高效液相色谱法研究瘤胃甲烷菌共存对厌氧真菌代谢产生有机酸特性的影响

本试验旨在利用建立的高效液相色谱法研究瘤胃甲烷菌共存对厌氧真菌代谢产有机酸特性的影响。根据6种有机酸的化学特性确定紫外检测波长、缓冲液浓度、p H、流速、柱温和进样量等液相色谱条件;利用建立的高效液相色谱法检测厌氧真菌纯培养及厌氧真菌与甲烷菌共培养上清液中6种有机酸的含量。结果表明,高效液相色谱采用的条件为:5 mmol/L磷酸二氢钾-磷酸缓冲液(p H=2.4)作为流动相,流速为0.5 m L/min,柱温25℃,进样量为20μL,214 nm波长检测。6种有机酸能够在30 min内得到良好分离。各种有机酸的线性相关系数均大于0.999,检测限为0.20~1.00μmol/L,定量限为0.667~3.333μmol/L,回收率为92.17%~101.61%。甲烷菌共存影响了厌氧真菌的代谢产物。厌氧真菌利用葡萄糖的主要水溶性代谢产物为甲酸、乳酸、乙酸,以及微量琥珀酸、柠檬酸、α-酮戊二酸及乙醇;甲烷菌共存显著降低了上清液中甲酸和乳酸的含量(P0.05),显著增加了乙酸含量(P0.05)。综上,本试验利用建立的高效液相色谱法可快捷、灵敏、有效地检测厌氧真菌代谢葡萄糖产生的6种有机酸的含量,并发现甲烷菌共存显著促进了厌氧真菌氢化酶体对碳水化合物的代谢。

Effects of Associated Methanogen on Organic Acid Profile of Metabolism by Anaerobic Fungus Revealed Using High Performance Liquid Chromatography

The objective of the present study was to investigate the effects of associated methanogen on organic acid profile of metabolism by anaerobic fungus using the established analytic method of high-performance liquid chromatography ( HPLC) . The wavelength of detector, concentration of buffer, pH, flow rate, column tem-perature and volume of injection were determined for HPLC method based on the chemical properties of the or-ganic acids. The established method was then applied to detecting the concentrations of formate, lactate, ace-tate, α-ketoglutarate, citric acid and succinic acid in the supernatant of fungal mono-culture and co-culture of anaerobic fungi with methanogens. The results showed that the operation condition for HPLC was as follows:5 mmol/L KH2PO4-H3PO4 buffer solution (pH=2.4) at a flow rate of 0.5 mL/min, the column temperature at 25 ℃, injection volume 20 μL and wavelength of detector at 214 nm. The organic acids could be separated and identified by their retention times within 30 min. All the correlation coefficients were no less than 0.999. The limit of detection ranged from 0.20 to 1.00 μmol/L and the limit of quantification ranged from 0.667 to 3.333 μmol/L. The recovery rate varied from 92.17% to 101.61%. The analysis of metabolites in the superna-tant of cultures showed that the metabolic profiles of anaerobic fungi were shifted by associated methanogens. The dominant water soluble metabolites of anaerobic fungal pure culture were formate, lactate and acetate. α-ketoglutarate, citric acid, succinic acid and ethanol were detected as well. With the presence of associated methanogens, the concentrations of formate and lactate were significantly decreased ( P<0.05) , while the con-centration of acetate was significantly increased (P<0.05). In conclusion, the six organic acid contents pro-duced by anaerobic fungi can be rapidly and sensitively detected by the established HPLC method. The presence of associated methanogens increases the carbohydrate metabolism in the hydrogenosomes of anaerobic fungi.