Immobilization of Cyclodextrin glycosyltransferase onto three dimensional- hydrophobic and two dimensional- hydrophilic supports: A comparative study.

Cyclodextrin glycosyltransferase (CGTase) degrades starch into cyclodextrin via enzymatic activity. In this study, we immobilize CGTase from Thermoanaerobacter sp. on two supports, namely graphene nanoplatelets (GNP) consisting of short stacks of graphene nanoparticles and a calcium-based two-dimensional metal organic framework (Ca-TMA). The uptakes of CGTase on GNP and Ca-TMA reached 40 and 21 mg g-1 respectively, but immobilized CGTase on Ca-TMA showed a higher specific activity (38 U mg-1 ) than that on GNP (28 U mg-1 ). Analysis of secondary structures of CGTase, shows that immobilization reduces the proportion of ß-sheets in CGTase from 56% in the free to 49% and 51.3% for GNP and Ca-TMA respectively, α-helix from 38.5% to 18.1 and 37.5%, but led to increased ß-turns from 5.5 to 40% and 11.2% for GNP and Ca-TMA, respectively. Lower levels of conformational changes were observed over the more hydrophilic Ca-TMA compared to hydrophobic GNP, resulting in its better activity. Increased ß-turns were found to correlate with lower ß-CD production, while more ß-sheets and α-helix favored more ß-CD. Reusability studies revealed that GNP retains up to 74% of initial CGTase activity, while Ca-TMA dropped to 33% after eight consecutive uses. The results obtained in this work provide insight on the effect of support's surface properties on CGTase performance and can assist in developing robust CGTase-based biocatalysts for industrial application.

ZIF-8 as support for enhanced stability of immobilized lipase used with a thermoresponsive switchable solvent to simplify the microalgae-to-biodiesel process.

The zeolitic imidazole framework (ZIF)- 8 was tested as a support to enhance the stability of immobilized lipase. The lipase immobilized on ZIF-8, through surface attachment and encapsulation, was used for the simultaneous cell disruption and oil extraction from untreated, wet microalgal paste. The successful attachment of the enzyme to ZIF-8 was confirmed via Fourier-transform infrared spectroscopy. The attachment of the enzyme did not significantly affect the crystallinity or morphology of ZIF-8 crystals. The encapsulated lipase@ZIF-8 system showed higher stability than the adsorbed system, due to its reduced vulnerability to leaching. After five cycles, the encapsulated lipase@ZIF-8 retained 32% of its initial activity, whereas, for the adsorbed lipase@ZIF-8, it reduced to 21%. An increase in methanol amount greater than 0.2 mL was shown to have a negative effect on enzyme activity. The fatty acid methyl ester yield increased significantly with an increase in the extraction- duration (up to 3 h), after which the effect faded until 5 h, after which the equilibrium yield was reached. Changing the composition of the thermoresponsive switchable solvent (TSS) showed that a higher FAME yield could be achieved by increasing the percentages of Ionic Liquid (IL) and polypropylene glycol and reducing the water percentage. Further studies are needed to optimize the TSS composition and its effects on the process.

Simultaneous Enzymatic Cellulose Hydrolysis and Product Separation in a Radial-Flow Membrane Bioreactor.

Hydrolysis is the heart of the lignocellulose-to-bioethanol conversion process. Using enzymes to catalyze the hydrolysis represents a more environmentally friendly pathway compared to other techniques. However, for the process to be economically feasible, solving the product inhibition problem and enhancing enzyme reusability are essential. Prior research demonstrated that a flat-sheet membrane bioreactor (MBR), using an inverted dead-end filtration system, could achieve 86.7% glucose yield from purified cellulose in 6 h. In this study, the effectiveness of flat-sheet versus radial-flow MBR designs was assessed using real, complex lignocellulose biomass, namely date seeds (DSs). The tubular radial-flow MBR used here had more than a 10-fold higher membrane surface area than the flat-sheet MBR design. With simultaneous product separation using the flat-sheet inverted dead-end filtration MBR, a glucose yield of 10.8% from pretreated DSs was achieved within 8 h of reaction, which was three times higher than the yield without product separation, which was only 3.5% within the same time and under the same conditions. The superiority of the tubular radial-flow MBR to hydrolyze pretreated DSs was confirmed with a glucose yield of 60% within 8 h. The promising results obtained by the novel tubular MBR could pave the way for an economic lignocellulose-to-bioethanol process.

Immobilization of Lipase from Thermomyces lanuginosus in Magnetic Macroporous ZIF-8 Improves Lipase Reusability in Biodiesel Preparation.

In recent years, metal-organic frameworks (MOFs) have emerged as a promising support for immobilizing enzymes due to their high designability and structural diversity. Previous studies show that MOFs with single-crystal-ordered macroporous structures can effectively improve the accessibility of large-size enzyme and reduce the mass transfer resistance compared to conventional MOFs. In order to further enhance the reusability of lipase immobilized on macroporous MOFs, modification of MOFs through some magnetic particles could be an efficient approach. In this work, magnetic macroporous zeolitic imidazolate framework-8 (ZIF-8), referred to as m-M-ZIF-8 (with an average macropore size of about 140 nm), was synthesized and used for the immobilization of Thermomyces lanuginosus lipase (TLL). It was found that enzyme loading and the specific enzyme activity of the immobilized lipase were greatly enhanced through this magnetic modification. The enzyme loading of TLL@C-ZIF-8, T LL@M-ZIF-8, and TLL@m-M-ZIF-8 was 0.060, 0.074, and 0.076 mg/mg respectively. Besides, the activity of 93.5% was maintained after the immobilized lipase being repeatedly used for five batches, which was much higher than that of the immobilized lipase without magnetic modification, which was only 73.4%.

Removal of Bromine from the non-metallic fraction in printed circuit board via its Co-pyrolysis with alumina.

The effectiveness of a recycling approach of the printed circuit board (PCBs), and, thus, the quality of polymeric constituents, primarily rests on the capacity to eliminate the bromine content (mainly as HBr). HBr is emitted in appreciable quantities during thermal decomposition of PCB-contained brominated flame retardants (BFRs). The highly corrosive, yet relatively reactive HBr, renders recovery of bromine-free hydrocarbons streams from brominated polymers in PCBs very challenging. Via combined experimental and theoretical frameworks, this study explores the potential of deploying alumina (Al2O3) as a debromination agent of Br-containing hydrocarbon fractions in PCBs. A consensus from a wide array of characterization techniques utilized herein (ICP-OES, IC, XRD, FTIR, SEM-EDX, and TGA) clearly demonstrates the transformation of alumina upon its co-pyrolysis with the non-metallic fractions of PCBs, into aluminum bromides and oxy-bromides. ICP-OES measurements disclose the presence of high concentration of Cu in the non-metallic fraction of PCB, along with minor levels of selected valuable metals. Likewise, elemental ionic analysis by IC demonstrates an elevated concentration of bromine in washed alumina-PCBs pyrolysates, especially at 500 °C. The Coats-Redfern model facilitates the derivation of thermo-kinetic parameters underpinning the thermal degradation of alumina-PCB mixtures. Density functional theory calculations (DFT) establish an accessible reaction pathway for the HBr uptake by the alumina surface, thus elucidating chemical reactions governing the observed alumina debromination activity. Findings from this study illustrate the capacity of alumina as a HBr fixation agent during the thermal treatment of e-waste.

Advances in Enzyme and Ionic Liquid Immobilization for Enhanced in MOFs for Biodiesel Production.

Biodiesel is a promising candidate for sustainable and renewable energy and extensive research is being conducted worldwide to optimize its production process. The employed catalyst is an important parameter in biodiesel production. Metal-organic frameworks (MOFs), which are a set of highly porous materials comprising coordinated bonds between metals and organic ligands, have recently been proposed as catalysts. MOFs exhibit high tunability, possess high crystallinity and surface area, and their order can vary from the atomic to the microscale level. However, their catalytic sites are confined inside their porous structure, limiting their accessibility for biodiesel production. Modification of MOF structure by immobilizing enzymes or ionic liquids (ILs) could be a solution to this challenge and can lead to better performance and provide catalytic systems with higher activities. This review compiles the recent advances in catalytic transesterification for biodiesel production using enzymes or ILs. The available literature clearly indicates that MOFs are the most suitable immobilization supports, leading to higher biodiesel production without affecting the catalytic activity while increasing the catalyst stability and reusability in several cycles.

MOFs as Potential Matrices in Cyclodextrin Glycosyltransferase Immobilization.

Cyclodextrins (CDs) and their derivatives have attracted significant attention in the pharmaceutical, food, and textile industries, which has led to an increased demand for their production. CD is typically produced by the action of cyclodextrin glycosyltransferase (CGTase) on starch. Owing to the relatively high cost of enzymes, the economic feasibility of the entire process strongly depends on the effective retention and recycling of CGTase in the reaction system, while maintaining its stability. CGTase enzymes immobilized on various supports such as porous glass beads or glyoxyl-agarose have been previously used to achieve this objective. Nevertheless, the attachment of biocatalysts on conventional supports is associated with numerous drawbacks, including enzyme leaching prominent in physical adsorption, reduced activity as a result of chemisorption, and increased mass transfer limitations. Recent reports on the successful utilization of metal-organic frameworks (MOFs) as supports for various enzymes suggest that CGTase could be immobilized for enhanced production of CDs. The three-dimensional microenvironment of MOFs could maintain the stability of CGTase while posing minimal diffusional limitations. Moreover, the presence of different functional groups on the surfaces of MOFs could provide multiple points for attachment of CGTase, thereby reducing enzyme loss through leaching. The present review focuses on the advantages MOFs can offer as support for CGTase immobilization as well as their potential for application in CD production.

Lipase Immobilization on Macroporous ZIF-8 for Enhanced Enzymatic Biodiesel Production.

Immobilization of enzyme on metal-organic frameworks (MOFs) has drawn increasing interest owing to their many well-recognized characteristics. However, the pore sizes of MOFs (mostly micropores and mesopores) limit their application for enzyme immobilization to a great extent owing to the large size of enzyme molecules. Synthesis of MOFs with macropores would therefore solve this problem, typically encountered with conventional MOFs. In this work, macroporous zeolitic imidazolate frameworks (ZIF-8), referred to as M-ZIF-8, were synthesized and used for immobilization of Aspergillus niger lipase (ANL). Immobilization efficiency using M-ZIF-8 and enzymatic catalytic performance for biodiesel preparation were investigated. The immobilized ANL on M-ZIF-8 (ANL@M-ZIF-8) showed higher enzymatic activity (6.5-fold), activity recovery (3.8-fold), thermal stability (1.4- and 3.4-fold at 80 and 100 °C, respectively), reusability (after five cycles, 68% of initial activity was maintained), and porosity than ANL on conventional ZIF-8 (ANL/ZIF-8). In addition, by using ANL@M-ZIF-8 for catalyzing a biodiesel production reaction, a higher fatty acid methyl ester yield was achieved.

Using microalgae for remediation of crude petroleum oil-water emulsions.

Crude petroleum oil spills are among the most important organic contaminations. While the separated oils accumulated on the surface water are relatively easily removed, the emulsified portions are more difficult to remove and pose significant threats to the environment. Bioremediation using bacteria has proven to be an effective method, but the biomass produced in this case does not have any significant remunerative value. In this work, microalgae were proposed to combine emulsified oil remediation process with the potential of microalgae as a biofuel feedstock, thus enhancing the economic and environmental benefits of the process. A freshwater strain of Chlorella vulgaris was grown in water containing different concentrations of emulsified crude oil at different temperatures. The specific growth rate (µmax ) of the microalgae for each initial oil concentration was determined and was found to increase with the increase in initial oil concentration. For example, at 30°C, the specific growth rate, µ increased from 0.477 to 0.784 per day as the oil concentration increased from 57 to 222 mg/L. At 30°C, the effect of substrate concentration agreed with that of the microalgae growth, whereas at 40°C, the drop in oil concentration decreased with the increase in concentration. The results were fitted to a modified Monod kinetics model that used specific interfacial area as the influential substrate instead of the actual concentration. The results of this study clearly show the potential of using microalgae for emulsified oil remediation at relatively high concentrations.

Immobilization of formate dehydrogenase in metal organic frameworks for enhanced conversion of carbon dioxide to formate.

Hydrogenation of carbon dioxide (CO2) to formic acid by the enzyme formate dehydrogenase (FDH) is a promising technology for reducing CO2 concentrations in an environmentally friendly manner. However, the easy separation of FDH with enhanced stability and reusability is essential to the practical and economical implementation of the process. To achieve this, the enzyme must be used in an immobilized form. However, conventional immobilization by physical adsorption is prone to leaching, resulting in low stability. Although other immobilization methods (such as chemical adsorption) enhance stability, they generally result in low activity. In addition, mass transfer limitations are a major problem with most conventional immobilized enzymes. In this review paper, the effectiveness of metal organic frameworks (MOFs) is assessed as a promising alternative support for FDH immobilization. Kinetic mechanisms and stability of wild FDH from various sources were assessed and compared to those of cloned and genetically modified FDH. Various techniques for the synthesis of MOFs and different immobilization strategies are presented, with special emphasis on in situ and post synthetic immobilization of FDH in MOFs for CO2 hydrogenation.

Simultaneous and rapid quantification of microalga biomolecule content using electrochemical impedance spectroscopy.

Lipids, proteins, and carbohydrates are the major constituents found in microalga cells, in varying proportions, and these biomolecules find applications in different industries. During microalga cultivation, to efficiently manipulate, control, and optimize the productivity of a specific compound for a specific application, real-time monitoring of these three cell components is essential. In this study, a method using measurement of electrical capacitance was developed to simultaneously determine the lipid, protein, and carbohydrate content of microalga cells without the requirement for any pre-processing steps. The marine microalga Nannochloropsis oculata was cultivated under nitrogen starvation conditions to induce lipid accumulation over a period of 22 days. The correlation between the electrical capacitance of the microalga culture and the intracellular biomolecule content (determined by standard techniques) was investigated, enabling subsequent deduction of microalga intracellular content from electrical capacitance of the culture. The accuracy and precision of the technique were proven by validating an independent sample. The main advantage of the proposed technique is its capability of quantifying microalga composition within a few minutes, significantly faster than currently available conventional techniques.

Gold extraction from biosolid sludge obtained by sewage treatment.

Treatment of municipal wastewater, which involves multiple steps, produces large amounts of biosolid sludge, which is either incinerated or disposed in landfills. This sludge contains carbon, nitrogen, and phosphorous in appreciable amounts, and hence, it is being recently suggested that it should be used as a fertilizer. However, the biosolid sludge also contains large amounts of heavy metals, which exert harmful effects on the plantation and therefore, they must be removed before it can be used as a fertilizer. In addition, some of these heavy metals are precious such as gold. In this work, heavy metals present in the biosolid sludge produced from municipal wastewater plants were extracted using acidic solutions of different strengths. The method of selected gold extraction using tributyl phosphate (TBP) in kerosene solution from the metal rich acidic solution was also tested. The rate and yield of gold extraction increase with the increase in the acidic strength. The highest extracted gold yield was 0.012 mg/g of biomass. The amount of gold recovery into the TBP solution was 26%, which was much higher than that of other metals extracted into the acid solution. The importance of removing the metals from the biosolid is obvious, as it allows the latter to be used as a fertilizer. In addition, using only one additional step, a valuable product, gold, can be selectively separated, despite being present in lower amounts that other metals found in the sludge.

Microalgae cultivation for phenolic compounds removal.

Microalgae are promising sustainable and renewable sources of oils that can be used for biodiesel production. In addition, they contain important compounds, such as proteins and pigments, which have large applications in the food and pharmaceutical industries. Combining the production of these valuable products with wastewater treatment renders the cultivation of microalgae very attractive and economically feasible. This review paper presents and discusses the current applications of microalgae cultivation for wastewater treatment, particularly for the removal of phenolic compounds. The effects of cultivation conditions on the rate of contaminants removal and biomass productivity, as well as the chemical composition of microalgae cells are also discussed.

Bilirubin detoxification using different phytomaterials: characterization and in vitro studies.

BACKGROUND: Activated carbon (AC) is a common adsorbent that is used in both artificial and bioartificial liver devices. METHODS: Three natural materials - date pits of Phoenix dactylifera (fruit), Simmondsia chinensis (jojoba) seeds, and Scenedesmus spp. (microalgae) - were used in the present investigation as precursors for the synthesis of AC using physical activation. The chemical structures and morphology of AC were analyzed. Then, AC's bilirubin adsorption capacity and its cytotoxicity on normal liver (THLE2) and liver cancer (HepG2) cells were characterized. RESULTS: Compared with the other raw materials examined, date-pit AC was highly selective and showed the most effective capacity of bilirubin adsorption, as judged by isotherm-modeling analysis. MTT in vitro analysis indicated that date-pit AC had the least effect on the viability of both THLE2 and HepG2 cells compared to jojoba seeds and microalgae. All three biomaterials under investigation were used, along with collagen and Matrigel, to grow cells in 3D culture. Fluorescent microscopy confirmed date-pit AC as the best to preserve liver cell integrity. CONCLUSION: The findings of this study introduce date-pit-based AC as a novel alternative biomaterial for the removal of protein-bound toxins in bioartificial liver devices.

Evaluation of an activated carbon packed bed for the adsorption of phenols from petroleum refinery wastewater.

The performance of an adsorption column packed with granular activated carbon was evaluated for the removal of phenols from refinery wastewater. The effects of phenol feed concentration (80-182 mg/l), feed flow rate (5-20 ml/min), and activated carbon packing mass (5-15 g) on the breakthrough characteristics of the adsorption system were determined. The continuous adsorption process was simulated using batch data and the parameters for a new empirical model were determined. Different dynamic models such as Adams-Bohart, Wolborsko, Thomas, and Yoon-Nelson models were also fitted to the experimental data for the sake of comparison. The empirical, Yoon-Nelson and Thomas models showed a high degree of fitting at different operation conditions, with the empirical model giving the best fit based on the Akaike information criterion (AIC). At an initial phenol concentration of 175 mg/l, packing mass of 10 g, a flow rate of 10 ml/min and a temperature of 25 °C, the SSE of the new empirical and Thomas models were identical (248.35) and very close to that of the Yoon-Nelson model (259.49). The values were significantly lower than that of the Adams-Bohart model, which was determined to be 19,358.48. The superiority of the new empirical model and the Thomas model was also confirmed from the values of the R 2 and AIC, which were 0.99 and 38.3, respectively, compared to 0.92 and 86.2 for Adams-Bohart model.

Effect of Enzymatic pre-treatment of microalgae extracts on their anti-tumor activity.

BACKGROUND: There is an increasing need to find natural bioactive compounds for pharmaceutical applications, because they have less harmful side effects compared to their chemical alternatives. Microalgae (MA) have been identified as a promising source for these bioactive compounds, and this work aimed to evaluate the anti-proliferative effects of semi-purified protein extracted from MA against several tumor cell lines. METHODS: Tested samples comprised MA cell extracts treated with cellulase and lysozyme, prior to extraction. The effect of dialysis, required to remove unnecessary small molecules, was also tested. The anti-cancer efficacies of the dialyzed and undialyzed extracts were determined by measuring cell viability after treating four human cancer cell lines, specifically A549 (human lung carcinoma), MCF-7 (human breast adenocarcinoma), MDA MB-435 (human melanoma), and LNCap (human prostate cancer cells derived from a metastatic site in the lymph node). This was compared to the effects of the agents on the human BPH-1 cell line (benign human prostate epithelial cells). The t-test was used to statistically analyze the results and determine the significance. RESULTS: Against LNCap and A549 cells, the performance of cellulase-treated extracts was better (with p-values < 0.05, as compared to the control) than that of lysozyme-treated preparations (with p-values mainly > 0.05, as compared to the control); however, they had similar effects against the other two tumor cell lines (with p-values mainly < 0.05, as compared to the control). Moreover, based on their effect on BPH-1 cells, extracts from lysozyme-treated MA cells were determined to be safer against the benign prostate hyperplasia cells, BPH-1 (with p-values mainly > 0.05, as compared to the control). After dialysis, the performance of MA extracts from lysozyme-treated cells was enhanced significantly (with p-values dropping to < 0.05, as compared to the control). CONCLUSIONS: The results of this work provide important information and could provide the foundation for further research to incorporate MA constituents into pharmaceutical anti-cancer therapeutic formulations.

Enzymatic pre-treatment of microalgae cells for enhanced extraction of proteins.

Crude proteins and pigments were extracted from different microalgae strains, both marine and freshwater. The effectiveness of enzymatic pre-treatment prior to protein extraction was evaluated and compared to conventional techniques, including ultrasonication and high-pressure water extraction. Enzymatic pre-treatment was chosen as it could be carried out at mild shear conditions and does not subject the proteins to high temperatures, as with the ultrasonication approach. Using enzymatic pre-treatment, the extracted proteins yields of all tested microalgae strains were approximately 0.7 mg per mg of dry cell weight. These values were comparable to those achieved using a commercial lytic kit. Ultrasonication was not very effective for proteins extraction from Chlorella sp., and the extracted proteins yields did not exceed 0.4 mg per mg of dry cell weight. For other strains, similar yields were achieved by both treatment methods. The time-course effect of enzymatic incubation on the proteins extraction efficiency was more evident using laccase compared to lysozyme, which suggested that the former enzyme has a slower rate of cell disruption. The crude extracted proteins were fractionated using an ion exchange resin and were analyzed by the electrophoresis technique. They were further tested for their antioxidant activity, the highest of which was about 60% from Nannochloropsis sp. The total phenolic contents in the selected strains were also determined, with Chlorella sp. showing the highest content reaching 17 mg/g. Lysozyme was also found to enhance the extraction of pigments, with Chlorella sp. showing the highest pigments contents of 16.02, 4.59 and 5.22 mg/g of chlorophyll a, chlorophyll b and total carotenoids, respectively.

RF microalgal lipid content characterization.

Most conventional techniques for the determination of microalgae lipid content are time consuming and in most cases are indirect and require excessive sample preparations. This work presents a new technique that utilizes radio frequency (RF) for rapid lipid quantification, without the need for sample preparation. Tests showed that a shift in the resonance frequency of a RF open-ended coaxial resonator and a gradual increase in its resonance magnitude may occur as the lipids content of microalgae cells increases. These response parameters can be then calibrated against actual cellular lipid contents and used for rapid determination of the cellular lipids. The average duration of lipid quantification using the proposed technique was of about 1 minute, which is significantly less than all other conventional techniques, and was achieved without the need for any time consuming treatment steps.

A review of enzymatic transesterification of microalgal oil-based biodiesel using supercritical technology.

Biodiesel is considered a promising replacement to petroleum-derived diesel. Using oils extracted from agricultural crops competes with their use as food and cannot realistically satisfy the global demand of diesel-fuel requirements. On the other hand, microalgae, which have a much higher oil yield per hectare, compared to oil crops, appear to be a source that has the potential to completely replace fossil diesel. Microalgae oil extraction is a major step in the overall biodiesel production process. Recently, supercritical carbon dioxide (SC-CO(2)) has been proposed to replace conventional solvent extraction techniques because it is nontoxic, nonhazardous, chemically stable, and inexpensive. It uses environmentally acceptable solvent, which can easily be separated from the products. In addition, the use of SC-CO(2) as a reaction media has also been proposed to eliminate the inhibition limitations that encounter biodiesel production reaction using immobilized enzyme as a catalyst. Furthermore, using SC-CO(2) allows easy separation of the product. In this paper, conventional biodiesel production with first generation feedstock, using chemical catalysts and solvent-extraction, is compared to new technologies with an emphasis on using microalgae, immobilized lipase, and SC-CO(2) as an extraction solvent and reaction media.