Basal Medium Research Articles

Reappraisal of different agro-industrial waste for the optimization of cellulase production from Aspergillus niger ITV02 in a liquid medium using a Box-Benkhen design.

High-value metabolites, such as enzymes and biofuels, can be produced from various agro-industrial waste containing high percentages of cellulose and hemicellulose. Aspergillus niger ITV02 demonstrates high potential in cellulases production, the key enzyme for converting lignocellulosic materials into fermentable sugars to produce second-generation bioethanol (bioethanol 2G). This study evaluated five lignocellulosic residues of agricultural importance: sugarcane bagasse (SCB), sorghum bagasse (SB), corn stubble (CS), barley straw (BS) and rice husk (RH) as substrates for cellulase production. The temperature, pH and stirring conditions were optimized using a Box-Behnken design to identify the most suitable conditions for cellulase production while minimizing nitrogen concentrations. The results indicate that the best way of propagation A. niger ITV02 is through the use of spores as an inoculant, in conjunction with the use of materials with a high cellulose/lignin ratio, such as CS and SB for the generation of cellulases. These conditions promote the expression of cellulases towards β-glucosidase production, unlike materials with lower cellulose/lignin ratios like BS and RH, which exhibited lower cellulase activity. The optimal conditions for cellulase production by A. niger ITV02 were determined to be 33 °C, pH 5.3, and 200rpm, resulting in a 1.7-fold increase in Exoglucanase (FPase activity) (from 0.127 to 0.215 U/mL). These findings demonstrate the potential to enhance FPase activity by utilizing substrates with high cellulose/lignin content and implementing optimal operational conditions without the need to raise the nitrogen content of the basal medium, thus mitigating the economic impact of cellulase production.

Pollen viability and in vitro pollen germination in chrysanthemum (<i>Dendranthema </i>x <i>grandiflora </i>Tzvelv.)

Chrysanthemum (Dendranthema x grandiflora Tzvelv.), is one of the commercially grown flower crops worldwide, valued for its cut flower, loose flower, pot mums, bedding etc. To overcome the challenges for cross breeding in chrysanthemum, the access to viable pollen, cultivar distant in location and difference in flowering, are required. Low pollen viability along with self-incompatibility are mainly responsible for low seed set in modern chrysanthemum cultivars. In the present study, pollen viability was tested by staining pollen with Alexander staining solution and examined under light microscope (20X). In vitro pollen germination was investigated in 10 chrysanthemum genotypes with different concentrations of sucrose (0, 5%, 10%, and 15%), PEG4000 (0, 10%, 20% and 30%) and their combination, as basal medium. Significant differences were observed among genotypes with respect to pollen viability, which was recorded highest in Red Stone (95.02%) followed by IIHR6-26 (88.20%), IIHR9-3 (87.44%), Sweta Singar (85.59%), Kalpana (79.97%), IIHR2-7 (73.50%), and White Andaman (64.14%), while, lowest pollen viability was recorded in IIHR6-29 (49.55%). Negligible in vitro pollen germination was observed among the genotypes when sucrose was used as pollen germination medium. However, highest germinability of pollen was recorded at 20% PEG4000 in IIHR9-3 (43.57%) followed by Kalpana (19.43%) and IIHR6-29 (19.34%). The best media for in vitro pollen germination was 15% sucrose + 30% PEG. Irrespective of different media combination, the genotypes namely, IIHR9-3, IIHR2-7 and IIHR6-26 showed better in vitro pollen germination. These results provide a valuable background to theconventional breeding to create hybrids through cross-pollination.

Optimisation of Lactobacillus fermentation conditions and its application in the fermentation of salt-free sauerkraut

To identify what are the dominant lactic acid bacteria (LAB) involved in the fermentation of salt-free sauerkraut, and optimize its industrial culture conditions, we isolated and identified a strain of LAB, which is referred to as Lactobacillus sp. DF_001, with the preservation number CCTCC NO: M20232593, from five different regions in Guizhou Province. Industrial culture conditions were optimized using Plackett-Burman and Central Composite design experiments, and the potential role of this LAB in salt-free sauerkraut fermentation was validated. Bioproduction was optimal with a culture time of 66 h, starch/water ratio of 1.7% and inoculum of 0.02%, which gave approximately three-fold higher yield than the basal culture medium DeMan-Rogosa-Sharpe medium (MRS). The LAB was used in small-scale industrial experiments. The Dafang LAB significantly enhanced the sensory score of the salt-free sauerkraut products by about 32% compared to the control group. The total acid content increased by about 32% and the sugar and nitrite contents were reduced by 67.27 and 69.58%, respectively. The total number of bacterial colonies decreased by 37.5%. All other indicators complied with the national standard, providing overall the basis to improve salt-free sauerkraut fermentation.

A convenient and versatile culturomics platform to expand the human gut culturome of Lachnospiraceae and Oscillospiraceae.

The human gut microbiota is increasingly being recognised to play an important role in maintaining health. The families Lachnospiraceae and Oscillospiraceae in particular, are often reduced in disease states but are relatively poorly represented in culture collections. Cultured representatives are required to investigate the physiology and host interactions of gut microbes. Establishing cultured isolate collections can be laborious and expensive owing to the fastidious growth requirements of these organisms and the costs associated with taxonomic classification. This study proposes a culturomics platform combining a single basal culture medium with matrix-assisted laser adsorption/ionisation coupled to time-of-flight mass spectrometry (MALDI-TOF MS) for fast and reliable isolation and identification of hundreds of novel isolates. In this study, basal YCFA medium supplemented with either glucose, apple pectin, or porcine mucin was used to cultivate a total of 724 different isolates derived from only 11 different faecal samples from healthy volunteers, of which 389 isolates belonged to the Lachnospiraceae and Oscillospiraceae families. Moreover, 27 isolates could not be assigned to known species based on their 16S rRNA gene, 17 of which may even represent novel genera. To aid MALDI-TOF MS identification of gut bacteria, the commercial database was complemented with the MaldiGut database presented here, containing a collection of 132 different Main Spectrum Profiles, including the profiles of 125 Firmicutes species, 3 Bacteroidetes species, 3 Actinobacteria species, and one Verrucomicrobia species. The culturomics platform and MaldiGut database presented here will enable further expansion of the gut culturome, especially within the understudied Lachnospiraceae and Oscillospiraceae families.

Isolation, Extraction of Oil and Production of Biodiesel from Microalgae

This work is aimed at producing biodiesel from microalgae using Bold Basal Medium. After a month of cultivation, isolation, and harvesting, the microalgae were processed (oil was extracted using a chemical extraction procedure including alcohol with a 96% alcohol content and n-hexane). Transesterification was employed to produce the biodiesel from the extracted algal oil, and the resulting fuel was characterized. The results show that Chlorella sp, Desmid sp and Ankistrodesmus sp were isolated, density, viscosity, acid value, saponification, free fatty acid, peroxide value, iodine value, impurity level, and moisture content of Chlorella sp, Desmid sp and Ankistrodesmus sp oil were (0.805, 0.702, 0.716), (4.92, 4.45, 4.22), (1.51, 1.61, 1.62), (179.8, 178.61, 186.60), (0.86, 0.87, 0.88), (28.6, 28.9, 28.8), (0.31, 0.35, 0.33), (0.90, 0.80, 0.88) and (0.30, 0.36, 0.24) respectively, biodiesel had cetane number (126,125,127), density (0.79, 0.88, 0.87), flash point (126, 127, 125), calorific value (38.39, 38.68, 38.72), and viscosity (2.43, 2.66, 2.33) respectively, the quantity of biodiesel produced varied across all the replicates as the mean difference were less than the standard mean of 2.074. The production of biodiesel is practically possible in Nigeria, and the government should set up companies so as to diversify the oil sector and reduce pollution. The major limitation in this research work was using the traditional method for the isolation and identification of dominant microalgae instead of the molecular method.

In Vitro Rooting of <em>Gyrinops walla</em> in Activated Charcoal-Containing Semi-Solid Culture and Filter Paper-Bridged Liquid Culture Systems

Plant tissue culture is the best option in producing planting material for cultivation programmes of Gyrinops walla. Perfection of in vitro rooting protocol for this species is the most decisive step in micropropagation. In the present study the effect of physical state of substrate, different concentrations of sucrose, IBA and NAA in media containing activated charcoal on in vitro rooting was investigated. In experiment-1, 0.2% (w/v) activated charcoal was added in to the ½ MS medium fortified with 30 or 40 g/L sucrose and varying concentrations of IBA or NAA (0, 0.1, 0.5, 1.0 and 2.0 mg/L). In experiment-2, filter paper-bridged liquid medium was used with 40 g/L sugar and varying concentrations of IBA or NAA (0, 0.1, 0.5, 1.0 and 2.0 mg/L). The treatments were arranged in completely randomized design with three replicates. Percentage of rooted microshoots and number of roots per shoot were recorded. The ½ MS semi-solid medium with 0.2% activated charcoal, 40 g/L sucrose and 0.1 mg/L NAA resulted in the significantly highest rooting percentage (50%)while the significantly highest number of roots (6.0) were noted in ½ MS charcoal added (0.2%) basal medium with 40 g/L sucrose and 0.5 mg/L NAA. Shoots supported with filter paper bridge on ½ MS liquid medium, supplemented with 0.5 mg/L IBA, had the highest rooting percentage (66.7%) and the highest mean number of roots per shoot (3.0) (P<0.05). Results confirmed that in vitro rooting of G. walla could be perfected by optimizing the culture medium and its constituents.

Chemical profiling of fungal metabolites via the OSMAC approach: Novel identification of Brianthein W from an endophytic fungus, Hypomontagnella monticulosa Zg15SU

In the previous report, we reported that Hypomontagnella monticulosa originating from the rhizome of Zingiber griffithii was known to produce a marine-derived natural product. An OSMAC-based approach was designed by modifying the nutritional composition of the growth medium to investigate any possible new metabolites produced by the strain. The culture filtrate and biomass were conditioned through the use of three basal media, namely, Czapek-dox, potato dextrose, and Wickersham broth medium. GC-MS and multivariate analysis was performed to distinguish the chemicals and determine their composition in the tested extracts. Antimicrobial activity was tested against selected human pathogenic microbes using the disk-diffusion method. The MeOH extract of both culture filtrate and biomass from different fermentation media revealed that the majority of identified compounds (n=40) were found in Wickerham medium (n=23), which is later termed as MeOH-Wi. The chemical composition of MeOH-Wi was fatty acids (21.74%), followed by terpenoids (17.38%), cyclosiloxane (13.04%), aldehydes (13.04%), alkenes (8.7%), hydrocarbons (8.7%), esters (8.7%), alkaloids (4.35%), and an unclassified compound, the 9,9-Dimethyl-3,7-diazabicyclo[3.3.1]nonane. The numerous chemical compounds in MeOH-Wi corresponded with its antimicrobial activities against Micrococcus luteus NBRC 13867 (±11 mm), Candida maltosa NBRC 1977 (±9 mm), and Escherichia coli JM 109 (±5 mm) which were higher than the other tested extracts. Further purification using HPLC RP-C18 using NP-SiO2/n-Hex–EtOAc (10:2) to yield compound (1). Compound (1) was determined as an analog of briarian W—a diterpene mostly found in marine sponge, Briareum spp.—as a pure compound in the MeOH extract of H. monticulosa based on the 1D and 2D NMR analysis. The anticancer activity (IC50) of compound (1) was 0.077, 0.080, and 0.102 µg/mL against the growth of HCT-116, NBT-T2, and Panc-1, respectively. As far as we are aware, this is the first report on finding a briarian diterpene that originates from an endophytic fungus, especially by H. monticulosa.

Maximizing resource efficiency with dairy and municipal wastewater as nutrient media for Chlorella vulgaris with simultaneous CO2 sequestration

Addressing critical environmental challenges such as greenhouse gas emissions, global warming, and eutrophication demands urgent and innovative solutions. In recent years, microalgae have emerged as a promising avenue for addressing these pressing issues. In this study, the combination of dairy and municipal wastewater is proposed as a culture medium for cultivating microalgae strains capable of sequestering atmospheric CO2. Specifically, the growth of Chlorella vulgaris was investigated using Bold's basal medium, along with varying concentrations of municipal and dairy wastewater, both with and without CO2 supplementation, to assess their CO2 capture potential. Concurrently, the efficiency of chemical oxygen demand (COD), total nitrogen, potassium, and phosphate removal from the wastewater was evaluated. Additionally, the combination of wastewater media with CO2 supplementation yielded the highest CO2 uptake rates, indicating the feasibility of simultaneous CO2 capture during microalgae cultivation. Media composition with 25% municipal wastewater: 75% dairy wastewater supplemented with CO2 demonstrated superior COD elimination with a higher percentage of nutrient removal from wastewater compared to other wastewater proportions. The nutrient removal capacity of aforementioned media also comes in line with high CO2 sequestration rate (13.57 mg L−1 h−1). These findings underscore the potential of utilizing wastewater from diverse sources as a viable culture medium for microalgae cultivation, facilitating concurrent CO2 capture and wastewater treatment.

Impact of yeast extract and basal salts medium on 1,4-dioxane biodegradation rates and the microorganisms involved in carbon uptake from 1,4-dioxane

Conventional physical and chemical treatment technologies for 1,4-dioxane can be ineffective and consequently attention has focused on bioremediation. Towards this, the current research investigated the impact of basal salts medium (BSM) and yeast extract on 1,4-dioxane biodegradation rates in microcosms with different soil or sediment (agricultural soil, wetland sediment, sediment from an impacted site). Phylotypes responsible for carbon uptake from 1,4-dioxane were determined using stable isotope probing (SIP), both with and without BSM and yeast extract. Further, putative functional genes were investigated using 1) soluble di-iron monooxygenase (SDIMO) based amplicon sequencing, 2) qPCR targeting propane monooxygenase (large subunit, prmA) and 3) a predictive approach (PICRUSt2). The addition of BSM and yeast extract significantly enhanced 1,4-dioxane removal rates the agricultural soil and impacted site sediment microcosms. The phylotypes associated with carbon uptake varied across treatments and inocula. Gemmatimonas was important in the heavy SIP fractions of the wetland sediment microcosms. Unclassified Solirubacteraceae, Solirubrobacter, Pseudonocardia and RB4 were dominant in the heavy SIP fractions of the agricultural soil microcosms. The heavy SIP fractions of the impacted site microcosms were dominated by only two phylotypes, unclassified Burkholderiaceae and oc3299. SDIMO based amplicon sequencing detected three genes previously associated with 1,4-dioxane. The predicted functional gene analysis suggested the importance of propane monooxygenases associated with Solirubrobacter and Pseudonocardia. Overall, more microorganisms were involved in carbon uptake from 1,4-dioxane in both the wetland and agricultural soil microcosms compared to the impacted site sediment microcosms. Many of these microorganisms have not previously been associated with 1,4-dioxane removal.

Effect of konjac glucomannan on gut microbiota from hyperuricemia subjects in vitro: fermentation characteristics and inhibitory xanthine oxidase activity.

The disorder of uric acid metabolism is closely associated with gut microbiota and short-chain fatty acids (SCFAs) dysregulation, but the biological mechanism is unclear, limiting the development of uric acid-lowering active polysaccharides. Konjac glucomannan (KGM) could attenuate metabolic disturbance of uric acid and modulate the gut microbiota. However, the relationship between uric acid metabolism and gut microbiota is still unknown. In this study, The fecal samples were provided by healthy volunteers and hyperuricemia (HUA) patients. Fecal samples from healthy volunteers was regarded as the NOR group. Similarly, 10% HUA fecal suspension was named as the HUA group. Then, fecal supernatant was inoculated into a growth basal medium containing glucose or KGM, and healthy fecal samples were designated as the NOR-GLU and NOR-KGM groups, while HUA fecal samples were designated as the HUA-GLU and HUA-KGM groups. All samples were cultured in an anaerobic bag system. After fermentation for 24 h, the samples were collected for further analysis of composition of intestinal microbiota, SCFAs concentration and XOD enzyme activity. The results showed that KGM could be utilized and degraded by the gut microbiota from HUA subjects, and it could modulate the composition and structure of their HUA gut microbiota to more closely resemble that of a healthy group. In addition, KGM showed a superior modulated effect on HUA gut microbiota by increasing Megasphaera, Faecalibacterium, Lachnoclostridium, Lachnospiraceae, Anaerostipes, and Ruminococcus levels and decreasing Butyricicoccus, Eisenbergiella, and Enterococcus levels. Furthermore, the fermentation solution of KGM showed an inhibitory effect on xanthine oxidase (XOD) enzyme activity, which might be due to metabolites such as SCFAs. In conclusion, the effect of KGM on hyperuricemia subjects was investigated based on the gut microbiota in vitro. In the present study. It was found that KGM could be metabolized into SCFAs by HUA gut microbiota. Furthermore, KGM could modulate the structure of HUA gut microbiota. At the genus level, KGM could decrease the relative abundances of Butyricicoccus, Eisenbergiella, and Enterococcus, while Lachnoclostridium and Lachnospiraceae in HUA gut microbiota were significantly increased by the addition of KGM. The metabolites of gut microbiota, such as SCFAs, might be responsible for the inhibition of XOD activity. Thus, KGM exhibited a superior probiotic function on the HUA gut microbiota, which is expected as a promising candidate for remodeling the HUA gut microbiota.

Multipurpose Impacts of Silver Nitrate on Direct Organogenesis of Begonia rex cv. DS-EYWA via Transverse Thin Cell Layering (tTCL) Technique

Begonia rex cv. DS-EYWA is an important plant for indoor and outdoor cultivation, and cv. DS-EYWA is a rare unique cultivar with curly, colorful leaves. Due to their importance, applying plant tissue culture techniques for mass and healthy production in a short period of time without seasonal limitation is of immense economic value. Applying several concentrations of silver nitrate (AgNO3) in combination with varied concentrations of cytokinins including 6-benzylaminopurine (BAP), thidiazuron (TDZ) (0, 0.5, 1, 1.5 mgL−1), and 1-naphthaleneacetic acid (NAA) auxin (0, 0.5, 1 mgL−1) via focusing on transverse thin cell layer (tTCL) petiole explants for high-scale production was used to establish an efficient in vitro propagation protocol. Our results showed that even low concentrations (25 mgL−1) can control internal bacterial infection and increase shoot direct regeneration efficiency. A combination of 1.5 mgL−1 BAP, 0.5 mgL−1 NAA, and 25 mgL−1 AgNO3 was the best treatment to increase the number of direct regenerated shoots, and a lower concentration of BAP (0.5 mgL−1) can be suggested for shoot elongation. Elongated shoots were successfully rooted in MS basal medium and acclimatized in a 1:1 peat moss/perlite sterilized pot mixture.

Evaluation of chicken embryo extract and egg yolk extract as alternatives to basic cell culture medium supplement

BackgroundFetal calf serum (FCS), an existing cell culture supplement, is effective but has several drawbacks, including being expensive, requiring a lengthy process of production, and requiring a hard currency. With this in mind, we planned to evaluate chick embryo extract and egg yolk extracts in cell culture as alternatives to fetal calf serum (FCS).MethodsSpecific pathogen-free eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Egg yolk extract (EYE) and chick embryo extract (CEE) were collected after the egg was opened with caution not to destroy the yolk sack or the chick embryo itself. Chick fibroblasts and Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract at ratios of 0:10, 1:9, 2.5:7.5, and 5:5% fetal calf serum.ResultsFibroblast cell attachment was better in media supplemented with 5% CEE and 5% FCS. The confluency was also greater than 50% at this concentration. Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70%. Viability and confluency were also observed at 5%:5% ratios of 50 and 70%, respectively.ConclusionThis investigation evaluated these two extracts as cell culture supplements and revealed promising results as alternatives to fetal calf serum. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested. With the above findings, further research using different cell lines, ratios and conditions is warranted.

Efficient Plantlet Regeneration from Branches in Mangifera indica L.

Mango (Mangifera indica L.) is one of the most significant tropical and subtropical fruit species, with high ecological and economic value. However, research on the in vitro culture of mangoes is relatively weak, so establishing an efficient and stable mango plant regeneration system is of great significance. In this study, a preliminary mango regeneration system was established with Mangifera indica L. cv. Keitt from young branches as the starting explants. The results showed that the optimal plant growth regulator (PGR) formula for direct adventitious shoot induction on the branches was 1 mg/L 6-benzylaminopurine (6-BA) + 0.1 mg/L a-naphthaleneacetic acid (NAA), with an adventitious shoot induction rate of 73.63% and an average of 6.76 adventitious shoots. The optimal basal medium for adventitious shoot induction was wood plant medium (WPM), with an adventitious shoot induction rate of 63.87% and an average of 5.21 adventitious shoots. The optimal culture medium for adventitious shoot elongation was WPM + 1 mg/L 6-BA + 0.5 mg/L NAA, with an adventitious shoot elongation rate of 89.33% and an average length of 5.17 cm. The optimal formula for the induction of mango rooting was Douglas fir cotyledon revised medium (DCR) + 3 mg/L indole-3-butyric acid (IBA), with a maximum rooting rate of 66.13% and an average rooting quantity of 6.43. The genetic fidelity of the in vitro-regenerated plants was evaluated using inter-simple sequence repeat (ISSR) molecular markers. There was no difference between the in vitro-regenerated plants and the parent plant. This study provides an efficient and stable propagation system for Mangifera indica L., laying the foundation for its rapid propagation and genetic improvement.

Effect of gamma irradiation on proliferation and growth of friable embryogenic callus and in vitro nodal cuttings of ugandan cassava genotypes.

Cassava (Manihot esculenta Crantz) production and productivity in Africa is affected by two viral diseases; cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Induced mutagenesis of totipotent/embryogenic tissues or in vitro plant material can lead to the generation of CMD and/or CBSD tolerant mutants. To massively produce non-chimeric plants timely and with less labor, totipotent cells or tissues are a pre-requisite. This study aimed to determine the effect of gamma radiation on the proliferation and growth of friable embryogenic callus (FEC) and in vitro nodal cuttings respectively. To obtain FEC, 2-6 mm sized leaf lobes of nine cassava genotypes were plated on Murashige and Skoog (MS) basal media supplemented with varying levels (37, 50, 70, 100) μM of picloram for production of organized embryogenic structures (OES). The OES of five cassava genotypes (Alado, CV-60444, NASE 3, NASE 13 and TME 204) were crushed and plated in Gresshoff and Doy (GD) basal media in combination with the amino acid tyrosine in varying concentrations for FEC production. FEC from five cassava genotypes and in vitro nodal cuttings of nine genotypes were irradiated using five different gamma doses (0, 5, 10, 15, 20 and 25 Gy) at a dose rate of 81Gy/hr. The lethal dose (LD)50 was determined using the number of roots produced and flow cytometry was done to determine the ploidy status of plants. The highest production of OES was noted in Alado across varying picloram concentrations, while TME 204 obtained the highest amount of FEC. The irradiated FEC gradually died and by 28 days post irradiation, FEC from all five cassava genotypes were lost. Conversely, the irradiated in vitro nodal cuttings survived and some produced roots, while others produced callus. The LD50 based on number of roots varied from genotype to genotype, but plants remained diploid post-irradiation. Accordingly, the effect of gamma irradiation on Ugandan cassava genotypes (UCGs) was genotype-dependent. This information is foundational for the use of in vitro tissues as target material for cassava mutation breeding.

Optimized medium conditions maximize colony regeneration from a single cell of Botryococcus braunii NIES836

Botryococcus braunii is a colonial alga recognized for its slow growth but high hydrocarbon accumulation. Although using genetic engineering to increase the growth rate and hydrocarbon yield of B. braunii is desirable, the presence of an extracellular matrix (ECM) significantly hinders the emergence of a homogeneous colony from a single DNA-transformed cell. Previously, we developed a method to isolate single cells without ECM from colonies. However, following the isolation of single cells, several months are required to regenerate colonies with a sufficient cell mass for subsequent analysis. To shorten the colony regeneration period, we investigated basal media and medium components, along with growth-promoting additives, in a series of single-factor experiments and optimized the concentrations of the medium constituents via response surface methodology (RSM). The results of the single-factor experiments revealed that the nitrogen source (a mixture of NaNO3 and NH4NO3), 1-naphthylacetic acid (NAA) and Fe(III)-citrate significantly increased the growth of B. braunii single cells into colonies. The optimal medium composition identified by RSM included 151.6 mg/L nitrogen source, 2.419 mg/L NAA and 15.3 mg/L Fe(III)-citrate. Verification experiments showed that the optimized medium resulted in a 1.75-fold increase in colony size compared with that of colonies grown in nonoptimized AF6 medium. This is the first report of the optimal medium composition for the regeneration of B. braunii colonies from single cells.

Liquefied capsules containing nanogrooved microdiscs and umbilical cord-derived cells for bone tissue engineering.

Surface topography has been shown to influence cell behavior and direct stromal cell differentiation into distinct lineages. Whereas this phenomenon has been verified in two-dimensional cultures, there is an urgent need for a thorough investigation of topography's role within a three-dimensional (3D) environment, as it better replicates the natural cellular environment. A co-culture of Wharton's jelly-derived mesenchymal stem/stromal cells (WJ-MSCs) and human umbilical vein endothelial cells (HUVECs) was encapsulated in a 3D system consisting of a permselective liquefied environment containing freely dispersed spherical microparticles (spheres) or nanogrooved microdiscs (microdiscs). Microdiscs presenting 358 ± 23 nm grooves and 944 ± 49 nm ridges were produced via nanoimprinting of spherical polycaprolactone microparticles between water-soluble polyvinyl alcohol counter molds of nanogrooved templates. Spheres and microdiscs were cultured in vitro with umbilical cord-derived cells in a basal or osteogenic medium within liquefied capsules for 21 days. WJ-MSCs and HUVECs were successfully encapsulated within liquefied capsules containing spheres and microdiscs, ensuring high cellular viability. Results show an enhanced osteogenic differentiation in microdiscs compared to spheres, even in basal medium, evidenced by alkaline phosphatase activity and osteopontin expression. This work suggests that the topographical features present in microdiscs induce the osteogenic differentiation of adhered WJ-MSCs along the contact guidance, without additional differentiation factors. The developed 3D bioencapsulation system comprising topographical features might be suitable for bone tissue engineering approaches with minimum in vitro manipulation.

Soy whey and brewer’s spent grain hydrolysates wholly replace conventional medium for microalgae growth: Process performance and economic considerations

To enhance circularity in heterotrophic microalgal bioprocesses, this study completely substituted glucose and Bold’s basal medium (BBM) with brewer’s spent grain (BSG) and soy whey (SW) hydrolysates. Mild acid hydrolysis conditions of BSG (0.2 M H2SO4, 130 °C, 36 min) and SW (0.1 M HCl, 95 °C, 30 min) were optimised for glucose release, and their hydrolysates were optimally mixed (15 % SW-85 % BSG) to obtain a medium that best supported Auxenochlorella protothecoides growth. Maximum biomass production (Xmax) and productivity (PXmax) obtained in the hydrolysate medium containing 50.75 g/L endogenous glucose (Xmax: 22.17 g/L; PXmax: 7.06 g/L/day) were comparable to that in BBM containing 50.44 g/L exogenous glucose (Xmax: 20.02 g/L; PXmax: 6.34 g/L/day). Moreover, estimated hydrolysate medium production costs were within an order of magnitude to BBM. Overall, the integrated approach of tailored hydrolytic treatments and complementary side-streams presents a promising technical and economic feasibility, with applications extending beyond A. protothecoides.

Selection of the Most Suitable Culture Medium for Patient-Derived Lung Cancer Organoids

Introduction: Patient-derived organoids have emerged as a promising in vitro model for precision medicine, particularly in cancer, but also in noncancer-related diseases. However, the optimal culture medium for culturing patient-derived lung organoids has not yet been agreed upon. This study aimed to shed light on the optimal selection of a culture media for developing studies using patient-derived lung organoids. Methods: Tumor and normal paired tissue from 71 resected non-small cell lung cancer patients were processed for organoid culture. Lung cancer organoids (LCOs) were derived from tumor tissue and normal lung organoids (LNOs) from nonneoplastic lung tissue. Three different culture media were compared: permissive culture medium (PCM), limited culture medium (LCM), and minimum basal medium (MBM). We assessed their effectiveness in establishing organoid cultures, promoting organoid growth and viability, and compared their differential phenotypic characteristics. Results: While PCM was associated with the highest success rate and useful for long-term expansion, MBM was the best option to avoid normal organoid overgrowth in the organoid culture. The density, size, and viability of LNOs were reduced using LCM and severely affected with MBM. LNOs cultured in PCM tend to differentiate to bronchospheres, while alveolosphere differentiation can be observed in those cultured with LCM. The morphological phenotype of LCO was influenced by the culture media of election. Mesenchymal cell overgrowth was observed when LCM was used. Conclusion: This work highlights the importance of considering the research objectives when selecting the most suitable culture medium for growing patient-derived lung organoids.

In Vitro Micropropagation of Eria Lasiopetala (Willd.) Ormerod- A Native Medicinal Orchid in Bangladesh

The present study focuses on the asymbiotic germination of seeds through in vitro culture of Eria lasiopetala, a native medicinal orchid in Bangladesh. Out of the four basal media, MS was found to be the most effective medium, where 93.58±0.374% seed germinations were recorded. Different plant growth regulators (PGRs), e.g. BAP, Kin, NAA and were used for secondary protocorm proliferation, plantlet development, and multiplication of shoot buds through in vitro conditions. The maximum number of secondary protocorms (12.80±0.115) were developed that were derived from primary protocorms on MS medium fortified with 0.5 mg/l BAP and 1.0 mg/l NAA. Data revealed that 0.5 mg/l BAP + 0.25 mg/l NAA showed the best combination for height elongation of plantlets (3.12±0.012 cm) in MS medium. While for multiple shoot buds (MSBs) 1.0 mg/l BAP + 0.5 mg/l NAA showed better (6.8±0.127) performance than other combinations of PGRs. The single shoots were isolated from the multiple shoots and sub-cultured on ½MS medium supplemented with NAA (0.5 - 2.0 mg/l), IAA (0.5 - 2.0 mg/l) and IBA (0.5 - 2.0 mg/l) for root induction. The maximum number of roots (7.53±0.176) and its growth (4.61±0.01 cm) were found in ½MS medium supplemented with 1.0 mg/l IAA. The well rooted plantlets were hardened and successfully transferred after acclimatization in pots containing the mixture of coconut husk, charcoal and brick pieces in the ratio of 2:1:1 and eventually established under natural condition. J. Bio-Sci. 32(1): 45-60, 2024

Perspectives on optimizing microalgae cultivation: Harnessing dissolved CO2 and lactose for sustainable and cost-efficient protein production

Microalgae, known for their land-sparing cultivation and remarkable photosynthetic efficiency, play a crucial role in advancing sustainable development goals. This study explored the use of cost-effective inorganic carbon (the ionic form of CO2) and organic carbon sources (lactose, a primary sugar in whey wastewater) to support the growth of Chlorellasorokiniana UTEX 1230, and improved protein content through nitrogen supply optimization. NaHCO3 proved to be more effective than Na2CO3, resulting in a 3.4-fold increase in biomass concentration, with an initial 5 mM HCO3‾ concentration compared to the basal medium. Further addition of 0.75 % lactose led to a 4.05-fold increase in biomass concentration, with the maximum specific growth rate at 1.28/d, maximum biomass concentration at 695.88 mg/L, and maximum biomass productivity at 250.91 mg/L/d. Nitrogen supplementation greatly increased protein concentration from 16.41 % (250 mg/L NaNO3) to 52.95 % (1000 mg/L NaNO3). These results support the potential for utilizing whey wastewater bioremediation to produce high-value protein via microalgal cultivation.