Bioreactor studies on the endophytic fungus Entrophospora infrequens for the production of an anticancer alkaloid camptothecin

2006 ◽  
Vol 52 (3) ◽  
pp. 189-196 ◽  
Author(s):  
Touseef Amna ◽  
Satish C Puri ◽  
V Verma ◽  
Jai P Sharma ◽  
Rajinder K Khajuria ◽  
...  
Keyword(s):  
Cell Mass ◽  
Scale Up ◽  
Maximum Yield ◽  
Dry Mass ◽  
Spectroscopic Studies ◽  
Physicochemical Analysis ◽  
Culture Broth ◽  
Nothapodytes Foetida ◽  
Bark Tissue ◽  
Shake Flasks

Twigs (young and old) from Nothapodytes foetida growing in the Jammu and Mahabaleshwar regions in India were used for the isolation of 52 strains of endophytic fungi and were tested for their ability to produce the anticancer alkaloid camptothecin. One of the isolates from the inner bark tissue of the N. foetida plant growing in the Jammu region of J&K state, India, was found to produce detectable quantities of camptothecin and its derivatives when grown in a semi-synthetic liquid medium. Camptothecin was identified by physicochemical analysis and further confirmed by spectroscopic studies. No camptothecin was detected in zero time cultures or in uninoculated culture broth. The maximum yield of camptothecin was 0.575 ± 0.031 mg/100 g of dry cell mass in 96 h in shake flasks, whereas 4.96 ± 0.73 mg/100 g of dry mass was recorded in 48 h in a bioreactor.Key words: endophytes, camptothecin, anticancer drug, Nothapodytes foetida, Entrophospora infrequens, bioreactor, scale-up.

scholarly journals Highly efficient production of transfructosylating enzymes using low-cost sugarcane molasses by A. pullulans FRR 5284

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Most Sheauly Khatun ◽  
Morteza Hassanpour ◽  
Mark D. Harrison ◽  
Robert E. Speight ◽  
Ian M. O’Hara ◽  
...  
Keyword(s):  
Yeast Extract ◽  
Production Systems ◽  
Low Cost ◽  
Scale Up ◽  
Maximum Yield ◽  
Extracellular Enzyme ◽  
Efficient Production ◽  
Sugarcane Molasses ◽  
Transfructosylating Enzyme ◽  
Shake Flasks

AbstractFructooligosaccharides (FOS) are a type of important prebiotics and produced by transfructosylating enzymes. In this study, sugarcane molasses was used as the substrate for production of transfructosylating enzymes by Aureobasidium pullulans FRR 5284. NaNO3 was a superior nitrogen source to yeast extract for production of transfructosylating enzymes by A. pullulans FRR 5284 and decreasing the ratio of NaNO3 to yeast extract nitrogen from 1:0 to 1:1 resulted in the reduction of the total transfructosylating activity from 109.8 U/mL to 82.5 U/mL. The addition of only 4.4 g/L NaNO3 into molasses-based medium containing 100 g/L mono- and di-saccharides resulted in total transfructosylating activity of 123.8 U/mL. Scale-up of the A. pullulans FRR 5284 transfructosylating enzyme production process from shake flasks to 1 L bioreactors improved the enzyme activity and productivity to 171.7 U/mL and 3.58 U/mL/h, 39% and 108% higher than those achieved from shake flasks, respectively. Sucrose (500 g/L) was used as a substrate for extracellular, intracellular, and total A. pullulans FRR 5284 transfructosylating enzymes, with a maximum yield of 61%. Intracellular, extracellular, and total A. pullulans FRR 5284 transfructosylating enzymes from different production systems resulted in different FOS profiles, indicating that FOS profiles can be controlled by adjusting intracellular and extracellular enzyme ratios and, hence prebiotic activity.

UTILIZATION OF FISH OIL BY CANDIDA LIPOLYTICA AND GEOTRICHUM CANDIDUM

1974 ◽  
Vol 37 (10) ◽  
pp. 522-528 ◽  
Author(s):  
H. H. Hottinger ◽  
T. Richardson ◽  
C. H. Amundson ◽  
D. A. Stuiber
Keyword(s):  
Fish Oil ◽  
Crude Protein ◽  
Cell Mass ◽  
Geotrichum Candidum ◽  
Inoculum Size ◽  
Candida Lipolytica ◽  
Cell Biomass ◽  
Oil In Water ◽  
Shake Flasks ◽  
Triton X

Attempts were made to maximize biomass production in the oxidation of fish oil by Candida lipolytica Y1094 and Geotrichum candidum Y552 in shake flasks. Maximum biomass was produced on 5% w/v oil in water containing 1.5% (NH4)2 SO4, 2.2% KH2PO4, 0.12% MgSO4, 0.03% fish stickwater solids, 10 ppm FeSO4 · 7H2O, 1.5 ppm MnSO4 · H2O, and adjusted to pH 7.5. For G. candidum 40 ppb biotin were added to the medium. When inoculum size was varied from 1 to 8% v/v, the most appropriate inoculum size was about 2%. Aeration rates were increased by decreasing the medium volume per 500-ml shake flask. Maximum yields were obtained with 25 ml of medium. Cell recoveries were complicated by formation of oil-media emulsions. Several methods of biomass recovery were studied. Warming the acidified cell suspension in the presence of Triton X-100 allowed removal of oil from the cell mass. Extraction of the dried cell mass with ethyl ether yielded highest recovery of cell material compared to other methods. Dry cells contained 40.1 to 49.3% crude protein. These studies suggest the feasibility of obtaining 800 g of single cell biomass with a crude protein yield of 320 g from 1 kg of fish oil using batch or continuous culture systems.

Scale-up from shake flasks to fermenters in batch and continuous mode withCorynebacterium glutamicum on lactic acid based on oxygen transfer and pH

2007 ◽  
Vol 98 (4) ◽  
pp. 800-811 ◽  
Author(s):  
Juri M. Seletzky ◽  
Ute Noak ◽  
Jens Fricke ◽  
Eike Welk ◽  
Werner Eberhard ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Oxygen Transfer ◽  
Scale Up ◽  
Continuous Mode ◽  
Shake Flasks

scholarly journals Productivity of potato seed submitted to different doses of potassium in hydroponic system

2019 ◽  
pp. 555
Author(s):  
Camila B.A. Tufik
Keyword(s):  
Seed Potato ◽  
Block Design ◽  
Maximum Yield ◽  
Dry Mass ◽  
Potato Seed ◽  
Design Data ◽  
Fresh Matter ◽  
Hydroponic System ◽  
Randomized Block Design ◽  
Different Doses

The potato is one of the most economically important crops in Brazil, and among the items that most cost the production is seed potatoes. The deficiency of one nutrient can interfere with the absorption and accumulation of the others in plants. The aim of this work was to quantify the optimal potassium (K) dose for minituber basic seed potato yield in hydroponic system. The experiment was installed in a greenhouse with the Agata cultivar minitubers. The treatments consisted of five doses of K (0.0; 2.5; 5.0; 7.5 e 10.0 mmol L-1) with four repetitions. The experimental design was a randomized block design. Data were submitted to analysis of variance and regression. The number of tubers, fresh mass, classification and dry mass were measured. The content of K and content other nutrients in the seed potato tubers were also evaluated. In the hydroponic systems, the maximum yield per plant was 48.41 tubers obtained with 6.15 mmol L-1 of K and maximum mass of fresh matter was 646.6 g.

scholarly journals Enzymatic synthesis optimization of isoamyl butyrate from fusel oil

2021 ◽  
Vol 43 ◽  
pp. e54966
Author(s):  
Andréia Anschau ◽  
Katira da Mota Huerta ◽  
Tatiane Vieira Rêgo ◽  
Janaina Mardioni Gonçalves de Oliveira ◽  
Carina Molins Borba ◽  
...  
Keyword(s):  
Butyric Acid ◽  
Immobilized Lipase ◽  
Scale Up ◽  
Maximum Yield ◽  
Fractional Factorial ◽  
Molar Ratio ◽  
Lipozyme Tl Im ◽  
Fusel Oil ◽  
Alcohol Production ◽  
Pharmaceutical Industries

Many food, cosmetic and pharmaceutical industries have increased their interest in short-chain esters due to their flavor properties. From the industrial standpoint, enzyme reactions are the most economical strategy to reach green products with neither toxicity nor damage to human health. Isoamyl butyrate (pear flavor) was synthesized by isoamyl alcohol (a byproduct of alcohol production) and butyric acid with the use of the immobilized lipase Lipozyme TL IM and hexane as solvents. Reaction variables (temperature, butyric acid concentration, isoamyl alcohol:butyric acid molar ratio and enzyme concentration) were investigated in ester conversion (%), concentration (mol L-1) and productivity (mmol ester g-1 mixture . h), by applying a sequential strategy of the Fractional Factorial Design (FFD) and the Central Composite Rotatable Design (CCRD). High isoamyl butyrate conversion of 95.8% was achieved at 24 hours. At 3 hours, the highest isoamyl butyrate concentration (1.64 mol L-1) and productivity (0.19 mmol ester g-1 mixture . h) were obtained under different reaction conditions. Due to high specificity and selectivity of lipases, process parameters of this study and their interaction with the Lipozyme TL IM are fundamental to understand and optimize the system so as to achieve maximum yield to scale up. Results show that fusel oil may be recycled by the green chemistry process proposed by this study.

scholarly journals Bacterial surface charge in “layers”: revealed by wash buffers of different ionic strength

2018 ◽  
Author(s):  
Wenfa Ng ◽  
Yen-Peng Ting
Keyword(s):  
Ionic Strength ◽  
Zeta Potential ◽  
Surface Charge ◽  
Cell Envelope ◽  
Cell Mass ◽  
Culture Broth ◽  
Point Of Zero Charge ◽  
Bacterial Surface ◽  
Incomplete Removal ◽  
Wash Buffer

Bacterial surface charge derives its meaning from the cell’s environment such as the solution in contact with the cell. Determining the surface charge of bacteria in its native environment requires measuring the proxy variable, zeta potential, using cells obtained from field studies. However, lack of adequate cell mass and concerns over measurement of a mixed species consortia rather than a specific species meant that bacterial surface charge measurement require biomass obtained from pure culture. Often grown in rich medium where myriad proteins and ions nonspecifically adsorbed onto the cell envelope or peptidoglycan layer, standard procedures for preparing the cell mass incorporated repeated steps of washing and centrifugation with various wash buffers, the efficacies of which are poorly understood. This report describes the results of a systematic study on how wash buffers of different composition and ionic strength affect the efficiency of removing nonspecifically adsorbed biomolecules and ions from Escherichia coli DH5α (ATCC 53868) cultured aerobically (shake flask, 37 oC and 230 rpm) in LB Lennox medium with 2 g/L glucose and a formulated medium. Using zeta potential-pH profiles over pH 1 to 12 as readout, the results showed that efficiency of removing nonspecifically adsorbed ions and metabolites positively correlated with wash buffer ionic strength. More importantly, 0.15M ionic strength (i.e., 9 g/L NaCl) seemed to be the minimum below which there was incomplete removal of nonspecifically adsorbed biomolecules. On the other hand, high ionic strength of 0.6M (e.g., 0.1M sodium citrate) significantly changed the point of zero charge (pHzpc), a reference marker for removal of ions intrinsic to the cell envelope. Collectively, results obtained inform wash buffer choice with regards to preserving cell envelope integrity, and avoidance of adsorption of buffer ions such as citrate. But, is there a true cell surface charge? Yes, but how do we define it in number of “layers” of adsorbed biomolecules? Philosophically, cells in culture broth are coated with layers of metabolites, proteins and ions. Hence, desire to reveal the true surface charge is essentially a decoating process, where wash buffers of increasing ionic strength remove each layer via charge screening. However, where is the endpoint? This research offers a different perspective and answer. Imagine a single bacterium suspended in LB medium, where there is constant adsorption and desorption of biomolecules as the cell grows: what is its relevant surface charge? It is the one where the loosely associated ions and metabolites are removed while retaining the nonspecifically adsorbed ions and biomolecules. Thus, deionized water wash provides a good estimate of the bacterial surface charge as grown in specific medium.

scholarly journals Robust surface-to-mass coupling and turgor-dependent cell width determine bacterial dry-mass density

2021 ◽  
Vol 118 (32) ◽  
pp. e2021416118
Author(s):  
Enno R. Oldewurtel ◽  
Yuki Kitahara ◽  
Sven van Teeffelen
Keyword(s):  
Single Cell ◽  
Caulobacter Crescentus ◽  
Mass Density ◽  
Turgor Pressure ◽  
Cell Mass ◽  
Dry Mass ◽  
Model Organisms ◽  
Cell Width ◽  
Dependent Cell

During growth, cells must expand their cell volumes in coordination with biomass to control the level of cytoplasmic macromolecular crowding. Dry-mass density, the average ratio of dry mass to volume, is roughly constant between different nutrient conditions in bacteria, but it remains unknown whether cells maintain dry-mass density constant at the single-cell level and during nonsteady conditions. Furthermore, the regulation of dry-mass density is fundamentally not understood in any organism. Using quantitative phase microscopy and an advanced image-analysis pipeline, we measured absolute single-cell mass and shape of the model organisms Escherichia coli and Caulobacter crescentus with improved precision and accuracy. We found that cells control dry-mass density indirectly by expanding their surface, rather than volume, in direct proportion to biomass growth—according to an empirical surface growth law. At the same time, cell width is controlled independently. Therefore, cellular dry-mass density varies systematically with cell shape, both during the cell cycle or after nutrient shifts, while the surface-to-mass ratio remains nearly constant on the generation time scale. Transient deviations from constancy during nutrient shifts can be reconciled with turgor-pressure variations and the resulting elastic changes in surface area. Finally, we find that plastic changes of cell width after nutrient shifts are likely driven by turgor variations, demonstrating an important regulatory role of mechanical forces for width regulation. In conclusion, turgor-dependent cell width and a slowly varying surface-to-mass coupling constant are the independent variables that determine dry-mass density.

scholarly journals Quantitative Phase Imaging of Spreading Fibroblasts Identifies the Role of Focal Adhesion Kinase in the Stabilization of the Cell Rear

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1089
Author(s):  
Olga Ramaniuk ◽  
Zuzana Klímová ◽  
Tomáš Groušl ◽  
Tomáš Vomastek
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cell Mass ◽  
Dry Mass ◽  
Cell Polarization ◽  
Phase Imaging ◽  
Quantitative Phase Imaging ◽  
Quantitative Phase ◽  
Cell Front

Cells attaching to the extracellular matrix spontaneously acquire front–rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front–rear polarity in spreading cells.

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