This method generates and characterizes microparticle dependent morphological structures of filamentous fungus, aspergillus, Niger, to correlate fungal morphology with productivity. Cultivate eight Niger with or without microparticles in a three liter stirred tank, bioreactor for 72 hours to calculate specific productivity. Take samples at defined time points and determine biomass, dry weight, and veto fructo sase activity.
After 72 hours, examine the fungal morphology by microscopy and characterize by digital image analysis. Then combine relevant parameters of image analysis to a morphology number which is mathematically correlated with the specific productivity. Ultimately, this method can precisely generate and comprehensively characterize morphology of aspergillus Niger for a better understanding of the morphogenesis of filamentous microorganisms.
Aspergillus Nigel is an important industrial working horse in biotechnology since many decades. One of the most intriguing and often uncontrollable characteristics of aspers, Nigel, and related species is there complex morphology ranging from dense spherical pellets to viscous mycelia depending on culture conditions. The main advantage of our procedure is that through addition of microparticles, we are now able to tailor make fungal morphology specifically for process needs.
So far, no other method has been described for such a customization of fungal morphology. This method can help answer key questions about the cultivation of filamentous microorganisms. Because for industrial application, it is crucial to control fungal morphology and also to distinguish between a well and to poorly producing fungal morphology.
Our protocol provides the means for desirable customization and characterization of fungal morphology. Though this method can provide insight into aspergillus Nigel morphology, it can also be applied to other filamentous microorganisms such as penicillium or strepto strains. Set up four bioreactors for sterile cultures as detailed in the accompanying protocol.
Text based on Microparticle addition, cultivate aspergillus Niger with different morphology. After 72 hours transfer 50 milliliters. Samples of culture broth into a Falcon tube.
Biomass samples are to be taken at least in duplicate after drying in the desiccate, weigh a cellulose filter with the microscales. Place the filter in a buchner funnel with connected water jet vacuum pump filter 10 milliliters of sample followed by 10 milliliters. Deionized water to remove medium compounds from the biomass wrinkle the filter once in the middle.
Place it in a glass petri dish and into a compartment dryer until weight constancy. Transfer the filter to a desiccate and let it cool. Then measure the weight.
Calculate the biomass dry weight per liter by calculation of weight, difference of filters, and subsequent division by sample volume for the glucose oxidase and peroxidase enzyme assays. Store samples on ice using a syringe pass, 1.5 milliliters. Culture suspension through a cellulose acetate filter into a reaction tube Performs the beta fructo SASE assay.
Meticulously we ensure success with two triplicate samples for statistical robustness. To start the assay, add 20 microliters sample to the test tube to control for the pH and temperature dependent cleavage of sucrose to glucose, use 20 microliters deionized water instead of 20 microliters sample. Also, for each sample, prepare a negative control for residual glucose in the culture broth by assay.
20 microliters of heat inactivated sample To initiate the reaction from sucrose to glucose, add 200 microliters of 1.65 molar sucrose solution. At pH 5.4 to 20 microliters sample, incubate all reaction tubes in a 40 degree Celsius heating block for 20 minutes. Stop the reaction by heating at 95 degrees Celsius for 10 minutes after cooling tubes on ice.
Centrifuge samples when necessary, dilute samples for the assay such that the measured absorption is in the calibrated value range. Now for each reaction, add two microliters of sample into a microtiter plate. Well perform sample measurements in triplicate.
Also prepare a standard curve for 10 glucose solutions ranging from one millimolar to 15 millimolar. For the zero point calibration, use two microliters deionized water. Next, add 200 microliters of the reagent solution to each.
Well incubate for 10 minutes at room temperature. Measure the absorption at 450 nanometers using a 96 well sunrise Microplate reader and the Magellan data retrieval software. Open the result chart with a spreadsheet and construct a standard curve calibration line.
Calculate the activity for each sample. Then compute beta fructo tase activity by subtracting values of the appropriate negative control from the sample activity. Finally, calculate the specific productivity by factoring biomass dry weight and beta fructo sase activity.
Place three milliliters of culture suspension in a plastic Petri dish and dilute with physiological sodium chloride solution to separate morphological structures. The quality of the microscopic pictures is of exceptional importance. For the subsequent image analysis, create care should be taken during the dilution step.
Situate the Petri dish under a microscope, which features an integrated camera. Acquire and save about 100 images of morphological structures per sample, ensuring that at least one object is completely pictured on each image. Continue in the same manner with dishes containing samples from different reactors, acquiring new images each time.
Note the different morphological growth form of samples from different reactors grown with differing amounts of added talc powder. Then open all images of the same sample in the image processing program. Image J.Using the process tool, make binary.
Convert the images to black and white for applying the command to a whole series of images. Use a macro code next, open one of the pictures containing a scale bar. Construct a straight line across the scale bar to determine the number of pixels correlating to 2000 microns.
Select analyze tool, set measurement, and choose the shape factors, ferrets, diameter area, and perimeter. Use a macro code to process a series of images. Now open the spreadsheet that charts results of shape factor values.
For each image, calculate a morphology number for each image with all images of one sample. Calculate the mean value for the morphology number. Use a graphing and data analysis program to plot morphology number with the specific productivity.
Determine the mathematical relation by mathematical regression through addition of increasing concentrations of talc microparticles. Eight Niger SKAN 10 15. Morphology is changed from a true pellet morphology to a dispersed or even my morphology as expected pellet morphology is exhibited at standard conditions.
Interestingly, mycelial morphology is created by supplementation of medium with 10 grams per liter of talc microparticles. Concurrently the activity of Beto Fructo tase increases approximately threefold supplementation of one or three grams per liter of talc powder leads to a dispersed morphology with a doubled fructose activity using parameters determined by automatic image analysis. The microparticle dependent morphology can be comprehensively described by morphology.
Number note, perfectly round in smooth pellets will in microscopic images appear as perfect circles for such particles. The morphology number has a value approaches one at standard conditions. The morphology in reactor one exhibits a morphology number around 0.8.
The morphology in reactor four with 10 grams per liter. Talc powder features a morphology number around 0.1. The morphology number for reactors two and three with talc powder concentrations of one and three grams per liter lies between these extremes demonstrating a dispersed morphology.
Since microparticle dependent morphology is closely related with the beta fructose cytes productivity, there is a good mathematical correlation of morphology, number and productivity After its development. This technique paved the way for researchers in the field of biotechnology to further explore fungal morphology and especially its relation to productivity. Following this procedure of detailed creation of specific morphological, fungal cross types, other important aspects of filamentous microorganism cultivations can be explored.
Mycelial morphology, for instance, is known to be much more viscous than palate morphology. Therefore, overall productivity of the process is impaired through problems and purification of the desired product. Our morphology number will help to establish models concerning culture, pro theology, and further our understanding of fungal morphology in general.
