Real-time process analytical technology: Fluorescent dye-based miniaturized sensor for aggregate detection

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Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase Implications for NADPH recognition and structural stability

AbstractPhe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 Å is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 Å revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH

Combined bead milling and enzymatic hydrolysis for efficient fractionation of lipids, proteins, and carbohydrates of Chlorella vulgaris microalgae

A combined bead milling and enzymatic hydrolysis process was developed for fractionation of the major valuable biomass components, i.e., proteins, carbohydrates, and lipids from the microalgae Chlorella vulgaris. The cells were treated by bead milling followed by hydrolysis with different hydrolytic enzymes, including lipase, phospholipase, protease, and cellulase. Without enzymatic hydrolysis, the recovery yield of lipids, carbohydrates, and proteins for bead milled biomass was 75%, 31%, and 40%, respectively, while by applying enzymatic treatments these results were improved significantly. The maximum recovery yield for all components was obtained after enzymatic hydrolysis of bead milled biomass by lipase at 37 degrees C and pH 7.4 for 24 h, yielding 88% lipids in the solid phase while 74% carbohydrate and 68% protein were separated in the liquid phase. The recovery yield of components after enzymatic hydrolysis of biomass without bead milling was 44% lower than that of the milled biomass.publishedVersionPaid Open Acces

Understanding mild cell disintegration of microalgae in bead mills for the release of biomolecules

Cell disintegration is, in general, the first step in the biorefinery of algae, since it allows the release of biomolecules of interest from the cells into the bulk medium. For high-value commercial applications, the disintegration process must be selective, energy efficient and mild. Developing a process with such features would demand extensive experimental effort. In the present study, we attempt to provide a tool for developing an efficient disintegration process via bead milling, by proposing a modelling strategy that allows the prediction of the kinetics of cell disintegration while having as input not only process parameters but also strain-specific parameters like cell size and cell-wall strength. The model was validated for two different algal strains (Tetraselmis suecica and Chlorella vulgaris), at various values of bead size (0.3–1 mm) and bead fillings (2.5–75%) and at two different scales of 80 and 500 mL. Since the kinetics of disintegration is proportional to the kinetics of release of biomolecules, the model can be further used for scale-up studies and to establish a window of operation to selectively target cells or metabolites of interest. Furthermore, the energy consumption in the mill was evaluated and it was found that operating at high bead fillings (>65%) is crucial to ensure an energy efficient process.publishedVersionPaid Open Acces

Biorefinery of the macroalgae Ulva lactuca : extraction of proteins and carbohydrates by mild disintegration

publishedVersionPaid Open Acces

Design of value chains for microalgal biorefinery at industrial scale : process integration and techno-economic analysis

The objective of this work was to identify industrial scenarios for the most promising microalgal biorefinery value chains on the basis of product selection, yields, and techno-economic performance, using biological characteristics of algae species. The development, value creation, and validation of several new processing routes with applications in food, aquafeeds and non-food products were particularly considered in this work. The techno-economic performance of various single product value chains (SP) and multiproduct value chains (MP) was evaluated for four industrial microalgal strains. Cost-revenue optimization was done for a 10 kton microalgal dry weight y–1 simulated biorefinery plant, using flow sheeting software for equipment sizing, mass and energy flow modeling, and subsequent techno-economic evaluation. Data on yield, material and energy consumption were based on pre- and pilot size production plants (TRL 5–6). Revenue optimization was accomplished by first analyzing the performance of single product value chains of the microalgal strains. Subsequently, a strategy was developed to exploit almost all biomass based on the most promising microalgal strains. The cultivation costs are most of the time the major costs of the value chains. For the single product value chains common process bottlenecks are low product yields, especially for soluble proteins where only a small fraction of the biomass is leading to economic value. The biorefinery costs (excluding cultivation) vary significantly for various species, due to the species-specific operating conditions as well as differences in product yields. For the evaluated single product value chain scenarios the costs for utilities and other inputs were in general the highest contributing expenses. A biorefinery approach significantly increases the biomass utilization potential to marketable products from 7–28% to more than 97%. Although the cascading approach increases the total production costs of the multiproduct value chains significantly, this is more than compensated by the increased overall biomass revenue. For all selected multiproduct chains there is a significant potential to become profitable at a relevant industrial scale of 10 kton per year. Additional insights in the product functionality, quality, and their market size are needed to narrow down the wide range of foreseen product revenues and resulting profits.publishedVersio

Multistep Fractionation of Microalgal Biomolecules Using Selective Aqueous Two-Phase Systems

We aim to develop liquid-liquid extraction processes for the fractionation of microalgal components (proteins, pigments, lipids, and carbohydrates). The partitioning behavior of microalgal pigments and proteins in aqueous two-phase systems (ATPS) composed of the polymer polypropylene glycol with molecular weight 400 (PPG 400) + various cholinium based-ionic liquids was studied. A process for fractionation of multiple components from disrupted Neochloris oleoabundans was developed and evaluated. Results show that cholinium dihydrogen phosphate (Ch DHp) allows the fractionation of pigments in the PPG 400-rich phase and proteins in the Ch DHp-rich phase with high selectivity. It was demonstrated that a multiproduct approach can fractionate free glucose, and proteins in the ionic liquid-rich phase, pigments in the polymer-rich phase, while starch and lipids are recovered at the interface.</p

Selective fractionation of free glucose and starch from microalgae using aqueous two-phase systems

Microalgae are a promising source of lipids, pigments, proteins and carbohydrates, which are valuable compounds for many industries. However, optimal fractionation and valorization of all produced compounds is necessary to improve the economic viability of microalgae production. This paper aims to understand the fractionation of microalgae carbohydrates (free glucose and starch) in aqueous two-phase systems. Three aqueous two-phase systems were investigated to efficiently and mildly separate carbohydrates from disrupted Neochloris oleoabundans. This strain contains 16 w/w% of proteins, 48 w/w% total fatty acids and 27 w/w% carbohydrates when cultivated under saline water and nitrogen depletion conditions. The protein content decreases and the amount of fatty acids and carbohydrates increases notably under stress conditions and glucose becomes the main carbohydrate in this microalgae. Glucose is present in the disrupted microalgae as part of polymeric carbohydrates (starch) or in monomeric form (free glucose). With the aqueous two-phase system Polyethylene Glycol 400 - Cholinium dihydrogen phosphate (PEG400-ChDHp) microalgal free glucose is fractionated up to a recovery of 99% to the most hydrated bottom phase in a single step. Simultaneously, a recovery of 70% is reached for microalgal starch in the interface after two additional liquid-liquid extractions with PEG400-ChDHp. The final fractions obtained were free of pigments.publishedVersionPaid Open Acces

Pulsed Electric Field for protein release of the microalgae Chlorella vulgaris and Neochloris oleoabundans

publishedVersionPaid Open Acces

Lipid extraction from fresh Nannochloropsis oceanica using semi-hydrophobic eutectic solvents

Conventional lipid extraction from microalgae involves energy-intensive pretreatments and the use of non-renewable organic solvents. Eutectic solvents (ES), a new class of designer solvents, hold the potential to improve lipid extraction. Hydrophilic ES have been reported to impair the cell wall of microalgae, bypassing the need for pretreatments. However, other hydrophobic solvents were still required as extraction medium. Recently, ES imidazole and hexanoic acid was discovered to exhibit tuneable hydrophobicity, i.e., dissolving both water and lipids depending on their molar composition. In this work, we evaluated the feasibility of imidazole/hexanoic ES as a single solvent for lipid extraction from intact wet and dried microalga Nannochloropsis oceanica. Single-factor multilevel design of experiments is used to evaluate the yield under different conditions (ES composition, temperature, time, solvent/biomass ratio, and water content). Interestingly, the extractions from wet algae paste were higher than dried biomass, reaching a comparable yield to the traditional chloroform/methanol method. From wet biomass, >80 % lipids were extracted by the imidazole/hexanoic acid ES (15:85 mol/mol) at 50 °C within 2 h. Whereas, the extraction yield of dry biomass was lower, reaching only 65 % even after 12 h under the same condition. Supplementation of water during the dry extraction resulted in the same yield as the wet extraction. This research demonstrated that ES can be used to replace non-renewable organic solvents without the need of using mechanical disruption and can be applied directly on wet biomass