Immobilized Cells Research Articles

Immobilization of rough morphotype Mycolicibacterium neoaurum R for androstadienedione production.

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. 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 0.075g/L/h during the repeated-batch fermentation for 40days. 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.

Immobilization of microbial cells in a sol-gel matrix using a precursor with two non-hydrolyzed bonds

Immobilization of microbial cells in a sol-gel matrix using a precursor with two non-hydrolyzed bonds

Microstructure Based Flow Stress Modelling of Superalloy 718

In order to get the insights about microstructural changes that occurs under the thermo-mechanical processing conditions, the physics based modelling approach is very useful. Therefore, the flow curves of alloy 718 are theoretical simulated using a dislocation density dependent constitutive model for different conditions. Presented model considers the microstructural ingredients that are immobile dislocation density, effective grain size and dislocation cell size as the variables to address the creep. The simulated flow curves show a good agreement with the experimental flow curves. The magnitude of immobile dislocation density and dislocation cell size in between 3.87× 1014 - 3.87× 1014­ m-2 and 8.29-8.45 μm, respectively, at the completion of the simulation. Furthermore, this approach also provides the possibility to quantify and depict the variation in each strengthening contributions.

Hydro-Cracked Light Gas Oil Biodesulphurisation by Immobilised Pseudomonas aeruginosa Cells on Polyvinyl Alcohol

Abstract Refining petroleum fractions containing heterocyclic sulphur compounds to produce sulphur-free fuels, requires efficient desulphurisation methods. A new biocatalyst has been synthesised by immobilising Pseudomonas aeruginosa cells on polyvinyl alcohol by adsorption for hydro-cracked light gas oil bio-desulphurisation. The surface functional groups and biocatalyst morphology have been investigated by Fourier transform infrared spectroscopy and scanning electron microscopy. The bio-desulphurisation of dibenzothiophene as a heterocyclic sulphur model compound of gas oil was achieved with an aqueous-oil ratio (v/v) of 50 %, where the removed mass was 0.3826 mg per gram of biocatalyst at equilibrium condition, bio-desulphurisation rate of 0.375 h–1 and removal percentage was 95.65 %. The biodegradation of dibenzothiophene and its derivatives in hydro-cracked light gas oil has been determined after a batch process using 0.5 g of the biocatalyst after 5 h of contact time at 37 °C. According to gas chromatography - mass spectrometry, ethyl and trimethyl derivatives of dibenzothiophene have been degraded by higher efficiencies in comparison with other derivatives. Also, thiophenes and mercaptans of the gasoil sample have been degraded simultaneously to some extent. Equilibrium data have been observed to obey the pseudo-first-order kinetic model. The cell immobilisation facilitates the interaction of surface functional groups with sulphur compounds. The synergistic effect of cell immobilisation on the bio-activity of bacterial cells was due to the maintenance of the heterotrophic, bacillus morphology of the cells after immobilisation. This approach provides a simple, economical method with mild operating conditions to produce low-sulphur light gas oil through the biodegradation of heterocyclic sulphur compounds.

Treatment of 2-naphthalene sulfonate by free and immobilized cells of indigenous isolate Comamonas sp. C2 and toxicity evaluation of biotransformed sample

An indigenous bacterial strain Comamonas sp. C2, capable of utilizing 2-naphthalene sulfonate (2-NS) as a sole source of carbon, was isolated after enrichment of microbial diversity present in sample collected from sewage treatment plant (STP) receiving untreated or partially treated effluent from textile industries. The isolate was able to degrade 100 mg/L of 2-NS, when grown in sulfur free minimal salt medium (SFMSM), after an incubation of 8 h. The release of sulfite/sulfate ions during biotransformation, assay of protocatechuate 4, 5-dioxygenase activity and formation of metabolic intermediates like salicylaldehyde, protocatechuate and 2-hydroxy-4-carboxy-muconatesemialdehyde in the biologically treated sample indicated to the meta-cleavage of aromatic ring of 2-NS. An immobilized-cell plug flow bioreactor (PFR) was developed and evaluated for its efficiency to treat simulated feed supplemented with 100 mg/L of 2-NS. The cells of C2 were immobilized on pieces of polyurethane foam (PUF) and refractory brick pieces (RBP). The PFR, when operated at an HRT of 36 h, achieved complete degradation of 100 mg/L of 2-NS corresponding to 89% lowering of COD. The reduction in overall toxicity of 2-NS after biological treatment was evaluated using plant (Triticum aestivum and Vigna radiata) and animal (freshwater fish Channa punctatus) models, exposed to both untreated and PFR treated samples. To the best of our knowledge, there are no similar reports on exploitation of the metabolic potential of Comamonas sp. in 2-NS degradation and validation of the biological transformation by toxicity analysis.

A simple joint detection platform for high-throughput single-cell heterogeneity screening

Single cell heterogeneity plays an important role in many biological phenomena and distinguishing cells that exhibit certain mutation in sample could benefit clinical diagnose and drug screening. Typical single cell detection methods such as flow cytometry, in-situ hybridization, real-time amplification or sequencing test either protein or nucleic acid as target and usually require specialized instruments. Joint measurement of the both types of targets could be done by combining the above strategies precisely but also unwieldly. Methods for rapidly and parallelly screening single cells with target genotype and antigen is needed. In this study, we describe a gel plate platform to distinguish cell types based on their phenotypes on target gene and antigen with low equipment requirement. Integrated cell lysis and immobilization were done in the gel solidification step, after which antibody hybridization and real-time amplification were sequentially carried out without losing the original loci information of individual single cells so the three types of information of individual single cells could be combined to distinguished cells with expected genotype and phenotype. The easy-to-use gel platform has potential in point-of-care circumstances and single-cell stimulation response that have high requirements on efficiency and simplicity.

Microbial Upcycling of Waste PET to Adipic Acid.

Microorganisms can be genetically engineered to transform abundant waste feedstocks into value-added small molecules that would otherwise be manufactured from diminishing fossil resources. Herein, we report the first one-pot bio-upcycling of PET plastic waste into the prolific platform petrochemical and nylon precursor adipic acid in the bacterium Escherichia coli. Optimizing heterologous gene expression and enzyme activity enabled increased flux through the de novo pathway, and immobilization of whole cells in alginate hydrogels increased the stability of the rate-limiting enoate reductase BcER. The pathway enzymes were also interfaced with hydrogen gas generated by engineered E. coli DD-2 in combination with a biocompatible Pd catalyst to enable adipic acid synthesis from metabolic cis,cis-muconic acid. Together, these optimizations resulted in a one-pot conversion to adipic acid from terephthalic acid, including terephthalate samples isolated from industrial PET waste and a post-consumer plastic bottle.

Effects of electrospun nanofiber fabrications on immobilization of recombinant Escherichia coli for production of xylitol from glucose

A suitable nanofiber sheet was formulated and developed based on its efficacy in the immobilization of recombinant Escherichia coli (E. coli) to enhance xylitol production. The effects of different types of nanofibers and solvents on cell immobilization and xylitol production were studied. The most applicable nanofiber membrane was selected via preliminary screening of four types of nanofiber membrane, followed by the selection of six different solvents. Polyvinylidene fluoride (PVDF) nanofiber sheet synthesized using dimethylformamide (DMF) solvent was found to be the most suitable carrier for immobilization and xylitol production. The thin, beaded PVDF (DMF) nanofibers were more favourable for microbial adhesion, with the number of immobilized cells as high as 96 × 106 ± 3.0 cfu/ml. The attraction force between positively charged PVDF nanofibers and the negatively charged E. coli indicates that the electrostatic interaction plays a significant role in cell adsorption. The use of DMF has also produced PVDF nanofibers biocatalyst capable of synthesizing the highest xylitol concentration (2.168 g/l) and productivity (0.090 g/l/h) and 55–69% reduction in cell lysis compared with DMSO solvent and free cells. This finding suggests that recombinant E. coli immobilized on nanofibers shows great potential as a whole-cell biocatalyst for xylitol production.

Real-time monitoring of recombinant GFP expression in single-cell Komagataella phaffii through filamentous pelletization technology

Abstract Komagataella phaffii is an important cell factory for recombinant protein production under methanol induction. Tracking of recombinant protein expression by single K. phaffii cell is a hot topic to identify the variation of expression level in submerge cultivation system. In this study, a platform system harnessing Aspergillus niger pellets for K. phaffii cell immobilization in fabricated glass plate was developed to measure the green fluorescent protein (GFP) expression of single cell using fluorescence microscope. This system was optimized through pellets preparation, K. phaffii cell absorption, inducer comparison, methanol concentration, flow velocity of medium, and obtaining a successful platform to track GFP expression of single K. phaffii cell. This system provided an on-line analytical technology to discover the heterogeneous capability of various K. phaffii cells.

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 45days, a specific treatment was applied to each group. The group of rats, supplied with immobilized Lactobacillus casei, showed a serum glucose concentration of 137mg/dL, which was close to the one observed in the groups of healthy rats (117mg/dL) and rats treated with metformin (155mg/dL). The diabetic rats without treatment presented a higher serum glucose concentration (461mg/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.

Microbial immobilisation and adaptation to Cu2+ enhances microbial Fe2+ oxidation for bioleaching of printed circuit boards in the presence of mixed metal ions

A circular economy requires effective re-use of finite resources, such as metals from waste electrical and electronic equipment (WEEE). Bioleaching for extraction and recovery of base metals from printed circuit boards (PCBs) before recovering precious metals has potential to increase metal circularity. However, inhibition by base metals released from the PCBs and accumulated in PCB leachates on microbial Fe2+ oxidation, a critical bioleaching sub-process for Fe3+ regeneration, can limit this approach. Here, we explore the potential of microbial immobilisation on polyurethane foam (PUF) and adaptation to cupric ions to minimise inhibition by mixed metals released from PCBs, particularly zinc, nickel, and tin, and enhancing Fe2+ oxidation rates in PCB bioleaching systems. A mixed mesophilic culture dominant in Leptospirillum ferriphilum, Acidiplasma cupricumulans and Acidithiobacillus caldus was immobilised on PUF and adapted to 6 g/L Cu2+. Tolerance of Cu-adapted immobilised cells to the inhibitory metal ions Zn2+, Ni2+, and Sn2+, as individual (0–10 g/L) and mixed metal ions at concentrations typically leached from PCBs at solids loadings of 0–20% (mass/volume) was compared to that of non-adapted immobilised cells. Further, the impact of solutes from PCB leachates was evaluated. Inhibition by individual metal ions decreased in the order Sn2+ > Ni2+ > Zn2+. Inhibition of ferrous iron oxidation by mixed metal ions was synergistic with respect to individual metal ions. PCB leachates were more inhibitory than both mixed and individual metal ions even where metal concentration was low. Cu-adapted immobilised cells exhibited higher tolerance to increasing concentrations of inhibitory metal ions than non-adapted cells. These results are promising for the application of Cu-adapted cells in the bioleaching of PCBs and multi-metal concentrates.

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

The online version contains supplementary material available at 10.1007/s10616-023-00592-0.

An immobilized mixed microflora approach to enhancing hydrogen and methane productions from high-strength organic loading food waste hydrolysate in series batch reactors

The immobilized cells technology was used for enhancing the two stages batch bioreactors in series from high-strength food waste hydrolysate. Carrageenan and gelatin were used as support materials to create immobilized cells to improve microorganisms’ stability, activity, and productivity. It was found a promising advantages of a higher hydrogen and methane production rates, better stability, and resistance to shock loads to reduce the cost of producing biogaseous fuels from food waste. It was found that optimal biohydrogen production based on the working volume was 1.00 L H2/L which using 80 g COD/L of food waste hydrolysate and 20% of immobilized cells, and peak of methane production of 11.72 L CH4/L which using 120 g COD/L and 30% of immobilized cells. The yield of hydrogen and methane were 21.33 mL H2/g VS and 373.83 mL CH4/g VS respectively. TS, VS, and COD removal efficiency were 91.24, 90.64, and 86.35%, respectively. This study can support an efficient bioenergy production system development from a sustainable valuable feedstock such as a high-strength organic food waste.

Electrochemical properties of hydroxyapatite immobilization material for potential cytosensor fabrication

Aim: The biorecognition unit of an electrochemical biosensor requires molecules that are immobilised to serve as a bridge between the recognition unit and the transducing surface. Unique materials that enhance immobilisation of biorecognition molecules and improve electrochemical signal transduction are important in overcoming challenges based on the sensitivity of biosensors. In this regard, the electrochemical properties (EPs) of hydroxyapatite (HAp) material for the direct immobilisation of cells was investigated. Methods: Snail shell HAp (SHAp) material was synthesised from Achatina achatina snail shells and phosphate-containing solutions. The SHAp material was characterised using X-ray diffractometry (XRD), Fourier transform infrared (FTIR) spectroscopy, and Raman spectroscopy to determine the structural configuration, after which it was blended with a conductive polymer [poly(3,4-ethylenedioxythiophene): poly-4-styrene sulfonate (PEDOT: PSS)] to improve the electrochemical responses. The SHAp/PEDOT: PSS blend was used to modify a screen-printed carbon electrode (SPCE) by drop-casting, followed by seeding of pheochromocytoma (PC 12) and human embryonic kidney (HEK)-293T cells on the modified SPCE to record the EP using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Red blood cells (RBCs) were used as a control. Results: The CV analysis showed lower peak currents for HEK 293T (50 µA) and PC 12 (120 µA) compared to the RBC (230 µA). Also, the EIS showed impedance values of 0.70 for HEK 293T, 0.62 for PC 12, and 0.52 mΩ for RBC. The findings indicate that SHAp/PEDOT: PSS enables the differentiation of cell proliferation signals through voltammetric and impedimetric measurements. Conclusions: The unique current and impedance differences among the cells could serve as potential markers for rapid cell detection.

The Transcription Factor CsgD Contributes to Engineered Escherichia coli Resistance by Regulating Biofilm Formation and Stress Responses.

The high cell density, immobilization and stability of biofilms are ideal characteristics for bacteria in resisting antibiotic therapy. CsgD is a transcription activating factor that regulates the synthesis of curly fimbriae and cellulose in Escherichia coli, thereby enhancing bacterial adhesion and promoting biofilm formation. To investigate the role of CsgD in biofilm formation and stress resistance in bacteria, the csgD deletion mutant ΔcsgD was successfully constructed from the engineered strain E. coli BL21(DE3) using the CRISPR/Cas9 gene-editing system. The results demonstrated that the biofilm of ΔcsgD decreased by 70.07% (p < 0.05). Additionally, the mobility and adhesion of ΔcsgD were inhibited due to the decrease in curly fimbriae and extracellular polymeric substances. Furthermore, ΔcsgD exhibited a significantly decreased resistance to acid, alkali and osmotic stress conditions (p < 0.05). RNA-Seq results revealed 491 differentially expressed genes between the parent strain and ΔcsgD, with enrichment primarily observed in metabolism-related processes as well as cell membrane structure and catalytic activity categories. Moreover, CsgD influenced the expression of biofilm and stress response genes pgaA, motB, fimA, fimC, iraP, ompA, osmC, sufE and elaB, indicating that the CsgD participated in the resistance of E. coli by regulating the expression of biofilm and stress response. In brief, the transcription factor CsgD plays a key role in the stress resistance of E. coli, and is a potential target for treating and controlling biofilm.

Mucor cells passively immobilized on rose waste phyto-biomass: An eco-friendly hybrid material for the biotreatment of reactive dye contamination

The immobilization of microbial cells on eco-friendly supports is a promising technique to obtain effectively immobilized biomaterials for the biotreatment of aquatic pollution. For the biosorption of reactive dye-contaminated water, a new immobilized biosorbent, rose waste/Mucor plumbeus cells (RWMPC) composite was designed by immobilizing the fungal cells on a process waste of the rose oil industry. Batch and continuous mode biosorption features of RWMPC under different pH, initial dye concentration, contact time, biomaterial dose, and effluent flow rate conditions were examined. Zeta potential measurements, FT-IR, and SEM analysis were used to determine the morphological features of the biomaterial and possible Reactive Blue 49 (RB49)-biomaterial interactions. Kinetic investigations revealed that Ho's pseudo-second-order kinetic model could offer the best data fitting for biosorption, while the Freudlich model best matched the equilibrium data. Immobilized biomaterial displayed a high biosorption capacity of 120.88mg g−1. With an appropriate flow rate and amount of RWMPC packed into the column, dynamic treatment tests demonstrated >90% biosorption efficiency. The breakthrough curve of the RWMPC column adequately fits Chu's simplistic model. Regeneration tests revealed that after ten consecutive cycles, the biosorption yield and recovery efficacy of RWMPC were >65 and 85%, respectively. Zeta potential tests, SEM micrographs, and FT-IR studies also verified the dye uptake. Using synthetic wastewater in a continuous system, RWMPC could remove >90% of RB49. Breakthrough and exhaustion points were identified, and the maximum exhaustion capacity value was computed as 127.6 mg g−1. The results might inspire a fresh approach to the problem of reactive dye contamination in waters.

Optimization of Process Parameters of Immobilized Escherichia Coli for Cyclodextrin Production

The growing interest toward cylclodectrin (CD) application in industries is due to its unique structure that able to form inclusion complexes with substances. However, the problems arise during the CD synthesis using the commercial enzyme (cyclodextrin glucanotransferase) are low production rate and the process is expensive. Hence, cell immobilization system was used. The objective of this study is to optimize the process parameters using Response Surface Methodology to increase the CD production of the immobilized cell. High CD production was achieved after the optimization process. The reusability of immobilized cell also can be reuse up to six cycles using the optimized parameters. The findings propose that optimization process increased the CD production and made it more valuable for industrial application.

Immobilized Cell Bioreactor Industrialization in the Development of an Innovative Optical Biosensor Technology

This commentary shows the development of a new optical biosensor, based on cell immobilization of Pseudomonas Fluorescens HK44, in nano and micro-tubular cellulose (TC) and a mixture of carbohydrate nanotubes (CHNTs) and carbohydrate micro-tubes (CHMTs). Methodology follows, this biocatalyst can be industrialized with the use of a single tank immobilized cell bioreactor (ICB). A techno-economic analysis was conducted within the framework of it by designing a process flow sheet with mass and energy balance. According to its case study, the investment is 227,800 euros, and the daily production cost is 1434 euros, with a maximum daily added value of 25,000 euros. The discussion revealed that novel research proposals and a novel study concept are being developed in the field of biosensors. The results are supported by papers published on ICB area development. The problem that leads to this commentary is industrialization of ICB, in the case of a simple biosensor development using immobilized cells and it is the objective.

Hexavalent chromium detoxification by haloalkaliphilic Nesterenkonia sp strain NRC-Y immobilized in different matrices

To develop a bioprocess for Cr(VI) detoxification in industrial effluents, a previously isolated potent Cr(VI) reducing haloalkaliphilic Nesterenkonia sp strain NRC-Y was immobilized and investigated for Cr(VI) detoxification. The immobilization matrices included; natural, modified natural, synthetic, and mixtures of natural and synthetic polymers. Among the tested matrices and immobilization approaches, strain NRC-Y cells encapsulated in 1.5% (w/v) amidated pectin beads exhibited the highest Cr(VI) reduction efficiency (47.02% of initial Cr(VI) concentration 150 mg/L after 20 h) followed by alginate (34.4%), alginate-PVA-chitosan (29.6%), alginate-PVA (25.1%), PVA-PVP (23.2%), and PVA (9.5%). Chromate reductase enzyme was poorly immobilized by covalent binding on the tested materials. Therefore, entrapment in amidated pectin was selected for further investigation and immobilization of both whole cells and partially purified chromate reductase. Operational stability study revealed that the immobilized cells were more efficient and stable than the immobilized chromate reductase, and the free cells. For instance, about 60%, 27.0% and 11.5% of these samples initial activities were retained after four successive batches, respectively. The temperature and pH optima for the immobilized cells were 35°C and 7.0, respectively. The pH and thermal stability of strain NRC-Y cells were significantly enhanced upon immobilization in amidated pectin beads by up to 2.3- and 1.4-fold, respectively. The developed immobilized biocatalyst was applied for Cr(VI) reduction in industrial effluent samples, and it completely reduced Cr(VI) within 4 and 8 h for effluents of initial Cr(VI) concentrations 10 and 30 mg/L, respectively. The developed immobilized biocatalyst is promising and has the potential for large-scale Cr(VI) detoxification application.

Microbial Biofilms: Harnessing the Power of Complex Microbial Communities for Industrial Applications

Microbial biofilms are complex microbial colonies that attach to surfaces and grow inside an extracellular polymeric substance (EPS) matrix. These biofilms have great promise for industrial applications and play crucial roles in several natural and artificial systems. The use of microbial biofilms in industrial settings is examined in this abstract, which also places an emphasis on techniques to improve biofilm development and performance through surface modification and quorum sensing manipulation. Due to their versatility, durability, and cooperative behaviour, biofilms have attracted interest and are useful in a variety of industrial industries. They work in the food business, bioenergy generation, agriculture industry and biosensor, among other fields.Biofilms are more effective in industrial processes because of their intricate interactions, which also lead to higher metabolic capacities, increased stress tolerance, and improved retention of immobilised cells. The production and performance of microbial biofilms need to be improved in order to fully realise their potential. Techniques for surface modification provide a promising way to customise the characteristics of biofilms. It is possible to modify the topography, hydrophobicity, and charge of the substrate to affect how quickly biofilms form after initial microbial attachment. Additionally, functionalization and coatings based on nanomaterials provide novel approaches to improve biofilm adhesion, cohesiveness, and stability. The method of cell-to-cell communication known as quorum sensing (QS) directs the development and behaviour of biofilms. Controlling QS pathways enables fine-grained regulation of biofilm growth. QS may be controlled via genetic engineering and small molecule therapy, which affects the phenotypic and architecture of biofilms. With the use of these techniques, biofilms may be designed with the required properties, including greater thickness, higher resistance to shear pressures, and increased production of desirable chemicals. In conclusion, because of their cooperative nature and adaptable functioning, microbial biofilms show enormous promise for industrial applications. The importance of surface modification and quorum sensing modulation as tactics to improve biofilm performance is highlighted in this abstract. As science advances, a fuller comprehension of the ecology of biofilms, interspecies interactions, and synthetic biology technologies will make it easier to create biofilms that are specifically suited to a given industrial purpose. Understanding the complex principles underlying biofilm production and behaviour will allow for the full realisation of the promise for sustainable and effective industrial processes, ushering in a new age of biofilm-based technology.