Measurement of ethanol concentration using solvent extraction and dichromate oxidation and its application to bioethanol production process

Abstract

A method for measuring the ethanol concentration in a yeast culture broth was developed using both microtubes and a 96-deepwell microplate. The strategy involved first the solvent extraction of ethanol from the yeast culture broth and measurements of the ethanol concentration using the dichromate oxidation method. Particular focus was made on selecting the extraction solvent as well as determining the measurable range of ethanol concentrations using this solvent extraction-dichromate oxidation method. This method was developed as an assay format in 2.0-ml microtubes and 1.2-ml 96-deepwell microplates, and the ethanol concentration in the batch cultures and fed-batch fermentations was measured. Tri-n-butyl phosphate [non-alcoholic solvent, density = 0.9727, solubility in water = 0.028% (w/v)] was used for solvent extraction when measuring the ethanol concentration from the yeast culture broth. The maximum detectable ethanol concentration was 8% (v/v) when 10 g potassium dichromate in 100 ml of 5 M sulfuric acid was used. The concentrations determined from the solvent extraction-dichromate oxidation methods were remarkably similar to those of gas chromatography in which samples were prepared from seven experiments, such as four batch cultures and three fed-batch fermentations.

Inexpensive Procedure for Measurement of Ethanol: Application to Bioethanol Production Process

“…It was reported that the high content of ethanol even 8% (v/v) could be estimated by Solvent Extraction and dichromate oxidation method using both microtubes and a 96-deepwell microplate [5]. Therefore, this ethanol assay format is practically useful for the selection of a strain having high productivity, the development of a bioethanol production process, monitoring and control in alcoholic beverage production.…”

“…Moreover aliphatic alcohol like n-dodecanol or n-decanol is a representative solvent for extracting ethanol from a culture broth and benzyl alcohol had been used as an alternative to distillation [11] [12]. In this study, non-alcoholic solvent (TBP) was used for extracting ethanol from a culture broth, because the alcoholic solvent is oxidized by dichromate solution more efficiently than others [5]. Three yeast strains were used to check their bioethanol activity in two different methods and bioethanol estimation were compared and found more or less similar result are shown in the Figure 1 and Table 1.…”

“…The main aim of this study is to develop cost effective eco-friendly method to measure bioethanol content from our experimental samples. Our developed method was designed based on method published previously [5]. Ethanol was first extracted from the aqueous phase using the solvent dichromate, which works as a reagent for color development from orange to green, in which it oxidizes ethanol.…”

“…The dichromate reagent was prepared by dissolving fresh 10 gm potassium dichromate in 100 ml of 5 M sulfuric acid solution [5]. Tri-n-butyl phosphate [TBP, density = 0.975 to 0.976, solubility in water = 0.028% (w/v), Sigma] was used to extract ethanol in an aqueous solution.…”

“…Solvent extraction and dichromate oxidation method is a simpler, more convenient for ethanol measurement in many industries and research fields, such as the selec-Advances in Microbiology tion of a strain having high ethanol productivity, the development of a bioethanol production process etc. [5]. During fermentation, culture broth contains a variety of complex polysaccharides like starch, cellulose etc.…”