Quantification Of Glycerol Research Articles

Towards controlled and simple design of non-enzymatic amperometric sensor for glycerol determination in yeast fermentation medium

Glycerol is a widely used signaling bioanalyte in biotechnology. Glycerol can serve as a substrate or product of many metabolic processes in cells. Therefore, quantification of glycerol in fermentation samples with inexpensive, reliable, and rapid sensing systems is of great importance. In this work, an amperometric assay based on one-step designed electroplated functional Pd layers with controlled design was proposed for a rapid and selective measurement of glycerol in yeast fermentation medium. A novel assay utilizing electroplated Pd-sensing layers allows the quantification of glycerol in yeast fermentation medium in the presence of interfering species with RSD below 3% and recoveries ranged from 99 to 103%. The assay requires minimal sample preparation, viz. adjusting of sample pH to 12. The time taken to complete the electrochemical analysis was 3 min. Remarkably, during investigations, it was revealed that sensitivity and selectivity of glycerol determination on Pd sensors were significantly affected by its adsorption and did not depend on the surface structure of sensing layers. This study is expected to contribute to both fundamental and practical research fields related to a preliminary choice of functional sensing layers for specific biotechnology and life science applications in the future.Graphical

Development of a portable optical device with a multi-channel spectrometer sensor for quantification of glycerol in wine: a maker approach for on-site analysis.

In this work we present a novel and environmentally friendly approach for quantifying glycerol in wine samples using a portable optical device based on the maker concept and do-it-yourself (DIY) principles. This method offers significant advantages, including cost-effectiveness, reduced sample and reagent consumption, and the potential for integrating IoT (Internet of Things) technology. The chemical strategy involves the oxidation of glycerol using periodate, followed by the formation of the 3,5-diacetyl-1,4-dihydrolutidine (DDL) compound through a reaction with acetylacetone. The utilization of a cost-effective AS7341 color sensor as a detector enables accurate and sensitive detection of glycerol levels in wine samples. The optimized procedure demonstrates adequate analytical performance for glycerol determination in wine samples, encompassing a wide linear range (0.5 mg L-1 to 40.0 mg L-1), high correlation coefficient (r = 0.998), and low limits of detection (0.050 mg L-1). The method exhibits excellent precision, with the coefficient of variation estimated to be 0.1% for 10 independent measurements of a 20 mg L-1 solution. These features render it suitable not only for routine glycerol analysis in the wine industry, but also for addressing challenges related to wine adulteration and counterfeiting.

Electrochemical Analysis of Free Glycerol in Biodiesel Using Reduced Graphene Oxide and Gold/Palladium Core-Shell Nanoparticles Modified Glassy Carbon Electrode

Glycerol is a major byproduct obtained in the production of biodiesel, an important renewable fuel. The presence of free glycerol in fuel can have structural and performance consequences with respect to the engine, making fuel quality control important. The standard method to analyze glycerol in biodiesel is gas chromatography, a time-consuming and expensive technique. In this context, an electrode based on glassy carbon electrodes (GCEs) modified with reduced graphene oxide and core-shell gold@palladium nanoparticles was developed for the determination of glycerol in biodiesel. The free glycerol analysis was performed in the aqueous phase obtained by liquid–liquid extraction from a biodiesel sample. Cyclic voltammetry was chosen as the method for glycerol electrochemical analysis to regenerate active sites and promote greater sensor stability. The modified Au@Pd/rGO/GCE electrode showed an excellent performance, obtaining a linear range of 18.2 to 109 µmol L−1 with a correlation coefficient of 0.9895, limits of detection and quantification of 5.33 and 17.6 µmol L−1, respectively, high stability during 1000 cycles, and recovery values of 86% and 87% in the quantification of glycerol in biodiesel samples. The proposed method proved to be a great alternative for the analysis of glycerol in biodiesel, being a fast, sensitive, and low-cost technique due to its high stability and the use of small quantities of reagents.

DietSee: An on-hand, portable, strip-type biosensor for lipolysis monitoring via real-time amperometric determination of glycerol in blood

Glycerol is a clinical biomarker of lipolysis that is mainly produced by adipose tissues. Blood glycerol content increases in pathological conditions such as metabolic and cardiovascular diseases or cancer cachexia, but also in response to energetic stress such as physical exercise. Accurate glycerol monitoring is therefore important in a range of healthcare contexts. However, current methods available for the quantification of glycerol are expensive, time-consuming, and require the extraction of plasma from blood, from which blood glycerol content is then extrapolated. Here, we report the development of a new point-of-care glycerometer device, DietSee, based on a strip-type biosensor that enables the quantification of glycerol directly from whole blood in 6 s. The performance of the biosensor was first evaluated using buffer solutions and spiked human and mouse plasma samples, and its response was compared with that of the gold-standard colorimetric method. The results obtained using DietSee correlated strongly with those from the reference method and demonstrated a linear response to glycerol levels across a wide range of concentrations (40–750 μM) that were representative of those in the human body. Next, the biosensor was validated using spiked human blood samples over a range of 30–55% hematocrit; it also demonstrated a strong correlation with reference measurements under these conditions (R2 = 0.97). In addition, the biosensor was only minimally affected by a variety of potential interferents (endogenous and exogenous) and was highly stable in storage (more than 2 years when strips were stored dry at 4 °C). Finally, we investigated the application of the biosensor to real-time monitoring of lipolysis and found that the DietSee is well adapted for this purpose in both human and mouse samples. To conclude, the novel DietSee glycerometer is a sensitive, selective, and rapid tool that enables characterization of the metabolic status of an individual by measuring the glycerol concentration from a single fingertip blood drop.

A method for measuring glycerol-blanked triglyceride concentrations by using gas chromatography-isotope dilution mass spectrometry.

Serum triglyceride concentrations are measured as total glycerides content in many Western countries. In Japan, glycerol-blanked triglycerides (TG-GB) are measured to identify postprandial hypertriglyceridaemia and to minimize the influence of glycerol formulation on serum triglyceride values. However, TG-GB measurements have not been standardized. Therefore, we developed an efficient quantification system for total glycerides and free glycerol that allows the calculation of TG-GB concentrations. We measured total glycerides and free glycerol in human serum by using gas chromatography-isotope dilution mass spectrometry and compared its performance to the reference method of the US Centers for Disease Control and Prevention (CDC). Our practical method of total glycerides and free glycerol quantification achieved excellent precision for both within-run and among-run coefficients of variation (<1.5% and <2.7%, respectively), with an average recovery of 99.8% for free glycerol. However, we noted an average %bias of -0.26% for total glycerides and -3.15% for free glycerol between our TG-GB method and the CDC reference method. This practical method of total glycerides and free glycerol quantification enables traceability assessment of TG-GB measurements. Differences between the output values of TG-GB and the CDC reference method might result from the differences in free glycerol values.

Evaluation of Metabolites and Amino Acids Assimilation by Yeast FT-858 in Saccharine Substrates for the Production of Bioethanolroduction

Yeasts are exposed to stressing agents in the fermentation process. The study aims to analyze the production of ethanol and intracellular glycerol, as well as to evaluate the consumption of amino acids by yeast FT-858 in different culture conditions. The sugarcane and sorghum broth were used, at 22ºBx and pH of 5.0. A 2% YPD medium was pre-inoculated, and 0.10 g of lyophilised yeast were inoculated, incubated at 30 °C for 12 hours at 250 rpm. Cells were recovered by centrifugation and reinoculated in the fermentation medium at 30 °C and 40 °C at 250 rpm, this procedure was conducted with cell recycles, and 4 mL aliquots were withdrawn for analysis. Quantification of glycerol was accomplished by cell lysis and enzymatic kit of triglycerides, ethanol by gas chromatography and amino acid analysis by high performance liquid chromatography. The results showed that intracellular glycerol production and ethanol concentration were inversely proportional due to stress factors. The highest amount of amino acids available was in sorghum broth, and the most assimilated by the yeast were: serine, arginine, alanine and tryptophan and the high temperature accelerated their consumption of them. DOI: http://dx.doi.org/10.17807/orbital.v11i4.1220

Identification of volatile compounds, quantification of glycerol and trace elements in distilled spirits produced in Mozambique.

Distilled spirits, in addition to satisfying the legal requirements with respect to its composition, must possess a sensorial quality capable of satisfying its consumers. The present work describes the analysis of volatile compounds, trace elements and glycerol in thirteen samples of Mozambican spirits collected in different regions of the country. Extraction of volatile components was a accomplished using the headspace SPME technique, the quantification of glycerol was achieved by UV/Vis spectroscopy and the determination of trace elements by atomic absorption spectrometry. Of the 35 volatile compounds identified, 19 were esters, of which ethyl hexanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl laurate, ethyl hexadecanoate, ethyl tetradecanoate, ethyl benzenepropanoate, 3-methylbutyl acetate and isoamyl alcohol were the predominant compounds found in the different samples, contributing to the characteristics and aroma of the beverages. As for glycerol, concentrations below 5mgL-1 were observed in all the samples studied. The samples were found to be contaminated by metals such as copper, lead and zinc. In addition, the results provided information on the quality of Mozambican spirits regarding some aspects related to the determination of metals and glycerol. This information might stimulate producers to be more attentive to the conditions of production and the risks involved in achieving good quality spirits.

A methodological approach for the simultaneous quantification of glycerol and fatty acids from cork suberin in a single GC run.

Suberin, as part of plant protective barriers, is one of the most important natural polymers after cellulose and lignin. For a full elucidation of suberin structure the quantification of glycerol, fatty α,ω-diacids and ω-hydroxyacids, the major building blocks of suberin, is of primary importance. Glycerol is often lost in the most used analytical procedures or rarely determined by deficient or too laborious techniques. Propose a simple, accessible and reliable methanolysis work-up procedure for an accurate and simultaneous quantification of glycerol and suberin fatty monomers in the same GC run. Cork from Quercus suber L. was depolymerised by methanolysis. Glycerol was derivatised to an organic soluble form before the suberin monomers recovery in water/organic solvent partition. Gas chromatography flame ionisation detector (GC-FID) response factors were determined for glycerol, ferulic acid and one for each fatty monomer substructure. Additionally, 1,2,4-butanetriol and methyl nonadecanoate were used as internal standards. The proposed experimental approach allowed the glycerol and all the fatty suberin monomers in the same GC run to be quantified accurately. Glycerol represented 30.6 area%, 14.2 mass% and 38.4 molar% of suberin and the COOH/OH groups ratio was 0.6:1 in the proposed experimental approach in contrast with 0.10 area% and COOH/OH ratio of 3:1 in the most used protocol. Furthermore, ω-hydroxyacids/α,ω-diacids mass ratio was 1:1 as opposed to an area ratio of 1.5:1. The proposed work-up procedure revealed to be a reliable analytical tool for the complete analysis of suberin allowing the future knowledge to grow towards a better understanding of suberin structure throughout its range and variability.

Development of an electrochemical sensor for the determination of glycerol based on glassy carbon electrodes modified with a copper oxide nanoparticles/multiwalled carbon nanotubes/pectin composite

A very sensitive electrochemical sensor to determine glycerol (GLY) in biodiesel samples has been developed. It is based on the electrochemical oxidation of GLY at glassy carbon electrodes modified with copper oxide nanoparticles supported on multiwalled carbon nanotubes/pectin composite. The electrochemical oxidation of GLY was studied in pH 8 phosphate buffer solutions. The quantification of GLY was performed by amperometric measurements. The proposed electrochemical sensor improves the performance of others methods developed for the quantitation of GLY. A low limit of detection of 5.8×10−6g dm−3 and a percent coefficient of variation smaller than 10% was found, along with a reasonable linear range of 9×10−6–1×10−3g dm−3. The proposed electrochemical sensor exhibited good performance, stability, reproducibility, repetitiveness and a good detection limit and a linear concentration range to quantify GLY in biodiesel samples.

Towards Process Spectroscopy in Complex Fermentation Samples and Mixtures

AbstractSupernatants from a fermentation process of Pichia pastoris were investigated by Raman spectroscopy. Using partial least squares regression, the principal substrates glycerol and methanol could be predicted, however not the expressed protein. To gain further insight, a priori prepared calibration samples were studied by vibrational‐, UV/Vis‐, and fluorescence spectroscopy. For the quantification of glycerol and methanol, Raman spectroscopy was identified as the most sensitive technique, and superior to near‐infrared spectroscopy, but not for protein contents below 1 g L−1. Both UV/Vis absorption and fluorescence spectroscopy are well suited for the quantification of protein, however, best results were obtained with UV/Vis absorption.

A nonenzymatic approach for selective and sensitive determination of glycerol in biodiesel based on a PtRu-modified screen-printed edge band ultramicroelectrode

A nonenzymatic electrochemical method using PtRu-modified screen-printed edge band ultramicroelectrode has been developed for selective and sensitive detection of glycerol. It was based on the periodate oxidation of glycerol and the as-formed formic acid and formaldehyde was detected by the proposed electrochemical sensor in the gas phase. This approach simplified the two steps of sample preparation and detection into a 2-in-1 single step process. It also eliminated almost all of the interferants in the solution phase and hence resulted in a highly selective analysis of glycerol. The proposed approach improved the analytical performance of other electroanalytical methods for quantification of glycerol with a low detection limit of 0.51mgL−1 glycerol (S/N=3) and a wide linear range of 0.92–92mgL−1. Especially, for monitoring free glycerol in biodiesel, no extraction of glycerol from biodiesel to eliminate organic interferents was required. The accuracy was evaluated using a spike-and-recovery assay to prove its applicability as a valuable tool for practical glycerol analysis.

Potentiometric Glycerol Biosensor Based On Immobilization Of Glycerol-Dehydrogenase On Au Using A Molecular Wiring System

An intensive interest in glycerol determination exists due to its extensive applications in major industries including medical, food and biofuels. This has led to a quest for a simple, specific, rapid and highly sensitive method for glycerol quantification. This study attempts to develop an enzyme-based electrochemical biosensor for glycerol quantification using NAD+-dependent enzyme, Glycerol dehydrogenase (GlDH) immobilized on a modified gold (Au) electrode. The objective of this study is to fabricate a molecular wiring system to anchor GlDH enzyme onto a Au electrode and to evaluate the efficacy of the proposed wiring system to effectively transport generated charges (as a result of glycerol oxidation) from the enzyme active site to the supporting electrode. A novel concatenation of molecules was utilized to attach the enzyme onto the Au surface via layer-by-layer (LBL) self-assembly method. Surface characterization of the enzyme electrodes using cyclic voltammetry and SEM techniques confirmed attachment of enzyme on the Au electrode with the assistance of the tethering molecules. The enzymatic biosensor was assessed for its potentiometric response to glycerol concentrations ranging from low (0.001M) to very high (10 M). The effects of supporting electrolyte(s) with varying buffers and presence of enzyme stimulators on the electrical potential generated were analyzed and compared. Current sensing-Atomic force microscopy (CS-AFM) was used to elucidate the overall surface electrical conductance of the electrodes. The carrier electrolytes and glycerol concentrations had a significant impact on open circuit potentials produced by the cell (p-value<0.0001). The glycerol vs electrode open circuit voltage was highly correlated with a logarithmic relationship with a correlation coefficient of R2 = 0.9921 in a water solution with enzyme stimulants and R2=0.9643 in pure water (both at a (pH ~ 4.5). Water along with enzyme stimulators was found to be the suitable carrier electrolyte for producing high potentials. The potentiometric electrode showed high reproducibility and repeatability for the determination of glycerol concentrations ranging from 0.01 – 10M in 1M resolution. It was observed that attachment of the enzyme to the NAD+ layer caused a significant reduction in current flow, and thus the electron transport. The crosslinking agent, glutaraldehyde also seemed to negatively affect charge transport between the enzyme active site. The CS-AFM images obtained showed that the surface charge distribution in the electrode was unhindered as a result of enzyme attachment. Simple fabrication, high speed of measurement, high sensitivity and possibility of miniaturization are the main advantages of this biosensor. Additionally, glycerol being the backbone of triglycerides, the long-term goal of the study is to fabricate a lipid sensor using the concentration of glycerol produced during lipid hydrolysis as an indirect modality for measuring lipids. Sensing glycerol accurately with a simple set-up is the first step toward this longer-term goal.

Evaluation of Glycerol Profiles in Sugarcane Spirits (Cachaças)

A new method for glycerol quantification in sugarcane spirits (cachacas) is proposed, based on sample derivatization using benzoyl chloride followed by solid-phase extraction and high performance liquid chromatography with diode array detection analysis. The limits of detection and quantification for glycerol analysis are 0.25 mg L−1 and 0.74 mg L−1, respectively. The accuracy is of 97.5%, the precision 93.5% and the linearity r2 = 0.998 in the range of 0.56 mg L−1 to 112.0 mg L−1 of glycerol. Experiments with forty eight samples of aged and non-aged cachacas and seven alcoholic extracts of wood showed that the aging process contributes to increase the glycerol concentration. The average concentration of glycerol in aged cachacas were about 10-fold higher than in non-aged cachacas.

Development of a Sensor System for the Determination of Sanitary Quality of Grapes

An instrument for the automatic quantification of glycerol in grapes has been developed. We verify here that this analyte can be used as a benchmark of a serious disease affecting the grapevines, namely Botrytis cinerea. The core of the instrument is an amperometric biosensor consisting of a disposable screen printed electrode, generating the analytical signal thanks to a bi-enzymatic process involving glycerol dehydrogenase and diaphorase. The full automation of the analysis is realised by three micropumps and a microprocessor under control of a personal computer. The pumps allow the correct and constant dilution of the grape juice with a buffer solution also containing [Fe(CN)6]3− redox mediator and the injection of NAD+ cofactor when the baseline signal reaches a steady state; the instrument leads to automated reading of the analytical signal and the consequent data treatment. Although the analytical method is based on an amperometric technique that, owing to heavy matrix effects, usually requires an internal calibration, the analyses indicate that a unique external calibration is suitable for giving accurate responses for any grapes, both white and black ones.

말라카이트 그린의 색엷음 현상을 이용한 글리세롤의 정량: 바이오디젤 내 반응물 분석의 적용 가능성

Nowadays biodiesel (fatty acid methyl ester, FAME) has been becoming an important issue as a desired alternative of energy products because of non-toxic, biodegradable properties, and lower exhaust emissions. During esterification of fatty acids or transesterification of oils and fats with short chain alcohols by the alkali-catalyzed methanolysis, FAME and unrefined glycerol are generated. Quantification of glycerol as a by-product is important because of a determinant of biodiesel quality. However, the glycerol analysis by gas chromatography (GC) method has laborious works with sample preparation, long time and cost of sample analysis. Thus, there is a need to analyze glycerol more simply. Herein we demonstrate that the colorimetric assay for glycerol analysis conducted by UV-vis spectrophotometer at the wavelength 617 nm whose peak is maximum intensity of malachite green, resulting in the red-shift occurred proportionally as a function of glycerol amount. Thus, it is considered the solvent media for malachite green fading for biodiesel production: (1) water, (2) MeOH, and (3) EtOH. The resulting findings show that the peak intensity at 617 nm in glycerol-malachite green mixture had a relationship between glycerol concentration and degree of peak shift as increase in pure glycerol concentration approximately at pH 7.0. However,

Flow injection analysis of free glycerol in biodiesel using a copper electrode as an amperometric detector

The main goal of this work was to develop a simple analytical method for quantification of glycerol based on the electrocatalytic oxidation of glycerol on the copper surface adapted in a flow injection system. Under optimal experimental conditions, the peak current response increases linearly with glycerol concentration over the range 60–3200mgkg−1 (equivalent to 3–160mgL−1 in solution). The repeatability of the electrode response in the flow injection analysis (FIA) configuration was evaluated as 5% (n=10), and the detection limit of the method was estimated to be 5mgkg−1 in biodiesel (equivalent to 250μgL−1 in solution) (S/N=3). The sample throughput under optimised conditions was estimated to be 90h−1. Different types of biodiesel samples (B100), as in the types of vegetable oils or animal fats used to produce the fuels, were analysed (seven samples). The only sample pre-treatment used was an extraction of glycerol from the biodiesel sample containing a ratio of 5mL of water to 250mg of biodiesel. The proposed method improves the analytical parameters obtained by other electroanalytical methods for quantification of glycerol in biodiesel samples, and its accuracy was evaluated using a spike-and-recovery assay, where all the biodiesel samples used obtained admissible values according to the Association of Official Analytical Chemists.

Gene regulation in β-sitosterol-mediated stimulation of adipogenesis, glucose uptake, and lipid mobilization in rat primary adipocytes

The nutraceutical benefits of β-sitosterol (SIT) are well documented. The present study investigated the in vitro effects of SIT on adipogenesis, glucose transport, and lipid mobilization in rat adipocytes. Primary cultures of rat preadipocytes and differentiated adipocytes were used in this study. Glucose uptake was measured by the uptake of radio-labeled glucose. Adipogenesis and lipolysis were measured by oil-red-O and glycerol quantification methods, respectively. The expression of protein kinase B (Akt), glucose transporter 4 (GLUT4), hormone sensitive lipase (HSL), and phosphatidylinositol-3-kinase (PI3K) genes in SIT-treated adipocytes were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). The data showed that SIT induced glucose uptake in adipocytes. It also stimulated adipogenesis in differentiating preadipocytes. Interestingly, although SIT displayed general insulin-mimetic activity by stimulating glucose uptake and adipogenesis, it also induced lipolysis in adipocytes. Furthermore, the SIT-induced lipolysis was not attenuated by insulin and co-incubation of SIT with epinephrine improved epinephrine-induced lipolysis. GLUT4 gene expression was highly down-regulated in SIT-treated adipocytes, compared to insulin-treated adipocytes, which was up-regulated. Insulin- and SIT-treated adipocytes showed similar levels of Akt, HSL, and PI3K gene down-regulation. These observations suggest that the elevation of glucose uptake in SIT-treated adipocytes was unrelated to de novo synthesis of GLUT4 and the SIT-induced lipolysis is associated with the down-regulation of Akt and PI3K genes. The unique effects of SIT on the regulation of glucose uptake, adipogenesis, and lipolysis in adipocytes show that it has potential to be utilized in diabetes and weight management.

Determination of Free Glycerol in Biodiesel via Solid‐Phase Extraction and Spectrophotometric Analysis

AbstractTo date, the standardized method for glycerol quantification in biodiesel production utilizes gas chromatography (GC); however, availability to manufacturers and instrumentation cost limits GC as an analytical method for general quality enforcement among producers. The method developed here is a bench top technique for quantitative determination of glycerol in biodiesel, with practical application in pharmaceutical and environmental quality control. The method extracts the glycerol contaminant from biodiesel using a normal phase solid phase extraction column (SPE). The protocol proceeds by rinsing with hexane to remove residual methyl esters, then collecting the glycerol with water. The aqueous extract is analyzed spectrophotometrically by an anthrone coloring reagent. Use of 2‐g SPE columns and the solvent system developed has achieved 85% glycerol recovery. The assay applied has a detection range of 0.004–0.400% free glycerol, comparable to the established American Society of Testing and Materials (ASTM) D 6584‐07 GC technique. Results were confirmed by GC and high‐pressure liquid chromatography (HPLC). The bench top technique reduces the costs of operation relative to current methods, completes analysis in proficient time, requires minimal labor, and has analytical limits comparable to existing standard methods of biofuel analysis.

Diagnosis and Quantification of Glycerol Assimilating Denitrifying Bacteria in an Integrated Fixed-Film Activated Sludge Reactor via13C DNA Stable-Isotope Probing

Glycerol, a byproduct of biodiesel and oleo-chemical manufacturing operations, represents an attractive alternate to methanol as a carbon and electron donor for enhanced denitrification. However, unlike methanol, little is known about the diversity and activity of glycerol assimilating bacteria in activated sludge. In this study, the microbial ecology of glycerol assimilating denitrifying bacteria in a sequencing batch integrated fixed film activated sludge (SB-IFAS) reactor was investigated using (13)C-DNA stable isotope probing (SIP). During steady state SB-IFAS reactor operation, near complete nitrate removal (92.7 ± 5.8%) was achieved. Based on (13)C DNA clone libraries obtained after 360 days of SB-IFAS reactor operation, bacteria related to Comamonas spp. and Diaphorobacter spp. dominated in the suspended phase communities. (13)C assimilating members in the biofilm community were phylogenetically more diverse and were related to Comamonas spp., Bradyrhizobium spp., and Tessaracoccus spp. Possibly owing to greater substrate availability in the suspended phase, the glycerol-assimilating denitrifying populations (quantified by real-time PCR) were more abundant therein than in the biofilm phase. The biomass in the suspended phase also had a higher specific denitrification rate than the biofilm phase (p = 4.33e-4), and contributed to 69.7 ± 4.5% of the overall N-removal on a mass basis. The kinetics of glycerol based denitrification by suspended phase biomass were approximately 3 times higher than with methanol. Previously identified methanol assimilating denitrifying bacteria were not associated with glycerol assimilation, thereby suggesting limited cross-utilization of these two substrates for denitrification in the system tested.

Detection and Quantification of Free Glycerol in Virgin Olive Oil by 31P‐NMR Spectroscopy

Abstract31P‐Nuclear magnetic resonance (NMR) spectroscopy was employed to detect and quantify free glycerol in virgin olive oils originating from various regions of Greece. This analytical method was based on the derivatization of the hydroxyl groups of glycerol with the tagging reagent 2‐chloro‐4,4,5,5‐tetramethyldioxaphospholane, and identification of the phosphitylated compound on the basis of 31P chemical shifts. Quantification of glycerol in olive oils was accomplished by integration of the appropriate signals in the 31P NMR spectrum and the use of the phosphitylated cyclohexanol as internal standard. A linear correlation was observed between the glycerol content and 1,3‐diacylglycerols and free acidity indicating that glycerol is the final product of the partial hydrolysis of triacylglycerols.