Microorganisms and an Array of Their Compounds

Published: 2021-09-13 22:10:10
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Category: Biology

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Microorganisms are able to produce an array of compounds including biofuels, its precursors and other high value metabolites. However, accurate analysis of the specific compounds produced by microbes is vital since even a small variation can influence the quality, and thus the value, of the end product. Traditional methods of analysing metabolites are time intensive and prone to high degrees of variation amongst species and test conditions. Here, a method that allows fast and reliable extraction of alcohol with minimal effort and similar compounds from culture broth of fungi, followed by a low cost technique for ethanol quantification using gas chromatography flame-ionization-detector (GC-FID) chromatography is presented.. The method is robust, profoundly reproducible, and quick, permitting analysis of multiple samples. to be analysed throughout the time course of culturing, thus providing time resolved information with respect to ethanol quality and quantity. The total time from harvesting to obtaining analytical results is less than 1 h. This method offers wide range of detection and exhibited as appropriate for fermentation process.
Introduction
Utilization rates of traditional non-renewable energy sources have been steadily increasing worldwide. According to environmental impact assessment (EIA), 2012 in the United States surpass 18 million barrels for every day with a critical rate being imported from foreign sources (EIA, 2012). The political, economic and environmental contentions over these resources have brought high interest for cutting edge alternate, renewable, sustainable and ecofriendly fuels to the meet the ever increasing demand (Dismukes et al., 2008; Greenwell et al., 2010; Groom et al., 2008; Hill et al., 2006; Sheehan, 1998). Lately, biological production of ethanol based fuels have making it as environmentally beneficial energy source. In addition, toxicity of exhaust emissions is lower than that of petroleum sources. Energy crops grown for the production of ethanol absorbs huge amounts of greenhouse gases (GHG) released by the burning of fossil fuels and ethanol contains 35% oxygen that helps complete combustion of fuel and thus reduces particulate emission that pose severe health hazard (Hosuk et al., 2013; Hirshfeld et al., 2014; Thomas et al., 2015).With increasing interest for the utilization of sustainable resources for the production of ethanol, lignocellulose as one of the most plenteous natural resources has pulled in a lot of consideration for fermentation with microbes (Binod et al., 2010; Chandel et al., 2011; Saini et al., 2015; Kim, 2018). However, an effective method for the detection and quantification of produced ethanol is prerequisite . Despite the fact that there are several methods reported for the detection of ethanol, most of them are expensive, time consuming as well as are convoluted. Since the microbial production of ethanol by fermentation is important, the development of a method for the synchronous measurement of different alcohols is essential. . Here, we report an effective method using gas chromatography flame-ionization-detector (GC-FID) chromatography for the detection and quantification of ethanol produced during fermentation.
Materials and Methods
Chemicals
Ethanol and n-Butanol (BuOH) (analytical grade, 99.5%) were purchased from Sigma-Aldrich, USA. Ethyl acetate (EtAc)(analytical grade, 99.5%) was purchased from Merk. All remaining chemicals used in this study were either analytical grade or technical grade.
Instrumentation and operation conditions
For gas chromatography separation and quantification, analytical standards were prepared and diluted in ethyl acetate to obtain appropriate concentrations. The ethanol content was resolved utilizing a Varian 430-GC gas chromatograph (Varian Inc., USA) equipped with a Factor Four ™VF-1ms column from Varian (15 m length, 0.25 mm internal breadth, and 0.25 μm film thickness). The chromatography conditions were as follows: a split ratio of 1/100, injector was initially held at 80°C for 1 min and increased at 20°C per min till 120°C and the detector temperature was set to 280°C, and injection volume of 1 μL was injected to inlet and nitrogen (N2) was used as carrier gas at a rate of 0.5 ml/ min.
Preparation of standard ethanol samples
A serial dilution of the ethanol stock (1g/ml) solution with ethyl acetate water in GC vials serially to give a set of standards at 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/ml. Butanol was used as an internal standard. A standard curve was generated using serially diluted ethanol with following concentrations, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/ml. In all cases, butanol was used as an internal standard.
Monitoring of ethanol in a culture broth
Standardization of extraction of ethanol from culture broth
Procedure for extraction of ethanol from culture broth of the fungal isolates was initially standardized. For this purpose, MSM medium was dispensed in 250 ml Erlenmeyer conical flasks at the rate of 30 ml per flask. A known quantity of authentic ethanol (10 ml) was added to each flask. Two solvents – hexane and ethyl acetate were used for extraction of ethanol from MSM media. In all cases, only one of the solvent was added to each flask in 2: 1 ratio along with internal standard – Butanol. Suspension was mixed well for 5 min and spun in a refrigerated centrifuge (Remi Make; Model – C-24) at 10, 000 g to facilitate separation of layers. Of the two solvents used in this study, ethyl acetate was found to be better for extraction of ethanol. Hence, ethyl acetate was used for extraction of ethanol from culture broth.
Fermentation experiments
Previously reported fungal isolates (Ramanjaneyulu and Rajasekhar Reddy 2016) were tested for their ability to produce bioethanol up on glucose fermentation. For this purpose, MSM broth (Ramanjaneyulu and Rajasekhar Reddy, 2016) was dispensed in 250 ml Erlenmeyer conical flasks at 50 ml per flask. These flasks were amended with D – dextrose at concentration of 10g/L. The pH of the medium was adjusted to 5.0. Flasks were inoculated with 0.5 mm of 5 agar plugs from 5-day old culture of fungi grown on MSM medium. Subsequently, flasks were incubated at 30°C and 150 rpm in an incubator and withdrawn every 24 hrs. Aliquots of culture broth from flasks were used for extraction and estimation of bioethanol as specified.

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