This study presents an evaluation of integrating virtual laboratory simulations in assessment design of a biotechnology course at Taylor's University in Malaysia before, during and post-COVID recovery phases. The purpose was to investigate how virtual laboratory simulations were integrated as part of the assessments of a practical-embedded course-the aim being to evaluate students' acceptance and perception of using virtual simulation. A total of 46 students, across three different study cohorts (August 2019, March 2020, and August 2020) were evaluated different educational aspects of using virtual laboratory cases in a 4-week course within Animal Biotechnology. Overall, students regarded virtual laboratory simulation useful as part of their learning, and there is a significant increase in the level of acceptance before, during and post-COVID recovery phases. The study showed that across the different study cohorts, students perceived their confidence level in laboratory skills have been enhanced and that they can apply the skills in real-life situation. Interestingly, students (March and August 2020 cohort) who have not been exposed to the related laboratory session still perceived that the simulated activity provides clear explanation and realistic experience. Furthermore, it had been highlighted across the study cohorts that the quiz questions helped to enhance their understanding on the underlying principles of the laboratory techniques. The overall conclusion of this study was that structured simulation-based activities which provide clear instructions and explanation would support significant improvements in students learning.
This study was aimed to identify and optimize the culture conditions for gamma-aminobutyric acid (GABA) production by a lactic acid bacterium strain isolated from mam nem, a fermented fish sauce. Among the six isolates obtained from mam nem, the MN12 had the most potent GABA-producing capability. The strain was then identified to be Pedioccocus pentosaceus by employing MALDI-TOF-MS and phenylalanyl-tRNA synthase sequencing methods. The initial cell density of 5.106 CFU/mL, monosodium glutamate concentration of 60 mM, initial pH of 7, temperature of 45°C and cultivation time of 72 h were found to be the optimal culture conditions for highest production of GABA, reaching 27.9 ± 0.42 mM, by this strain. The cultivation conditions for GABA production by P. pentosaceus MN12 have been successfully optimized, providing a foundation for the development of fermented foods enriched with GABA.
The versatility of a well-known fibrous crop, Hibiscus cannabinus (kenaf) is still relatively new to many. Kenaf's potential applications, which can be extended even into critical industries such as pharmaceutical and food industries, have always been overshadowed by its traditionally grown fiber. Therefore, this study aimed to venture into the biotechnological approach in reaping the benefits of kenaf through plant cell suspension culture to maximize the production of kenaf callus biomass (KCB) and exopolysaccharide (EPS), which is deemed to be more sustainable. A growth curve was established which indicates that cultivating kenaf callus in suspension culture for 22 days gives the highest KCB (9.09 ± 1.2 g/L) and EPS (1.1 ± 0.02 g/L). Using response surface methodology (RSM), it was found that sucrose concentration, agitation speed, and naphthalene acetic acid (NAA) concentration can affect the production of KCB and EPS significantly (p cell suspension culture of kenaf callus serve as the blueprint for any sustainable large-scale production in the future and provide an alternative cultivating method to kenaf traditional farming.
Plant cell cultures could be used as an important tool for biochemical production, ranging from natural coloring (pigments) to pharmaceutical products. Anthocyanins are becoming a very important alternative to synthetic dyes because of increased public concern over the safety of artificial food coloring agents. Several factors are responsible for the production of anthocyanin in cell cultures. In the present study, we investigate the effects of different environmental factors, such as light intensity, irradiance (continuous irradiance or continuous darkness), temperature and medium pH on cell biomass yield and anthocyanin production in cultures of Melastoma malabathricum. Moderate light intensity (301 - 600 lux) induced higher accumulation of anthocyanins in the cells. The cultures exposed to 10-d continuous darkness showed the lowest pigment content, while the cultures exposed to 10-d continuous irradiance showed the highest pigment content. The cell cultures incubated at a lower temperature range (20 ± 2 ºC) grew better and had higher pigment content than those grown at 26 ± 2 ºC and 29 ± 2 ºC. Different medium pH did not affect the yield of cell biomass but anthocyanin accumulation was highest at pH 5.25 - 6.25.
Mesenchymal stems cells (MSCs) are currently the focus of numerous therapeutic approaches in tissue engineering/repair because of their wide multi-lineage potential and their ability to modulate the immune system response following transplantation. Culturing these cells, while maintaining their multipotency in vitro, currently relies on biological substrates such as gelatin, collagen and fibronectin. In addition, harvesting cells from these substrates requires enzymatic or chemical treatment, a process that will remove a multitude of cellular surface proteins, clearly an undesirable process if cells are to be used therapeutically. Herein, we applied a high-throughput 'hydrogel microarray' screening approach to identify thermo-modulatable substrates which can support hES-MP and ADMSC growth, permit gentle reagent free passaging, whilst maintaining multi-lineage potential. In summary, the hydrogel substrate identified, poly(AEtMA-Cl-co-DEAA) cross-linked with MBA, permitted MSCs to be maintained over 10 passages (each time via thermo-modulation), with the cells retaining expression of MSC associated markers and lineage potency. This chemically defined system allowed the passaging and maintenance of cellular phenotype of this clinically important cell type, in the absence of harsh passaging and the need for biological substrates.
Stem cell culture is typically based on batch-type culture, which is laborious and expensive. Here, we propose a continuous harvest method for stem cells cultured on thermoresponsive nanobrush surfaces. In this method, stem cells are partially detached from the nanobrush surface by reducing the temperature of the culture medium below the critical solution temperature needed for thermoresponse. The detached stem cells are harvested by exchange into fresh culture medium. Following this, the remaining cells are continuously cultured by expansion in fresh culture medium at 37 °C. Thermoresponsive nanobrush surfaces were prepared by coating block copolymers containing polystyrene (for hydrophobic anchoring onto culture dishes) with three types of polymers: (a) polyacrylic acid with cell-binding oligopeptides, (b) thermoresponsive poly-N-isopropylacrylamide, and (c) hydrophilic poly(ethyleneglycol)methacrylate. The optimal coating durations and compositions for these copolymers to facilitate adequate attachment and detachment of human adipose-derived stem cells (hADSCs) and embryonic stem cells (hESCs) were determined. hADSCs and hESCs were continuously harvested for 5 and 3 cycles, respectively, via the partial detachment of cells from thermoresponsive nanobrush surfaces.
Commonly, stem cell culture is based on batch-type culture, which is laborious and expensive. We continuously cultured human pluripotent stem cells (hPSCs) on thermoresponsive dish surfaces, where hPSCs were partially detached on the same thermoresponsive dish by decreasing the temperature of the thermoresponsive dish to be below the lower critical solution temperature for only 30 min. Then, the remaining cells were continuously cultured in fresh culture medium, and the detached stem cells were harvested in the exchanged culture medium. hPSCs were continuously cultured for ten cycles on the thermoresponsive dish surface, which was prepared by coating the surface with poly(N-isopropylacrylamide-co-styrene) and oligovitronectin-grafted poly(acrylic acid-co-styrene) or recombinant vitronectin for hPSC binding sites to maintain hPSC pluripotency. After ten cycles of continuous culture on the thermoresponsive dish surface, the detached cells expressed pluripotency proteins and had the ability to differentiate into cells derived from the three germ layers in vitro and in vivo. Furthermore, the detached cells differentiated into specific cell lineages, such as cardiomyocytes, with high efficiency.
Human induced pluripotent stem cells (hiPSCs) were generated on several biomaterials from human amniotic fluid in completely xeno-free and feeder-free conditions via the transfection of pluripotent genes using a nonintegrating RNA Sendai virus vector. The effect of xeno-free culture medium on the efficiency of the establishment of human amniotic fluid stem cells from amniotic fluid was evaluated. Subsequently, the effect of cell culture biomaterials on the reprogramming efficiency was investigated during the reprogramming of human amniotic fluid stem cells into hiPSCs. Cells cultured in laminin-511, laminin-521, and Synthemax II-coated dishes and hydrogels having optimal elasticity that were engrafted with specific oligopeptides derived from vitronectin could be reprogrammed into hiPSCs with high efficiency. The reprogrammed cells expressed pluripotency proteins and had the capability to differentiate into cells derived from all three germ layers in vitro and in vivo. Human iPSCs could be generated successfully and at high efficiency (0.15-0.25%) in completely xeno-free conditions from the selection of optimal cell culture biomaterials.
The current differentiation process of human pluripotent stem cells (hPSCs) into cardiomyocytes to enhance the purity of hPSC-derived cardiomyocytes requires some purification processes, which are laborious processes. We developed cell sorting plates, which are prepared from coating thermoresponsive poly(N-isopropylacrylamide) and extracellular matrix proteins. After hPSCs were induced into cardiomyocytes on the thermoresponsive surface coated with laminin-521 for 15 days, the temperature of the cell culture plates was decreased to 8-9 °C to detach the cells partially from the thermoresponsive surface. The detached cells exhibited a higher cardiomyocyte marker of cTnT than the remaining cells on the thermoresponsive surface as well as the cardiomyocytes after purification using conventional cell selection. The detached cells expressed several cardiomyocyte markers, such as α-actinin, MLC2a and NKX2.5. This study suggested that the purification of hPSC-derived cardiomyocytes using cell sorting plates with the thermoresponsive surface is a promising method for the purification of hPSC-derived cardiomyocytes without conventional laborious processes.
Recombinant vitronectin-grafted hydrogels were developed by adjusting surface charge of the hydrogels with grafting of poly-l-lysine for optimal culture of human embryonic stem cells (hESCs) under xeno- and feeder-free culture conditions, with elasticity regulated by crosslinking time (10-30 kPa), in contrast to conventional recombinant vitronectin coating dishes, which have a fixed stiff surface (3 GPa). hESCs proliferated on the hydrogels for over 10 passages and differentiated into the cells derived from three germ layers indicating the maintenance of pluripotency. hESCs on the hydrogels differentiated into cardiomyocytes under xeno-free culture conditions with much higher efficiency (80% of cTnT+ cells) than those on conventional recombinant vitronectin or Matrigel-coating dishes just only after 12 days of induction. It is important to have an optimal design of cell culture biomaterials where biological cues (recombinant vitronectin) and physical cues (optimal elasticity) are combined for high differentiation of hESCs into specific cell lineages, such as cardiomyocytes, under xeno-free and feeder-free culture conditions.
Current xeno-free and chemically defined methods for the differentiation of hPSCs (human pluripotent stem cells) into cardiomyocytes are not efficient and are sometimes not reproducible. Therefore, it is necessary to develop reliable and efficient methods for the differentiation of hPSCs into cardiomyocytes for future use in cardiovascular research related to drug discovery, cardiotoxicity screening, and disease modeling. We evaluated two representative differentiation methods that were reported previously, and we further developed original, more efficient methods for the differentiation of hPSCs into cardiomyocytes under xeno-free, chemically defined conditions. The developed protocol successively differentiated hPSCs into cardiomyocytes, approximately 90-97% of which expressed the cardiac marker cTnT, with beating speeds and sarcomere lengths that were similar to those of a healthy adult human heart. The optimal cell culture biomaterials for the cardiac differentiation of hPSCs were also evaluated using extracellular matrix-mimetic material-coated dishes. Synthemax II-coated and Laminin-521-coated dishes were found to be the most effective and efficient biomaterials for the cardiac differentiation of hPSCs according to the observation of hPSC-derived cardiomyocytes with high survival ratios, high beating colony numbers, a similar beating frequency to that of a healthy adult human heart, high purity levels (high cTnT expression) and longer sarcomere lengths similar to those of a healthy adult human heart.
The present study focused on lipopeptide biosurfactant production by Streptomyces sp. PBD-410L in batch and fed-batch fermentation in a 3-L stirred-tank reactor (STR) using palm oil as a sole carbon source. In batch cultivation, the impact of bioprocessing parameters, namely aeration rate and agitation speed, was studied to improve biomass growth and lipopeptide biosurfactant production. The maximum oil spreading technique (OST) result (45 mm) which corresponds to 3.74 g/L of biosurfactant produced, was attained when the culture was agitated at 200 rpm and aeration rate of 0.5 vvm. The best aeration rate and agitation speed obtained from the batch cultivation was adopted in the fed-batch cultivation using DO-stat feeding strategy to further improve the lipopeptide biosurfactant production. The lipopeptide biosurfactant production was enhanced from 3.74 to 5.32 g/L via fed-batch fermentation mode at an initial feed rate of 0.6 mL/h compared to that in batch cultivation. This is the first report on the employment of fed-batch cultivation on the production of biosurfactant by genus Streptomyces.
To determine the optimum light intensity per cell required for rapid growth regardless of cell density, continuous cultures of the microalga Chlorella zofingiensis were grown with a sufficient supply of nutrients and CO2 and were subjected to different light intensities in the range of 75-1000 μE m(-2) s(-1). The cell density of culture increased over time for all light conditions except for the early stage of the high light condition of 1000 μE m(-2) s(-1). The light intensity per cell required for the high specific growth rate of 0.5 day(-1) was determined to be 28-45 μE g-ds(-1) s(-1). The specific growth rate was significantly correlated to light intensity (y=0.721×x/(66.98+x), r(2)=0.85, p<0.05). A high specific growth rate was maintained over a range of light intensities (250-1000 μE m(-2) s(-1)). This range of light intensities suggested that effective production of C. zofingiensis can be maintained outdoors under strong light by using the optimum specific light intensity.
The objectives of this study were: (1) to investigate the role of mixed culture of biomass in the regeneration of mono-amine modified silica (MAMS) and granular activated carbon (GAC) loaded with Acid Orange 7 (AO7), (2) to quantify and compare the bioregeneration efficiencies of AO7-loaded MAMS and GAC using the sequential adsorption and biodegradation approach and (3) to evaluate the reusability of bioregenerated MAMS. The results show that considerably higher bioregeneration efficiency of AO7-loaded MAMS as compared to that of AO7-loaded GAC was achieved due to higher reversibility of adsorption of MAMS for AO7 and favorable pH factor resulting in more AO7 desorption. The progressive loss of adsorption capacity of MAMS for AO7 with multiple cycles of use suggests possible chemical and microbial fouling of the adsorption sites.
The performance of moving bed sequencing batch reactors (MBSBRs) added with 8 % (v/v) of polyurethane (PU) foam cubes as carrier media in nitrogen removal was investigated in treating low COD/N wastewater. The results indicate that MBSBR with 8-mL cubes achieved the highest total nitrogen (TN) removal efficiency of 37% during the aeration period, followed by 31%, 24% and 19 % for MBSBRs with 27-, 64- and 125-mL cubes, respectively. The increased TN removal in MBSBRs was mainly due to simultaneous nitrification and denitrification (SND) process which was verified by batch studies. The relatively lower TN removal in MBSBR with larger PU foam cubes was attributed to the observation that larger PU foam cubes were not fully attached by biomass. Higher concentrations of 8-mL PU foam cubes in batch reactors yielded higher TN removal.
Biosorption potential of mustard oil cake (MOC) for Ni(II) from aqueous medium was studied. Spectroscopic studies showed possible involvement of acidic (hydroxyl, carbonyl and carboxyl) groups in biosorption. Optimum biosorption was observed at pH 8. Contact time, reaction temperature, biosorbent dose and adsorbate concentration showed significant influence. Linear and non-linear isotherms comparison suggests applicability of Temkin model at 303 and 313 K and Freundlich model at 323K. Kinetics studies revealed applicability of Pseudo-second-order model. The process was endothermic and spontaneous. Freundlich constant (n) and activation energy (Ea) values confirm physical nature of the process. The breakthrough and exhaustive capacities for 5 mg/L initial Ni(II) concentration were 0.25 and 4.5 mg/g, while for 10 mg/L initial Ni(II) concentration were 4.5 and 9.5 mg/g, respectively. Batch desorption studies showed maximum Ni(II) recovery in acidic medium. Regeneration studies by batch and column process confirmed reutilization of biomass without appreciable loss in biosorption.
Moving bed sequencing batch reactors (MBSBRs) packed with 8% (v/v) of 8-, 27- and 64-mL polyurethane (PU) foam cubes, respectively, were investigated for simultaneous 4-chlorophenol (4-CP) and nitrogen removal at increasing 4-CP concentration. When the 4-CP concentration exceeded 300 mg L(-1), the MBSBR with 27-mL foam cubes was observed to outperform the other MBSBRs in removing 4-CP and nitrogen. The reasons were: (1) there were more biomass in inner layer of the 27-mL cubes, compared to that of the 8-mL cubes, which was more shielded from the inhibitory effect of 4-CP and (2) the 27-mL cubes were more mobile than the 64-mL cubes. Although increasing 4-CP concentration to 600 mg L(-1) resulted in incomplete removal of 4-CP in the MBSBRs, results of the batch reactor with 27-mL foam cubes showed that complete 4-CP removal within the REACT period could be achieved by increasing the packing volume to 20%.
The physical factors affecting the production of an organic solvent-tolerant protease from Pseudomonas aeruginosa strain K was investigated. Growth and protease production were detected from 37 to 45 degrees C with 37 degrees C being the optimum temperature for P. aeruginosa. Maximum enzyme activity was achieved at static conditions with 4.0% (v/v) inoculum. Shifting the culture from stationary to shaking condition decreased the protease production (6.0-10.0% v/v). Extracellular organic solvent-tolerant protease was detected over a broad pH range from 6.0 to 9.0. However, the highest yield of protease was observed at pH 7.0. Neutral media increased the protease production compared to acidic or alkaline media.
Aerobic granular sludge (AGS) was successfully cultivated at 27±1 °C and pH 7.0±1 during the treatment of rubber wastewater using a sequential batch reactor system mode with complete cycle time of 3 h. Results showed aerobic granular sludge had an excellent settling ability and exhibited exceptional performance in the organics and nutrients removal from rubber wastewater. Regular, dense and fast settling granule (average diameter, 1.5 mm; settling velocity, 33 m h(-1); and sludge volume index, 22.3 mL g(-1)) were developed in a single reactor. In addition, 96.5% COD removal efficiency was observed in the system at the end of the granulation period, while its ammonia and total nitrogen removal efficiencies were up to 94.7% and 89.4%, respectively. The study demonstrated the capabilities of AGS development in a single, high and slender column type-bioreactor for the treatment of rubber wastewater.
Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.