A simple,reproducible and sensitive spectrophotometric method to estimate microalgal lipids

Quantification of total lipids is a necessity for any study of lipid production by microalgae, especially given the current interest in microalgal carbon capture and biofuels. In this study, we employed a simple yet sensitive method to indirectly measure the lipids in microalgae by measuring the fatty acids (FA) after saponification. The fatty acids were reacted with triethanolamine–copper salts (TEA–Cu) and the ternary TEA–Cu–FA complex was detected at 260 nm using a UV–visible spectrometer without any colour developer. The results showed that this method could be used to analyse low levels of lipids in the range of nano-moles from as little as 1 mL of microalgal culture. Furthermore, the structure of the TEA–Cu–FA complex and related reaction process are proposed to better understand this assay. There is no special instrument required and the method is very reproducible. To the best of our knowledge, this is the first report of the use of UV absorbance of copper salts with FA as a method to estimate lipids in algal cultures. It will pave the way for a more convenient assay of lipids in microalgae and can readily be expanded for estimating lipids in other biological systems.     

Microalgae and bioremediation of domestic wastewater

Domestic wastewaters are produced in huge volumes and abundant with carbon, nitrogen and phosphorous, which are a promising source of nutrients for production of microalgae. Microalgae-based bioremediation of domestic wastewater offers various advantages over traditional treatment approaches because the process consumes CO₂, completely removes nitrogen and phosphorous for production of green biomass and oxygen. Moreover, the abundance of biochemical compositions (e.g., lipids, proteins, carbohydrates, bioactive compounds) of microalgae biomass is superior to terrestrial plant biomass in refining to multi-products having variety of commercial values. In this review, the most dominant microalgae used for simultaneous removal of pollutants and production of biomass and metabolites from domestic wastewater are presented. Biorefinery of microalgae biomass produced from domestic wastewater for production of multiple products is also explored. Finally, challenges and perspectives of successful microalgae-based bioremediation of domestic wastewater toward the biorefinery are briefly discussed.     

Application of surface plasmon resonance for the detection of carbohydrates,glycoconjugates, and measurement of the carbohydrate-specific interactions: A comparison with conventional analytical techniques. A critical review

Carbohydrates (glycans) and their conjugates with proteins and lipids contribute significantly to many biological processes. That makes these compounds important targets to be detected, monitored and identified. The identification of the carbohydrate content in their conjugates with proteins and lipids (glycoforms) is often a challenging task. Most of the conventional instrumental analytical techniques are time-consuming and require tedious sample pretreatment and utilising various labeling agents. Surface plasmon resonance (SPR) has been intensively developed during last two decades and has received the increasing attention for different applications, from the real-time monitoring of affinity bindings to biosensors. SPR does not require any labels and is capable of direct measurement of biospecific interaction occurring on the sensing surface. This review provides a critical comparison of modern analytical instrumental techniques with SPR in terms of their analytical capabilities to detect carbohydrates, their conjugates with proteins and lipids and to study the carbohydrate-specific bindings. A few selected examples of the SPR approaches developed during 2004–2011 for the biosensing of glycoforms and for glycan–protein affinity studies are comprehensively discussed.     

Determination of post-culture processing with carbohydrates by MALDI-MS and TMS derivatization GC-MS

Biological materials generally require stabilization to retain activity or viability in a dry form. A number of industrial products, such as vaccines, probiotics and biopesticides have been produced as dry preparations. The same methods and materials used for stabilizing commercial microbial products may be applicable to preserving biothreat pathogens in a dry form. This is a likely step that may be encountered when looking at samples from terrorism attempts since only spores, such as those from Bacillus anthracis, are inherently stable when dried. The stabilizers for microbial preparations generally include one or more small carbohydrates. Different formulations have been reported for different industrial products and are often determined empirically. However sugar alcohols (mannitol and sorbitol) and disaccharides (lactose, sucrose and trehalose) are the common constituents of these formulations. We have developed an analytical method for sample preparation and detection of these simple carbohydrates using two complementary analytical tools, MALDI-MS and GC-MS. The native carbohydrates and other constituents of the formulation are detected by MALDI-MS as a screening tool. A longer and more detailed analysis is then used to specifically identify the carbohydrates by derivatization and GC-MS detection. Both techniques were tested against ten different types of stabilization recipes with Yersinia pestis cell mass cultured on different media types used as the biological component. A number of additional components were included in these formulations including proteins and peptides from serum or milk, polymers (e.g. poly vinyl pyrrolidone - PVP) and detergents (e.g. Tween). The combined method was characterized to determine several figures of merit. The accuracy of the method was 98% for MALDI-MS and 100% for GC-MS. The repeatability for detection of carbohydrates by MALDI-MS was determined to be 96%. The repeatability of compound identification by GC-MS was determined by monitoring variation in retention time, which is vital for identification of isomeric carbohydrates. The figures of merit illustrate an effective and accurate method for mono and disaccharide detection independent of formulation. This meets our primary goal for method development as small carbohydrates are among the most common stabilizers employed.     

Ionic liquids for the fractionation of microalgae biomass

Microalgae have emerged as one of the most promising sources of renewable biomass. However, considerable challenges must be addressed in order to improve the commercial outlook for the production of commodity chemicals. The largest challenge remains the energy intensive and consequently costly process of microalgae harvesting and drying. Ionic liquids have found a niche application in this area by allowing the extraction of lipids from wet biomass at low temperatures in less time than traditional lipid extraction methods. A number of recent studies have advanced the study of wet extraction of microalgae using ionic liquids and elucidated some of the limitations of this process. However, the most promising avenue for ionic liquid-based wet extraction lies in the fractionation and recovery of multiple biomass products such as lipids, carbohydrates, and carotenoids, in a single process.     

Photoautotrophic and Mixotrophic Cultivation of Polyhydroxyalkanoate-Accumulating Microalgae Consortia Selected under Nitrogen and Phosphate Limitation

Microalgae consortia were photoautotrophically cultivated in sequencing batch photobioreactors (SBPRs) with an alteration of the normal growth and starvation (nutrient limitation) phases to select consortia capable of polyhydroxyalkanoate (PHA) accumulation. At the steady state of SBPR operation, the obtained microalgae consortia, selected under nitrogen and phosphate limitation, accumulated up to 11.38% and 10.24% of PHA in their biomass, which was identified as poly(3-hydroxybutyrate) (P3HB). Photoautotrophic and mixotrophic batch cultivation of the selected microalgae consortia was conducted to investigate the potential of biomass and PHA production. Sugar source supplementation enhanced the biomass and PHA production, with the highest PHA contents of 10.94 and 6.2%, and cumulative PHA productions of 100 and 130 mg/L, with this being achieved with sugarcane juice under nitrogen and phosphate limitation, respectively. The analysis of other macromolecules during batch cultivation indicated a high content of carbohydrates and lipids under nitrogen limitation, while higher protein contents were detected under phosphate limitation. These results recommended the selected microalgae consortia as potential tools for PHA and bioresource production. The mixed-culture non-sterile cultivation system developed in this study provides valuable information for large-scale microalgal PHA production process development following the biorefinery concept.     

Development of high throughput 96-blade solid phase microextraction-liquid chromatrography-mass spectrometry protocol for metabolomics

In metabolomics, the workflow for quantitative and comprehensive metabolic mapping of cellular metabolites can be a very challenging undertaking. Sampling and sample preparation play a significant role in untargeted analysis, as they may affect the composition of the analyzed metabolome. In the current work, different solid phase microextraction (SPME) coating chemistries were developed and applied to provide simultaneous extraction of a wide range of both hydrophobic and hydrophilic cellular metabolites produced by a model organism, Escherichia coli. Three different LC-MS methods were also evaluated for analysis of extracted metabolites. Finally, over 200 cellular metabolites were separated and detected with widely varying hydrophobicities ranging within −7 < log P < 15, including amino acids, peptides, nucleotides, carbohydrates, polycarboxylic acids, vitamins, phosphorylated compounds, and lipids such as hydrophobic phospholipids, prenol lipids, and fatty acids at the stationary phase of the E. coli life cycle using the developed 96-blade SPME-LC-MS method.     

Recovering Microalgal Bioresources: A Review of Cell Disruption Methods and Extraction Technologies

Microalgae have evolved into a promising sustainable source of a wide range of compounds, including protein, carbohydrates, biomass, vitamins, animal feed, and cosmetic products. The process of extraction of intracellular composites in the microalgae industry is largely determined by the microalgal species, cultivation methods, cell wall disruption techniques, and extraction strategies. Various techniques have been applied to disrupt the cell wall and recover the intracellular molecules from microalgae, including non-mechanical, mechanical, and combined methods. A comprehensive understanding of the cell disruption processes in each method is essential to improve the efficiency of current technologies and further development of new methods in this field. In this review, an overview of microalgal cell disruption techniques and an analysis of their performance and challenges are provided. A number of studies on cell disruption and microalgae extraction are examined in order to highlight the key challenges facing the field of microalgae and their future prospects. In addition, the amount of product recovery for each species of microalgae and the important parameters for each technique are discussed. Finally, pulsed electric field (PEF)-assisted treatments, which are becoming an attractive option due to their simplicity and effectiveness in extracting microalgae compounds, are discussed in detail.     

Influence of the preparation route on the supramolecular organization of lipids in a vesicular system

A confocal fluorescence microscopy-based assay was used for studying the influence of the preparation route on the supramolecular organization of lipids in a vesicular system. In this work, vesicles composed of cholesterol and CTAB (1/1 mol %) or cholesterol and DOPC (2/8 mol %) and incorporating two membrane dyes were prepared by either a compressed fluid (CF)-based method (DELOS-susp) or a conventional film hydration procedure. They were subsequently immobilized and imaged individually using a confocal fluorescence microscope. Two integrated fluorescence intensities, I(dye1) and I(dye2), were assigned to each tracked vesicle, and their ratio, I(dye1)/I(dye2), was used for quantifying the degree of membrane inhomogeneity between individual vesicles within each sample. A distribution of I(dye1)/I(dye2) values was obtained for all the studied vesicular systems, indicating intrasample heterogeneity. The degree of inhomogeneity (DI) was similar for Chol/DOPC vesicles prepared by both procedures. In contrast, DI was more than double for the hydration method compared to the CF-based method in the case of Chol/CTAB vesicles, which can suffer from lipid demixing during film formation. These findings reveal a more homogeneous vesicle formation path by CFs, which warranted good homogeneity of the vesicular system, independently of the lipid mixture used.     

Fabrication of Highly Stable Glyco‐Gold Nanoparticles and Development of a Glyco‐Gold Nanoparticle‐Based Oriented Immobilized Antibody Microarray for Lectin (GOAL) Assay

The design of high‐affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco‐gold nanoparticles (glyco‐AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco‐AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70 mol %) and an amphiphilic linker (30 mol %) provide long‐term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco‐AuNPs enable the detection of model plant lectins such as Concanavalin A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop in situ glyco‐AuNPs‐based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of glyconanoparticle‐based biosensors.     

Liquid chromatographic determination of simple phenolic compounds in waste waters from olive oil production plants.

A method was developed for the determination of simple phenolic compounds (PCs) in waste waters from olive oil production plants by liquid chromatography (LC). The sample under examination was acidified to pH 2 to precipitate proteins, acetone was added to eliminate the colloidal fraction, and hexane was used for extraction to eliminate lipidic substances. The solution obtained was filtered and injected into the LC system; the wavelength selected for the spectrophotometric detection was specific for PCs, so that carbohydrates, organic acids, and short-chain free fatty acids did not interfere. Recoveries of 9 PCs spiked to a real sample were 90-100% for concentrations ranging from 20 to 2000 mg/L for each analyte.     

Determination of tazobactam and piperacillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid chromatography

A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.     

Sampling hazelnuts for aflatoxin: uncertainty associated with sampling, sample preparation, and analysis

The variability associated with the aflatoxin test procedure used to estimate aflatoxin levels in bulk shipments of hazelnuts was investigated. Sixteen 10 kg samples of shelled hazelnuts were taken from each of 20 lots that were suspected of aflatoxin contamination. The total variance associated with testing shelled hazelnuts was estimated and partitioned into sampling, sample preparation, and analytical variance components. Each variance component increased as aflatoxin concentration (either B1 or total) increased. With the use of regression analysis, mathematical expressions were developed to model the relationship between aflatoxin concentration and the total, sampling, sample preparation, and analytical variances. The expressions for these relationships were used to estimate the variance for any sample size, subsample size, and number of analyses for a specific aflatoxin concentration. The sampling, sample preparation, and analytical variances associated with estimating aflatoxin in a hazelnut lot at a total aflatoxin level of 10 ng/g and using a 10 kg sample, a 50 g subsample, dry comminution with a Robot Coupe mill, and a high-performance liquid chromatographic analytical method are 174.40, 0.74, and 0.27, respectively. The sampling, sample preparation, and analytical steps of the aflatoxin test procedure accounted for 99.4, 0.4, and 0.2% of the total variability, respectively.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis maps: relevance to proteomics

Proteomics requires a large-scale, simultaneous separation of proteins from a mixture, assessment of the relative abundance of these molecules, and identification and characterization of each component. In 2-D PAGE separations, the best method of choice for protein analysis, separation of all the proteins present in the sample is still far to be achieved and comigrating proteins in the same spot are in general present. A statistical estimation of the degree of spot overlapping present in a 2-D PAGE separation is here described: for different conditions of spot overcrowding in the map, the degree of overlapping can be quantified in terms of purity degree of each spot or percentage of proteins that will appear in the map as a single spot. A computer simulation approach is described: it is based on the protein separation pattern present in the experimental maps. The results thus obtained are compared to a theoretical model (statistical degree of peak overlapping model) based on random spot position. The described procedures were applied to an experimental reference map of human plasma. The severity of spot overlapping in 2-D PAGE maps is estimated and the influence of different experimental conditions (strip dimension, detector system performance, pI range) is discussed. These informations are useful to quantitatively estimate the degree of error associated with identification and quantitation of each protein and to set-up experimental conditions which will increase resolution and greatly decrease the probability of spot overlapping.     

Application of different modes of thin-layer chromatography and mass spectrometry for the separation and detection of large and small biomolecules

Biomolecules are widespread throughout the world. A biomolecule is any organic molecule produced by a living organism, including large polymeric molecules such as proteins, polysaccharides and nucleic acids. Many sample preparation techniques are used in biomolecule analysis; the method selected depends on the complexity of the sample, the nature of the matrix and the analytes, and the analytical technique available. This review covers the current state of knowledge on thin-layer chromatography and mass spectrometry for qualitative analysis of biomolecules. In the first part of the paper the reader will gain useful information to avoid some problems about performing various modes of thin-layer chromatography combined with mass spectrometry experiments and in the second part he will find useful information for application of these techniques for separation, detection, and qualitative investigation of structures and quantitative determination of biomolecules such as proteins, peptides, oligonucleotides, amino acids, DNA, RNA, and lipids.     

A Method for Microalgae Proteomics Analysis Based on Modified Filter-Aided Sample Preparation

With the fast development of microalgal biofuel researches, the proteomics studies of microalgae increased quickly. A filter-aided sample preparation (FASP) method is widely used proteomics sample preparation method since 2009. Here, a method of microalgae proteomics analysis based on modified filter-aided sample preparation (mFASP) was described to meet the characteristics of microalgae cells and eliminate the error caused by over-alkylation. Using Chlamydomonas reinhardtii as the model, the prepared sample was tested by standard LC-MS/MS and compared with the previous reports. The results showed mFASP is suitable for most of occasions of microalgae proteomics studies.     

Characterization and discrimination of pollen by Raman microscopy

The chemical characterization and discrimination of allergy-relevant pollen (common ragweed (Ambrosia artemisiifolia), white birch (Betula pendula), English oak (Quercus robur), and European linden (Tilia cordata)) has been studied by Raman microscopy. Spectra were obtained at different excitation wavelengths (514, 633, and 780 nm) and various methods were examined to minimize the strong fluorescence background. The use of a He–Ne laser (633 nm) for excitation yields high-quality single pollen Raman spectra, which contain multiple bands due to pollen components such as carotenoids, proteins, nucleic acids, carbohydrates, and lipids. Multivariate classification, i.e. principal component analysis (PCA) and hierarchical cluster analysis, demonstrated the validity of the approach for discrimination between different pollen species.     

Simultaneous determination of iridoids, phenolic acids, flavonoids, and saponins in Flos Lonicerae and Flos Lonicerae Japonicae by HPLC-DAD-ELSD coupled with principal component analysis

A method, HPLC coupled with diode-array and evaporative light scattering detectors (HPLC-DAD-ELSD), was newly developed to evaluate the quality of Flos Lonicerae (FL) and Flos Lonicerae Japonicae (FLJ), through a simultaneous determination of multiple types of bioactive components. By employing DAD, the detection wavelengths were set at 240 nm for the determination of iridoids, 330 nm for phenolic acids, and 360 nm for flavonoids, respectively. While ELSD, connected in series after DAD, was applied to the determination of saponins. This assay was fully validated with respect to precision, repeatability, and accuracy. Moreover, principal component analysis (PCA) was used for the similarity evaluation of different samples, and it was proven straightforward and reliable to differentiate FL and FLJ samples from different origins. For PCA, two principal components have been extracted. Principal component 1 (PC1) influences the separation between different sample sets, capturing 54.598% variance, while principal component 2 (PC2) affects differentiation within sample sets, capturing 12.579% variance. In conclusion, simultaneous quantification of bioactive components by HPLC-DAD-ELSD coupled with PCA would be a well-acceptable strategy to differentiate the sources and to comprehensively control the quality of the medicinal plants FL and FLJ.     

Application of MOS Gas Sensors Coupled with Chemometrics Methods to Predict the Amount of Sugar and Carbohydrates in Potatoes

Five potato varieties were studied using an electronic nose with nine MOS sensors. Parameters measured included carbohydrate content, sugar level, and the toughness of the potatoes. Routine tests were carried out while the signals for each potato were measured, simultaneously, using an electronic nose. The signals obtained indicated the concentration of various chemical components. In addition to support vector machines (SVMs that were used for the classification of the samples, chemometric methods, such as the partial least squares regression (PLSR) method, the principal component regression (PCR) method, and the multiple linear regression (MLR) method, were used to create separate regression models for sugar and carbohydrates. The predictive power of the regression models was characterized by a coefficient of determination (R²), a root-mean-square error of prediction (RMSEP), and offsets. PLSR was able to accurately model the relationship between the smells of different types of potatoes, sugar, and carbohydrates. The highest and lowest accuracy of models for predicting sugar and carbohydrates was related to Marfona potatoes and Sprite cultivar potatoes. In general, in all cultivars, the accuracy in predicting the amount of carbohydrates was somewhat better than the accuracy in predicting the amount of sugar. Moreover, the linear function had 100% accuracy for training and validation in the C-SVM method for classification of five potato groups. The electronic nose could be used as a fast and non-destructive method for detecting different potato varieties. Researchers in the food industry will find this method extremely useful in selecting the desired product and samples.     

Sampling almonds for aflatoxin, part I: estimation of uncertainty associated with sampling, sample preparation, and analysis

Domestic and international regulatory limits have been established for aflatoxin in almonds and other tree nuts. It is difficult to obtain an accurate and precise estimate of the true aflatoxin concentration in a bulk lot because of the uncertainty associated with the sampling, sample preparation, and analytical steps of the aflatoxin test procedure. To evaluate the performance of aflatoxin sampling plans, the uncertainty associated with sampling lots of shelled almonds for aflatoxin was investigated. Twenty lots of shelled almonds were sampled for aflatoxin contamination. The total variance associated with measuring B1 and total aflatoxins in bulk almond lots was estimated and partitioned into sampling, sample preparation, and analytical variance components. All variances were found to increase with an increase in aflatoxin concentration (both B1 and total). By using regression analysis, mathematical expressions were developed to predict the relationship between each variance component (total, sampling, sample preparation, and analysis variances) and aflatoxin concentration. Variance estimates were the same for B1 and total aflatoxins. The mathematical relationships can be used to estimate each variance for a given sample size, subsample size, and number of analyses other than that measured in the study. When a lot with total aflatoxins at 15 ng/g was tested by using a 10 kg sample, a vertical cutter mixer type of mill, a 100 g subsample, and high-performance liquid chromatography analysis, the sampling, sample preparation, analytical, and total variances (coefficient of variation, CV) were 394.7 (CV, 132.4%), 14.7 (CV, 25.5%), 0.8 (CV, 6.1%), and 410.2 (CV, 135.0%), respectively. The percentages of the total variance associated with sampling, sample preparation, and analytical steps were 96.2, 3.6, and 0.2, respectively.