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

LI Yuanfei, SUN Meizhou, CHENG Yanfen, ZHU Weiyun

doi:10.3969/j.issn.1006-267x.2017.04.015

2017 , Vol. 29 >Issue 4: 1198 - 1204

DOI: https://doi.org/10.3969/j.issn.1006-267x.2017.04.015

Molecular Nutrition

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

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Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health, Laboratory of Gastrointestinal Microbiology, Nanjing Agricultural University, Nanjing 210095, China

Received date: 2016-10-01

Online published: 2017-04-14

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Abstract

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 temperature and volume of injection were determined for HPLC method based on the chemical properties of the organic acids. The established method was then applied to detecting the concentrations of formate, lactate, acetate, 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 supernatant 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 concentration of acetate was significantly increased (P<0.05). In conclusion, the six organic acid contents produced 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.

Key words： high performance liquid chromatography; organic acids; anaerobic fungi; methanogens; co-culture

Cite this article

LI Yuanfei, SUN Meizhou, CHENG Yanfen, ZHU Weiyun . Effects of Associated Methanogen on Organic Acid Profile of Metabolism by Anaerobic Fungus Revealed Using High Performance Liquid Chromatography[J]. Chinese Journal of Animal Nutrition, 2017 , 29(4) : 1198 -1204 . DOI: 10.3969/j.issn.1006-267x.2017.04.015

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