| 摘要: | 4-t-octylphenol (4-t-OP) 是廣泛使用的非離子表面活性劑 (辛基酚聚乙氧基化物) 的常見生物轉化產物. 然而, 與已廣泛用於許多工業領域 (包括紡織, 石油, 造紙和塑料工業) 的化合物相比, 4-t-OP 在環境中更持久, 普遍存在並具有雌激素活性. 微生物的生物降解是最重要的自然過程之一, 可以影響水生和陸地環境中污染物去除的速率. 4-t-OP 的降解途徑和其在細菌和白腐菌中酵素的分子研究已有很多文獻. 相比之下, 沒有研究調查非木質素分解真菌和酵母菌對於用 4-t-OP 作唯一碳源之降解潛力, 其降解機制仍然很大程度上未知.因此, 本研究旨在從污水處理廠和塑料工業廢物樣品中分離降解 4-t-OP 之非木質素分解真
菌和酵母, 並探索所涉及的降解途徑.通過使用 4-t-OP 作為唯一碳源和能源的培養技術, 從台北的污水處理廠中分離出具有降解 4-t-OP 能力的三種酵母菌種. 顯微鏡觀察和 ITS 和 LSU rRNA 基因序列的分子鑑定顯 示這些菌株分屬於兩個屬 , 命 名為 Candida rugopelliculosa RRKY5, Galactomyces
candidum RRK17 和 G. candidum RRK22. 在初步篩選中, 使用了不同的基質包括 4-t-OP, 4-
t-NP, OPEOn, NPEOn, 苯酚和異辛烷來研究分離的菌株的生長性質. 發現菌株 RRKY5 能利
用了所有測試的基質並且比其它菌株生長得更快. 在有無葡萄糖基質 (0.05％) 的情況下, 通
過分離的酵母菌種進行 4-t-OP 的生物降解的比較研究. HPLC 分析的結果表明, 無論有無葡
萄糖 (0.05％) 都會發生 4-t-OP 降解. 在 24 天後, 在含有或不含右旋糖的培養基中 93％或
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95％ 的 4-t-OP 會被 RRKY5 菌株降解, 而在兩種白色念珠菌菌株中降解較低. 這些結果表
明, RRKY5 是比其他酵母物種更好的候選物. 因此選擇菌株 RRKY5 用於進一步研究.
菌株 RRKY5 具有廣泛的生存基質範圍並且能夠利用各種支鏈烷基酚 (AP). 有趣的
是, 在掃描電子顯微鏡中顯示, 在 4-t-OP 存在下菌株 RRKY5 形成節孢子, 而在沒有 4-t-OP
的葡萄糖存在下, 菌株保持在出芽酵母樣形式, 暗示節孢子的產生可能是使用 4-t-OP 的指
標. 在使用不同的溫度, pH 和 4-t-OP 濃度的生長和生物降解實驗的結果顯示, 菌株 RRKY5
降解 4-t-OP 的最佳條件為 30℃, pH5.0, 初始 4-t-OP 濃度為 30mg L-1. 在這些條件下, 最大
生物降解速率常數為 0.107d-1, 相當於 9.6 d 的最小半衰期. 利用液相色譜法-質譜聯用檢測
和表徵降解過程中的不同代謝物. 基於所鑑定到的代謝物，酵母菌株 RRKY5 可以通過支
鏈烷基側鍊和芳香環裂解途徑降解 4-t-OP.
另一方面，通過使用具有 4-t-OP 的最小鹽培養基 (MMSM) 作為唯一的碳源, 從台
北污水處理廠的樣品中分離出 14 種非木質素分解的真菌. 根據 ITS 序列的分子鑑定, 這些
菌 株屬於如 Aspergillus (2 spp.), Fusarium (6 spp.), Nectria (2 spp.), Pseudallescheria,
Scedosporium 和 Trichoderma (2 spp.). 利用在不同基質作為唯一碳源的培養基上進行初始
篩選。發現除了 Nectria (2 種), Pseudallescheria 和 Scedosporium 以外, 剩餘 10 種菌株具有
在多種基質的培養基上有效生長. 比較了各個菌株液體培養基中降解 4-t-OP 的有效性. 14
天後, 6 種 Fusarium 菌株中的 3 種, 4-t-OP 的降解最高 (>70％), 其次是其他 Fusarium 菌株,
在 Aspergillus 和 Trichoderma 的兩種菌株中最低。由於 Fusarium sp. RRK20 擁有最有效的
4-t-OP 降解能力, 因此選擇該菌株進行進一步研究.
基於 tef-1α基因序列的分析, RRK20 進一步鑑定為 Fusarium falciforme RRK20. 通
過中心複合設計和響應表面方法評價和優化參數 (例如 pH, 溫度和乾重) 之間的影響和相
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互作用. 降解效率受溫度和乾重顯著的影響. 發現最佳值為 pH 6.5, 初始接種物密度為 0.6
gL-1, 溫度為 28 ℃. 預測值與實驗值具有令人滿意的相關性, 係數測定 (R2) 為 0.9788. 統
計模型進一步驗證了在優化條件下的後續實驗. 評價葡萄糖濃度對 RRK20 降解 4-t-OP 的
影響. 結果表明, 通過在相對低的劑量下加入葡萄糖可以加速 4-t-OP 降解. 此外, 通過質譜
儀研究 4-t-OP 的真菌代謝途徑. 基於所鑑定到的代謝物, 菌株 RRK20 通過兩種不同的機制
降解 4-t-OP, 包括烷基支鏈側鏈氧化和芳環羥基化. 代謝物通過烷基側鏈進行進一步降解,
隨後芳環裂解.
結論, 這是第一個以酵母和非木質素分解真菌降解 4-t-OP 的研究. 本研究還證明了
這些菌株對於 4-t-OP 降解的最佳條件以提高生物降解效率. 添加營養物也可以增加 4-t-OP
的降解效率. 酵母和非木質素分解真菌降解 4-t-OP 的途徑不同於目前已知的細菌和真菌的
降解途徑.;4-t-Octylphenol (4-t-OP) is a common biotransformation product of the widely used nonionic
surfactants, octylphenol polyethoxylates (OPEOn). However, compared to the parent compound that has been extensively used in many industrial sectors including textile, petroleum,
paper, and plastic industries, 4-t-OP is known to be more persistent, ubiquitous in the environment, and estrogenically active. Microbial biodegradation is one of the most important natural processes which can influence the fate and removal of pollutants in both aquatic and
terrestrial environments. Degradation pathways of 4-t-OP and their molecular studies of enzymes
in bacteria and white rot fungi have been well documented. By contrast, no study has
investigated the potential of non-ligninolytic fungi and yeasts for degradation of 4-t-OP used as
the sole carbon source and its degradation mechanisms remain largely unknown. Therefore, this
study aims to isolate 4-t-OP degrading non-ligninolytic fungi and yeasts from a sewage treatment plant and plastic industry waste samples and to explore the degradation pathways involved.
Three yeast species, with the ability to degrade 4-t-OP, were isolated from a sewage
treatment plant in Taipei by enrichment culture technique using 4-t-OP as the sole carbon and energy source. Microscopic observation and molecular identification by ITS and LSU rRNA gene sequences revealed that the isolates belonged to two genera and were designated as Candida rugopelliculosa RRKY5, Galactomyces candidum RRK17, and G. candidum RRK22.
For preliminary screening, growth properties of the isolated strains were explored using different substrates including 4-t-OP, 4-t-nonylphenol (4-t-NP), octylphenol polyethoxylates (OPEOn),
nonylphenol polyethoxylates (NPEOn), phenol, and isooctane. It was found that strain RRKY5
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utilized all the tested substrates and grew faster than the others. Comparative investigation on biodegradation of 4-t-OP both in the presence and absence of the co-substrate dextrose (0.05%)
was carried out by the isolated yeast species. Results of HPLC analysis indicated that 4-t-OP
degradation took place both in the presence and absence of dextrose (0.05%); after 24 d, 93% or 95% of 4-t-OP was degraded by C. rugopelliculosa RRKY5 in media with or without dextrose,
whereas degradation was lower in the two strains of G. candidum. These results indicate that the C. rugopelliculosa RRKY5 is a better candidate than the other yeast species in degrading 4-t-OP
in liquid culture. The strain RRKY5 was chosen for further studies to address the kinetics and degradation mechanism of 4-t-OP.
C. rugopelliculosa RRKY5 was tested with various alkylphenols and their derivatives including 4-methylphenol, bisphenol A (BPA), 4-ethylphenol (4-EP), 4-t-butylphenol (4-t-BP), 4-t-OP, 4-t-NP, isooctane, and phenol. Strain RRKY5 had a broad substrate range and was
capable of utilizing various branched chain alkylphenols (APs). Interestingly, morphological analysis using scanning electron microscopy revealed that the strain RRKY5 in the presence of
4-t-OP formed arthroconidia, whereas the strain remained in the budding yeast-like form in the presence of glucose without 4-t-OP, implicating that arthroconidium production might be an
indicator for the utilization of 4-t-OP. Results of growth and biodegradation experiments with varying temperature, pH, and 4-t-OP concentrations showed that the optimum conditions for 4-t-
OP degradation by strain RRKY5 were 30 oC, pH 5.0, and an initial 4-tert-OP concentration of
30 mg L-1. Under these conditions, the maximum biodegradation rate constant was 0.107 d-1
equivalent to a minimum half-life of 9.6 d. Liquid chromatography-hybrid mass spectrometry
was used to detect and characterize different metabolites during the degradation. Based on the
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identified metabolites, the yeast strain RRKY5 can degrade 4-t-OP via both branched alkyl side
chain and aromatic ring cleavage pathways.
On the other hand, 14 non-ligninolytic fungi were isolated from soil, water, and sludge
samples from the sewage treatment plant in Taipei and plastic industry waste in Taoyuan, by using the modified minimal salt medium (MMSM) with 4-t-OP as the sole carbon source. According to the molecular identification by ITS sequences, the isolates belonged to taxa such as Aspergillus (2 spp.), Fusarium (6 spp.), Nectria (2 spp.), Pseudallescheria, Scedosporium, and
Trichoderma (2 spp.). Initial screening was conducted based on the growth properties on solid media with different substrates including 4-t-OP, 4-t-NP, OPEOn, NPEOn, estrone, 17β-estradiol
and 17α-ethnylestradiol as the sole carbon source. It was found that 10 strains, except the Nectria
(2 spp.), Pseudallescheria, and Scedosporium, grew effectively on media with a wide variety of
substrates. The effectiveness for the degradation of 4-t-OP in liquid media by the individual fungal isolates was compared. After 14 days, the degradation of 4-t-OP was the highest (>70%) by three of the six strains of Fusarium, followed by the other Fusarium strains and the lowest in
the two strains of Aspergillus and Trichoderma. Given that the most effective 4-t-OP degradation
was observed in Fusarium sp. RRK20, this strain was chosen for further studies.
Based on the analysis of the tef-1α gene sequence, FSSC RRK20 was further identified as Fusarium falciforme RRK20. The effects and the interactions between the parameters such as pH, temperature, and dry weight, were evaluated and optimized by a central composite design and a response surface methodology. The degradation efficiency was significantly affected by the temperature and dry weight. The optimal values were found to be a pH of 6.5, an initial
inoculum density of 0.6 g L-1, and a temperature of 28 oC. The predicted values were in
satisfactory correlation with experimental values with a coefficient determination (R2) of 0.9788.
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The statistical model was further validated for subsequent experimentation under optimized conditions. The effect of glucose concentrations on 4-t-OP degradation by F. falciforme RRK20
was evaluated. The results revealed that 4-t-OP degradation could be accelerated through the addition of glucose at relatively low dosage. Furthermore, the fungal metabolic pathway of 4-t-
OP was investigated by the Orbitrap elite mass spectrometer. Based on the identified metabolites, strain RRK20 degraded 4-t-OP via two different mechanisms including the alkyl branched side chain oxidation and the aromatic ring hydroxylation. Further degradation of the metabolites proceeded through the alkyl side chain followed by the aromatic ring cleavage.
In summary, this is the first study focusing on the degradation of 4-t-OP as the sole
carbon source by yeast and a non-ligninolytic fungus from sewage treatment plant and plastic industry waste samples. This study also demonstrated their optimum conditions for 4-t-OP
degradation to improve biodegradation efficiency. The addition of nutrients can increase
degradation efficiency of 4-t-OP. The pathways of degradation of 4-t-OP by the yeast and nonligninolytic fungi are different from the reported degradation pathways shown for bacteria and
fungi. |
