Processing Method for the Quantification of Methanol and Ethanol from Bioreactor Samples Using Gas Chromatography–Flame Ionization Detection

Abstract

Methanol, a simple polar solvent, has been widely identified as an attractive carbon source to produce chemicals and fuels in bioprocesses. Specifically, to achieve recombinant protein production from methylotrophic yeasts, such as Pichia pastoris , this organic solvent can be used as a sole carbon source for growth and maintenance as well as an inducer for protein expression. However, if methanol feeding is not controlled well in such a fermentation process, accumulation of the solvent in the growth media will have a detrimental effect on the cells. Hence, monitoring the levels of methanol in these fermentation processes is a crucial step to ensure a healthy culture and maximum protein production. There are various techniques elaborated in the literature for monitoring methanol in cell cultures, but often, they appear to be expensive methods that are less affordable for many laboratories. This is because, in addition to the sophisticated equipment that is required for the analysis, the complexity of the samples retrieved from the bioprocesses necessitates laborious processing steps often involving expensive tools. In this study, a fast, simple, and sensitive method is developed to process biological samples by using the salting-out-assisted liquid–liquid extraction technique to quantify the concentration of methanol and ethanol using gas chromatography. On comparing the combinations of widely available salts and solvents, it was noticed that salting out using potassium carbonate followed by the liquid–liquid extraction of the analyte using ethyl acetate showed the best recovery. Followed by this, a validation test for the developed method was performed, which resulted in good peak resolution, linearity, and limit of detection for the quantitation of methanol and ethanol. By further assessing the tested combination, it was confirmed that its application could be extended to other matrices. Such an approach facilitates the possibility to monitor and control the methanol levels in fermentation and aids in bioprocess optimization.

Macroscopic modeling of the growth and substrate consumption of wild type and genetically modified Pichia pastoris

“…The methanol present in the samples was quantified using an Agilent 8860 gas chromatography (GC) system (Agilent Technologies, California, USA) equipped with a Flame Ionization Detector (Agilent technologies) as detailed in Joseph et al. [ 34 ] Prior to the analysis, the methanol present in the complex mixture was separated by using the salting‐out assisted liquid–liquid extraction (SALLE) technique. SALLE was conducted by adding 0.772 g of potassium carbonate into 700 µL of filtered sample for salting out followed by 700 µL of ethyl acetate.…”

“…The methanol present in the samples was quantified using an Agilent 8860 gas chromatography (GC) system (Agilent Technologies, California, USA) equipped with a Flame Ionization Detector (Agilent technologies) as detailed in Joseph et al [34] Prior to the analysis, the methanol present in the complex mixture was separated by using the…”

Determination of Ethanol in Vinegar and Beverage by Gas Chromatography: A Validated Method for Halal Verification

“…The resolution value indicates the degree of separation of mixture components in gas chromatography analysis with high selectivity at optimum conditions [9]. The selectivity value is categorized as good with Rs ≥ 1.5, shown in the chromatogram, where the mixture is separated [9,18,19]. The addition of propanol as an internal standard was modified and used for peak separation and quantitative determination of ethanol [6].…”

Interfacial Electronic Interactions within the Pd-CeO2/Carbon Onions Define the Efficient Electrocatalytic Ethanol Oxidation Reaction in Alkaline Electrolytes

“…Conventionally, ethanol is determined using a hydrometer, which is fraught with inaccuracies and human errors during reading. Other methods include the use of spectroscopy, and chromatography, which are bulky, expensive, complex, and difficult to miniaturize. Electrochemical techniques, on the other hand, are getting more attention due to their simplicity, accuracy, low cost, and ease of miniaturizing the sensor for use by nonskilled workers …”