Immobilization Conditions Research Articles

Preparation of highly stable immobilized Candida antarctica lipase B (CALB) through adjusting the surface properties of carrier: Preparation, characterization and performance evaluation

The stability of the immobilized lipase is the key factor that determines the economy and feasibility of its industrial application. Here, two robust immobilized Candida antarctica lipase B (CALB) were prepared through adjusting the surface properties of ECR1030 resin. Silane coupling agent (SCA) and dialdehyde cellulose (DAC) were employed to modify the carrier surface. Contact angle measurement showed that the hydrophobicity of the modified carrier increased first, and then decreased with the increase of the chain length of SCA. FTIR results showed that Si-O-Si bond and aldehyde group were attached to ECR1030, respectively, indicating that the ECR1030 resin was successfully modified. Meanwhile, the NH and CN bond were observed in the corresponding immobilized CALB, suggesting CALB was immobilized onto the modified carriers. The effects of immobilization conditions on CALB immobilization was further investigated, and the C8-ECR1030-CALB and DAC-ECR1030-CALB with the activity of 12,736 U/g and 11,962 U/g were obtained. Moreover, the stability of the immobilized lipases was evaluated and compared with the commercial Novozym 435. The C8-ECR1030-CALB and DAC-ECR1030-CALB exhibited comparable or superior stability to Novozym 435 and showed better deacidification effect than Novozym 435. This study paves road for further study involving preparation of highly stable immobilized lipase.

Application of Spectrogel clay as a support for immobilization of lipase from Burkholderia cepacia

AbstractThe interest in maximizing the production of ethyl esters in a sustainable way and with lower energy costs has increased the use of immobilized enzymes as catalysts. This study aimed to apply the commercial clay Spectrogel® as a support for the immobilization of lipase from Burkholderia cepacia by adsorption and covalent bonding methods. The immobilizations were carried out using a 23 factorial design to study the effects of the activity offered (U g−1), pH, and the molar concentration of the enzyme solution buffer (mol L−1) on the enzyme activity obtained (U g−1). From the statistical analysis of the results, the best conditions for immobilization were pH 7.0 and 0.1 mol L−1 for both tested immobilization methods, with the best offered activity being 5709 and 7600 U g−1. The activities obtained were 1219.81 ± 7.51 and 1274.89 ± 14.99 U g−1 for adsorption and covalent bonding, respectively. The biocatalysts exhibited protein leaching of 33.55 ± 1.08% and 19.44 ± 2.43% when immobilized by adsorption and covalent bonding, respectively. The optimal activity temperature and thermal stability were obtained at 40°C. Additionally, the immobilization of lipase in Spectrogel® by both methods was efficient, showing higher thermal stability than the free enzyme. Thus, this work contributed scientifically to the development of a new and economical biocatalyst for ethyl ester production.

Improvement of Thermo-Stability and Solvent Tolerant Property of Streptomyces sp. A3301 Lipase by Immobilization Techniques with Application in Poly (lactic acid) Polymerization by Using Biological Process

The thermo-solvent-tolerant lipase-producing actinomycete, Streptomyces sp. A3301, was utilized as a biocatalyst for poly (lactic acid) or PLA polymerization. The study aimed to optimize lipase immobilization conditions, characterize the immobilized lipase and apply it for PLA polymerization. The results showed using a sponge as the immobilizing matrix was the most effective method, achieving a maximum activity of 277 U/g of sponge. The optimal sponge size was determined to be 0.125 cm³ and pre-soaking the sponge in 0.1 M phosphate buffer at pH 7.0 for 24 h before use proved advantageous. Immobilization significantly enhanced the thermo-stability of the enzyme, with a relative activity ranging from 140 to 190 % within the temperature range of 30 to 60 °C. In contrast, the crude lipase exhibited thermo-stability only within the 30 - 50 °C range. The immobilized lipase demonstrated stability under PLA polymerization conditions, which involved a reaction mixture containing toluene and lactic acid and performed at 60 °C for 8 h. The immobilized lipase maintained its activity under this condition for 5 h, retaining a relative activity of 230 %, which was 1.2 times higher than the activity of the crude lipase. When the immobilized lipase was used in PLA polymerization, the resulting PLA product exhibited a molecular weight of 5,333 ± 0.02 Da, and the degree of polymerization was approximately 72. These findings underscore the potential of the immobilization technique to enhance lipase activity for PLA polymerization. HIGHLIGHTS PLA polymerization via a biological process is demonstrated. PLA can be polymerized by enzyme lipase produced by Streptomyces sp. A3301. Improving PLA polymerization with enzyme immobilization techniques. Sponge is the best immobilizer for PLA polymerization. PLA can be synthesized under mild conditions. GRAPHICAL ABSTRACT

Zr-based MOF as a support for lipase immobilization to enhance enzymatic transesterification for biodiesel production

The development of novel biocatalysts is essential to promote the commercialization of biodiesel production by transesterification reaction. In this paper, Rhizopus oryzae lipase (ROL) was immobilized on an amino-functionalized zirconium-based metal organoskeleton by interfacial adsorption. The immobilization conditions were optimized and the enzymatic properties were tested, and the resulting novel biocatalysts exhibited higher stability and heat resistance. SEM, XRD and BET analyses were used to characterize the biocatalysts and carrier materials. The catalytic performance of ROL@UiO-66-NH2 in the production of biodiesel by transesterification reaction was explored, and the production process was optimized by response surface method. The results showed that the conversion rate of FAEE reached 82.05% at molar ratio of ethanol/oil of 15.43:1, reaction temperature of 50.28°C, reaction time of 120.9 min, DES addition of 48.08 wt%, biocatalyst addition of 3 wt%, and ultrasonic power of 90 W. In addition, ROL@UiO-66-NH2 demonstrated good recyclability, with the catalytic efficiency remaining at 71.87% after five cycles.

Combination of the degrading bacterium Bacillus cereus MZ-1 and corn straw biochar enhanced the removal of imazethapyr from water solutions

Long residual herbicide imazethapyr has caused a serious threat to subsequent sensitive crop plants and ecological security. Efficient immobilized microorganism technology offers a sustainable solution for remediating pesticides contamination. In this study, three corn straw biochars produced at 300℃, 500℃, and 700℃ were used as carriers to immobilize the imazethapyr degrading strain Bacillus cereus MZ-1, aiming to efficiently remove imazethapyr from the water environment. Response surface methodology was employed to optimize the best immobilization conditions based on the efficiency of imazethapyr removal. The results indicate that the biochar produced at 500 ℃ was the most effective carrier for immobilization, despite having a lower imazethapyr removal capacity compared to the biochar produced at 700 ℃. Additionally, the optimal strain addition amount was a suspension of strain MZ-1 (OD600=1) in a 3.5-fold biochar solution, and the best immobilized time was 18 h. The prepared immobilized MZ-1 achieved a maximum imazethapyr removal efficiency of 79.85 %. Moreover, the immobilized MZ-1 demonstrated enhanced detoxification efficacy against imazethapyr-induced harm in sensitive oilseed rape crops, surpassing the effects of using MZ-1 or biochar alone. Results of this work suggests biochar immobilized degradation strain MZ-1 is a highly effective in-situ remediation strategy for bioremediation of imazethapyr contamination.

Establishment and evaluation of haloalkane dehalogenase tagged α1A-adrenergic receptor chromatography

Receptor chromatography is an efficient analytical technique that combines the high separation ability of chromatography with the high specificity of receptors for drug recognition. In addition, this technique offers the advantages of active recognition, online separation, and convenient multidimensional target tracking. This strategy allows target active ingredients in complex systems, such as traditional Chinese medicines, to be efficiently screened and accurately identified. Furthermore, the interactions between ligands and immobilized proteins can be studied. To avoid a loss in function, receptor chromatography requires efficient, mild, and simple immobilization methods that do not damage the structure of the immobilized receptors. Improvements in the activity, stability, and ligand-recognition specificity of immobilized functional proteins can be achieved by selecting appropriate immobilization conditions. Notably, the protein immobilization method is not only closely related to the recognition ability of receptor chromatography but also determines the accuracy of the technique. Common methods for immobilizing functional proteins include physical adsorption, chemical reactions, biological affinity reactions, and click chemistry. Despite being easy to operate under mild reaction conditions, these methods have shortcomings, including poor reaction specificity and the necessity of using high-purity functional proteins to prepare chromatography columns. Maintaining the high activity of immobilized receptors and ensuring excellent identification and separation abilities are key challenges in the further development of receptor chromatography. In this work, these issues were addressed by introducing a specific bioorthogonal reaction involving haloalkane dehalogenase (Halo) and 6-chlorohexanoic acid for the immobilization of the α1A-adrenergic receptor (α1A-AR). Specifically, Halo-α1A-AR was immobilized on the surface of 6-chlorohexanoic acid-modified aminopropyl silica gel in one step. The stationary phase with immobilized Halo-α1A-AR was characterized using scanning electron microscopy. Moreover, the activity of the Halo-α1A-AR chromatographic column was evaluated using specific ligands (terazosin hydrochloride, phentolamine mesylate, tamsulosin hydrochloride, and urapidil) and nonspecific ligands (yohimbe and metoprolol) for α1A-AR. Halo-α1A-AR was successfully immobilized on the silica gel surface with good stability over 30 days, and the Halo-α1A-AR chromatographic column exhibited good ligand-recognition activity. The nonlinear chromatography results indicated that prazosin hydrochloride, terazosin hydrochloride, and urapidil interacted with immobilized Halo-α1A-AR through one type of binding site, with association constants of 3.85×105, 5.00×105, and 5.90×105L/mol, respectively. In contrast, phentolamine mesylate and tamsulosin hydrochloride interacted with immobilized Halo-α1A-AR through two types of binding site. The association constants with the high- and low-affinity binding sites were 3.12×106 and 6.01×105L/mol, respectively, for phentolamine mesylate and 9.98×105 and 0.21×105L/mol, respectively, for tamsulosin hydrochloride. Compared with the traditional carbonyldiimidazole method, the immobilization method developed in this work did not require receptor purification and thus minimized the loss of receptor activity. The affinity constants obtained with immobilized Halo-α1A-AR were consistent with the values determined for receptor-ligand binding in solution, indicating that the Halo-α1A-AR chromatography column is suitable for studying drug-protein interactions. This approach also provides a foundation for the efficient screening and accurate determination of target active ingredients in complex systems.

Enhancing rhamnolipid production via immobilized Pseudomonas stutzeri lipase: A comparative study

Rhamnolipids (RLs) are widely studied biosurfactants with significant industrial potential in cosmetics, pharmaceuticals, and bioremediation due to their excellent surface activity, emulsifying properties and bioactive characteristics. However, high production costs impede their mass production. This study investigates the immobilization of Pseudomonas stutzeri lipase (PSL) on various supports to enhance RL synthesis efficiency, focusing on yield and regioselectivity in the enzymatic synthesis of 4-O-lauroylrhamnose by the transesterification of rhamnose with vinyl laurate. Three immobilization methods were compared: covalent binding, adsorption on Celite, and adsorption on hydrophobic supports. The immobilization efficiency varied depending on the method used, with the lowest observed for adsorption on Celite (56 %), followed by covalent immobilization on Sepabeads (EC-EP/S 78 % and EC-EP/L 70 %), and the highest for adsorption on hydrophobic supports (83–97 %, with EC-OD being the best at 97 %). For the enzymatic synthesis of 4-O-lauroylrhamnose, covalent immobilization on Sepabeads™ EC-EP yielded low conversions due to restricted conformational freedom of the enzyme. Celite® 545 adsorption resulted in moderate conversion rates, limited by the electrostatic interactions restricting enzyme activity. The most promising results were obtained with hydrophobic supports, particularly Purolite® ECR8806F, achieving nearly complete conversion and maintaining high regioselectivity at the 4-position of rhamnose in both THF and the green solvent 2-methyltetrahydrofuran (2-MeTHF). The study highlights the critical role of support hydrophobicity and active surface area in the immobilized enzyme performance. PSL immobilized on Purolite® ECR8806F demonstrated significant potential for sustainable RLs production, showing excellent reusability, stability and productivity across multiple reaction cycles. This study presents a significant advancement in RLs production by optimizing PSL immobilization and reaction conditions, facilitating the way for more cost-effective and sustainable industrial applications.

Long-Term Survival of Enterococcus faecium under Varied Stabilization and Cell Immobilization Conditions

Long-Term Survival of Enterococcus faecium under Varied Stabilization and Cell Immobilization Conditions

Cellulose / waste Cu2+-activated carbon composite: A sustainable and green material for boosting laccase activity and degradation of bisphenol A in wastewater

Low enzyme activity is one of the disadvantages of immobilized laccase. In this study, waste Cu2+-loaded activated carbon (Cu-AC) was successfully used in preparing a novel composite support，cellulose / Cu2+-loaded activated carbon beads (C / Cu-AC)， and effectively boosted immobilized laccase activity. To achieve optimum conditions for immobilization of laccase, the immobilization time, pH and laccase concentration were examined. The highest immobilized laccase activity (34.21 U/g) was achieved under optimum conditions (T = 4 h, pH = 4, C = 5 g/L), which was increased by 35.86 % compared to control. In addition, the immobilized laccase showed an outstanding performance in thermostability and reusability compared to free laccase. Moreover, the degradation of BPA by immobilized laccase was carried out, and the optimum degradation conditions were explored. Under such conditions: concentration of BPA was 75 mg / L and pH = 4, t = 1 h, T = 50 °C，the removal yield of BPA reached a maximum of 79.88 %. Therefore, the utilization of waste Cu-CA is a powerful method to boost immobilized laccase activity and creating a new way to high value treatment of waste Cu-CA.

Emergency services at sports places in Rio de Janeiro: intervention of the sports management, activity and tourism

Emergency services in sports environments are crucial to ensuring the safety of those participating. Although Rio de Janeiro boasts a large and varied number of sports facilities, little is known about the dynamics of care provided in these locations. The primary objective of this study was to analyze the emergency response data from sports environments attended by the Military Fire Brigade of the State of Rio de Janeiro (CBMERJ). Secondly, the study aimed to compare the factors associated with emergency care at sports facilities, identify the most common injury sites, infer the causes of these accidents, and observe the frequency of fatalities during sports activities. For this study, 615 data points from the first emergency rescue group (1GSE) of the Rio de Janeiro Military Fire Brigade were analyzed, divided into North (n = 215), South (n = 145), West (n = 214), and Central (n = 30) zones. The dependent variables were: gender, outcome types, care types, transport types, immobilization, care characterization, care strategy, death details, emergency occurrences, and injury distribution by region. The zones were compared using the Kruskal-Wallis test and Dunn’s post hoc analysis, with a significance level of p≤0.05. Statistical analysis revealed differences between the zones when comparing outcomes, with the northern zone showing higher values (~75%) of treated and removed victims compared to the western zone (60%). There were also differences between the zones in the type of care provided, where the northern zone showed higher values (~35%) of intermediate care compared to the western (~20%), southern (~25%), and central (%) zones. The type of transport used varied between zones, with the southern zone showing higher usage of rapid response vehicles (ASE L) than the northern and central regions. The analysis further revealed a difference in immobilization conditions, with the central zone showing a lower frequency (~20%) of immobilization, that is, a higher proportion of "other" outcomes compared to the northern (~40%), western (%), and southern (~50%) zones. Keywords: Emergency care; sports practices; injury; Fire Department.

Immobilization of Lipase from Thermomyces Lanuginosus and Its Glycerolysis Ability in Diacylglycerol Preparation.

In the glycerolysis process for diacylglycerol (DAG) preparation, free lipases suffer from poor stability and the inability to be reused. To address this, a cost-effective immobilized lipase preparation was developed by cross-linking macroporous resin with poly (ethylene glycol) diglycidyl ether (PEGDGE) followed by lipase adsorption. The selected immobilization conditions were identified as pH 7.0, 35 °C, cross-linking agent concentration 2.0%, cross-linking time 4 h, lipase amount 5 mg/g of support, and adsorption time 4 h. Enzymatic properties of the immobilized lipase were analyzed, revealing enhanced pH stability, thermal stability, storage stability, and operational stability post-immobilization. The conditions for immobilized enzyme-catalyzed glycerolysis to produce DAG were selected, demonstrating the broad applicability of the immobilized lipase. The immobilized lipase catalyzed glycerolysis reactions using various oils as substrates, with DAG content in the products ranging between 35 and 45%, demonstrating broad applicability. Additionally, the changes during the repeated use of the immobilized lipase were characterized, showing that mechanical damage, lipase leakage, and alterations in the secondary structure of the lipase protein contributed to the decline in catalytic activity over time. These findings provide valuable insights for the industrial application of lipase.

Pancreatic lipase immobilization on cellulose filter paper for inhibitors screening and network pharmacology study of anti-obesity mechanism

The discovery of pancreatic lipase (PL) inhibitors is an essential route to develop new anti-obesity drugs. In this experiment, chitosan was used to add amino groups to cellulose filter paper (CFP) and then glutaraldehyde was used to covalently combine PL with amino-modified CFP through the Schiff base reaction. Under optimal immobilization conditions, CFP immobilized PL has a wide range of pH and temperature tolerance, as well as excellent reproducibility, reusability and storage stability. Subsequently, 26 natural products (NPs) were screened by immobilized PL with black tea extract having the highest inhibition rate. Three compounds with binding effects on PL (epigallocatechin gallate, theaflavin-3-gallate and theaflavin-3,3′-digallate) were captured. Molecular docking proved that these three compounds have a strong binding affinity for PL. Fluorescence spectra further revealed that theaflavin-3,3′-digallate could statically quench the intrinsic fluorescence of pancreatic lipase. The molecular docking and thermodynamic parameters indicated that electrostatic interaction was considered as the main interaction force between PL and theaflavin-3,3′-digallate. Finally, the potential anti-obesity targets and pathways of the three compounds were discussed through network pharmacology. This study not only proposes a simple and efficient method for screening PL inhibitors, but also sheds light on the anti-obesity mechanism of active compounds in black tea.

Co-immobilization of magnesium precursor and Candida rugosa lipase on alumina via covalent bonding for biodiesel production

Catalyst development is one of the most challenging aspects in biodiesel production due to the compatibility issue with the feedstocks and catalyst poisoning upon exposure to process conditions. In the present research, chemoenzymatic catalyst was prepared by impregnation of magnesium aluminate (MA) on alumina support, modifying the support using activating agent, and immobilization of Candida rugosa lipase (CRL) onto the support. The optimum immobilization condition was resulting in 85.07 % of activity recovery of immobilized lipase, equivalent to 30.53 % of immobilization efficiency. The catalysts were characterized through XRD, FTIR, FESEM-EDX, NAA and CO2-TPD confirming the mixture of AlO(OH), magnesium aluminate (MA), (MgNO3)2 and Mg2+ on the support. CRL/L-lysine/MA showed remarkable improvement in thermal stability (40 − 70℃), solvents tolerance (ethanol, methanol, isopropanol, tert-butanol), and storage stability at 30 ℃, as compared to free CRL. CRL/L-lysine/MA successfully produced biodiesel yield up to 87.10 % at 200.71 rpm of agitation speed, 13.58 % (w/w) of water content, 10 h reaction time, 15 % (w/w) of catalyst loading, at 50 ℃, using 1:12 of oil to methanol molar ratio, and retaining 46.60 % of biodiesel yield after six cycle. The CRL/L-lysine/MA demonstrated novel catalysts characteristics comprising chemical and biological active species on a single support for biodiesel production from waste cooking oil (WCO).

New Design and Characteristics of Probiotics Immobilized on a Clinoptilolite-Containing Tuff

Increasing the biological effectiveness of probiotic preparations requires the development of new stable forms in the gastrointestinal tract. Live bacteria immobilized on a sorbent belong to the latest, fourth generation of probiotics, which ensures a prolonged effect. This study is devoted to developing a new method of preparing active lactobacilli on a natural mineral carrier, a tuff containing zeolite of the clinoptilolite group, which is among the most common authigenic silicate minerals that occur in sedimentary rocks and is known as a safe ion-exchange and adsorbing detoxicant. Among the characterized lactobacilli, strains of L. plantarum, L. acidophilus, and L. crispatus possessed a high level of acid formation and stability in gastrointestinal fluids. The protective effect of the clinoptilolite-containing tuff was registered when the samples were incubated in gastric juice. The optimal technological conditions for immobilization and lyophilization were determined, and the preservation of the viability and probiotic properties of bacteria was confirmed during 8 months of storage. The release of bacteria from the carrier occurred gradually over 12 h. The data obtained show how promising the new preparation is, combining the ability to detoxify harmful intestinal metabolites and the prolonged release of probiotics.

Goat Manure Potential as a Substrate for Biomethane Production—An Experiment for Photofermentation

This article presents the current state of biogas (biomethane) production technology—an example of the use of goat manure in terms of photofermentation efficiency. The theoretical and experimental potential of biomethane using biodegradability for anaerobic fermentation of goat manure was indicated. Goat manure was tested for its elemental composition to determine the suitability of this raw material for biogas production. The quality of biogas produced under atmospheric conditions from goat manure placed in a reactor (photodigester) was assessed. An attempt was made to determine the process conditions for immobilization on a goat manure bed (depending on the research material collected), which allows for demonstrating the activity of the fermentation bacterial flora, thus influencing the amount of biogas (biomethane) produced in the reactor. A mechanism for the photofermentation process involving the production of biomethane was developed. The novelty of this article is the development of the use of goat manure in an innovative way, pointing to the development of the biomethane industry. When comparing goat manure, active group (compact bed), it should be noted that K 3.132%, Na 0.266%, Ca 1.909% and Mg 0.993% are lower values compared to the material with values of K 3.397%, Na 0.284%, Ca 1.813% and Mg 0.990% which are higher. This is undoubtedly due to the presence of nutrients in the deposit that support the biomethane production process. The active group (compact bed) material A shows a dynamic increase in biomethane production with lower nutrient values. However, material B, having a higher percentage of ingredients, shows stabilization of biomethane production after the sixth month of the process. Technological trends and future prospects for the biomethane sector were initiated.

Immobilized Burkholderia cepacia lipase and its application in selective enrichment of EPA by hydrolysis of fish oil

Eicosapentaenoic acid (EPA), an orally active Omega-3 long-chain polyunsaturated fatty acid, has attracted much attention for its medical and healthcare value. Due to a large amount of polyunsaturated fatty acids (PUFA) including docosahexanoic acid (DHA) in fish oil, the highly specific enrichment of EPA from fish oil is difficult to achieve. In our study, we conducted immobilization studies on Burkholderia cepacia lipase (BCL) and its application in the selective enrichment of EPA in fish oil. BCL was immobilized on resin LX201A by covalent crosslinking, and immobilization conditions including glutaraldehyde concentration, pH, time were optimized. The optimal immobilization conditions were 0.5% glutaraldehyde, 4 h, and pH 8.0. The loading rate of BCL reached to 93.5%, and its activity was up to 68263 U/g under the optimal immobilization conditions. After hydrolysis reaction of fish oil for 15h catalyzed by immobilized BCL, EPA content showed a more increase by 33.2%, while a slight increase by 0.78% in DHA content. Moreover, after recycling use for 10 times, the catalytic efficiency of immobilized BCL remained at 93.0%. These indicated that immobilized BCL possessed good selectivity, reusability and stability, providing great application potential in EPA selective enrichment in fish oil industry.

Body weight and morphological profile of blood of deer (Cervus elaphus) in the conditions of Polissia Volyn

The homeostasis of the whole organism and the analysis of hematological parameters in red deer (Cervus elaphus) play an essential role in comparative physiology for health assessment and disease diagnosis. The obtained blood results can be the basis for assessing the condition of wild deer populations with an existing clinical picture or characterize the nutritional status, habitat, or other stress factors, as the hematopoietic system is susceptible to any changes in the animal body. In the course of the research, it was found that the body weight of adult deer varied depending on the population. Female deer of the Latvian and Lithuanian crossbreeds and the Eastern European population had the highest weight. As for the males, the animals of the Hungarian population had a higher weight than the English population. A high level of growth intensity in the early postnatal period was characteristic of both young females (body weight gain was in the range of 44.8–45.8 kg) and males (42.6–44.12 kg). The analysis of hematological parameters confirmed that the number of red blood cells and hemoglobin content increased in the blood of animals of the English population under technological stress (by 5.6 and 4.4 %). Regarding individual populations, it was noted that under the same conditions of immobilization of animals (medication), the number of red blood cells in the blood of Latvian crossbred deer was 8.5 % higher than the English population. The hemoglobin content was 5.3 % lower, and the saturation of red blood cells with hemoglobin was 12.9 % lower. It was found that the hematological parameters of 3–4 month-old fawns (females and males) of the English population were characterized by relative stability. Against the background of a tendency to a higher number of red blood cells in females (by 12.8 %), the hemoglobin content was 3.3 % lower than in males. The number of leukocytes in the blood of females was slightly higher than that of males, although no significant deviations were noted in some forms of white blood cells.

Rederiving kinetics to model biohydrogen production from immobilized microalgae alginate beads at various polymerization degrees of alginate under dark fermentative environment

The performance of immobilized microalgae-alginate beads on biohydrogen production and its stability across several dark fermentation cycles is influenced by the sodium alginate concentrations. Thus, it is vital to determine the optimal condition for immobilization to achieve maximum encapsulation efficiency, stability and biohydrogen production. In this work, different sodium alginate concentrations (2 to 8 w/v%) were used to immobilize microalgae in influencing the dark fermentative biohydrogen productions from municipal wastewater, and its reusability was also investigated. The immobilized microalgae-alginate beads with sodium alginate concentration of 2 % showed the lowest stability and biohydrogen production in all cycles. The highest biohydrogen production was achieved by immobilized microalgal-alginate beads prepared from 8 % sodium alginate, followed by 6 % and 4 % in the first and second cycles. However, 8 % of immobilized microalgae-alginate beads generated the lowest biohydrogen volume during the third cycle. Besides, a model was rederived from the modified Gompertz model and Fick's law of diffusion equation to describe the relationship between polymeric viscosity and biohydrogen production from immobilized microalgae-alginate beads with different sodium alginate concentrations. As the sodium alginate concentrations increased, the viscosity also increased which significantly affected the properties of immobilization matrix formed such as encapsulation efficiency, growth of microalgae, diffusion of substrate and biohydrogen yield. The rederived model managed to fit the experimental data with a coefficient of determination values of >0.95 for all the polymerization degrees of alginate. The kinetic parameters, namely, yield of biohydrogen and specific microalgae growth rate of sodium alginate concentration of 6 % were 129.80 L kg−1 and 4.69 h−1, respectively, which were considered as maximum results as compared with other sodium alginate concentrations. Besides, the difference in biohydrogen productions for all cycles and kinetic data of biohydrogen yields obtained from the rederived model between sodium alginate concentration of 6 % and 8 % only exhibited a slight variance (<3 %). Thus, sodium alginate concentration of 6 % was considered to be an optimal for immobilizing microalgae in performing the dark fermentation. Overall, the results revealed the significance of suitable sodium alginate concentration in maximizing the immobilization of microalgae for performing the dark fermentative biohydrogen production.

A new UiO-66-NH2 MOF-based nano-immobilized DFR enzyme as a biocatalyst for the synthesis of anthocyanidins

Anthocyanidins and anthocyanins are one subclass of flavonoids in plants with diverse biological functions and have health-promoting effects. Dihydroflavonol 4-reductase (DFR) is one of the important enzymes involved in the biosynthesis of anthocyanidins and other flavonoids. Here, a new MOF-based nano-immobilized DFR enzyme acting as a nano-biocatalyst for the production of anthocyanidins in vitro was designed. We prepared UiO-66-NH2 MOF nano-carrier and recombinant DFR enzyme from genetic engineering. DFR@UiO-66-NH2 nano-immobilized enzyme was constructed based on covalent bonding under the optimum immobilization conditions of the enzyme/carrier ratio of 250 mg/g, 37 °C, pH 6.5 and fixation time of 10 min. DFR@UiO-66-NH2 was characterized and its catalytic function for the synthesis of anthocyanidins in vitro was testified using UPLC-QQQ-MS analysis. Compared with free DFR enzyme, the enzymatic reaction catalyzed by DFR@UiO-66-NH2 was more easily for manipulation in a wide range of reaction temperatures and pH values. DFR@UiO-66-NH2 had better thermal stability, enhanced adaptability, longer-term storage, outstanding tolerances to the influences of several organic reagents and Zn2+, Cu2+ and Fe2+ ions, and relatively good reusability. This work developed a new MOF-based nano-immobilized biocatalyst that had a good prospect of application in the green synthesis of anthocyanins in the future.

Cellulase entrapment into alginate-PEG beads cross-linked with glutaraldehyde: Optimization of the immobilization conditions and kinetic characterization of the immobilized enzyme

Immobilization methods for cellulase entrapped into alginate beads with glutaraldehyde (GA) cross-linking were investigated. Cellulase entrapped into alginate-polyethylene glycol beads with glutaraldehyde cross-linking (SA-PEG-E) showed more superiority both in terms of catalytic performance and reusability than other methods. Several conditions during SA-PEG-E immobilization such as cellulase concentration, GA amount, pH of immobilization, cross-linking time and hardening temperature were evaluated to study their effect on immobilized enzyme activity, immobilization yield and immobilization efficiency. Based on the results, GA amount, pH of immobilization and hardening temperature were selected for process optimization of SA-PEG-E using the Box-Behnken design of response surface methodology. The results showed that the optimum immobilized conditions were obtained with a GA amount of 0.8 mL, pH of immobilization 4.7, and hardening temperature of 47.5 °C when the cellulase concentration was 7.5 mg/mL and cross-linking time was 2 h. Then, the characterization of SA-PEG-E was studied. The optimum pH of SA-PEG-E was observed to be 4, 1 unit lower than that of the free enzyme (pH 5). The optimum temperature was the same for both free and immobilized cellulase at 50 °C. In addition, the SA-PEG-E exhibited broad pH and temperature adaptability.