Determination of organic acids and ethanol in fermentation broth by liquid-liquid extraction and gas chromatography

Abstract: The degradation of cellulose by thermophilic bacteria is a growing trend in the 2nd generation bioethanol. The determination of the contents of ethanol and organic acids in the fermentation broth is of great significance to the control of the fermentation process. A gas chromatographic method for the determination of ethanol, acetic acid, propionic acid, and butyric acid contents in fermentation broth was established, using n-propanol as internal standard. The pH value of the aqueous phase was adjusted to 3 to prevent the dissociation of organic acids. Mixed with internal standard, the compounds were then extracted into the organic phase using ethyl acetate, and the organic phase was injected to the inlet of gas chromatograph. The mass concentration range of the standard curve was 0.050-2.000 g/L, the correlation coefficients were in the range of 0.9990-0.9999, the detection limits of analytes in this method were in the range of 10-45 mg/L, the recovery rates at 3 spiking levels were in the range of 80.65%-107.94%, and the relative standard deviation (RSD) of spiked sample were in the range of 1.71%-4.98%, the RSD of the actual sample was less than 5%. The method could be used for the detection of organic acids and ethanol in fermentation broth. The effects of temperature (0 or 20 ℃) and ion intensity (saturated with sodium chloride or not) on the variability of the extraction efficiency were investigated. The results showed that saturated with sodium chloride increased the partition ratio of ethanol in ethyl acetate and decreased the partition ratio of acetic acid, propionic acid and butyric acid in ethyl acetate; lower temperature decreased the partition ratio of ethanol in ethyl acetate and increased the partition ratio of acetic acid, propionic acid and butyric acid in ethyl acetate. The minimum extraction variability was obtained at 20 ℃ and sodium chloride saturated solution, so this condition was set as the extraction condition. The liquid-liquid extraction experiments were carried out under the conditions of the volumetric ratio of aqueous phase to organic phase being 2:1 and 1:1. Ratios of peak area under the two conditions were used to calculate the partition coefficients of the compounds. The partition coefficients of ethanol, n-propanol, acetic acid, propionic acid, and butyric acid in ethyl acetate and sodium chloride saturated solution at 20 ℃ were measured and calculated to be 0.28, 1.64, 1.37, 2.51, and 3.29, respectively. The ethanol content of the extract solution of fermented sweet sorghum bagasse was determined by this method. The detection value was compared to that of the traditional distillation-gas chromatography method. The differences between two methods were less than the RSD of actual samples in this method. The pretreating process could be simplified by using liquid-liquid extraction instead of distillation. The detection method can also be used in the detection of organic acids and ethanol in other aqueous systems, such as detecting volatile fatty acids in anaerobic digestion waste water and detecting ethanol in fermented liquors. The injection of organic phase instead of aqueous phase can prevent the corrosion of the syringe by acid.