Fumaric acid production from fermented oil palm empty fruit bunches using fungal isolate K20: a comparison between free and immobilized cells.

This study investigated the potential of using steam-exploded oil palm empty fruit bunches (EFB) as a renewable feedstock for producing fumaric acid (FA), a food additive widely used for flavor and preservation, through a separate hydrolysis and fermentation process using the fungal isolate K20. The efficiency of FA production by free and immobilized cells was compared. The maximum FA concentration (3.25 g/L), with 0.034 g/L/h productivity, was observed after incubation with the free cells for 96 h. Furthermore, the production was scaled up in a 3-L air-lift fermenter using oil palm EFB-derived glucose as the substrate. The FA concentration, yield, and productivity from 100 g/L initial oil palm EFB-derived glucose were 44 g/L, 0.39 g/g, and 0.41 g/L/h, respectively. The potential for scaling up the fermentation process indicates favorable results, which could have significant implications for industrial applications.

Improving mass transfer rates in microbial cell immobilization system for environmental applications: Synergistic interaction of cells on crude oil biodegradation.

Among the various techniques used to clean up polluted environments, bioremediation is the most cost-effective and eco-friendly option. The diversity of microbial communities in a consortium can significantly affect the biodegradability of hazardous organic pollutants, particularly for in situ bioremediation processes. This is largely attributed to interactions between members of a consortium. In this study, the effect of internal diffusion limitations in substrate model biodegradation was firstly examined by immobilized bacterial cells at different particle sizes produced by the electrospray technique. According to the obtained results, for particles with large size, the effectiveness factors (η) were about 0.58-0.67, and the resistance to diffusive on the biodegradation rate was significant, while with decreasing the particle size, η increases and approaches about 1. After selection of suitable bead size, heavy crude oil biodegradation was investigated using a consortium consisting of three oil-degrading bacterial strains at different treatment systems. The removal rate in the suspended co-culture system stands at minimum value of 38% with all three strains which is an indicator of negative interactions among consortium members. Independent immobilization of microorganisms minimizes the competition and antagonistic interactions between strains and leads to more crude oil removal, so that, the biodegradation rate reached 60%.

Biodegradation of crude oil by immobilized Exiguobacterium sp. AO-11 and shelf life evaluation.

Exiguobacterium sp. AO-11 was immobilized on bio-cord at 109 CFU g-1 carrier for the removal of crude oil from marine environments. To prepare a ready-to-use bioremediation product, the shelf life of the immobilized cells was calculated. Approximately 90% of 0.25% (v/v) crude oil removal was achieved within 9 days when the starved state of immobilized cells was used. The oil removal activity of the immobilized cells was maintained in the presence of oil dispersant (89%) and at pH values of 7-9. Meanwhile, pH, oil concentration and salinity affected the oil removal efficacy. The immobilized cells could be reused for at least 5 cycles. The Arrhenius equation describing the relationship between the rate of reaction and temperature was validated as a useful model of the kinetics of retention of activity by an immobilized biocatalyst. It was estimated that the immobilized cells could be stored in a non-vacuum bag containing phosphate buffer (pH 7.0) at 30 °C for 39 days to retain the cells at 107 CFU g-1 carrier and more than 50% degradation activity. These results indicated the potential of using bio-cord-immobilized crude oil-degrading Exiguobacterium sp. AO-11 as a bioremediation product in a marine environment.

Immobilization of metribuzin degrading bacterial consortium MB3R on biochar enhances bioremediation of potato vegetated soil and restores bacterial community structure.

Metribuzin (MB) is a triazinone herbicide used for the eradication of weeds in agriculture. Presence of its residues in agricultural soil can potentially harm the establishment of subsequent crops and structure of soil microbial populations. In this study, remediation potential of an MB degrading bacterial consortium MB3R immobilized on biochar was evaluated in potato vegetated soil. In potato vegetated soil augmented with MB3R alone and MB3R immobilized on biochar, 82 and 96% MB degradation was recorded respectively as compared to only 29.3% in un-augmented soil. Kinetic parameters revealed that MB3R immobilized biochar is highly proficient as indicated by significant increase in the rate of biodegradation and decrease in half-life of MB. Enhanced plant growth was observed when augmented with bacterial consortium either alone or immobilized on biochar. Presence of herbicide negatively affected the soil bacterial community structure. However, MB3R immobilized on biochar proved to be helpful for restoration of soil bacterial community structure affected by MB. This is the very first report that reveals improved remediation of contaminated soil and restoration of soil bacterial populations by use of the MB degrading bacterial consortium immobilized on biochar.

Cell-based biosensors: Recent trends, challenges and future perspectives.

Existing at the interface of biology and electronics, living cells have been in use as biorecognition elements (bioreceptors) in biosensors since the early 1970s. They are an interesting choice of bioreceptors as they allow flexibility in determining the sensing strategy, are cheaper than purified enzymes and antibodies and make the fabrication relatively simple and cost-effective. And with advances in the field of synthetic biology, microfluidics and lithography, many exciting developments have been made in the design of cell-based biosensors in the last about five years. 3D cell culture systems integrated with electrodes are now providing new insights into disease pathogenesis and physiology, while cardiomyocyte-integrated microelectrode array (MEA) technology is set to be standardized for the assessment of drug-induced cardiac toxicity. From cell microarrays for high-throughput applications to plasmonic devices for anti-microbial susceptibility testing and advent of microbial fuel cell biosensors, cell-based biosensors have evolved from being mere tools for detection of specific analytes to multi-parametric devices for real time monitoring and assessment. However, despite these advancements, challenges such as regeneration and storage life, heterogeneity in cell populations, high interference and high costs due to accessory instrumentation need to be addressed before the full potential of cell-based biosensors can be realized at a larger scale. This review summarizes results of the studies that have been conducted in the last five years toward the fabrication of cell-based biosensors for different applications with a comprehensive discussion on the challenges, future trends, and potential inputs needed for improving them.

Therapeutic Regeneration of Lymphatic and Immune Cell Functions upon Lympho-organoid Transplantation.

Lymph nodes (LNs) are secondary lymphoid tissues that play a critical role in filtering the lymph and promoting adaptive immune responses. Surgical resection of LNs, radiation therapy, or infections may damage lymphatic vasculature and compromise immune functions. Here, we describe the generation of functional synthetic lympho-organoids (LOs) using LN stromal progenitors and decellularized extracellular matrix-based scaffolds, two basic constituents of secondary lymphoid tissues. We show that upon transplantation at the site of resected LNs, LOs become integrated into the endogenous lymphatic vasculature and efficiently restore lymphatic drainage and perfusion. Upon immunization, LOs support the activation of antigen-specific immune responses, thus acquiring properties of native lymphoid tissues. These findings provide a proof-of-concept strategy for the development of functional lympho-organoids suitable for restoring lymphatic and immune cell functions.

Oxygen generating biomaterial improves the function and efficacy of beta cells within a macroencapsulation device.

Tissue-engineered devices have the potential to significantly improve human health. A major impediment to the success of clinically scaled transplants, however, is insufficient oxygen transport, which leads to extensive cell death and dysfunction. To provide in situ supplementation of oxygen within a cellular implant, we developed a hydrolytically reactive oxygen generating material in the form of polydimethylsiloxane (PDMS) encapsulated solid calcium peroxide, termed OxySite. Herein, we demonstrate, for the first time, the successful implementation of this in situ oxygen-generating biomaterial to support elevated cellular function and efficacy of macroencapsulation devices for the treatment of type 1 diabetes. Under extreme hypoxic conditions, devices supplemented with OxySite exhibited substantially elevated beta cell and islet viability and function. Furthermore, the inclusion of OxySite within implanted macrodevices resulted in the significant improvement of graft efficacy and insulin production in a diabetic rodent model. Translating to human islets at elevated loading densities further validated the advantages of this material. This simple biomaterial-based approach for delivering a localized and controllable oxygen supply provides a broad and impactful platform for improving the therapeutic efficacy of cell-based approaches.

Pectin Methacrylate (PEMA) and Gelatin-Based Hydrogels for Cell Delivery: Converting Waste Materials into Biomaterials.

The emergence of nontoxic, eco-friendly, and biocompatible polymers derived from natural sources has added a new and exciting dimension to the development of low-cost and scalable biomaterials for tissue engineering applications. Here, we have developed a mechanically strong and durable hydrogel composed of an eco-friendly biopolymer that exists within the cell walls of fruits and plants. Its trade name is pectin, and it bears many similarities with natural polysaccharides in the native extracellular matrix. Specifically, we have employed a new pathway to transform pectin into a ultraviolet (UV)-cross-linkable pectin methacrylate (PEMA) polymer. To endow this hydrogel matrix with cell differentiation and cell spreading properties, we have also incorporated thiolated gelatin into the system. Notably, we were able to fine-tune the compressive modulus of this hydrogel in the range ∼0.5 to ∼24 kPa: advantageously, our results demonstrated that the hydrogels can support growth and viability for a wide range of three-dimensionally (3D) encapsulated cells that include muscle progenitor (C2C12), neural progenitor (PC12), and human mesenchymal stem cells (hMSCs). Our results also indicate that PEMA-gelatin-encapsulated hMSCs can facilitate the formation of bonelike apatite after 5 weeks in culture. Finally, we have demonstrated that PEMA-gelatin can yield micropatterned cell-laden 3D constructs through UV light-assisted lithography. The simplicity, scalability, processability, tunability, bioactivity, and low-cost features of this new hydrogel system highlight its potential as a stem cell carrier that is capable of bridging the gap between clinic and laboratory.

Immobilization of Saccharomyces cerevisiae on Perlite Beads for the Decontamination of Aflatoxin M1 in Milk.

Aflatoxin M1 (AFM1) contamination presents one of the most serious concerns in milk safety. In this study, the immobilization of Saccharomyces cerevisiae was used to detoxify AFM1-contaminated milk. The yeasts were immobilized on perlite for 24 and 48 hr, and the best immobilization time was achieved at 48 hr. Microscopic examination confirmed successful immobilization. The milk samples with 0.08, 0.13, 0.18, and 0.23 ppb AFM1 contamination were passed through the biofilter for 20, 40, and 80 min. The results showed a significant reduction in AFM1 concentration for all the milk samples with various initial AFM1 contents. The contaminated milk with 0.08 ppb AFM1 was completely cleared after 40 min of circulation while for the milk solution with 0.23 ppb, the highest AFM1 reduction was obtained at about 81.3% after 80 min circulation. In addition, the biofilter was saturated after the third step of milk circulation, containing 0.23 ppb AF, in which each step duration was 20 min. This study showed the excellent capability of the immobilized cells on the perlite beads to detoxify the AFM1-contaminated milk without any side effects on its physicochemical properties. PRACTICAL APPLICATION: The immobilization of Saccharomyces cerevisiae cells on perlite beads can be used to detoxify AFM1-contaminated milk. The perlite can provide a perfect support for immobilization. With respect to qualitative properties, 20 min, was suggested as the optimum time for milk decontamination. This study indicated that the detoxification of contaminated milk using immobilized S. cerevisiae cells on the perlite support did not affect the different properties of detoxified milk. This method can lead to a practical solution to address aflatoxin contamination in dairy products considered high-risk foods.

Study on immobilization of marine oil-degrading bacteria by carrier of algae materials.

This study investigated the immobilizations with of bacteria two kinds of algal materials, Enteromorpha residue and kelp residue. The lipophilicity of them were compared by diesel absorption rates. The immobilization efficiency of Bacillus sp. E3 was measured to evaluate whether these carriers would satisfy the requirement for biodegradation of oil spills. The bacteria were immobilized through adsorption with the sterilized and non-sterilized carriers to compare the differences between the two treatments. Oil degradation rates were determined using gravimetric and GC-MS methods. Results showed the absorption rates of Enteromorpha residue and kelp residue for diesel were 411 and 273% respectively and remained approximately 105 and 120% after 2 h of erosion in simulated seawater system. After immobilized of Bacillus sp. E3, the oil degradation rates of them were higher than 65% after 21 days biodegradations. GC-MS analysis showed that two immobilizations degraded higher than 70% of the total alkane and the total PAHs, whereas the free bacteria degraded 63% of the total alkane and 66% the total PAHs. And the bacteria immobilized with the carriers degraded more HMW-alkanes and HMW-PAHs than the free bacteria. The bacteria immobilized by non-sterilized kelp residue showed a considerably higher degradation rate than that using sterilized kelp residue. A considerably higher cells absorption rate of immobilization was obtained when using kelp residue, and the preparation of immobilization was low cost and highly efficient. The experiments show the two algae materials, especially the kelp residue, present potential application in bioremediation of marine oil spills.

Alginate-perlite encapsulated Pseudomonas putida A (ATCC 12633) cells: Preparation, characterization and potential use as plant inoculants.

Microbial immobilization can be used to prepare encapsulated inoculants. Here, we characterize and describe the preparation of Ca-alginate-perlite microbeads loaded with cells of plant growth-promoting Pseudomonas putida A (ATCC 12633), for their future application as agricultural inoculants. The microbeads were prepared by dropwise addition of a CaCl2-paraffin emulsion mixture to an emulsion containing alginate 2% (w/v), perlite 0.1-0.4% (w/v) and bacterial suspension in 0.9% NaCl (1010 CFU/mL). For all perlite concentrations used, microbead size was 90-120 µm, the trapped population was 108 CFU/g microbeads and the increase in mechanical stability was proportional to perlite concentration. Microbeads containing 0.4% (w/v) perlite were able to release bacteria into the medium after 30 days of incubation. When we evaluated how P. putida A (ATCC 12633) entrapped in Ca-alginate-perlite (0.4% (w/v)) microbeads colonized the Arabidopsis thaliana rhizosphere, an increase in colonization over time was detected (from an initial 2.1 × 104 to 9.2 × 105 CFU/g soil after 21 days). With this treatment, growth promotion of A. thaliana occurred with an increase in the amount of proteins, and in root and leaf biomass. It was concluded that the microbeads could be applied as possible inoculants, since they provide protection and a controlled release of microorganisms into the rhizosphere.

Degradation of cationic surfactants using immobilized bacteria: Its effect on adsorption to activated sludge.

Adsorption of cationic surfactants (QACs) Br-tetradecyltrimethylammonium (TTAB), Cl-tetradecylbenzyldimethylammonium (C14BDMA) and Cl-hexadecylbenzyldimethylammonium (C16BDMA) to activated sludge from a wastewater treatment plant was tested. Adsorption equilibrium was reached after 2 h, and for initial 200 mg L-1 81%, 90% and 98% of TTAB, C14BDMA and C16BDMA were respectively adsorbed. After six successive desorption cycles, 21% of TTAB and 12.7% of C14BDMA were desorbed from the sludge. In agreement with the percentage of QACs pre-adsorbed, the more hydrophobic the compound, the lesser the extent of desorption. Wastewater samples with activated sludge were supplemented with TTAB 200 mg L-1 and Ca-alginate beads containing the QACs-degrading microorganisms Pseudomonas putida A (ATCC 12633) and Aeromonas hydrophila MFB03. After 24 h, 10 mg L-1 of TTAB were detected in the liquid phase and 6-8 mg L-1 adsorbed to the sludge. Since without Ca-alginate beads or with empty beads total TTAB amount (phase solid and liquid) did not change, the 90% reduction of the initial 200 mg L-1 after treatment with immobilized cells was attributed to the bacterial consortium's capacity to biodegrade QACs. The results show the advantages of using immobilized bacteria to achieve complete QACs elimination from wastewater systems, thus preventing them from reaching the environment.

Continuous Production of Isomalto-oligosaccharides by Thermo-inactivated Cells of Aspergillus niger J2 with Coarse Perlite as an Immobilizing Material.

The coarse perlite 40-80 mesh was selected as an immobilizing material and put into a packed bed reactor (PBR) to continuously convert maltose to isomalto-oligosaccharides (IMOs). The PBR was prepared by mixing the thermo-inactivated cells (TIC) from Aspergillus niger J2 strain with the coarse perlite, then the mixture was put into an overpressure-resistant column. Compared with diatomite 40-80 mesh and thin perlite 80-120 mesh in PBR, coarse perlite was chosen as the best filtration aid, when the ratio of coarse perlite versus TIC was 1:1. The thermal and pH stability of the free and immobilized TIC and the optimum conditions for the transglycosylation reactions were determined. The results show that approximately 75 and 82% and 87 and 91% of α-glucosidase activity were reserved for free and immobilized TIC at temperatures from 30 to 60 °C and pH from 3.00 to 7.00 for 12 h, respectively. With 30% malt syrup under the conditions of 50 °C and pH 4.00, a mini-scale packed bed reactor (Mi-PBR) and medium-scale packed bed reactor (Me-PBR) could continuously produce IMO over 25 and 34 days with the yield of effective IMO (eIMO) ≥ 35% and total IMO (tIMO) ≥ 50%, respectively. The strategy of mixing the coarse perlite with TIC in PBR is a novel approach to continuously produce IMO and has great application potential in industry.

Bio-reinforced self-healing concrete using magnetic iron oxide nanoparticles.

Immobilization has been reported as an efficient technique to address the bacterial vulnerability for application in bio self-healing concrete. In this study, for the first time, magnetic iron oxide nanoparticles (IONs) are being practically employed as the protective vehicle for bacteria to evaluate the self-healing performance in concrete environment. Magnetic IONs were successfully synthesized and characterized using different techniques. The scanning electron microscope (SEM) images show the efficient adsorption of nanoparticles to the Bacillus cells. Microscopic observation illustrates that the incorporation of the immobilized bacteria in the concrete matrix resulted in a significant crack healing behavior, while the control specimen had no healing characteristics. Analysis of bio-precipitates revealed that the induced minerals in the cracks were calcium carbonate. The effect of magnetic immobilized cells on the concrete water absorption showed that the concrete specimens supplemented with decorated bacteria with IONs had a higher resistance to water penetration. The initial and secondary water absorption rates in bio-concrete specimens were 26% and 22% lower than the control specimens. Due to the compatible behavior of IONs with the concrete compositions, the results of this study proved the potential application of IONs for developing a new generation of bio self-healing concrete.

Yeast screening and cell immobilization on inert supports for ethanol production from cheese whey permeate with high lactose loads.

Eight yeast strains of the genera Saccharomyces and Kluyveromyces were screened to ferment high lactose-load cheese whey permeate (CWP) (>130 g/L lactose) without nutrient supplementation. The fermentation conditions (temperature, pH and time) were optimized to maximize the fermentation performance (ethanol titer, ethanol yield and lactose consumption) for the two preselected strains, K. marxianus DSM 5422 and S. cerevisiae Ethanol Red, using a response surface methodology (RSM). Under optimized conditions, K. marxianus DSM 5422 attained ethanol titers of 6% (v/v) in only 44 h. Moreover, the feasibility of immobilizing this strain on four different inorganic supports (plastic, glass and Tygon silicone Raschig rings and alumina beads) was assessed. Glass Raschig rings and alumina beads showed a more stable performance over time, yielding ethanol titers of 60 g/L during 1,000 hours, which remarkably reduces yeast cultivation costs. Results demonstrate the feasibility of using CWP for successful ethanol production in a simple and economical process, which represents an attractive alternative for waste treatment in dairy industries.

Individually immobilized and surface-modified hydrocarbon-degrading bacteria for oil emulsification and biodegradation.

Effective emulsification plays an important role in the treatment of marine oil spills. The negative effects of chemical surfactants have necessitated a search for alternative dispersant that are sustainable and environmentally-friendly. To identify alternate dispersants, oil-in-seawater emulsions stabilized by hydrocarbon-degrading bacteria were investigated. After individual immobilization and surface-modification, the hydrocarbon-degrading bacteria, Bacillus cereus S-1, was found to produce a stable oil-in-seawater Pickering emulsion, which was similar to particle emulsifiers. The individual immobilization and surface-modification process improved the surface hydrophobicity and wettability of the bacterial cells, which was responsible for their effective adsorption at the oil-water interface. Through effective emulsification, the biodegradation of oil was remarkably facilitated by these treated bacteria, because of the increased interfacial area. By combining the emulsification and biodegradation, the results of this reported work demonstrated a novel approach for developing environmentally-friendly bioremediation technology in the field of oil treatment.

Application of magnetically immobilized edible fungus for the biotransformation of panax notoginseng saponin Rb1 to Rd and Rg3.

In this study, we developed a new magnetically immobilized edible fungus for the biotransformation of panax notoginseng saponins Rb1 to Rd and Rg3. The optimum biotransformation conditions were as follows: temperature 32°C, pH 6.5, time 48h and liquid-solid ratio 25:1(mL/g). The yields of Rd and Rg3 reached 41.35±0.12mg/g and 6.35±0.08mg/g which increased 6.97-fold and 3.23-fold to that of untreated control, respectively. Additionally, SEM demonstrated that vestured pits and cell walls of samples were destroyed obviously which was beneficial to the Rb1 biotransformation and target Rd and Rg3 release. Meanwhile, the reusability of magnetically immobilized microorganism was tested and the activity of the magnetically immobilized microorganism remained 83.8% after 15 runs. The recycling experiments demonstrated that magnetically immobilized fungus exhibited higher efficiency than the non-magnetical one. These results proved that this new magnetically immobilized microorganism could be applied for industrial production and pharmaceutical industry with good efficiency.

Biohydrogen production from sugarcane bagasse hydrolysate: effects of pH, S/X, Fe2+, and magnetite nanoparticles.

Batch dark fermentation experiments were conducted to investigate the effects of initial pH, substrate-to-biomass (S/X) ratio, and concentrations of Fe2+ and magnetite nanoparticles on biohydrogen production from sugarcane bagasse (SCB) hydrolysate. By applying the response surface methodology, the optimum condition of steam-acid hydrolysis was 0.64% (v/v) H2SO4 for 55.7 min, which obtained a sugar yield of 274 mg g-1. The maximum hydrogen yield (HY) of 0.874 mol (mol glucose-1) was detected at the optimum pH of 5.0 and S/X ratio of 0.5 g chemical oxygen demand (COD, g VSS-1). The addition of Fe2+ 200 mg L-1 and magnetite nanoparticles 200 mg L-1 to the inoculum enhanced the HY by 62.1% and 69.6%, respectively. The kinetics of hydrogen production was estimated by fitting the experimental data to the modified Gompertz model. The inhibitory effects of adding Fe2+ and magnetite nanoparticles to the fermentative hydrogen production were examined by applying Andrew's inhibition model. COD mass balance and full stoichiometric reactions, including soluble metabolic products, cell synthesis, and H2 production, indicated the reliability of the experimental results. A qPCR-based analysis was conducted to assess the microbial community structure using Enterobacteriaceae, Clostridium spp., and hydrogenase-specific gene activity. Results from the microbial analysis revealed the dominance of hydrogen producers in the inoculum immobilized on magnetite nanoparticles, followed by the inoculum supplemented with Fe2+ concentration. Graphical abstract ᅟ.

Calcium gluconate as cross-linker improves survival and shelf life of encapsulated and dried Metarhizium brunneum and Saccharomyces cerevisiae for the application as biological control agents.

Calcium chloride (CC) is the most common cross-linker for the encapsulation of biocontrol microorganisms in alginate beads. The aim of this study was to evaluate if calcium gluconate (CG) can replace CC as cross-linker and at the same time improve viability after drying and rehydration, hygroscopic properties, shelf life and nutrient supply. Hence, the biocontrol fungi Metarhizium brunneum and Saccharomyces cerevisiae were encapsulated in Ca-alginate beads supplemented with starch. Beads were dried and maximum survival was found in beads cross-linked with CG. Beads prepared with CG showed lower hygroscopic properties, but a higher shelf life for encapsulated fungi. Moreover, we demonstrated that gluconate has a nutritive effect on encapsulated fungi, leading to increased mycelium growth of M. brunneum and to enhanced CO2 release from beads containing Saccharomyces cerevisiae. The application of CG as cross-linker will pave the way towards increasing drying survival and shelf life of various, especially drying-sensitive microbes.

Bioenergetics of lactate vs. acetate outside TCA enhanced the hydrogen evolution levels in two newly isolated strains of the photosynthetic bacterium Rhodopseudomonas.

Two local hydrogen-evolving strains of purple nonsulfur bacteria have been isolated, characterized, and identified as Rhodopseudomonas sp. TUT (strains Rh1 and Rh2). Lactate followed by succinate and malate supported the highest amounts of H2 production, growth (O.D.660nm, proteins and bacteriochlorphyll contents), nitrogenase activity, and uptake hydrogenase; the least of which was acetate. Alginate-immobilized cells evolved higher hydrogen amounts than free cell counterparts. Rh1 was more productive than Rh2 at all circumstances. Lactate-dependent hydrogen evolution was more than twice that of acetate, due to ATP productivity (2/-1, respectively), which is limiting to the nitrogenase activity. The preference of lactate over other acids indicates the feasibility of using these two strains in hydrogen production from dairy wastewater.