Dynamic immobilization of bacterial cells on biofilm in a polyester nonwoven chemostat.

Cell immobilization plays an important role in biocatalysis for high-value products. It is necessary to maintain the viability of immobilized cells for bioconversion using viable cells as biocatalysts. In this study, a novel polyester nonwoven chemostat was designed for cell immobilization to investigate biofilm formation and the dynamic balance between adsorption and desorption of cells on polyester nonwoven. The polyester nonwoven was suitable for cell immobilization, and the cell numbers on the polyester nonwoven can reach 6.5 ± 0.38 log CFU/mL. After adding the polyester nonwoven to the chemostat, the fluctuation phenomenon of free bacterial cells occurred. The reason for this phenomenon was the balance between adsorption and desorption of bacterial cells on the polyester nonwoven. Bacterial cells could adhere to the surface of polyester nonwoven via secreting extracellular polymeric substances (EPS) to form biofilms. As the maturation of biofilms, some dead cells inside the biofilms can cause the detachment of biofilms. This process of continuous adsorption and desorption of cells can ensure that the polyester nonwoven chemostat has lasting biological activity.

Conversion of carbon dioxide in biogas into acetic acid by Clostridium thailandense immobilized on porous support materials.

This study aimed to convert CO2 in biogas into acetic acid using immobilized Clostridium thailandense cells on various support materials, including activated carbon, expanded clay, and coir. Immobilized cells and free cells were evaluated for their CO2 conversion ability into acetic acid using H2 as an electron donor at an H2 to CO2 in biogas ratio of 2:1 (v/v), 30 °C, 150 rpm. Results showed that immobilized cells on activated carbon increased CH4 content to 96.9% (v/v), and acetic acid production to 15.65 mmol/L within 96 h. These values outperformed free cells. The activated carbon-immobilized cells could be reused two times without losing efficacy in the purification of biogas and acetic acid production. This work indicates that using the immobilized cells offers a sustainable approach to biogas upgrading, reducing the environmental footprint of biogas production by increasing its energy content and purity.

Programmed immobilization of living cells using independent click pairs.

Therapeutic devices incorporating living cells or tissues have been intensively investigated for applications in tissue engineering and regenerative medicine. Because many biological processes are governed by spatially dependent signals, programmable immobilization of materials is crucial for manipulating multiple types of cells. In this study, click chemistry substrates were introduced onto the surfaces of cells and cover glass, and the cells were fixed on the cover glass via covalent bonds for selective cell deposition. Azide group (Az)-labeled living cells were prepared by metabolic labeling with azido sugars. Following the introduction of Az, TCO (trans-cyclooctene) was metabolically labeled into the living cells by reacting with TCO-DBCO (dibenzocyclooctyne). Az and TCO in the cells were detected using DBCO-FAM (fluorescein)and tetrazine-Cy3, respectively. The mixture of Az-labeled green fluorescent protein HeLa cells and TCO-labeled red fluorescent protein HeLa cells was reacted in a culture dish in which three different cover glasses, DBCO-, tetrazine-, or methyl-coated, were added. Az- or TCO-labeled cells could be immobilized in a functional group-dependent manner. Next, tetrazine-labeled cells were incubated on TCO- or Az-labeled cell layers instead of cover glass. Functional group-dependent immobilization was also achieved in the cell layer. Introducing substrates for the click reaction could achieve cell-selective immobilization on different patterned glass surfaces, as well as cell-cell immobilization.

Rapid Degradation of Hazardous Amides by Immobilized Engineered Pseudomonas putida KT2440 Based on a Novel Gene Expression Vector.

The amides 4-trifluoromethylnicotinamide, acrylamide, and benzamide are widely used in agriculture and industry, posing hazards to the environment and animals. Immobilized bacteria are preferred in wastewater treatment, but degradation of these amides by immobilized engineered bacteria has not been explored. Here, engineered Pseudomonas putida KT2440 pLSJ15-amiA was constructed by introducing a new amidase gene expression vector into environmentally safe P. putida KT2440. P. putida KT2440 pLSJ15-amiA had high amidase activity, even at 80 °C. P. putida KT2440 pLSJ15-amiA immobilized with calcium alginate exhibited a greater environmental tolerance than free cells. The amides were rapidly degraded by the immobilized cells, but the activity was inhibited by high concentrations of substrates. The substrate inhibition model revealed that the optimum initial concentrations of 4-trifluoromethylnicotinamide, acrylamide, and benzamide for degradation by immobilized cells were 197.65, 350.76, and 249.40 µmol/L, respectively. This study develops a novel and excellent immobilized biocatalyst for remediation of wastewater containing hazardous amides.

Immobilization of rough morphotype Mycolicibacterium neoaurum R for androstadienedione production.

OBJECTIVES: Enhance the androstadienedione (Androst-1,4-diene-3,17-dione, ADD) production of rough morphotype Mycolicibacterium neoaurum R by repeated-batch fermentation of immobilized cells. RESULTS: M. neoaurum R was a rough colony morphotype variant, obtained from the routine plating of smooth M. neoaurum strain CICC 21097. M. neoaurum R showed rougher cell surface and aggregated in broth. The ADD production of M. neoaurum R was notably lower than that of M. neoaurum CICC 21097 during the free cell fermentation, but the yield gap could be erased after proper cell immobilization. Subsequently, repeated-batch fermentation of immobilized M. neoaurum R was performed to shorten the production cycle and enhance the bio-production efficiency of ADD. Through the optimization of the immobilization carriers and the co-solvents for phytosterols, the ADD productivity of M. neoaurum R immobilized by semi-expanded perlite reached 0.075 g/L/h during the repeated-batch fermentation for 40 days. CONCLUSIONS: The ADD production of the rough-type M. neoaurum R was notably enhanced by the immobilization onto semi-expanded perlite. Moreover, the ADD batch yields of M. neoaurum R immobilized by semi-expanded perlite were maintained at high levels during the repeated-batch fermentation.

Effective methods for immobilization of non-adherent Pv11 cells while maintaining their desiccation tolerance.

Pv11 was derived from embryos of the sleeping chironomid Polypedilum vanderplanki, which displays an extreme form of desiccation tolerance known as anhydrobiosis. Pre-treatment with a high concentration of trehalose allows Pv11 cells to enter anhydrobiosis. In the dry state, Pv11 cells preserve transgenic luciferase while retaining its activity. Thus, these cells could be utilized for dry-preserving antibodies, enzymes, signaling proteins or other valuable biological materials without denaturation. However, Pv11 cells grow in suspension, which limits their applicability; for instance, they cannot be integrated into microfluidic devices or used in devices such as sensor chips. Therefore, in this paper, we developed an effective immobilization system for Pv11 cells that, crucially, allows them to maintain their anhydrobiotic potential even when immobilized. Pv11 cells exhibited a very high adhesion rate with both biocompatible anchor for membrane (BAM) and Cell-Tak coatings, which have been reported to be effective on other cultured cells. We also found that Pv11 cells immobilized well to uncoated glass if handled in serum-free medium. Interestingly, Pv11 cells showed desiccation tolerance when trehalose treatment was done prior to immobilization of the cells. In contrast, trehalose treatment after immobilization of Pv11 cells resulted in a significant decrease in desiccation tolerance. Thus, it is important to induce anhydrobiosis before immobilization. In summary, we report the successful development of a protocol for the dry preservation of immobilized Pv11 cells. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00592-0.

Attenuation of Hyperglycemia in Diabetic Rats Assisted by Immobilized Probiotic in Sodium Alginate.

Diabetes mellitus type 2 (DM2) is the most common chronic disease worldwide, characterized mainly by increased glucose concentration in the blood and affecting several organs' functionality. The daily consumption of probiotic bacteria can help control diabetes and reduce the damage caused. Cell immobilization techniques are a powerful tool that provides physical cell protection to such probiotic bacteria against gastrointestinal conditions. We suggest that cell immobilization could be a significant vector for delivering a high quantity of viable probiotics to the gut, helping attenuate hyperglycemia in diabetic rats. Seventy male Wistar rats were used in this work. Nicotinamide was administrated via intraperitoneal injection 15 minutes before inducing type 2 diabetes (DM2), followed by a second intraperitoneal injection of streptozotocin to induce DM2. Rats were divided into seven groups. For 45 days, a specific treatment was applied to each group. The group of rats, supplied with immobilized Lactobacillus casei, showed a serum glucose concentration of 137 mg/dL, which was close to the one observed in the groups of healthy rats (117 mg/dL) and rats treated with metformin (155 mg/dL). The diabetic rats without treatment presented a higher serum glucose concentration (461 mg/dL). In the rats treated with immobilized L. casei, there was no biochemical parameter alteration, and the cell morphology of the analyzed tissues was similar to those of the healthy group. The consumption of immobilized L. casei could allow a high quantity of viable probiotics to be delivered to the gut, reducing serum glucose concentration by up to 70% compared to diabetic rats and reducing organ damage caused by diabetes.

A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins.

Introducing the flow through the bioreactor has revolutionized in-cell NMR spectroscopy by prolonging the measurement time available to acquire spectral information about biomacromolecules in metabolically active cells. Bioreactor technology relies on immobilizer matrices, which secure cells in the active volume of the NMR coil and enable uniform perfusion of the growth medium, supplying fresh nutrients to the cells while removing toxic byproducts of their metabolism. The main drawbacks of commonly used matrices include the inability to recover intact cells post-measurement for additional analyses and/or requirements for specific operating temperatures. Here, we report on the development and characterization of a set of thermosensitive and nontoxic triblock copolymers based on poly(D,L-lactide)-b-poly(ethylene glycol)-b-poly(D,L-lactide) (PLA-PEG-PLA). Here, we show for the first time that these copolymers are suitable as immobilizer matrices for the acquisition of in-cell NMR spectra of nucleic acids and proteins over a commonly used sample temperature range of 15-40 °C and, importantly, allow recovery of cells after completion of in-cell NMR spectra acquisition. We compared the performances of currently used matrices in terms of cell viability (dye exclusion assays), cellular metabolism (1D 31P NMR), and quality of in-cell NMR spectra of two model biomacromolecules (hybrid double-stranded/i-motif DNA and ubiquitin). Our results demonstrate the suitability and advantages of PLA-PEG-PLA copolymers for application in bioreactor-assisted in-cell NMR.

Immobilization of Rhodococcus by encapsulation and entrapment: a green solution to bitter citrus by-products.

Debittering of citrus by-products is required to obtain value-added compounds for application in the food industry (e.g., dietary fiber, bioactive compounds). In this work, the immobilization of Rhodococcus fascians cells by encapsulation in Ca-alginate hollow beads and entrapment in poly(vinyl alcohol)/polyethylene glycol (PVA/PEG) cryogels was studied as an alternative to chemical treatments for degrading the bitter compound limonin. Previously, the Rhodococcus strain was adapted using orange peel extract to increase its tolerance to limonoids. The optimal conditions for the encapsulation of microbial cells were 2% Na-alginate, 4% CaCl2, 4% carboxymethylcellulose (CMC), and a microbial load of 0.6 OD600 (optical density at 600 nm). For immobilization by entrapment, the optimal conditions were 8% PVA, 8% PEG, and 0.6 OD600 microbial load. Immobilization by entrapment protected microbial cells better than encapsulation against the citrus medium stress conditions (acid pH and composition). Thus, under optimal immobilization conditions, limonin degradation was 32 and 28% for immobilization in PVA/PEG gels and in hollow beads, respectively, in synthetic juice (pH 3) after 72 h at 25 °C. Finally, the microbial cells entrapped in the cryogels showed a higher operational stability in orange juice than the encapsulated cells, with four consecutive cycles of reuse (runs of 24 h at 25 °C). KEY POINTS: • Increased tolerance to limonoids by adapting R. fascians with citrus by-products. • Entrapment provided cells with favorable microenvironment for debittering at acid pH. • Cryogel-immobilized cells showed the highest limonin degradation in citrus products.

Co-culture of Acinetobacter sp. and Scedosporium sp. immobilized beads for optimized biosurfactant production and degradation of crude oil.

The widespread exploration and exploitation of crude oil has increased the prevalence of petroleum hydrocarbon pollution in the marine and coastal environment. Bioremediation of petroleum hydrocarbons using cell immobilization techniques is gaining increasing attention. In this study, the crude oil degradation performance of bacterial and fungal co-culture was optimized by entrapping both cells in sodium-alginate and polyvinyl alcohol composite beads. Results indicate that fungal cells remained active after entrapment and throughout the experiment, while bacterial cells were non-viable at the end of the experimental period in treatments with the bacterial-fungal ratio of 1:2. A remarkable decrease in surface tension from 72 mN/m to 36.51 mN/m was achieved in treatments with the bacterial-fungal ratio of 3:1. This resulted in a significant (P < 0.05) total petroleum hydrocarbon (TPH) removal rate of 89.4%, and the highest degradation of n-alkanes fractions (from 2129.01 mg/L to 118.53 mg/L), compared to the other treatments. Whereas PAHs removal was highest in treatments with the most fungal abundance (from 980.96 µg/L to 177.3 µg/L). Furthermore, enzymes analysis test revealed that catalase had the most effect on microbial degradation of the target substrate, while protease had no significant impact on the degradation process. High expression of almA and PAH-RHDa genes was achieved in the co-culture treatments, which correlated significantly (P < 0.05) with n-alkanes and PAHs removal, respectively. These results indicate that the application of immobilized bacterial and fungal cells in defined co-culture systems is an effective strategy for enhanced biodegradation of petroleum hydrocarbons in aqueous systems.

Synthesis and molecular characterization of levan produced by immobilized Microbacterium paraoxydans.

In this study, we report high molecular weight (HMW) levan production by whole cells of Microbacterium paraoxydans, previously reported to be a good producer of fructooligosaccharides. Structural analysis of the extracellularly produced fructan indicated the glycosidic bonds between the adjacent fructose to be of ß-(2, 6) linkage with over 90% of the fructan to have molecular weight around 2 × 108 Da and 10% with a molecular weight of ∼20 kDa. Immobilization of the cells in Ca-alginate led to the production of 44.6 g/L levan with a yield of 0.29 g/g sucrose consumed. Factors affecting the conversion rate were identified by One-Factor-At-a-Time (OFAT) analysis and the combination of these (initial sucrose concentration of 400 g/L, 100 mM buffer pH 7, the temperature of 37 °C and 20 mM CaCl2) led to the production of ∼129 g/L of levan with a yield of ∼0.41 g/g sucrose consumed and volumetric productivity of 1.8 g/L/h.

Biodegradation Capabilities of Paraquat-Degrading Bacteria Immobilized on Nanoceramics.

The biodegradation of paraquat was investigated using immobilized microbial cells on nanoceramics fabricated from nanoscale kaolinite. Pseudomonas putida and Bacillus subtilis, which degrade paraquat, were immobilized separately on nanoceramics (respectively called ICnc-P and ICnc-B). The attachment of bacteria to nanoceramics resulted from electrostatic force interactions, hydrogen bonding, and covalent bonding (between the cells and the support materials). The initial 10 mg L-1 concentration of paraquat in water was removed by the adsorption process using nanoceramics at 68% and ceramics at 52%, respectively. The immobilized cells on the nanoceramics were able to remove approximately 92% of the paraquat within 10 h, whereas the free cells could only remove 4%. When the paraquat was removed, the cell-immobilized nanoceramics exhibited a significant decrease in dissolved organic nitrogen (DON). ICnc-B was responsible for 34% of DON biodegradation, while ICnc-P was responsible for 22%. Ammonia was identified as the end product of ammonification resulting from paraquat mineralization.

Yeast cell vacuum infusion into fungal pellets as a novel cell encapsulation methodology.

Immobilized yeast cells are used industrially in winemaking processes such as sparkling wine and Sherry wine production. Here, a novel approach has been explored for the infusion and immobilization of yeast cells into filamentous fungal pellets, which serve as a porous natural material. This was accomplished through vacuum application to force the yeast cells towards the core of the fungal pellets followed by culture in YPD medium to promote their growth from the interior. This method represents an improved variation of a previous approach for the assembly of "yeast biocapsules," which entailed the co-culture of both fungal and yeast cells in the same medium. A comparison was made between both techniques in terms of biocapsule productivity, cell retention capacity, and cell biological activity through an alcoholic fermentation of a grape must. The results indicated a substantial increase in biocapsule productivity (37.40-fold), higher cell retention within the biocapsules (threefold), and reduction in cell leakage during fermentation (twofold). Although the majority of the chemical and sensory variables measured in the produced wine did not exhibit notable differences from those produced utilizing suspended yeast cells (conventional method), some differences (such as herbaceous and toasted smells, acidity, bitterness, and persistence) were perceived and wines positively evaluated by the sensory panel. As the immobilized cells remain functional and the encapsulation technique can be expanded to other microorganisms, it creates potential for additional industrial uses like biofuel, health applications, microbe encapsulation and delivery, bioremediation, and pharmacy. KEY POINTS: • New approach improves biocapsule productivity and cell retention. • Immobilized yeast remains functional in fermentation. • Wine made with immobilized yeast had positive sensory differences.

Tissue-Adhesive Hydrogel Spray System for Live Cell Immobilization on Biological Surfaces.

Gelatin hydrogels are used as three-dimensional cell scaffolds and can be prepared using various methods. One widely accepted approach involves crosslinking gelatin amino groups with poly(ethylene glycol) (PEG) modified with N-hydroxysuccinimide ester (PEG-NHS). This method enables the encapsulation of live cells within the hydrogels and also facilitates the adhesion of the hydrogel to biological tissues by crosslinking their surface amino groups. Consequently, these hydrogels are valuable tools for immobilizing cells that secrete beneficial substances in vivo. However, the application of gelatin hydrogels is limited due to the requirement for several minutes to solidify under conditions of neutral pH and polymer concentrations suitable for live cells. This limitation makes it impractical for use with biological tissues, which have complex shapes or inclined surfaces, restricting its application to semi-closed spaces. In this study, we propose a tissue-adhesive hydrogel that can be sprayed and immobilized with live cells on biological tissue surfaces. This hydrogel system combines two components: (1) gelatin/PEG-NHS hydrogels and (2) instantaneously solidifying PEG hydrogels. The sprayed hydrogel solidified within 5 s after dispensing while maintaining the adhesive properties of the PEG-NHS component. The resulting hydrogels exhibited protein permeability, and the viability of encapsulated human mesenchymal stem/stromal cells (hMSCs) remained above 90% for at least 7 days. This developed hydrogel system represents a promising approach for immobilizing live cells on tissue surfaces with complex shapes.

Improving rhamnolipids production using fermentation-foam fractionation coupling system: cell immobilization and waste frying oil emulsion.

This work focused on the development of an inexpensive carbon source and the improvement of the fermentation-foam fractionation coupling system. The rhamnolipids production capacity of waste frying oil (WFO) was evaluated. The suitable bacterial cultivation of seed liquid and the addition amount of WFO was 16 h and 2% (v/v), respectively. A combined strategy of cell immobilization and oil emulsion avoid cell entrainment inside foam and improves the oil mass transfer rate. The immobilization conditions of bacterial cells into alginate-chitosan-alginate (ACA) microcapsules were optimized using the response surface method (RSM). Under the optimal conditions, rhamnolipids production using batch fermentation with immobilized strain reached 7.18 ± 0.23% g/L. WFO was emulsified into a fermentation medium using rhamnolipids as emulsifier (0.5 g/L). By monitoring dissolved oxygen, 30 mL/min was selected as a suitable air volumetric flow rate for fermentation-foam fractionation coupling operation. The total production and recovery percentage of rhamnolipids were 11.29 ± 0.36 g/L and 95.62 ± 0.38%, respectively.

Optimized enantioselective (S)-2-hydroxypropiophenone synthesis by free- and encapsulated-resting cells of Pseudomonas putida.

BACKGROUND: Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction. RESULTS: The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL- 1. 2-HPP titer, yield and productivity using the free cells were 1.2 g L- 1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g- 1 DCW h- 1, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)-polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product. CONCLUSIONS: Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones.

Extended Biosynthesis of Rhamnolipid by Immobilized Pseudomonas aeruginosa USM-AR2 Cells in a Fluidized Bed Bioreactor.

This study aims to evaluate rhamnolipid production by immobilized Pseudomonas aeruginosa USM-AR2 cells using waste cooking oil (WCO) as the carbon source. P. aeruginosa USM-AR2 cells were entrapped in polyvinyl alcohol (PVA)-alginate hydrogel beads. The performance of entrapped cells was compared with free cells in shake flasks before cultivation in a custom-designed fluidized bed reactor (FBR). A mass of 1 g wet cells entrapped in PVA-alginate hydrogel beads were successfully recycled 3 times in shake flasks at 200 rpm, producing between 0.66-1.34 g L-1 rhamnolipid after 120 h. Meanwhile, cultivation of entrapped cells in FBR with broth recirculation showed that the suitable hydrogel beads to medium ratio was 1:20 ratio at an aeration rate of 0.5 vvm, producing between 0.77 to 1.58 g L-1 rhamnolipid, degrading 8.67 to 20.93 g L-1 of waste cooking oil in 15 cycles of repeated batch cultivation. Entrapped P. aeruginosa USM-AR2 cells achieved a higher rhamnolipid production, by 1.03-fold during cycle 3 in shake flasks and 1.19-fold during cycle 11 in an FBR, compared to free cells. These results show that entrapment enables reusability of viable cells and maintains stability of rhamnolipid production throughout the extended cultivation, increasing cell tolerance to perturbations in fermentation conditions.

Nanoarchitectonics to entrap living cells in silica-based systems: encapsulations with yolk-shell and sepiolite nanomaterials.

In the present work, the bottom-up fabrication of biohybrid materials using a nanoarchitectonics approach has been applied to entrap living cells. Unicellular microorganisms, that is, cyanobacteria and yeast cells, have been immobilized in silica and silicate-based substrates organized as nanostructured materials. In a first attempt, matrices based on bionanocomposites of chitosan and alginate incorporating sepiolite clay mineral and shaped as films, beads, or foams have been explored for the immobilization of cyanobacteria. It has been observed that this type of biohybrid substrates leads to serious problems regarding the long-time survival of the encapsulated microorganisms. Alternative procedures using silica-based matrices with low sodium content, generated by sol-gel methods, as well as pre-synthesised yolk-shell bionanohybrids have been studied subsequently. Optical microscopy and SEM confirm that the silica shell microstructures provide a reduced contact between cells. The inorganic matrix increases the survival of the cells and maintains their bioactivity. Thus, the encapsulation efficiency is improved compared to the approach using a direct contact of cells in a silica matrix. Encapsulated yeast produced ethanol over a period of several days, pointing out the useful biocatalytic potential of the approach and suggesting further optimization of the present protocols.

Photobiohydrogen Production and Strategies for H2 Yield Improvements in Cyanobacteria.

Hydrogen gas (H2) is one of the potential future sustainable and clean energy carriers that may substitute the use of fossil resources including fuels since it has a high energy content (heating value of 141.65 MJ/kg) when compared to traditional hydrocarbon fuels [1]. Water is a primary product of combustion being a most significant advantage of H2 being environmentally friendly with the capacity to reduce global greenhouse gas emissions. H2 is used in various applications. It generates electricity in fuel cells, including applications in transportation, and can be applied as fuel in rocket engines [2]. Moreover, H2 is an important gas and raw material in many industrial applications. However, the high cost of the H2 production processes requiring the use of other energy sources is a significant disadvantage. At present, H2 can be prepared in many conventional ways, such as steam reforming, electrolysis, and biohydrogen production processes. Steam reforming uses high-temperature steam to produce hydrogen gas from fossil resources including natural gas. Electrolysis is an electrolytic process to decompose water molecules into O2 and H2. However, both these two methods are energy-intensive and producing hydrogen from natural gas, which is mostly methane (CH4) and in steam reforming generates CO2 and pollutants as by-products. On the other hand, biological hydrogen production is more environmentally sustainable and less energy intensive than thermochemical and electrochemical processes [3], but most concepts are not yet developed to production scale.

Hierarchical Emulsion-Templated Monoliths (polyHIPEs) as Scaffolds for Covalent Immobilization of P. acidilactici.

The immobilized cell fermentation technique (IMCF) has gained immense popularity in recent years due to its capacity to enhance metabolic efficiency, cell stability, and product separation during fermentation. Porous carriers used as cell immobilization facilitate mass transfer and isolate the cells from an adverse external environment, thus accelerating cell growth and metabolism. However, creating a cell-immobilized porous carrier that guarantees both mechanical strength and cell stability remains challenging. Herein, templated by water-in-oil (w/o) high internal phase emulsions (HIPE), we established a tunable open-cell polymeric P(St-co-GMA) monolith as a scaffold for the efficient immobilization of Pediococcus acidilactici (P. acidilactici). The porous framework's mechanical property was substantially improved by incorporating the styrene monomer and cross-linker divinylbenzene (DVB) in the HIPE's external phase, while the epoxy groups on glycidyl methacrylate (GMA) supply anchoring sites for P. acidilactici, securing the immobilization to the inner wall surface of the void. For the fermentation of immobilized P. acidilactici, the polyHIPEs permit efficient mass transfer, which increases along with increased interconnectivity of the monolith, resulting in higher L-lactic acid yield compared to that of suspended cells with an increase of 17%. The relative L-lactic acid production is constantly maintained above 92.9% of their initial relative production after 10 cycles, exhibiting both its great cycling stability and the durability of the material structure. Furthermore, the procedure during recycle batch also simplifies downstream separation operations.