Optimization Of Medium Components Research Articles

A bioprocess engineering approach for the production of hydrocarbons and fatty acids from green microalga under high cobalt concentration as the feedstock of high-grade biofuels

Botryococcus braunii, a colonial green microalga which is well-known for its capacity to synthesize hydrocarbons, has significant promise as a long-term source of feedstock for the generation of biofuels. However, cultivating and scaling up B. braunii using conventional aqua-suspended cultivation systems remains a challenge. In this study, we optimized medium components and light intensity to enhance lipid and hydrocarbon production in a multi-cultivator airlift photobioreactor. BBM 3N medium with 200 μmol/m2/s light intensity and a 16 h light–8 h dark regimen yielded the highest biomass productivity (110.00 ± 2.88 mg/L/day), as well as the highest lipid and hydrocarbon content. Cultivation in a flat-panel bioreactor resulted in significantly higher biomass productivity (129.11 ± 2.74 mg/L/day), lipid productivity (32.21 ± 1.31 mg/L/day), and hydrocarbon productivity (28.98 ± 2.08 mg/L/day) compared to cultivation in Erlenmeyer flasks and open 20-L raceway pond. It also exhibited 20.15 ± 1.03% of protein content including elevated levels of chlorophyll a, chlorophyll b, and carotenoids. This work is noteworthy since it is the first to describe fatty acid and hydrocarbon profiles of B. braunii during cobalt treatment. The study demonstrated that high cobalt concentrations (up to 5 mg/L of cobalt nitrate) during Botryococcus culture affected hydrocarbon synthesis, resulting in high amounts of n-alkadienes and trienes as well as lipids with elevated monounsaturated fatty acids concentration. Furthermore, pyrolysis experiments on microalgal green biomass and de-oiled biomass revealed the lipid and hydrocarbon compounds generated by the thermal degradation of B. braunii that facilitate extra economical value to this system.

Optimization of fermentation conditions and medium components for chrysomycin a production by Streptomyces sp. 891-B6

BackgroundChrysomycin A (CA) is a promising antibiotic for treatment of Gram-positive bacterial infections and cancers. In order to enhance CA yield, optimization of fermentation conditions and medium components was carried out on strain Streptomyces sp. 891-B6, an UV-induced mutant with improved CA titer compared with its wide-type marine strain 891.ResultsUsing one-way experiment, the optimal fermentation conditions for CA production in 1-L shake flask were obtained as follows: 12 days of fermentation time, 5 days of seed age, 5% of inoculum volume ratio, 200 mL of loading volume and 6.5 of initial pH. By response surface methodology, the optimal medium components determined as glucose (39.283 g/L), corn starch (20.662 g/L), soybean meal (15.480 g/L) and CaCO3 (2.000 g/L).ConclusionValidation tests showed that the maximum yield of CA reached 1601.9 ± 56.7 mg/L, which was a 60% increase compared to the initial yield (952.3 ± 53.2 mg/L). These results provided an important basis for scale-up production of CA by strain 891-B6.

Integrated Microbiome and Metabolomics Analysis of the Effects of Dietary Supplementation with Corn-Steep-Liquor-Derived Candida utilis Feed on Black Pigs.

In this experiment, glucose master liquor and corn steep liquor were used as carbon and nitrogen sources, and Candida utilis was used as a strain to ferment yeast feed. The OD value and number of yeast cells were used as response values to optimize the medium components of the yeast feed through a response surface methodology. The optimal medium components were a glucose master liquor concentration of 8.3%, a corn steep liquor concentration of 1.2%, and a KH2PO4 concentration of 0.14%. Under this condition of fermentation, the OD value was 0.670 and the number of yeast cells was 2.72 × 108/mL. Then, we fed Candida utilis feed to Dongliao black piglets, and the effects of the yeast feed on the piglets' growth performance, fecal microbiota, and plasma metabolic levels were investigated through 16S rDNA sequencing and metabolomics. In total, 120 black piglets with an average initial weight of 6.90 ± 1.28 kg were randomly divided into two groups. One group was fed the basic diet (the CON group), and the other was supplemented with 2.5% Candida utilis add to the basic diet (the 2.5% CU group). After a pre-feeding period, the formal experiments were performed for 21 days. The results showed that the addition of Candida utilis to the diet did not affect growth performance compared with the control group. Meanwhile, no significant differences were observed in the serum biochemical indices. However, piglets in the 2.5% CU group had a significantly altered fecal microbiota, with an increased abundance of Clostridium_sensu_stricto_1, Lactobacillus, and Muribaculaceae_unclassified. Regarding the plasma metabolome, the 12 differential metabolites detected were mainly enriched in the histidine, tryptophan, primary bile acid, and caffeine metabolic pathways. Regarding the integrated microbiome-metabolome analysis, differential metabolites correlated with fecal flora to variable degrees, but most of them were beneficial bacteria of Firmicutes. Collectively, dietary Candida utilis feed had no adverse effect on growth performance; however, it played an important role in regulating fecal flora and maintaining metabolic levels.

Enhancing β-farnesene production in engineered Yarrowia lipolytica: A new process control strategy

β-Farnesene, a sesquiterpene with diverse applications, is applied in fields ranging from bioenergy and agriculture to medicine. In this study, a series of fermentation conditions and process control strategies were employed to enhance the accumulation of β-farnesene. Through the optimization of medium components, a suitable environment was created for β-farnesene biosynthesis in Yarrowia lipolytica, using only three inorganic salts as the initial medium. On this basis, cell growth and citrate supply were enhanced by adjusting the feeding time point, which begins when dissolved oxygen level below 40%. Additionally, initial cell density and glucose concentration during fermentation were separately optimized. Ultimately, nitrogen-limited conditions were applied to maximize carbon flux towards β-farnesene synthesis and the ideal condition was found to be C/N ratio of 100. Under the optimal condition, the titer of β-farnesene was increased by 227%, reaching 22.04 g/L in a 2 L bioreactor. This study optimizes terpenoid biosynthesis fermentation control and establishes a platform for the potential utilizing of waste biomass in Y. lipolytica.

Polyporus ulleungus mycelia cultured in MEB medium produce metabolites with anticancer property.

Cancer cells are characterized by apoptosis evasion and uncontrolled cell cycle progression. To combat these characteristics, efforts have been made to find novel natural-source anticancer compounds. The aim of this work is to find new anticancer compounds in Polyporus ulleungus (P. ulleungus) mycelial culture extracts. P. ulleungus mycelium was cultured on four individual media (DYB, MEB, MYB, and PDB) and four extracts were generated from the mycelium culture media. Extracts of P. ulleungus mycelium cultured in MEB medium (pu-MEB) significantly reduced cancer cell growth by triggering apoptosis and S phase arrest. Furthermore, the anticancer effects of pu-MEB were not confined to one type of cancer. Taken together, our results confirmed that P. ulleungus mycelia cultured in MEB medium produce metabolites that exhibit anticancer properties. Development of an optimal medium for P. ulleungus mycelium through optimization of medium components will enable P. ulleungus mycelium to produce metabolites with more anticancer efficacy.

Isolation and characterization of a bisphenol A-degrading strain, Pseudomonas aeruginosa DU2, from soil containing decaying plants.

Bisphenol A (BPA) is a toxic compound with broad applications in the plastics industry. BPA has harmful effects on various organisms and its efficient removal is necessary. The microbial degradation of BPA is a safe and economical approach. In this research, soil samples containing decaying plants were screened to isolate a BPA-degradable bacterial strain. Soil samples were collected from different locations in Damghan, Semnan province, Iran. To enrich BPA-degrading bacteria, the samples were cultured in a stepwise manner in a mineral medium containing increasing BPA concentrations (5 to 40 mg/L). The ability of isolated bacteria in degrading BPA was assayed by Folin-Ciocalteu and high-performance liquid chromatography methods. The biodegradation efficiency of the most efficient isolate was assayed under distinct conditions and it was identified through the sequencing of the 16S rRNA gene. Among the isolated bacteria, Pseudomonas aeruginosa DU2 (GenBank accession number: OP919484) showed the most BPA biodegradation ability. The highest BPA degradation (52.98%) was observed in the mineral medium containing 5 mg/L BPA and the inoculum size of 6 × 107 CFU/mL at pH 9 and in the presence of 0.05% (w/v) NaCl during 10 days. These results offer soil containing decaying plants as a promising source for finding BPA-degrading bacteria. P. aeruginosa DU2 has basal BPA removal ability, which could be improved by optimization of medium components and growth conditions.

Optimization of Fermentation Conditions for Bacillus pumilus LYMC-3 to Antagonize Sphaeropsis sapinea

Bacillus pumilus is widely used as a biocontrol agent. To further develop the biological control potential of B. pumilus LYMC-3 against pine blight, a statistical experimental design was used to optimize a liquid medium using low-cost substrates to improve its antagonistic activity against Sphaeropsis sapinea. Through the plate antagonism test and greenhouse control effect test, this study determines the antifungal effect of strain LYMC-3 against S. sapinea and pine blight. Moreover, response surface optimization methodologies were used to systematically optimize the medium composition and culture conditions of the LYMC-3 strain. The plate antagonism test showed that the inhibition rate of LYMC-3 fermentation filtrate (diluted 5-fold) was 66.09%. The greenhouse control effect test showed that the control effect of its fermentation filtrate on shoot blight reached 89.99%. The response surface optimization test ultimately determined that a higher inhibition rate can be achieved under these conditions: the optimal medium components were 7.2 g/L glucose, 15 g/L peptone, and 7.1 g/L magnesium sulfate; the optimal culture conditions were 52% liquid volume, 28 °C culture temperature, an initial pH of 7, and 1% bacterial inoculation volume. Under the optimized system, the five-fold diluted LYMC-3 fermentation filtrate inhibition rate against S. sapinea was 81.23%, which was 15.84% higher than that before optimization. Meanwhile, optimize the selection of lower-cost and more commonly used glucose instead of beef paste as the carbon source for the culture medium, and choose cheaper magnesium sulfate instead of sodium chloride as the nitrogen source.

Bioconversion of Sargassum wightii to ethanol via consolidated bioprocessing using Lachnoclostridium phytofermentans KSM 1203

Contemporary studies suggest that macroalgal biomass is an attractive and alternate source for ethanol production to replace fossil fuel. Sargassum wightii, a brown macroalgae, rich in cellulose is suitable for bioethanol production. This investigation focused on the pretreatment, enzyme saccharification and fermentation for bioethanol production through consolidated bioprocessing (CBP) using Lachnoclostridium phytofermentans KSM 1203 isolate. The cellulose extraction from S. wightii biomass and essential medium components for L. phytofermentans KSM 1203 were optimized using Response Surface Methodology. The alkali pretreatment on S. wightii biomass removed lignin and hemicelluloses with the cellulose recovery of 82.35%. The sodium hyphochlorite treatment removes other contaminants, results the semi crystalline to amorphous cellulose structure. The optimized medium components for L. phytofermentans KSM 1203 was found to be pre-treated biomass 3.92 g/L, peptone 2.21 g/L, dipotassium hydrogen phosphate 1.84 g/L and sodium chloride 2.03 g/L and for the growth and co-production of cellulase activity and ethanol productivity. The maximum bioethanol yield was obtained under the optimal conditions to be 13.75 ± 0.687 g/L for L. phytofermentans KSM 1203 from pre-treated S. wightii biomass. Hence, this investigation suggests that wild type L. phytofermentans KSM 1203 strain could be exploited for the utilization of cellulose for production ethanol in a single-step CBP procedure.

Production of Taxol by Endophytic Fungi Isolated from Roots of Himalayan Yew (Taxus wallichiana Zucc.)

Taxol® (generic name – Paclitaxel), the most promising chemotherapeuticagent was isolated from bark of different Taxus sp. As Taxus species arethreatened with extinction (endangered in Himalaya), thus it is imperativeto develop alternate and sustainable method for commercialization and scaleup production of paclitaxel. In this respect, physical and chemical parametersare effective and important key points for active compound production par-ticularly by using endophytic microbes. In the present study, five endophyticfungi isolated from the roots of Taxus wallichiana, are tested for paclitaxelproduction using biochemical and molecular methods. Subsequently, effectof physico-chemical parameters like temperature, pH, incubation time, andmedium constituents i.e., salt concentration, carbon and nitrogen sources onpaclitaxel production were also analyzed. Among isolates, two of the fungiviz. GBPI TWR F1 (Penicillium sp.) and GBPI TWR F5 (Aspergillus sp.)were found to be paclitaxel producing. The genomic DNA samples were sequenced to confirm the presence of two genes viz. 10-deacetylbaccatinIII-10-O-acetyl transferase (DBAT) and C-13 phenylpropanoid side chain-CoA acyltransferase (BAPT), implicated in paclitaxel biosynthesis. Boththe endophytes showed the amplicons of DBAT and BAPT genes. Resultsrevealed that after optimization of medium components and physical condi-tion, paclitaxel production was increased in both the endophytes, maximumpaclitaxel production i.e., 5.45 ± 0.98 mg/L was obtained by GBPI TWR F5(Aspergillus sp.) following 10 days of incubation at 15◦C in optimized S7liquid medium composition

Phosphate-Solubilizing Capacity of Paecilomyces lilacinus PSF7 and Optimization Using Response Surface Methodology.

Phosphorus-solubilizing microorganisms release organic acids that can chelate mineral ions or reduce the pH to solubilize insoluble phosphates for use by plants; it is important to study potential phosphorus-solubilizing microorganisms for use in agriculture. In this study, PSF7 was isolated from the soil of the Wengfu Phosphorus Tailings Dump in Fuquan City, Guizhou Province, China. PSF7 was identified as Paecilomyces lilacinus, based on morphological characterization and ITS sequencing analysis. The relationship between the phosphorus-solubilizing capacity and pH variation of PSF7 under liquid fermentation was studied. The results showed that there was a significant negative correlation (-0.784) between the soluble phosphorus content of PSF7 and the pH value. When PSF7 was placed under low phosphorus stress, eight organic acids were determined from fermentation broth using HPLC, of which tartaric acid and formic acid were the main organic acids. Different optimization parameters of medium components were analyzed using response surface methodology. The optimized medium components were 23.50 g/L sucrose, 1.64 g/L ammonium sulfate and soybean residue, 1.07 g/L inorganic salts, and 9.16 g/L tricalcium phosphate, with a predicted soluble phosphorus content of 123.89 mg/L. Under the optimum medium composition, the actual phosphorus-solubilizing content of PSF7 reached 122.17 mg/L. Moreover, scanning electron microscopy analysis of the sample was carried out to characterize the phosphate-solubilizing efficiency of PSF7 on mineral phosphate. The results provide useful information for the future application of PSF7 as a biological fertilizer.

Production and bioprocessing of camptothecin from Aspergillus terreus, an endophyte of Cestrum parqui, restoring their biosynthetic potency by Citrus limonum peel extracts

The metabolic potency of fungi as camptothecin producer elevates their prospective use as an industrial platform for commercial production, however, the loss of camptothecin productivity by fungi with the storage and subculturing are the major obstacle. Thus, screening for endophytic fungal isolates inhabiting ethnopharmacological plants with an obvious metabolic stability and sustainability for camptothecin biosynthesis could be one of the most feasible paradigms. Aspergillus terreus ON908494.1, an endophyte of Cestrum parqui was morphologically and molecularly verified, displaying the most potent camptothecin biosynthetic potency. The chemical identity of A. terreus camptothecin was confirmed from the HPLC, FTIR and LC–MS/MS analyses, gave the same molecular structure and mass fragmentation patterns of authentic one. The purified putative camptothecin displayed a strong anticancer activity towards HepG-2 and MCF-7 with IC50 values 0.96 and 1.4 µM, respectively, with no toxicity to OEC normal cells. As well as, the purified camptothecin displayed a significant antifungal activity towards fungal human pathogen Candida albicans, Aspergillus flavus, and A. parasiticus, ensuring the unique structural activity relationships of A. terreus camptothecin, as a powerful dually active anticancer and antimicrobial agent. The camptothecin productivity of A. terreus was maximized by bioprocessing with Plackett–Burman design, with an overall 1.5 folds increment (170.5 µg/L), comparing to control culture. So, the optimal medium components for maximum yield of camptothecin by A. terreus was acid why (2.0 mL/L), Diaion HP20 (2.0 g/L), Amberlite XAD (2.0 g/L), dextrin (5.0 g/L), glucose (10.0 g/L), salicylic acid (2.0 g/L), serine (4.0 g/L), cysteine (4.0 g/L) and glutamate (10.0 g/L), at pH 6 for 15 days incubation. By the 5th generation of A. terreus, the camptothecin yield was reduced by 60%, comparing to zero culture. Interestingly, the productivity of camptothecin by A. terreus has been completely restored and over increased (210 µg/L), comparing to the 3rd generation A. terreus (90 µg/L) upon addition of methanolic extracts of Citrus limonum peels, revealing the presence of some chemical signals that triggers the camptothecin biosynthetic machinery. The feasibility of complete restoring of camptothecin biosynthetic-machinery of A. terreus for stable and sustainable production of camptothecin, pave the way for using this fungal isolate as new platform for scaling-up the camptothecin production.

Enhanced l-Methioninase production by Methylobacterium sp. JUBTK33 through RSM and its anticancer potential

l-Methioninase (E.C.4.4.1.11) has gained greater significance due to its anticancer potential. In the present study, high l-Methioninase activity was seen in a newly isolated bacterium, Methylobacterium sp. JUBTK33. Enhancing enzyme production and activity is crucial for exploring the strain towards industrial applications. Hence, optimization of media components was carried out using one factor at a time method followed by statistical approach of Response Surface Methodology (RSM)– CCD matrix using Design-Expert software. After conducting triplicates of all thirty runs, Glucose 1%, Yeast 1%, and media of pH 7.0, and Tapioca peel at 10%, were found to be the optimal parameters for enhancing enzyme production from 1.26 U/mL/min to 2.10 U/mL/min (1.7 folds). The regression model analysis R2 value 95.8% indicated the best fitness model in explaining relationship between the responses and the variables. Further, the enzyme produced from the bacteria was analyzed for its cytotoxicity to Breast cancer (MCF-7), Hepatocellular carcinoma (Hep-G2), cervical cancer (HeLa), and Human embryonic Kidney (HEK-293) cell lines, with Doxorubicin as the positive control. This enzyme was effective on the Breast cancer cell line with 11% cell viability at 400 μg/mL, while Doxorubicin showed 4% viability. Toxicity of the enzyme on healthy cells seen as 44%, whereas Doxorubicin showed 83%. The study demonstrates the suitability of statistical methods in enhancing the enzyme productivity and promising anticancer activity of the l-Methioninase from Methylobacterium sp., with lesser toxicity towards healthy cells. l-Methioninase from the newly isolated Methylobacterium sp. has promising characteristics towards anticancer drug developmental studies.

Optimization of Triterpene Yield from Ganoderma atrum HBSD Z19 (Agaricomycetes) by Optimization of Medium Components and Evaluation of Bioactivity Under Simulated Gastrointestinal Digestion In Vitro.

The intracellular triterpene yield from Ganoderma atrum was enhanced by optimization based on single-factor experiments, Plackett-Burman experimental design (PBED) and response surface methodology (RSM) under liquid fermentation conditions. The optimal medium composition (g·L-1) was glucose (46.0), bean cake powder (30.2), KH2PO4 (2.0), CaCl2 (3.0), MgSO4 (1.5), FeSO4 (0.2), and pH 6.0. Under the optimal conditions, the highest triterpene yield of 0.527 g·L-1 was obtained, which was 4.705-fold higher than before optimization. The fermented powder that was collected from the optimal medium was subjected to simulated gastrointestinal digestion, with differences resulting from extraction in different digestive juices (purified water, simulated gastric digestive juice, simulated gastrointestinal digestive juice). The content of triterpenes and polysaccharides increased, except for total phenol content. In terms of the antioxidant activity, the 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH+⋅) scavenging activity gradually decreased whereas the 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+⋅) scavenging activity first decreased and then increased. In terms of enzyme viability, the activity of α-amylase (α-AL) and α-glucosidase (α-GC) in the digestive juices decreased dramatically. The main bioactive components of G. atrum and their bioactivity in digestive juices were evaluated, providing a reference for the effective use of fermented power from G. atrum.

An endophytic Streptomyces sp. DARP-7 isolated from coastal lichen and enhancement of its antibacterial metabolite production using response surface methodology

Actinomycete DARP-7 was isolated from the coastal foliose lichen Dirinaria aegilita (Afzel.ex Ach.) B.J.Moore and identified as Streptomyces sp. using 16S rRNA sequencing. Among the eight culture media used for antimicrobial metabolite production, the peptone yeast extract iron medium (ISP-6) showed higher production with the maximum zone of inhibition (12 mm) against Staphylococcus aureus . Further, the ISP-6 medium components were optimized through the Plackett-Burman design (PBD) followed by Response Surface Methodology (RSM). Yeast extract, sodium thiosulphate, and ferric ammonium citrate would play a significant role in antibiotic production. Further, the significant variables from PBD were optimized through Central Composite Design (CCD) of RSM for enhanced antibiotic production. The optimal medium components were found to be peptone - 15 (g/L), protease peptone - 5 (g/L), yeast extract - 2.915 (g/L), ferric ammonium citrate - 0.651 (g/L), K 2 HPO 4 – 1 (g/L) and Na 2 S 2 O 3 – 0.155 (g/L), respectively. The optimized result was validated and the antibacterial activity against S. aureus increased upto 19.9 mm (66.7%). The resazurin dye based quantification of minimum inhibitory concentration of the DARP-7 crude extract was found to be 41.34 µg and 46.44 µg against S. aureus and E.coli , respectively. Further, the compound profiling was done using UV-VIS, FTIR, EEM and GC-MS confirming the presence of polyene conjugates. Thus, the study delivers coastal lichen could be promising source for isolating bioactive metabolites producing rare actinobacteria. Furthermore, the use of a statistical optimization tool to improve bioactive metabolite yield is discussed.

Statistical optimization of the fermentation factors for increased biorefinery of corn-steep water effluent to lactic acid by Enterococcus faecium strain WH51-1

AbstractOrganic waste bioprocessing for production of useful products are increasing due to fossil fuel depletion, problems with waste management, and the requirement for biodegradable plastics. Lactic acid (LA) is important in foods, polymer beverages, cosmetics, and medicine applications. However, the main challenge in the large-scale processing of LA is the enhanced production and productivity using low-cost resources. In this study, Enterococcus faecium WH51-1, a newly obtained thermo-alkali tolerant LA bacterium, was used for LA production from corn-steep water (CSW) as raw material. The optimal culture conditions and medium components were established. Different concentrations of yeast extract (YE) and pH values were investigated with highest LA production of 48.2 ± 2.7 g/L at a yield of 0.89 ± 0.04 g/g-consumed sugars and a productivity of 0.50 ± 0.03 g/L.h at pH 8.5 without YE supplementation. A central composite design (CCD) was utilized to determine the high LA concentrations at optimal values for the process variables (sugar concentrations, temperature, inocula sizes, pH value, and YE concentration). The model was realized with an acceptable fit. The optimization of the factors was carried out by normal probability plots, interaction-plots, incorporating effect plots, analysis of variance (ANOVA), surface plots, Pareto charts, and contour plots. All the parameters were influenced at a 5% significance level. Additionally, some of the possible interactions between these parameters also influenced the production process. A regression model was suggested and fitted the experimental data very well. The results of this work investigated that LA synthesized of the optimized-fermentation conditions was 10% greater than classical optimization methods. This study presented a green “free nutrient” and cost-effective utilization of corn steep effluent as a cheap alternative substrate for biorefinery to lactic acid. Graphical Abstract

Pentostatin Biosynthesis Pathway Elucidation and Its Application

Pentostatin (PNT), a nucleoside antibiotic with a 1,3-diazo ring structure, is distributed in several actinomycetes and fungi species. Its special structure makes PNT possess a wide spectrum of biological and pharmacological properties, such as antibacterial, antitrypanosomal, anticancer, antiviral, herbicidal, insecticidal, and immunomodulatory effects. Because of the promising adenosine deaminase inhibitory activity of PNT, its extensive application in the clinical treatment of malignant tumors has been extensively studied. However, the fermentation level of microbial-derived PNT is low and cannot meet medical needs. Because the biosynthesis pathway of PNT is obscure, only high-yield mutant screening and optimization of medium components and fermentation processes have been conducted for enhancing its production. Recently, the biosynthesis pathways of PNT in actinomycetes and fungi hosts have been revealed successively, and the large-scale production of PNT by systematic metabolic engineering will become an inevitable trend. Therefore, this review covers all aspects of PNT research, in which major advances in understanding the resource microorganisms, mechanism of action, and biosynthesis pathway of PNT were achieved and diverse clinical applications of PNT were emphasized, and it will lay the foundation for commercial transformation and industrial technology of PNT based on systematic metabolic engineering.

Production of rosmarinic acid and biomass from adventitious root cultures of Ocimum basilicum by optimization of medium components in airlift bioreactors

Production of rosmarinic acid and biomass from adventitious root cultures of Ocimum basilicum by optimization of medium components in airlift bioreactors

P-804 Microfluidic and static organotypic culture systems to support ex vivo spermatogenesis from prepubertal mammalian testicular tissue: a comparative study

Abstract Study question What is the impact of different static and dynamic organotypic culture systems on the outcome of prepubertal porcine immature testicular tissue (ITT) culture? Summary answer Using a hybrid silicone-agarose static culture system improves germ cell survival and differentiation in ITT ex-vivo. What is known already In mice, organotypic culture of ITT led to the birth of healthy fertile pups. In other larger mammals, including humans, it remains challenging to efficiently reproduce spermatogenesis ex-vivo. With the advent of tissue engineering, culture systems are becoming more complex to better mimic the in vivo environment by modifying and/or improving physical culture conditions (i.e. tissue perfusion, oxygen diffusion, direct/indirect contact with medium). Recent studies in rodents showed that by using hybrid static and dynamic silicone-based organotypic culture systems, the efficiency of spermatogenesis can be significantly improved. Such culture systems have not been reported in large mammals yet. Study design, size, duration Testes were collected from three 5-7 days old piglets as a by-product of castration for a Belgian farm. The fresh ITT fragments were immediately used for culture experiments. Fragments were cultured for a 30 days duration in the different static i.e. polytetrafluoroethylene membrane inserts (CT), agarose gel (AG) and agarose gel with polydimethylsiloxane cover (AGPC) and dynamic i.e. microfluidic (MF) systems using a single medium. The CT system served as control. Participants/materials, setting, methods Cultured ITT fragments were monitored every 4-5 days by serial imaging. Analyses were performed at days 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity and diameters were evaluated on Periodic Acid Schiff-stained slides. Immunohistochemistry was used to identify: germ (PGP9.5, VASA, SYCP3, CREM), somatic (SOX9, INSL3) and proliferating (Ki67) cells and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured using ELISA. A two-way ANOVA was used for statistical analyses. Main results and the role of chance All systems supported the survival, growth and maturation of ITT fragments in vitro. Between days 0 and 30, ST diameters increased in all systems (p < 0.000) (day 0: 61.2±6.5μm; day 30: CT: 75.3±18.1μm, AG: 77.7±15.7μm, AGPC: 84.9±13.6μm, 66.9±12.9μm). Sertoli cell (SOX9) numbers also increased while spermatogonia (PGP9) numbers per ST decreased (day 0: 6.94±0.31, day 30: CT: 1.84±0.64, AG: 2.65±0.88, AGPC: 3.47±0.7, MF: 2.11±0.84). Meiotic germ cells (SYCP3, CREM) were absent at day 0 and were generated starting day 5 (SYCP3) and 10 (CREM) in the different systems and maintained till day 30 (SYCP3 per ST; CT: 0.31±0.21, AG: 0.5±0.31, AGPC: 0.69±0.20, MF: 0.38±0.20 and CREM per ST: CT: 0.18±0.18, AG: 0.25±0.13, AGPC: 0.34±0.32, MF: 0.14±0.14). When compared to control (CT), significantly larger STs (days 10 to 30), a higher number of spermatogonia (PGP9) (day 30; AGPC: 3.72±0.70, CT: 1.84±0.64, p = 0.04), meiotic SYCP3 + (day 20, AGPC: 0.95±0.49, CT: 0.46±0.39, p = 0.01) and CREM+ cells (day 20, AGPC: 0.49±0.21, CT: 0.23±0.64, p = 0.02) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cells (Ki67/VASA), Leydig cells (INSL3) and testosterone levels were not significantly different between the different systems over the culture period. Limitations, reasons for caution The small sample size and inter-tissue variability could have limited the results of this study. Also it is not excluded that further differences, in terms of tissue growth, germ cell survival and differentiation, could be observed between the different systems if a more complex culture medium is used. Wider implications of the findings In contrast to rodents, modifying physical culture conditions minimally impacts the outcome of porcine ITT maturation ex-vivo. Due to anatomo-physiological similarities between pigs and humans, it is likely for similar results to be observed if applied to human ITT. Studies aiming at determining optimal culture medium components are rather needed. Trial registration number not applicable

Statistical optimization of medium components and growth conditions to enhance Polyhydroxybutyrate yield by Bacillus endophyticus MTCC 13038

Polyhydroxybutyrate (PHB) is a biodegradable polymer accumulated as carbon and energy reserve granules by several bacteria and is ideal substitute for petroleum-based polymers. The present study aimed to isolate PHB producing bacteria and optimize medium components and growth conditions to enhance PHB yield. Potent PHB accumulating bacterial strain was isolated from oil spilled soil and identified as Bacillus endophyticus based on its 16S rRNA analysis. Sudan Black B staining method was used to detect PHB granules and affirmed by using Fourier Transform Infrared Spectroscopy. Differential scanning calorimetry analysis was conducted for thermal analysis of extracted PHB polymer. Bacillus endophyticus showed highest PHB accumulation utilizing glucose as the carbon and potassium nitrate as the nitrogen source which were further optimized along with physical parameters like pH and temperature using Box and Behnken method. The highest biomass (7.49 g/L) and PHB (3.67 g/L) were observed at pH 7, temp. 37ºC, glucose 25 g/L and potassium nitrate 0.1 g/L. Bacillus endophyticus was found to accumulate up to 49% PHB under optimized conditions. These results indicate the potential application of Bacillus endophyticus at the industrial level for PHB production.

Successive approach of medium optimization using one-factor-at-a-time and response surface methodology for improved β-mannanase production from Streptomyces sp.

β-Mannanase is an enzyme catalyzing the β-1,4-mannosidic linkages of the hemi-cellulolytic structure. Optimization of medium components was studied for β-mannanase production from Streptomyces sp. RDA1496 using One-Factor-At-a-Time and resulting activity was found to be 244.54 IU/mL. Subsequently, Plackett-Burman (PB) design was adopted to evaluate the effect of guar gum, coprameal, peptone, yeast extract, fructose, inoculum size, NaCl, K2HPO4, KH2PO4 and CaCl2. Analysis of variance revealed the correlation coefficient (R2) of 0.9996 for the performed model at a significant level (p < 0.05). Four significant variables were optimized using central composite design of response surface methodology (RSM) at five different coded levels. The optimum concentration of significant factors namely fructose, guar gum, peptone, and K2HPO4 were found to be 4.81 g/L, 7.09 g/L, 4.91 g/L, and 2.05 g/L respectively. PB and RSM-guided experimental design, resulted in maximum β-mannanase production of 415.96 IU/mL, which is 5.55-fold increase from un-optimized condition.