Free Cell Culture Research Articles

Statistical modelling and optimization of medium for toluene degradation by Brevibacillus parabrevis ATHH40 immobilized cells

ABSTRACT Response surface methodology was used to optimise toluene degradation by immobilised Brevibacillus parabrevis ATHH40. The pH, temperature and toluene concentration were the most significant factors in toluene degradation. Central composite design was applied to determine optimal concentrations of these factors and the effect of their mutual interactions. The interaction between pH and temperature with toluene concentration was found to enhance toluene degradation, whereas pH, temperature and toluene concentration exhibited an independent influence. Using statistical optimisation, a maximum toluene degradation was achieved when pH, temperature and toluene concentration were adjusted to 6.93, 34.76°C and 774.76 mg L−1. This optimised medium was employed for toluene degradation by Brevibacillus parabrevis immobilised by multi-walled carbon nanotubes that improved toluene degradation to 93.87%, much higher than achieved by cultures of free cells.

Fermentative processes for the upcycling of xylose to xylitol by immobilized cells of Pichia fermentans WC1507.

Xylitol is a pentose-polyol widely applied in the food and pharmaceutical industry. It can be produced from lignocellulosic biomass, valorizing second-generation feedstocks. Biotechnological production of xylitol requires scalable solutions suitable for industrial scale processes. Immobilized-cells systems offer numerous advantages. Although fungal pellet carriers have gained attention, their application in xylitol production remains unexplored. In this study, the yeast strain P. fermentans WC 1507 was employed for xylitol production. The optimal conditions were observed with free-cell cultures at pH above 3.5, low oxygenation, and medium containing (NH4)2SO4 and yeast extract as nitrogen sources (xylitol titer 79.4g/L, YP/S 66.3%, and volumetric productivity 1.3g/L/h). Yeast cells were immobilized using inactive Aspergillus oryzae pellet mycelial carrier (MC) and alginate beads (AB) and were tested in flasks over three consecutive production runs. Additionally, the effect of a 0.2% w/v alginate layer, coating the outer surface of the carriers (cMC and cAB, respectively), was examined. While YP/S values observed with both immobilized and free cells were similar, the immobilized cells exhibited lower final xylitol titer and volumetric productivity, likely due to mass transfer limitations. AB and cAB outperformed MC and cMC. The uncoated AB carriers were tested in a laboratory-scale airlift bioreactor, which demonstrated a progressive increase in xylitol production in a repeated batch process: in the third run, a xylitol titer of 63.0g/L, YP/S of 61.5%, and volumetric productivity of 0.52g/L/h were achieved. This study confirmed P. fermentans WC 1507 as a promising strain for xylitol production in both free- and entrapped-cells systems. Considering the performance of the wild strain, a metabolic engineering intervention aiming at further improving the efficiency of xylitol production could be justified. MC and AB proved to be viable supports for cell immobilization, but additional process development is necessary to identify the optimal bioreactor configuration and fermentation conditions.

Enhanced growth and lipid productivity by living Chlorella sorokiniana immobilized in Ca-alginate beads

The bottleneck for the production of biofuels from microalgae consists on costly harvesting processes and low lipid production, immobilization technology could play a part on making the production of biofuels more feasible. The aim of this study was to evaluate the effect of alginate immobilization on the growth and lipid productivity of the microalgae Chlorella sorokiniana, so far, the main focus of immobilization technology has been its use for wastewater treatment and nutrient removal from effluents. The microalgae Chlorella sorokiniana was cultured in both free and immobilized forms under optimal autotrophic growth conditions. Microalgae were immobilized in calcium alginate beads generated by mixing algal cells with a sodium alginate solution, followed by extrusion into a CaCl2 solution. The results obtained in this study showed that the growth of the microalgae immobilized in alginate beads, was enhanced and achieved a dry cell weight 1.4-fold higher than that of a free cell culture, a higher light transmittance was also achieved in the alginate immobilized culture, and the lipid productivity was increased from 54.21 ± 2.48 mg l−1 d in the free cell culture to 82.22 ± 8.48 mg l−1 d in the immobilized culture. These results demonstrate the effectiveness of immobilization technology for promoting growth and lipid productivity in the microalgae Chlorella sorokiniana.

Alginate immobilization as a strategy for improving succinate production during autofermentation using cyanobacteria Synechocystis sp. PCC 6803

This study focused on immobilization of a cyanobacterial strain for succinate production. Recombinant cyanobacterium Synechocystis sp. PCC 6803, with an overexpressed endogenous PEPC gene, was immobilized on calcium alginate beads. This immobilization technology improved succinate production during a continuous autofermentation cycle compared with that produced using a free-cell culture. In addition, the immobilized cells achieved a higher dry cell weight than the free cells. Furthermore, this process permitted cultivation within a shorter timeframe than in a free-cell culture. During continuous succinate production, the immobilized cells increased the succinate yield by 1.4-fold, whereas the use of free-cells resulted in a 50 % decrease in the succinate yield. Continuous batch culture offers a convenient strategy for the use of immobilized cyanobacteria, resulting in a simpler and faster process that achieves a higher rate of succinate production with each cycle.

MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF FOWL AVIADENOVIRUS ISOLATED FROM COMMERCIAL CHICKEN FARMS

Fowl Adenovirus (FAdV) is a virus that causes disease in poultry with characteristics such as inclusionsbody found in organ lesions, especially the liver. The disease causes economic losses, in the form of chickendeaths and immunosuppression effects. Since 2018, outbreaks of FAdV or known as inclusion body hepatitis(IBH) have been reported in broiler farms, in several provinces in Indonesia. This study aims to isolate,and characterize molecularly, and biologically of embryonic chicken eggs and liver cell cultures of chickenembryos. Liver samples, bursa fabricius, and spleen were obtained from 23 commercial chicken farms inIndonesia that were diagnosed with IBH disease from 2018-2020. Molecular detection was carried out bypolymerase chain reaction (PCR) technique, using hexon gene-specific primers. The amplification resultwas positive, then sequencing was performed. Isolation was carried out using embryonic chicken eggs(ECE) specific pathogen free (SPF) and chicken embryo liver (CEL) cell cultures. Detection and molecularcharacterization showed that all FAdV isolated from poultry obtained from cases diagnosed with IBHdisease confirmed as FAdV genotype E, including strain FAdV-8b. Isolation on TAB showed embryonicbleeding lesions and swelling of the liver. Propagation of the FADV isolate using chicken embryo liver cellculture, showed cytopathic lesions that could be observed in the first passage, in the form of swelling androunding of infected cells. IBH disease outbreaks in commercial chickens in this study, confirmed the causeis FAdV genotype E, which is included in serotype 8b. FAdV virus isolated on SPF TAB showed embryonicbleeding lesions and hepatitis. FAdV virus isolated in this study was assessed as pathogenic in SPFchicken embryos.

Immobilization of Clostridium perfringens type D in calcium alginate beads: toxin production mimics free cell culture.

Cell-immobilization is used to maintain microbial culture to produce metabolites in repeated-batch or continuous fermentations, thereby reducing the time and resources spent on delivering mass production of microbe. The technique also enables shortening of the detoxification phase and the amount of formaldehyde required due to low incidence of viable bacteria in the extract. A solution of sodium alginate containing Clostridium perfringens cells was dropped into stirring CaCl solution via a sterile syringe needle. Optimizations resulted in reasonably uniform beads containing C. perfringens. Beads were externally stabilized by poly L-lysine, followed by immersion in a solution of Na-alginate to coat them with a new layer of alginate forming an alginate-PLL-alginate cortex. This study proved successful in immobilizing C. perfringens cells inside uniform alginate microspheres. Cell loading and cell propagation inside the beads were measured. The cell loaded beads were cultivable in liquid media producing 550 minimum lethal doses per milliliter (MLD/ml) in a 72 h. The research paved the way for further investigations to optimize and establish an efficient bacterial encapsulation method. Thus, it seems possible to produce toxins from beads engulfing C. perfringens on larger scales via repeated-batch or continuous fermentation processes.

AAV vector characterization best practices

David McCall, Commissioning Editor, Cell & Gene Therapy Insights, speaks to Gaël Stephant, Senior CMC Project Leader, Vivet Therapeutics.Gaël Stéphant graduated from the Arts et Métiers CNAM, Paris III in 2008 as Engineer in biology, after working a few years as a technician. M. Stéphant joined innovative cell therapy company, Celogos in 2005 to develop an animal component free cell culture media designed to increase cell growth as well as reducing risk for patient. In 2008, he set up the cell culture laboratory Xentech (CRO company) for preclinical in vitro/ex vivo cancer model development. In 2012, he was hired as head of vector bioproduction unit at Theravectys. He then started in 2015, a first CMC Project leader rôle at Yposkezy (formerly Genethon CDMO) followed by 3 years at Gensight Biologics, a biotech company where he was in charge of the production of AAV vector batches to be used in phases I and II clinical trials, as well as the preparation towards commercial phase. He then changed environment and joigned the pharmaceutical company Servier, to lead the CMC team of the CART cell/gene therapy development. Since 2021, M.Stéphant works for Vivet Therapeutics, a biotech company, where he manages the CMC activities for recombinant proteins and gene therapy products.

Effects of Constant Electric Field on Biodegradation of Phenol by Free and Immobilized Cells of Bradyrhizobium japonicum 273

It is shown that bacteria Bradyrhizobium japonicum 273 were capable of degrading phenol at moderate concentrations either in a free cell culture or by immobilized cells on granulated activated carbon particles. The amount of degraded phenol was greater in an immobilized cell preparation than in a free culture. The application of a constant electric field during cultivation led to enhanced phenol biodegradation in a free culture and in immobilized cells on granulated activated carbon. The highest phenol removal efficiency was observed for an anode potential of 1.0 V/S.H.E. The effect was better pronounced in a free culture. The enzyme activities of free cells for phenol oxidation and benzene ring cleavage were very sensitive to the anode potential in the first two steps of the metabolic pathway of phenol biodegradation catalyzed by phenol hydroxylase—catechol-1,2-dioxygenase and catechol-2,3-dioxygenase. It was observed that at an anode potential of 0.8 V/S.H.E., the meta-pathway of cleavage of the benzene ring catalyzed by catechol-2,3-dioxygenase became competitive with the ortho-pathway, catalyzed by catechol-1,2-dioxygenase. The obtained results showed that the positive effect of constant electric field on phenol biodegradation was rather due to electric stimulation of enzyme activity than electrochemical anode oxidation.

Delivery of extracellular matrix-enriched stem cells encapsulated with enzyme-free pH-sensitive polymer for enhancing therapeutic angiogenesis

At clinical transplantation of human adipose derived stem cells (hADSCs), a proteolytic enzyme treatment for harvesting hADSCs must be used but usually causes the disruption of extracellular matrices (ECMs) interconnection. As ECMs-poor hADSCs partially receive the cellular signals, hADSCs deactivated or undergo cell death. Herein, we have proposed a method for delivering ECMs-enriched hADSCs encapsulated with a new polymer for transplanting into ischemic wound sites to enhance the therapeutic angiogenic efficacy. Enzyme free pH-sensitive polymer (EFP) was synthesized and coated on tissue culture plate for hADSCs culture. When the hADSCs were cultured on EFP-coated surfaces, they were easily detached with pH 6.0 buffer solution and dissolved EFP wrapped hADSCs with more ECMs for 48 h. When ECMs enriched-hADSCs were re-adhered, the cells secreted more angiogenic factors under serum free and hypoxic cell culture condition. When injecting ECMs-enriched hADSCs to mouse hindlimb ischemia models, the blood perfusion and blood vessel regeneration rate were significantly higher. Our method for delivering ECMs-enriched stem cells using EFP significantly improved the therapeutic angiogenesis with hADSCs compared to proteolytic enzymatic application.

Development of diatom entrapped alginate beads and application of immobilized cells in aquaculture

The objective of this study was entrapment of freshwater diatom Nitzschia palea in a Ca-alginate hydrogel bead through the gelation method. The study further aimed at remediating aquaculture water and its efficient utilization for diatom productivity and futuristic aquaculture applications. Results demonstrate that entrapped diatom in Ca-alginate hydrogel bead showed no swelling behavior​ and they were quite stable during analysis. The maximum cell density was 71.4 × 105 cell mL −1 which was 3.5 times higher than the cell density achieved in free cell culture. The maximum nitrate, phosphate, ammonia removal efficiency was 86.93 ± 3.46%, 57.51 ± 3.8%, 63.83 ± 1.18% which signifies the efficiency of diatom entrapped Ca-alginate hydrogel bead in removing the nutrient load from the aquaculture water. The entrapped diatom cells were quite successful in controlling the water hardness (52.33 ± 2.51%) to a significant level. This work demonstrates that diatom entrapped in Ca-alginate hydrogel bead is a cost-effective method in aquaculture water remediation which can be a long-lasting solution towards sustainability and diverse aquaculture applications.

Effect of FLT3-ITD Length on 32D Cell Proliferation, Apoptosis and Sensitivity to FLT3 Inhibitor

To study the effects of FLT3-ITD length on 32D cell proliferation, apoptosis and sensitivity to FLT3 inhibitor, so as to provide references for stepwise therapy of FLT3-ITD mutated acute myeloid leukemia patients. Three different FLT3-ITD mutants with same or adjacent insert sites were selected and constructed in an eukaryotic expression vector. FLT3-ITD mutants stably expressed 32D cell strains were selected with the help of lentivirus system and IL3 free cell culture medium. The proliferation and apoptosis of 32D cell strains after AC220 treatment were detected. FLT3-ITD mutants (ITD1, ITD2 and ITD3) stably expressed 32D cell strains were constructed successfully. In the absence of IL3 factor, the proliferation number of ITD1, ITD2 and ITD3 cell strains were mounted up to 2.3 folds, 3.7 folds, and 4.3 folds after 48 hours, respectively. Under the exposure of FLT3 inhibitor AC220, the IC50 values was 0.183, 0.446 and 0.836 nmol/L, and apoptosis rates was 88.6%, 34.2% and 16.1%, respectively. FLT3-ITD mutant expressed cell strains with longer ITD show higher capacity of proliferation and higher tolerance to AC220 treatment.

Enhancing the recombinant protein productivity of Yarrowia lipolytica using in situ fibrous bed bioreactor

In this study, the ability of Yarrowia lipolytica to produce the recombinant lipase CalB from Candida antarctica, used as a model protein has been compared across different bioreactor processes using glycerol, a byproduct from the biodiesel industry as the main carbon source. Batch, pulsed fed-batch (PFB), and continuous fed-batch (CFB) strategies were first compared using classical stirred tank (STR) bioreactors in terms of biomass production, carbon source uptake, and lipase production. Additionally, an in situ fibrous bed bioreactor (isFBB) was developed using sugarcane bagasse as a cell immobilization support. The maximum lipase titer achieved using the isFBB culture mode was 38%, 33%, and 49% higher than those obtained using the batch, PFB, and CFB cultures, respectively. The lipase productivity in isFBB mode (142U/mL/h) was 1.4-fold higher than that obtained using batch free cell cultures. These results highlight that isFBB is an efficient technology for the production of recombinant enzymes.

Enhanced bioremediation of diesel oil-contaminated seawater by a biochar-immobilized biosurfactant-producing bacteria Vibrio sp. LQ2 isolated from cold seep sediment

This study investigated the effect of immobilized biosurfactant-producing bacteria on the bioremediation of diesel oil-contaminated seawater. Initially, a biosurfactant-producing bacterium, LQ2, was isolated from a marine cold-seep region, and identified as Vibrio sp. The biosurfactant produced by LQ2 was characterized as a phospholipid, exhibiting high surface activity with strong stability. Meanwhile, the inoculation of biochar-immobilized LQ2 demonstrated superior efficiency in removing diesel oil (94.7%, reduction from 169.2 mg to 8.91 mg) over a seven-day period compared to free-cell culture (54.4%), through both biodegradation and adsorption. In addition, the microbial growth and activity were greatly enhanced with the addition of immobilized LQ2. Further experiment showed that degradation-related genes, alkB and CYP450–1, were 3.8 and 15.2 times higher in the immobilized LQ2 treatment, respectively, than those in the free cell treatment. The findings obtained in this study suggest the feasibility of applying immobilized biosurfactant-producing bacteria, namely LQ2, in treating diesel oil-contaminated seawater.

Properties and aerogel applications of a marine algal origin biocellulose produced by the immobilized Gluconacetobacter xylinus ATCC 10245

Algae are known to produce biomass faster as compared with lignocellulosic biomass. BC was produced by using reducing sugar from algae as a cheaper carbon source. The effect of immobilization on the production of biocellulose(BC) by Gluconacetobacter xylinus ATCC 10245 was studied. The BC production of all entrapped cultures within different gel materials was lower than that of free cell cultures but the entrapped G. xylinus cells in alginate beads recorded the highest BC productivity (12.5 g/l). The adsorption of G. xylinus cells on luffa pulp (LP) and ceramics particles (CP) gave the highest BC production (14.5 g/l), which represents increasing of 1.14 and 1.05- fold, respectively from the free cells. This attempt clarified that LP was the best immobilizing support as it gave the highest BC production. The effect of repeated reuse of LP was confirmed. Different physical properties of BC including; pH, water absorption capacity, dissolving in several solvents, and chemicals were determined. Observation of BC through SEM analysis showed the fine cellulose ribbons (fibrils). The crystallinity for the BC produced from X-ray diffraction (XRD) spectra was 84%. Upon the applicable level, the BC aerogel was formed to be applied in rising up the water and oil absorption capability by developing its hydrophilic properties. Data introduced strong affinities to the motor, olive, and cooking oils with high oil uptake contents of 7.5, 5.2, and 3.7 gg−1, respectively. Moreover, the BC aerogel exhibited potential adsorption for different heavy metal ions Pb2+, Cd2+, and Cu2+. The highest capability of Cd2+ removal was 95.7% at pH 6. The biodegrability of our produced BC and then its safety was proven.

Abstract 6276: Brain penetrant MDM2 inhibitor RG7388 extends survival benefit of radiation treatment in select glioblastoma patient-derived xenograft models

Abstract Despite aggressive treatment that involves surgery, radiation and temozolomide therapy, a significant morbidity from glioblastoma (GBM) recurrence highlights the pressing unmet medical need to develop effective novel therapies for GBM. Murine Double Minute 2 (MDM2) is an important regulator of the p53 tumor suppressor, which promotes cell cycle arrest and apoptosis in response to DNA damage. Here we have assessed the efficacy of RG7388, a purported brain penetrant MDM2-inhibitor, alone or combined with radiation therapy (RT) in patient-derived xenograft (PDX) models of GBM. In vitro, RG7388 suppressed viability of GBM PDX short-term stem cell cultures of p53 wildtype lines with MDM2-amplification (GBM46 and 108, IC50 25 and 11 nM, respectively) or without MDM2-amplification (GBM10 and 14, IC50 43.5 and 9.1 nM, respectively). Serum proteins appear to reduce efficacy of RG7388, which is supported by the notion that adding 2.5% bovine serum albumin to a serum free stem cell cultures increased the IC50 by 3 to 5 fold. At the molecular level, both RG7388 and RT could induce p53 signaling in GBM10, but a more robust induction was observed with the combination. In athymic nude mice, RG7388 readily distributes into normal brain as measured by LCMS-MS at 1 hour after five daily oral doses (70 mg/kg) the average RG7388 concentrations in brain and plasma were 2167 ng/g and 4423 ng/ml, respectively. In an initial efficacy study using flank tumors established from GBM10, one week of dosing at 70 mg/kg/day RG7388 alone was ineffective, while combining RG7388 with focal radiation (4 daily doses of 5 Gy each) had a moderate 15 day delay in tumor progression. However, in an MDM2-amplified GBM108 line with a relatively intact blood-brain barrier, one week of daily RG7388 dosing, either alone or in combination with RT was remarkably effective (median survival 71 days with RG7388 alone vs. 29 days with vehicle, p<0.0001; and 262 days for RG7388/RT vs. 74 days with RT, p=0.017). Taken together, RG7388 alone or in combination with RT is highly effective in an MDM2-amplified pre-clinical model of GBM. Determining impact of serum protein binding on drug pharmacokinetics and further evaluation of pharmacodynamic effects and orthotopic therapy in additional p53 wildtype PDX lines will help to increase our understanding if RG7388/RT therapy can be used in a larger population of GBM tumors. Citation Format: Shiv K. Gupta, Ann C. Mladek, Surabhi Telele, Afroz S. Mohammad, Lihong He, Zeng Hu, Katrina K. Bakken, Danielle M. Burgenske, Brett L. Carlson, William F. Elmquist, Jann N. Sarkaria. Brain penetrant MDM2 inhibitor RG7388 extends survival benefit of radiation treatment in select glioblastoma patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6276.

Investigation of fungal treatment potential for bulgur cooking process wastewater

The objective of this study was to evaluate chemical oxygen demand (COD), protein, and carbohydrate removal from bulgur processing industry wastewater with fungal treatment. In the first stage, white rot and filamentous fungi species were tested with free cell culture at the original pH (4.5) of wastewater. Among the tested species A. parasiticus supplied the highest COD, protein, and carbohydrate removal efficiencies at the end of the incubation period (9 d). In the second stage, the removal efficiencies were compared to free A. parasiticus and immobilized A. parasiticus on waste plastic bath fiber. Moreover, the effect of initial pH of wastewater was tested on the removal efficiency of COD, protein, and carbohydrate. The results showed that immobilized A. parasiticus supplied higher removal efficiencies than the free one. Maximum removal efficiency of COD, carbohydrate, and protein was determined as 82.5 ± 1.8%, 67.9 ± 1.9% and 61.2 ± 2.3 at pH 5.5 for 12 d, respectively.

Potential of immobilized Chlorella minutissima for the production of biomass, proteins, carotenoids and fatty acids

Abstract This work investigated the potential of Chlorella minutissima immobilized in alginate beads for biomass and bioactive compounds production. The effects of nitrogen concentration in the culture medium (48 to 96 mg L-1 N–NO3) and the nitrogen addition profile during fed-batch culture were evaluated. In batch cultures, the initial nitrogen concentration of 96 mg L-1 N–NO3 produced the highest chlorophyll and protein contents in biomass, about 2.8 and 1.5-fold higher than the control (48 mg L-1 N–NO3), respectively. The biomass concentration (1.65 ± 0.01 g L-1) and carotenoid content (11.0 ± 0.8 mg g-1) were 21.3% and 32.6% higher than the control (48 mg L-1 N–NO3), but similar to the other nitrogen concentrations tested (64 and 80 mg L-1 N–NO3). Fed-batch cultures were performed in order to optimize the productivity of immobilized C. minutissima. The fed-batch culture with three additions of 96 mg L-1 of N–NO3 resulted in the highest biomass concentration (2.13 ± 0.02 g L-1) and an increase in chlorophylls (1.7-fold), carotenoids (1.3-fold) and proteins (1.6-fold) content in biomass compared to batch culture. The contents of all-trans-lutein and polyunsaturated fatty acids were 5.31 ± 0.46 mg g-1 (dry cell weight) and 52.0 ± 0.9% of total fatty acids, respectively, similar or higher than free cell cultures. The culture of immobilized C. minutissima cells showed to be a promising technique towards decreasing microalgae production costs. Through fed-batch cultures, it was possible to increase the production of biomass and compounds of biotechnological interest.

Injectable thermoresponsive hydrogel/nanofiber hybrid scaffolds inducing human adipose-derived stem cell chemotaxis

Hydrogels with three-dimensional networks similar to extracellular matrix (ECM) have been widely investigated as biomaterials in tissue engineering. Injectable hydrogels have been studied for less invasive treatments, and moreover, the endogenous cell recruitment capability can create synergy with these hydrogels for repairing damaged tissues. However, their low mechanical properties or difficult process has been a disadvantage for their use in load-bearing cartilage such as intervertebral disc. Therefore, high-modulus injectable hydrogels which have the potential in cell recruitment are required for fundamental and less invasive treatments. In this study, we fabricated injectable and thermoresponsive hybrid scaffolds comprising chitosan/gelatin hydrogel and polycaprolactone nanofibers, and we added platelet-derived growth factor-BB (PDGF-BB) within the hybrid scaffolds to recruit human adipose-derived stem cells (hADSCs). The modulus of the hybrid scaffolds was approximately 0.9MPa after gelation at 37℃ with structural similarity to the ECM. Also the modulus could be managed by changing the concentration of the components in the hybrid scaffolds. The release of PDGF-BB from the hybrid scaffolds induced hADSC chemotaxis under rough condition which chemoattractant was diluted by serum free cell culture medium. Our proposed hybrid scaffolds are promising candidates for less invasive tissue engineering strategies to induce endogenous hADSC recruitment.

Strain improvement and immobilization technique for enhanced production of the anticancer drug paclitaxel by Aspergillus fumigatus and Alternaria tenuissima.

UV and gamma irradiation mutagenesis was applied on Aspergillus fumigatus and Alternaria tenuissima in order to improve their producing ability of paclitaxel. Among the screened mutants, two stable strains (designated TXD105-GM6 and TER995-GM3) showed the maximum paclitaxel production. Paclitaxel titers of the two respective mutants were dramatically intensified to 1.22- and 1.24-fold, as compared by their respective parents. Immobilization using five different entrapment carriers of calcium alginate, agar-agar, Na-CMC, gelatin, and Arabic gum was successfully applied for production enhancement of paclitaxel by the two mutants. The immobilized cultures were superior to free-cell cultures and paclitaxel production by the immobilized mycelia was much higher than that of the immobilized spores using all the tried carriers. Moreover, calcium alginate gel beads were found the most conductive and proper entrapment carrier for maximum production of paclitaxel. The feasibility of the paclitaxel production by the immobilized mycelia as affected by incubation period, medium volume, and number of beads per flask was adopted. Under the favorable immobilization conditions, the paclitaxel titers were significantly intensified to 1.31- and 1.88-fold by the respective mutants, as compared by their free cultures. The obtained paclitaxel titers by the immobilized mycelia of the respective mutants (694.67 and 388.65μgL-1) were found promising in terms of fungal production of paclitaxel. Hence, these findings indicate the future possibility to reduce the cost of producing paclitaxel and suggest application of the immobilization technique for the biotechnological production of paclitaxel at an industrial scale.

Comparison of acid-, alkaline-, and ionic liquid–treated Napier grass as an immobilization carrier for butanol production by Clostridium beijerinckii JCM 8026

Immobilization is a simple technique in which microorganisms adhere and agglomerate onto the porous material’s surface, resulting in a higher cell density, cell tolerance, and productivity. Acetone-butanol-ethanol (ABE) fermentation using immobilized Clostridium beijerinckii JCM 8026 on Napier grass (Pennisetum purpureum) chemically pretreated with H2SO4, NaOH, and 1-ethyl-3-methylimidazolium acetate (EMIM-OAc) was compared in order to investigate the effect of the treatment on butanol production. X-ray diffraction, Fourier-transform infrared spectroscopy, and surface area measurement analyses indicated that the alkaline-pretreated Napier grass sample had the highest crystalline level with the lowest lignin content (lignin/cellulose) and a high surface area compared with other pretreated samples, suggesting its potential as an immobilized material. The fermentation was operated in a batch system for 5 days with an initial glucose level of 60 g/L. Clostridium beijerinckii JCM 8026 immobilized on the NaOH-treated Napier grass gave the highest butanol concentration (8.99 g/L), which corresponded to a 24.7% and 25.6% higher concentration than that when the cells were immobilized on untreated Napier grass and free cell culture, respectively. It is likely that immobilization on NaOH-treated Napier grass increased the cells’ protection from environmental stresses and prevented their washing out due to its swollen structure within an enlarged surface area.