劑來進行生物復育。其中，實施例D2之油分解優勢菌種係 由叢毛單胞菌株（Ct)、戈登氏菌（Ga)、金橙黃微小桿菌（Ea) 組合而成，各菌株之構成比例，以菌落數比計係爲C t : G a : Ed = 1 : 1 : 1。又，實施例D2之油分解優勢菌種之添加量爲 106 (CFU/g dry soil)，而在實施例D3中，油生物分解促進 劑的添加量爲10(mg/kg dry soil)。 200909590 然後，使用和實施例A1〜A3同樣之微生物晶片，測定 待生物復育土壤中原生菌，並利用GC-FID分析法，依照環 保署公告之NIEAS703.60B標準方法，分別對於實施例B1 〜B3測定經進行生物復育第21天、第56天與第84天於土 壤中所殘留的油污染成分含量進行測定，結果分別如表5、 表6所示。 表5Analytical project test remarks texture sandy loam sand S and (%) 64 bismuth Silt (%) 22 viscous CI ay (%) 14 cation exchange capacity (c mol / kg) 5.4 total nitrogen N (%) 0.04 iron Fe (mg/kg) 723 0.1 N HCI Olsen Phosphorus P (mg/kg) 5.7 Exchangeable Calcium Ca (mg/kg) 1686 Exchangeable Magnesium Mg (mg/kg) 76 -26 - 200909590 Next, use and Example A1~ The same microbial wafer of A3 'measures the protobacteria in the soil to be revived, and the results are shown in the following table. l.Acinetobacter sp. 2.Pseudomonas sp. 1 m / TM • ······························································· The method of 'Plate count' is used to determine the number of native bacteria in the soil to be reconstituted. After counting, the soil to be revived contains about 1. 〇x1〇6 CFU/g soil soil protobacteria. In addition, in the embodiment B1, the oil biodegradation accelerator is not added, and in the biological re-cultivation soil of the examples B2 and B3, an oil biodegradation accelerator containing rhamnolipid as a main component is additionally added, by the soil. The primary bacteria present in the organism are subjected to biological re-cultivation. The amounts of the oil biodegradation accelerators of Examples B2 and B3 were 10 and 80 (mg/kg dry soil), respectively. Then, using the GC-FID analysis method, according to the NIEAS703.60B standard method announced by the Environmental Protection Agency, the residuals in the soil on the 21st, 56th, and 84th day of the biological rehabilitation are measured for the examples B1 to B3, respectively. The oil contamination component content was determined, and the results are shown in Table 3. -27 - 200909590 Table 3 Contents of oil-contaminated components ((TPH-d(mg/kg soil) )__ After bio-remediation in the initial stage of biological re-cultivation, after biopsy for 56 days, after biological re-emergence for 84 days, Example B1 3,276 2,891 1,820 1.780 Example B2 3,276 2,168 348 173 Example B3 3,276 2,436 552 247 [Examples C1 to C4] In Examples C 1 to C4, soils contaminated with diesel were respectively subjected to the following practices (Example C1~ C2), fuel oil contaminated soil (Examples C3 to C4) for biological re-cultivation. In addition, the content of oil-contaminated components in the soil before bioremediation is determined by GC-FID analysis according to the NIEAS703.60B standard announced by the Environmental Protection Agency. The method and the content of the oil-contaminated component contained in the measurement result were respectively 16962, 16385, 17820, and 1 0826 TPH (mg/kg soil) in the examples C1 to C4. Further, the texture of the oil-contaminated land was loamy. The basic physical properties of sand are detailed in the following table. Analytical items test remarks texture loamy sand sand S and (%) 95.2 S S S i 11 (%) 4.8 Clay Clay (%) 0 cation exchange capacity ( c mol/kg) 3. 7 total nitrogen N (%) 0.03 iron Fe (mg / kg) 61 0 0.1 N HCI Olsen phosphorus P (%) 0.046 exchangeable calcium C a (mg / kg > 251 0 exchangeable magnesium Mg (mg / kg) 45 -28 - 200909590 Next, using the same microbial wafer as in Examples A 1 to A3, the primary bacteria in the soil to be re-fed were determined, and the results are shown in the following table: 1. Acinetobacter sp. 2. A. junii 3. G alkanivorans 4. G desulfuricans 5. Pseudomonas sp. 6. R aeruginosa 7. Ralstonia picketti 8· R· erythropolis • 1 5 2 6 3 8 pc 参 f Next, the same procedure as in Examples A1 to A3, using the number of bacteria (Plate count) Determines the number of native bacteria in the soil to be re-fostered. After counting, the soil to be bioremediated contains about 1.0x106 CFU/g soil soil protobacteria content. In addition, it is not added in Examples C1 and C3. The oil decomposes the dominant bacteria, and in the biological re-cultivation soils of Examples C2 and C4, 106 CFU/g soil oil is used to decompose the dominant bacteria, and the primary bacteria and the oil-decomposing dominant bacteria in the soil are used for biological re-cultivation. . Among them, the added oil decomposition dominant strains are composed of Rhodococcus erythropolis (Re), Gordonia (Ga), Acinetobacter junii (Aj), and Polygonum aeruginosa (Ea), with colony ratio The composition ratio of each strain was calculated as Re : Ga: Aj: Ea = 1: 1:1:1. Then, using the GC-F丨D analysis method, according to the NIEAS703.60B standard method announced by the Environmental Protection Agency, respectively, the samples B1 to B3 were tested for bioremediation on the 7th day 'the 42nd day and the 69th day in the soil. The content of the remaining oil contaminated components was measured, and the results are shown in Table 4. -29- 200909590 Table 4 Contents of oil-contaminated components ((TPH-d(mg/kg soil)) After bio-remediation in the initial stage of biological re-cultivation, bioremediation for 42 days after bioremediation for 69 days, Example C1 16,962 11,061 3,923 3,864 Example C2 16,385 9,133 3,365 3,346 Oil Contaminant Content (TPH (mg/kg soil)) Example C3 17,820 13,693 6,431 6,930 Example C4, 10,826 12,902 5,648 7,998 [Examples D1 to D3] In Example D1~ In D3, a soil gas collecting pipe with a hole diameter of 1 预留 is reserved at different heights (10cm, 30cm, 5〇cm), and a drain hole is provided at the bottom of the tank body to collect about 1 m long x1 m of the oozing water. In the bioremediation tank with a width of x50 cm, respectively, according to the method described below, the acid-base enthalpy contaminated by diesel fuel leaked from a diesel pipeline is 6.9, and the cation exchange capacity (CEC) is 5.4 cmol/ Biological re-cultivation of soil with a total volume of about 〇5 m3. Further, the content of oil-contaminated components in the soil before bioremediation is determined by GC-FID analysis according to the NIEAS703.60B standard method announced by the Environmental Protection Agency. The firewood contained in the result The content of the contaminated components, Examples D1 to D3 are separated! J- is 6557, 4549, 4770 TPH-d (mg/kg soil). Moreover, the texture of the oil-contaminated land is sandy soil, and its basic physics The properties of the compounds are detailed in the following table. -30- 200909590 Analytical project test remarks texture sandy loam ------- sand S and (%) 64 __---- Si grain Silt (%) 22 _______ cosmid Clay(%) 14 _______ Cation exchange capacity (c mol/kg) 5.4 ----- Total nitrogen N (%) 0.04 - Iron Fe (mg/kg) 723 0.1 N HCI — Olsen P (mg/kg) 5.7 Exchangeable calcium C a ( mg / kg) 1686 ----- exchangeable magnesium M g (mg / kg ) 76 --1 - Next, use the same microbial wafer as in Examples A 1 to A3 to determine the biological complex Protozoa in the soil. Then, in the same manner as in Examples A1 to A3, the number of protobacteria in the soil to be reconstituted was determined by Plate Count. After counting, the soil to be reconstituted was included in the soil. Soil native bacteria content of about 1.00x1 〇6 〇FU/g soil. In addition, in the example D1, the oil biodegradation accelerator and the oil-decomposing dominant species were not added, but in the example D 2, only the oil-decomposing dominant species was added, and in the example D3, only the rhamnolipid was added. The oil biodegradation accelerator of the component is used for biological re-cultivation. Among them, the oil-decomposing dominant strain of Example D2 is composed of C. elegans strain (Ct), Gordonia (Ga), and Phellodendron triticum (Ea), and the proportion of each strain is determined by the number of colonies. The ratio is C t : G a : Ed = 1 : 1 : 1. Further, the addition amount of the oil-decomposing dominant species of Example D2 was 106 (CFU/g dry soil), and in Example D3, the amount of the oil biodegradation accelerator added was 10 (mg/kg dry soil). 200909590 Then, using the same microbial wafer as in Examples A1 to A3, the primary bacteria in the soil to be re-fostered were determined, and the GC-FID analysis method was used according to the NIEAS703.60B standard method announced by the Environmental Protection Agency, respectively, for Example B1~ B3 The content of the oil-contaminated component remaining in the soil on the 21st, 56th, and 84th day after the biological re-cultivation was measured, and the results are shown in Table 5 and Table 6, respectively. table 5
Day 30 實 施 例 D1 LAcinetobacter sp. 2.Pseudomonas sp. LAcinetobacter sp. 2. Pseudomonas sp. LAcinetobacter sp. 2. Pseudomonas sp. 實施例 * —Day 30 Example D1 LAcinetobacter sp. 2. Pseudomonas sp. LAcinetobacter sp. 2. Pseudomonas sp. LAcinetobacter sp. 2. Pseudomonas sp. Example *
•«Mi V m 1. Acinetobacter sp. 2. Pseudomonas sp. 3. Pseudomonas putida 4. G alkJG-1 1. Pseudomonas sp. 2. Pseudomonas fluorescens 3· G alkJG-1 G. alkJG-2 G alk2-l 1. Pseudomonas sp. 2. G alkJG-1 G alkJG- 2 G. alk2-l 實施例• «Mi V m 1. Acinetobacter sp. 2. Pseudomonas sp. 3. Pseudomonas putida 4. G alkJG-1 1. Pseudomonas sp. 2. Pseudomonas fluorescens 3· G alkJG-1 G. alkJG-2 G alk2-l 1 Pseudomonas sp. 2. G alkJG-1 G alkJG-2 G. alk2-l Example
D 1. Pseudomonas sp. 1, Pseudomonas sp. 1. Pseudomonas sp. 200909590 表6 油污染成分含量((TPH-d(mg/kg soil)) 進行生物復育初期 生物復育7天後 生物復育14天後 生物復育30天後 實施例D1 6,557 6,809 2,711 5,601 實施例D2 4,549 3,656 972 1,063 實施例D3 4,770 4,891 3,539 2,843 【實施例E1〜E5】 於實施例E 1〜E 5中，按照以下之作法分別對於5 m X 5 m X 1 m區塊大小之已受到柴油污染土壤進行生物復育。 又，在進行生物復育前土壤中油污染成分含量，經利用 GC-FID分析法，依照環保署公告之NIEAS703.60B標準方 法測定結果，實施例E 1〜E 5中所含之柴油污染成分的含量 爲 9059、846、9242、10042、10279 TPH-d (mg/kg soil)。 又，該受油污染土地之質地係爲砂質性土壤，其基本物 理化物性質詳如下表所示。 -33 - 200909590 分析項目 測値 ---*~ 備註 有機碳（％) 2.6 濕化法 有機質（％) 4.6 濕化法 質地 粘壤土 砂粒 S a n d (% ) 22 玢粒 Silt(°/〇) 50 粘粒 C I a y (% ) 28 陽離子交換能力（c mol/kg) 9.1 全氮N(%) 0.097 鐵 Fe(mg/kg) 620 0.1 N HCI Brayl 磷 P (mg/kg) 4.1 交換性鈣 C a (m g / k g ) 2585 交換性鎂 M g (m g / k g ) 180 鹼度 C a 0 ( % ) 0.55 土壤原生菌含量 CFU/g soil 1.0x1 Ο8 其次’使用和實施例A 1〜A3同樣之微生物晶片，測定 待生物復育土壤中原生菌。接著，和實施例A1〜A3同樣的 I" 作法，以菌數塗碟法（Plate count)測定待生物復育土壤中原 生菌數。經計數後，待生物復育土壤中含有約1 .0x1 〇8 CFU/ 克土壤之土壤原生菌含量。 另外，於實施例E1〜E5中，除了在實施例E1中不添 加油生物分解促進劑、油分解優勢菌種及營養鹽以外’在實 施例E 2〜E 4則分別按照表7所示之劑量添加各種油分解優 勢菌、以鼠李糖脂（1 g/L)爲主成分之油生物分解促進劑 '營 養鹽。又，油分解優勢菌種係由叢毛單胞菌株 -34 - 200909590 testosterone )CC-CF3 ' |E Μ M {Rhodococcus erythropolis) CC-BCll ' 登氏菌（fl/A:am_v0ram·) CC-JG39、戈登氏去硫菌（Gorifom'a DSM44462組合而成，各菌株之構成比例，以菌落數 比計係爲 Ct: Re: Ga: Gd = 1: 1: 1: 1。 表7 油分解優勢菌 添加量 (CFU/ g dry soil) 油生物分解促進劑 添加量 (mg/kg dry soil) 營養鹽添加量 氮 (mg/ kg dry soil) 磷 (mg/ kg dry soil) 實施例E1 — 一 0 0 實施例E2 — 101·5(ΒΗ 媒質〉 10.8 12.5 實施例E3 — 3.5 0 0 實施例E4 106 一 0 0 實施例E5 106 3.5 0 0 註：B H 媒質之組成：MgS04-7H20 (0.2 g/L)、K2HP〇4 (1 g/L)、KH2P〇4 (1 g，L)、FeCI3(0.05g/L)、NH4N03(1 g/L)、CaCI2 (0.05 g/L) 然後，使用和實施例A1〜A3同樣之微生物晶片，測定 待生物復育土壤中原生菌，並利用GC-FID分析法，依照環 保署公告之NIEAS703.60B標準方法，分別對於實施例B1 〜B3測定經進行生物復育第14天、第59天與第98天於土 壤中所殘留的油污染成分含量進行測定，結果分別如表8、 表9所示。 -35 - 200909590 表8 \ 實施例E1 實施例EE2 實施例E3 生物 _ • 參 復育 Φ ， mmm • 初期 • • * # • • • # '參. • 參 春 • Φ C. testosteroni G. alkanivorans C. testosteroni G. alkanivorans G. desulfuricans G. alkanivorans G. desulfuricans Pseudomonas spp. G. desulfuricans Pseudomonas spp. P. aeruginosa Pseudomonas spp. P. aeruginosa P. stutzeri P. aeruginosa P. stutzeri R. erythropolis P. mendocina R. erythropolis R. picketti P. stutzeri R. picketti R. erythropolis R. picketti 生物 • •蓼 •. • •暴 m •.籌 復育 # •. # 14天 f,' ... Λ • 中期 5 • S m m ♦ • 益 · G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. mendocina P. stutzeri R. erythropolis R. picketti 生物 • 參 e Φ · 參 Φ 籲铋’ 復育 59天 ·. • 後期 • 0 · • • • · • • • «蠹 G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti -36 - 200909590 表8(續） 實施例E4 實施例E5 生物 t · · 龜邊 攀 • 着 •- 復育 初期 • • 參 • • 參 m % φ G. alkanivorans G. alkanivorans G. desulfuricans G. desulfuricans Pseudomonas spp. Pseudomonas spp. P. aeruginosa P. aeruginosa P. stutzeri P. stutzeri R. erythropolis R. erythropolis R. picketti R. picketti 生物 • •參 φ • 參.擊 φ 復育 • · 14天 • 中期 • 參 A' • • 響 ·· 參 魯 拳 G. alkanivorans G. alkanivorans G. desulfuricans G. desulfuricans Pseudomonas spp. Pseudomonas spp. P. aeruginosa P. aeruginosa P. mendocina P. stutzeri P. stutzeri R. erythropolis R. erythropolis R. picketti R. picketti 生物 • · # • 鲁奪 復育 • # • 59天 • # 後期 • 參 癱 鲁 • 參 • 嫌 · m G. alkanivorans G. alkanivorans G. desulfuricans G. desulfuricans Pseudomonas spp. Pseudomonas spp. P. aeruginosa P. aeruginosa P. mendocina P. stutzeri P. stutzeri R. erythropolis R. erythropolis R. picketti R. picketti -37 - 200909590 表9 油污染成分含量((TPH-d(mg/kg soil)) 進行生物復育初期 生物復育14天後 生物復育59天後 生物復育98天後 實施例Ε1 9,059 7,203 1,170 696 實施例Ε2 8,461 6,990 1,096 580 實施例Ε3 9,242 5,398 1,185 518 實施例Ε4 10,042 6,764 669 696 實施例Ε5 10,279 7,449 1,155 715 文中使用之紅呈球菌(Rhodococcus erythropolis) CC-BC11 是獲自新竹食品工業發展硏究所寄存編號BCRC910241，戈 登氏菌（Gor而mlWar) CC-JG39是獲自新竹食品工業發展 硏究所寄存編號BCRC910234。叢毛單胞菌株 ieWosierom·) CC-CF3、填氏不動桿菌CC-FH2 及金檀黃微小桿菌（五幻CC-LSH4-1、銅 綠假單胞菌（Piewi/omoniw aerwgiwosa) CC-RS1皆獲自國立中興大 學楊秋忠教授實驗室。此外，戈登氏去硫菌 desulfuricans) DSM44462 是購自 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany ° 由上述之表2、3、4、5、7、9內殘留於土壤中的油污 染成分含量之測定結果，可明白：依照本發明之生物復方 法，藉由導入系統化環境分子生物復育技術（S Ε Μ BT)，以偵 測原生菌群社會結構，藉以決定整治油污染土壤及/或地下 -38 - 200909590 水之最佳解決方案來進行生物復育，無論是利用生物優植 法、添加促進微生物活性之助劑之生物促進法、或者結合兩 種策略進行柴油污染土壤之生物復育，結果顯示實施例A 1 〜A3、實施例 B1〜B3、實施例C1〜C4、實施例D1〜D3 及實施例E1〜E5均可以在一年工程整治期限內有效地將土 壤中所殘留的柴油（TPH-d)或燃料油（TPH)量降至法規標準 1〇〇〇(毫克TPH/公斤土壤乾重）以下。 從而，依照本發明的話，可以提供一種融合分子生物、 微矩陣生物晶片等之整合型系統化環境分子生物監測與復 育技術，利用生物促進與生物優植原理並藉由分生監控策略 而整合成可顯著縮短污染物處理、復育時程之整合技術。同 時，根據本發明亦可以提供生態相容性極高之生物界面活性 助劑型的油油分解促進劑、及油降解效率極強之油分解優勢 菌。另外，本發明可以提供一種具有簡便、經濟、快速等特 點之土壤及/或地下水之生物復育方法，配合污染場址之微 生物社會群族結構形態，藉由將具有優異效能的油分解促進 劑及/或油分解菌，添加於油污染土 _及/或地下水中，而有 效地提昇利用生物來進行土壤及/或地下水之生物復育速率 而得到令人滿意之復育功效。 此外，雖然本發明已藉由上述詳細說明以及較佳實施 例來予以闡釋，本發明仍不應被解釋爲受以上例示所限 制，相對地，本發明實係涵蓋那些熟知此項技藝者依照本 發明說明書所揭示的技術內容與實施方式所衍生出的多種 -39- 200909590 在不偏離本發 範圍及發明說 皆應仍應屬於 等效變化例及與其他不同之修飾例。因此 明之精神與精義下，大凡依本發明申請專 明書內容所作之簡單的等效變化與修飾例 本發明所涵蓋之範圍。 【圖式簡單說明】 te /ns 【元件符號對照表】 -40 -D 1. Pseudomonas sp. 1, Pseudomonas sp. 1. Pseudomonas sp. 200909590 Table 6 Contents of oil-contaminated components ((TPH-d(mg/kg soil)) Bioremediation after 7 days of biological rehabilitation in the early stage of biological rehabilitation After days of bioremediation for 30 days, Example D1 6,557 6,809 2,711 5,601 Example D2 4,549 3,656 972 1,063 Example D3 4,770 4,891 3,539 2,843 [Examples E1 to E5] In Examples E 1 to E 5, according to the following procedure Bioremediation of diesel-contaminated soils for 5 m X 5 m X 1 m block size. Also, the content of oil-contaminated components in the soil before bioremediation is reported by the Environmental Protection Agency by GC-FID analysis. As a result of the NIEAS 703.60B standard method measurement, the contents of the diesel-contaminated components contained in Examples E 1 to E 5 were 9059, 846, 9242, 10042, and 10279 TPH-d (mg/kg soil). The texture of the contaminated land is sandy soil, and its basic physical properties are shown in the following table. -33 - 200909590 Analytical project test---*~ Remarks organic carbon (%) 2.6 Wet method organic matter (%) 4.6 Humidification method S and (% ) 22 Si Grain Silt (° / 〇) 50 Cosmid CI ay (%) 28 Cation exchange capacity (c mol / kg) 9.1 Total nitrogen N (%) 0.097 Iron Fe (mg / kg) 620 0.1 N HCI Brayl Phosphorus P (mg/kg) 4.1 Exchangeable calcium C a (mg / kg ) 2585 Exchangeable magnesium M g (mg / kg ) 180 Alkalinity C a 0 ( % ) 0.55 Soil native bacteria content CFU / g soil 1.0 X1 Ο8 Next, using the same microbial wafer as in Examples A 1 to A3, the protobacteria in the soil to be re-fed were determined. Then, the same I/quoting method as in Examples A1 to A3, the plate number method was used. The number of native bacteria in the soil to be re-conserved is determined. After counting, the soil to be reconstituted contains about 1.00×1 〇8 CFU/g of soil native bacteria content. In addition, in Examples E1 to E5, In Example E1, no oil biodegradation accelerator, oil-decomposing dominant species, and nutrient salt were added. In the examples E 2 to E 4, various oil-decomposing dominant bacteria were added at the doses shown in Table 7, respectively, and buckthorn was added. Glycolipid (1 g / L) as the main component of the oil biodegradation accelerator 'nutrient salt. Also, the dominant strain of oil decomposition is from the monoclonal strain -34 - 200909590 testosterone )CC-CF3 ' |E Μ M {Rhodococcus erythropolis) CC-BCll 'Deng's bacteria (fl/A:am_v0ram·) CC-JG39 Gordon's desulfurization bacteria (Gorifom'a DSM44462 combination, the composition ratio of each strain, based on the number of colonies is Ct: Re: Ga: Gd = 1: 1: 1: 1. Table 7 oil decomposition advantages CFU/ g dry soil Addition amount of oil biodegradation accelerator (mg/kg dry soil) Nutrient addition amount of nitrogen (mg/kg dry soil) Phosphorus (mg/kg dry soil) Example E1 - one 0 0 Example E2 - 101·5 (ΒΗ Medium > 10.8 12.5 Example E3 - 3.5 0 0 Example E4 106 A 0 0 Example E5 106 3.5 0 0 Note: Composition of BH medium: MgS04-7H20 (0.2 g/L ), K2HP〇4 (1 g/L), KH2P〇4 (1 g, L), FeCI3 (0.05 g/L), NH4N03 (1 g/L), CaCI2 (0.05 g/L), then used and implemented The microbial wafers of the same examples A1 to A3 were used to measure the protobacteria in the soil to be re-conserved, and the GC-FID analysis method was used to carry out the determination of the examples B1 to B3 according to the NIEAS703.60B standard method announced by the Environmental Protection Agency. The content of the oil-contaminated component remaining in the soil on the 14th day, the 59th day, and the 98th day of the re-cultivation was measured as shown in Table 8 and Table 9. -35 - 200909590 Table 8 \ Example E1 Example EE2 Example E3 Biology _ • Participation Φ, mmm • Initial • • * # • • • # '参. • 参春• Φ C. testosteroni G. alkanivorans C. testosteroni G. alkanivorans G. desulfuricans G. alkanivorans G Desulfuricans Pseudomonas spp. G. desulfuricans Pseudomonas spp. P. aeruginosa Pseudomonas spp. P. aeruginosa P. stutzeri P. aeruginosa P. stutzeri R. erythropolis P. mendocina R. erythropolis R. picketti P. stutzeri R. picketti R. erythropolis R. picketti Biology • •蓼•. • • violence m •. 养育育# •. # 14天f,' ... Λ • Mid 5 • S mm ♦ • Yi·G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. mendocina P. stutzeri R. erythropolis R. picketti Creatures • e e Φ · Φ Φ 铋 铋 'Recommendation 59 days ·. • Late • 0 · • • • • • • • «蠹G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti G. alkanivorans G. desulfuricans Pseudomonas spp. P. aeruginosa P. stutzeri R. erythropolis R. picketti -36 - 200909590 Table 8 (continued) Example E4 Example E5 Biot · · Turtle climbing • At the beginning of re-education • • 参 • • 参m % φ G. alkanivorans G. alkanivorans G. desulfuricans G. desulfuricans Pseudomonas spp. Pseudomonas spp. P. aeruginosa P. aeruginosa P. stutzeri P. stutzeri R. erythropolis R. erythropolis R. picketti R. Picketti Creature • • φ φ • 参. φ φ Reproduction • · 14 days • Mid • Participate in A' • • 响······················································································································································································· Erythropolis R. picketti R. picketti Biology • · # • 鲁夺复育 • # • 59天 • #后• 瘫 瘫 • • 参 • · · m G. alkanivorans G. alkanivorans G. desulfuricans G. desulfuricans Pseudomonas spp. Pseudomonas Spp. P. aeruginosa P. aeruginosa P. mendocina P. stutzeri P. stutzeri R. erythropolis R. erythropolis R. picketti R. picketti -37 - 200909590 Table 9 Oil Contaminant Content ((TPH-d(mg/kg soil) After the biological re-inflation for 14 days, the biological re-cultivation after 59 days, after the biological re-cultivation for 98 days, the example Ε1 9,059 7,203 1,170 696 Example 8,2 8,461 6,990 1,096 580 Example 9,3 9,242 5,398 1,185 518 Example Ε4 10,042 6,764 669 696 Example Ε 5 10,279 7,449 1,155 715 Red Rhodococcus erythro (Rhodococcus erythro) Polis CC-BC11 is the number BCRC910241 obtained from the Hsinchu Food Industry Development Research Institute, and Gordon's bacteria (Gor and mlWar) CC-JG39 is obtained from the Hsinchu Food Industry Development Research Institute registered number BCRC910234. C. elegans strain IEWosierom·) CC-CF3, Acinetobacter baumannii CC-FH2 and P. sinensis (five phantom CC-LSH4-1, P. aeruginosa (Piewi/omoniw aerwgiwosa) CC-RS1 From the laboratory of Professor Yang Qiuzhong of National Chung Hsing University. In addition, the Desulfuricans of Gordon's desulfuricans is purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany ° from Tables 2, 3, 4, 5, 7, 9 above. As a result of measuring the content of the oil-contaminated component remaining in the soil, it can be understood that the biological complex method according to the present invention detects the native flora society by introducing a systematic environmental molecular biological remediation technology (S Ε Μ BT) Structure to determine the best solution for remediation of oil-contaminated soils and/or underground -38 - 200909590 for bioremediation, whether using bio-optimal methods, bio-promoting methods that add auxiliaries that promote microbial activity, or Two strategies for bioremediation of diesel contaminated soil showed the results of Examples A 1 to A3, Examples B1 to B3, Examples C1 to C4, Examples D1 to D3, and Examples E1 to E5. Can soil in the remaining diesel (TPH-d) or fuel oil (TPH) will be reduced to an amount effective to regulatory standards 1〇〇〇 within the period of one year renovation project (mg TPH / kg soil dry weight) or less. Thus, according to the present invention, it is possible to provide an integrated systemic environmental molecular biological monitoring and re-cultivation technique for merging molecular organisms, micromatrix biochips, etc., utilizing the principles of bio-promoting and bio-optimal planting and integrating by a sub-monitoring strategy Cheng can significantly shorten the integration technology of pollutant treatment and re-cultivation time. At the same time, according to the present invention, it is also possible to provide an oil-oil decomposition accelerator having a highly biocompatible organic interface active type and an oil decomposition-producing bacteria having an extremely high oil degradation efficiency. In addition, the present invention can provide a biological and remediation method for soil and/or groundwater which is simple, economical, and rapid, and cooperates with the microbial social group structure of the contaminated site, and has an excellent oil decomposition accelerator. And / or oil-decomposing bacteria, added to oil-contaminated soil _ and / or groundwater, and effectively improve the use of organisms for soil and / or groundwater bioremediation rate to obtain satisfactory re-efficiency. In addition, the present invention is not limited by the above description, and the present invention should not be construed as being limited by the above description. The technical content and the embodiments disclosed in the present specification are intended to be equivalent to variations and modifications of the inventions without departing from the scope of the invention and the invention. Therefore, in light of the spirit and essence of the present invention, the scope of the present invention is as simple as the equivalent changes and modifications of the present invention. [Simple diagram of the diagram] te / ns [component symbol comparison table] -40 -