Name: Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles (Video) | JoVE Journal
Uploaded: 2012-03-15T13:00:00.000+00:00
Duration: 10 min 51 s
Description: Learn to customize Aspergillus niger morphology using talc microparticles in bioreactor cultivation. This method enables precise correlation between fungal morphology and productivity through image analysis.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors gratefully acknowledge financial support provided by the German Research Foundation (DFG) through the collaborative research center SFB 578 "From Gene to Product" at the Technische Universit&auml;t Braunschweig, Germany.

1. Reactor Setup and Start of Cultivation
4 bioreactor cultivations in total are conducted.
<ol>
 <li>Use a 3 L stirred tank bioreactor with a working volume of 2.2 L for the cultivation of A. niger SKAn1015.</li>
 <li>Pour 72.6 g glucose monohydrate into the reactor and fill it up with 1.9 L deionized water.</li>
 <li>Install the reactor equipment such as 3 baffles, a two six-bladed disk turbine impeller, a pH electrode, a gas inlet with air filter, a cooling finger, an exhaust air cooler with air filter, an immersion tube for sterile sampling with air filter and inlet hoses for medium, inoculum, acid and base.</li>
 <li>Autoclave the reactor at 121 °C for 20 min.</li>
 <li>Connect the reactor with the acid-base reservoir (2 M HCl, 2 M NaOH) and the corresponding pumps and set up a pH-value of 5.0 ± 0.05 with the pH control unit.</li>
 <li>Link the reactor with the cooling-water system and put the heating jacket around the reactor vessel.</li>
 <li>Install the temperature sensor with corresponding control unit and set up a temperature of 37 ± 0.1 °C at the temperature control unit.</li>
 <li>Fit the agitation motor atop of the reactor and bring it into service with an agitation speed of 200 min-1.</li>
 <li>Add the sterile minimal growth medium11 trough one of the sterile inlet hoses (250 mL). For medium preparation all components were autoclaved at 121 °C for 20 min and mixed together. For three cultivations the components include talc powder (3MgO•4SiO2•H2O) in the concentrations of 1 g/L (reactor 2), 3 g/L (reactor 3) and 10 g/L (reactor 4). Prior to use, re-suspend the micro particles in 50 mM sodium acetate buffer (pH 6.5) and add it to the sterile medium. In control cultures (without particles) replace the micro particle suspension by 50 mM sodium acetate buffer (pH 6.5).</li>
 <li>Add the spore suspension (inoculum) as prepared in Wucherpfennig et. al (2011)11 trough one of the sterile inlet hoses (50 mL) so that the concentration of spores amounts to 1X106 mL-1. The inoculation marks the start of cultivation (h = 0 h).</li>
 <li>Start the aeration with a rate of 1.0 L min-1.</li>
</ol>
2. Sterile Sampling after 24, 48 and 72 h of Cultivation
<ol>
 <li>Take 50 mL of sterile culture broth into a flacon tube.</li>
 <li>Use sample for determination of biomass dry weight, β-fructofuranosidase activity and microscopic analysis.</li>
</ol>
3. Determination of the Biomass Dry Weight after 24, 48 and 72 h of Cultivation
<ol>
 <li>Biomass samples are to be taken at least in duplicate.</li>
 <li>Weight a cellulose filter with a micro scales after drying in the desiccator and place the filter in a Büchner funnel with connected water-jet vacuum pump.</li>
 <li>Filter a defined sample volume (e. g. 10 mL) and rinse the filter with 10 mL deionized water to remove medium compounds from the biomass.</li>
 <li>Wrinkle the filter once in the middle, place it in a glass Petri dish and put it into a compartment dryer until weight constancy (at least 24 h).</li>
 <li>Cool the filter in a desiccator and measure the weight.</li>
 <li>Calculate the biomass dry weight as the difference between the weight of the filter with and without dried biomass divided by the used sample volume.</li>
</ol>
4. Determination of the Extracellular Enzymatic Activity of β-fructofuranosidase by GOD/POD-essay after 24, 48 and 72 h of Cultivation
Store the samples on ice constantly while working with them.
<ol>
 <li>Filter 1.5 mL culture suspension trough a cellulose acetate filter by pressing the culture suspension with a syringe through the filter into a reaction tube. Store the reaction tube at -20 °C until usage.</li>
 <li>For the reaction mixture use 20 μL sample and add 200 μL of 1.65 M sucrose dissolved in 0.05 M phosphate buffer (pH 5.4) to initiate the reaction from sucrose to glucose. Cary out the reaction at least in duplicate.</li>
 <li>Incubate the reaction mixture at 40 °C for 20 min in a heating block. Stop the reaction by incubating at 95 °C for 10 min in a heating block. Cool the reaction mixture by storing it on ice and spin down the condensed water by centrifuge at 13.000 g for 10 min at 4 °C.</li>
 <li>To account the pH and temperature dependent cleavage of sucrose to glucose, carry out a blank value by using 20 μL deionized water instead of 20 μL sample.</li>
 <li>To account for residual glucose in the culture broth, carry out a negative control for each sample. To that end use 20 μL of the sample and inactivate β-fructofuranosidase by heating at 95 °C for 10 min prior to add the sucrose and incubate as described in step 4.3.</li>
 <li>Dilute the samples such that the following measured adsorption is in the calibrated value range.</li>
 <li>Carry out all enzymatic assays in triplicate. Apply 2 μL diluted sample in a micro titer plate well. Apply a standard range for calibration on the micro titer plate by each using 2 μL of ten glucose solutions with ten different know concentrations (from 1 mM to 15 mM) instead of 2 μL sample. For zero point calibration use 2 μL deionized water instead of 2 μL sample.</li>
 <li>Add 200 μL of the reagent solution to each well by using a multi pipette.</li>
 <li>Incubate the mixture for 10 min at room temperature.</li>
 <li>Store the micro titer plate at 6 °C until measurement (a few hours at most).</li>
 <li>Measure the absorption at 450 nm using a 96-well Sunrise micro plate reader and the Magellan data retrieval software. Set the mixing time up to 5 sec. and the rest period up to 1 sec.</li>
 <li>Open the result chart with a spreadsheet and construct a calibration line by using the standard range: 
 glucose concentration = a X absorption + b </li>
 <li>Calculate the activity: 
 activity = (absorption X dilution factor-blank value ) X a+b </li>
 <li>Figure out the β-fructofuranosidase activity by calculating the difference between the activities of the sample and the appropriate negative control.</li>
 <li>Calculate the specific productivity for following use by taking into account biomass dry weight and β-fructofuranosidase activity 11.</li>
</ol>
5. Microscopy and Automated Image Analysis after 72 h of Cultivation
<ol>
 <li>Place around 3 mL culture suspension in a plastic Petri dish and dilute them with physiological sodium chloride solution until the morphological structures are separated.</li>
 <li>Situate the Petri dish under a microscope, which features an integrated or connected camera. Acquire and save about 100 images (Figure 2) of morphological structures per sample. Pay attention that on each image at least one object is completely pictured.</li>
 <li>Open all images of the same sample with the image processing program ImageJ12. Convert the images to black and white by using the process tool "Make Binary" (Figure 2). For applying the command to the whole series of images use a macro code as follows. run(,,Make Binary") 
 </li>
 <li>Open the processed binary images with ImageJ again. Calculate the shape factors Feret's diameter, projected area, perimeter, circularity, aspect ratio, roundness und solidity for every image with the analyze tool "Set Measurement". For applying the command to a series of images use a macro code as follows. 
 run("8-bit"); 
 run("Make Binary"); 
 run("Set Scale...", "distance=X known=1000 pixel=1 unit=μm global"); 
 run("Set Measurements...", "area perimeter shape feret's limit display redirect=None decimal=3"); 
 run("Analyze Particles...", "size=10000-Infinity circularity=0.00-1.00 show=Outlines display"); 
 Determine X as the number of pixels which correlates to 1000 μm by constructing a straight line across a scale bar. Correlate the number of pixels of the straight line with the length of the scale bar in μm.</li>
 <li>Open the result chart containing shape factor values for each image with a spreadsheet. Calculate a Morphology number as follows for each image. 
 <img alt="Morphology number formula using area, solidity, Feret's diameter, and aspect ratio in physics diagram." src="/files/ftp_upload/4023/4023eq1.jpg" data-altupdatedat="1787630408000" data-alt="Equation 1"></li>
 <li>Calculate mean value and standard deviation for the Morphology number taking into account all images of one sample.</li>
 <li>Use a graphing and data analysis program for graphical correlation of the Morphology number with the specific productivity and determine the mathematical relation by mathematical regression.</li>
</ol>
6. Representative Results
Through addition of talc micro particles A. niger SKAn 1015 morphology is changed from a true pellet morphology to a dispersed or even mycelial morphology. Whereas pellet morphology is exhibited at standard conditions a mycelial morphology is created by supplementation of medium with 10 g/L of talc micro particles (Figure 4.). Concurrently the activity of β-fructofuranosidase increases around 3 fold 3-5. A supplementation of 1 or 3 g/L of talc powder leads to a dispersed morphology, with a doubled fructofuranosidase activity (Figure 4.).
The micro particle dependent morphology can be comprehensively described by the Morphology number which can be calculated using parameters determined by automatic image analysis. Perfectly round and smooth pellets will in microscopic images appear as perfect circles. For such particles the Morphology number has a value of 1. The smallest fragment of mycelial morphology can be simplified as a one-dimensional line yielding a Morphology number of 0. All intermediate morphological forms like elongated irregular pellets or clumps will therefore have values between 0 and 1. Fairly large particles will result in a high, fungal particles with a large surface or elongated particles, in a rather low Morphology number 11.
At standard conditions the morphology in reactor 1 exhibits a Morphology number around 0.8. The morphology in reactor 4 with 10 g/L talc powder features a Mn around 0.1. The Morphology number for reactors 2 and 3, with talc powder concentrations of 1 and 3 g/L, lies between these extremes, demonstrating a dispersed morphology. Since micro particle dependent morphology is closely related with the β-fructofuranosidase productivity, a mathematical correlation of Morphology number and productivity similar to Figure 5 is obtained.
<img alt="Bioreactor process diagram; dry weight, enzyme activity, microscopy; timepoints 24-72h." src="/files/ftp_upload/4023/4023fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Overall scheme of the experimental design and the analytic procedure. A. niger is cultivated (with or without micro particles) in an 3 L stirred tank bioreactor for 72 h. After 24, 48 and 72 h a sample is taken for determination of biomass dry weight and β-fructofuranosidase activity, which again are used for calculation of the specific productivity. After 48 h the fungal morphology is examined by microscope and characterized by digital image analysis. Relevant parameters of image analysis are combined to a Morphology number, which is mathematical correlated with the specific productivity.
<img alt="Image processing workflow diagram: acquisition, improvement, binarization, analysis using ImageJ." src="/files/ftp_upload/4023/4023fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Steps of image processing for microscopic-generated images of morphological structures from A. niger. Step 1: image acquisition by microscope. Step 2: image improvement if necessary. Step 3: image binarization, black-and-white (binary) image generated in ImageJ. Step 4: the binary image is processed by and unwanted object are cleared. Step 5: morphological analysis is conducted with the "Analyze particles" function of the open source program ImageJ.
<img alt="Microparticle talc effect on samples; optical microscopy; 6 μm particles; morphology analysis." src="/files/ftp_upload/4023/4023fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Different morphological forms of A. niger dependent on the concentration of added micro particles. With increasing concentration of micro particles added the pellet size can be precisely decreased down to small core-shell pellets, small flocks and even freely dispersed mycelium. Morphology engineering of Aspergillus niger SKAn1015 by micro particle supplementation in submerged culture. Without microparticles (A), 10 mg/L (B), 0.1 g/L (C), 0.2 g/L (D), 0.3 g/L (E), 0.6 g/L (F), 1.0 g/L (G), 1.5 g/L (H), 2.0 g/L (I), 2.5 g/L (J), 3.0 g/L (K), 3.5 g/L (L), 4.0 g/L (M), 4.5 g/L (N), 5.0 g/L (O), 10 g/L (P), 15 g/L (Q), 20 g/L (R), 30 g/L (S) and 40-50 g/L (T). Images were taken by light microscopy after 72 h of cultivation.
<img alt="Bar graph of fructofuranosidase activity vs. talc concentration, showing increased enzyme activity." src="/files/ftp_upload/4023/4023fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. Fructofuranosidase activity in dependence of talc micro particle concentration 1 g/L (reactor 2), 3 g/L (reactor 3) and 10 g/L (reactor 4). Reactor 1 is not supplemented with micro particles; here the cultivation is conducted under standard conditions.
<img alt="Specific productivity vs. morphology number graph; A. niger SKAn 1015; exponential decay fitting." src="/files/ftp_upload/4023/4023fig5.jpg" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
Figure 5. Representative good correlation (R2 = 0.91) of the Morphology number and the specific productivity. The Morphology number is plotted (abscissa) against the specific productivity (ordinate). Nonlinear regression yields the exponential correlation.

<ol><li>Wucherpfennig, T., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWucherpfennig%2C+T+%22Advances+in+Applied+Microbiology%22">Advances in Applied Microbiology</a>. 72, Academic Press. 89-136 (2010).</li>
<li>Driouch, H., Hänsch, R., Wucherpfennig, T., Krull, R., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+enzyme+production+by+bio-pellets+of+Aspergillus+niger%3A+Targeted+morphology+engineering+using+titanate+microparticles%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">Improved enzyme production by bio-pellets of Aspergillus niger: Targeted morphology engineering using titanate microparticles.</a> Biotechnology and Bioengineering. 109, 462-471 (2012).</li>
<li>Driouch, H., Roth, A., Dersch, P., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+bioprocess+for+production+of+fructofuranosidase+by+recombinant+Aspergillus+niger%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">Optimized bioprocess for production of fructofuranosidase by recombinant Aspergillus niger.</a> Applied Microbiology and Biotechnology. 87, 2011-2024 (2010).</li>
<li>Driouch, H., Roth, A., Dersch, P., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Filamentous+fungi+in+good+shape%3A+Microparticles+for+tailor-made+fungal+morphology+and+enhanced+enzyme+production%5BTitle%5D)+AND+%22Bioengineered+Bugs%22%5BJournal%5D)">Filamentous fungi in good shape: Microparticles for tailor-made fungal morphology and enhanced enzyme production.</a> Bioengineered Bugs. 2, 100-104 (2011).</li>
<li>Driouch, H., Sommer, B., Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphology+engineering+of+Aspergillus+niger+for+improved+enzyme+production%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">Morphology engineering of Aspergillus niger for improved enzyme production.</a> Biotechnology and Bioengineering. 105, 1058-1068 (2010).</li>
<li>Kaup, B. -A., Ehrich, K., Pescheck, M., Schrader, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microparticle-enhanced+cultivation+of+filamentous+microorganisms%3A+Increased+chloroperoxidase+formation+by+Caldariomyces+fumago+as+an+example%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">Microparticle-enhanced cultivation of filamentous microorganisms: Increased chloroperoxidase formation by Caldariomyces fumago as an example.</a> Biotechnology and Bioengineering. 99, 491-498 (2008).</li>
<li>Hirayama, M., Sumi, N., Hidaka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+characterization+of+a+fructooligosaccharide-producing+beta-fructofuranosidase+from+Aspergillus+niger+ATCC+20611%5BTitle%5D)+AND+%22Agricultural+and+Biological+Chemistry%22%5BJournal%5D)">Purification and characterization of a fructooligosaccharide-producing beta-fructofuranosidase from Aspergillus niger ATCC 20611.</a> Agricultural and Biological Chemistry. 53, 667-673 (2006).</li>
<li>Rajoka, M. I., Yasmeen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+productivity+of+beta-fructofuranosidase+by+a+derepressed+mutant+of+Aspergillus+niger+form+conventional+and+non-conventional+substrates%5BTitle%5D)+AND+%22World+Journal+of+Microbiology+and+Biotechnology%22%5BJournal%5D)">Improved productivity of beta-fructofuranosidase by a derepressed mutant of Aspergillus niger form conventional and non-conventional substrates.</a> World Journal of Microbiology and Biotechnology. 21, 471-478 (2005).</li>
<li>Zuccaro, A., GÃ¶tze, S., Kneip, S., Dersch, P., Seibel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tailor-made+fructooligosaccharides+by+a+combination+of+substrate+and+genetic+engineering%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">Tailor-made fructooligosaccharides by a combination of substrate and genetic engineering.</a> ChemBioChem. 9, 143-149 (2008).</li>
<li>Huggett, A. S. G., Nixon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+glucose+oxidase%2C+peroxidase%2C+and+o-dianisidin+in+determination+of+blood+and+urinary+glucose%5BTitle%5D)+AND+%22The+Lancet%22%5BJournal%5D)">Use of glucose oxidase, peroxidase, and o-dianisidin in determination of blood and urinary glucose.</a> The Lancet. , 270-368 (1957).</li>
<li>Wucherpfenning, T., Hestler, T., Krull, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphology+engineering+-+Osmolality+and+its+effect+on+Aspergillus+niger+morphology+and+productivity%5BTitle%5D)+AND+%22Microb.+Cell+Fact%22%5BJournal%5D)">Morphology engineering - Osmolality and its effect on Aspergillus niger morphology and productivity.</a> Microb. Cell Fact. 10, (2011).</li>
<li>Rasband, W. S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRasband%2C+WS+%22ImageJ%22">ImageJ</a>. , National Institutes of Health. Bethesda, Maryland, USA. (1997).</li>
<li>Papagianni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+the+fractal+nature+of+mycelial+aggregation+in+Aspergillus+niger+submerged+cultures%5BTitle%5D)+AND+%22Microbial+Cell+Factories%22%5BJournal%5D)">Quantification of the fractal nature of mycelial aggregation in Aspergillus niger submerged cultures.</a> Microbial Cell Factories. 5, 5(2006).</li>
</ol>

I have nothing to disclose.

The modification of fungal morphology has been of interest in biotechnology since many decades. Different studies have tried to vary selected process parameters such as pH value, power input, temperature, medium nutrients or inoculum concentration 1, but suffer from rather imprecise and incomplete control of morphology, high energy costs, inhibition effects or product instability, In contrast, the supplementation of micro particles allows a precise engineering of fungal morphology through fine-tuned variation of particle size and concentration. This opens new possibilities to use micro particles for optimization and for tailor-made design of high producing morphology in biotechnological production with A. niger and other filamentous microorganisms.
The digital image analysis is an easy reproducible method to characterize fungal macro morphology. However, the variety of parameters for size, shape and surface character of morphological structures described in literature makes quick assessment of fungal morphology complicated. The presented Morphology number as a combination of relevant parameters, avoids this deficiency and can be used not only for comprehensive characterization of morphological structures but also for direct mathematical correlation with productivity. This again renders an estimation of productivity by given morphology and therefore a customization of morphology for process needs possible.
Using the Morphology number, it is possible to distinguish between various pellet and clump morphologies 4,5. For further development of the Morphology number the consideration of the fractal dimension seems to be promising. A fractal dimension gives a measurement of the complexity and mass filling properties of an object 13 and is therefore predestinated for holistic characterization of mycelial morphology.
The creation of a high producing mycelial morphology, however, might lead to problems with process performance especially in large scale cultivation, because the mycelial growth form has been previously shown to exhibit much greater culture broth viscosities 2. This leads to problems with heat and mass transfer and formation of stagnant non mixed zones, which require a higher power input and make the cultivation more expensive to operate1. Therefore the relationship between fungal morphology and culture broth viscosity should be considered when changing the morphology and be incorporated in further models.

customization of aspergillus niger morphology through addition of talc micro particles

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

The filamentous fungus A. niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most intriguing and often uncontrollable characteristics of this filamentous organism is its complex morphology. It ranges from dense spherical pellets to viscous mycelia (Figure 1). Various process parameters and ingredients are known to influence fungal morphology 1. Since optimal productivity correlates strongly with a specific morphological form, the fungal morphology often represents the bottleneck of productivity in industrial production.
A straight forward and elegant approach to precisely control morphological shape is the addition of inorganic insoluble micro particles (like hydrous magnesium silicate, aluminum oxide or titanium silicate oxide) to the culture medium contributing to increased enzyme production 2-6. Since there is an obvious correlation between micro particle dependent morphology and enzyme production it is desirable to mathematically link productivity and morphological appearance. Therefore a quantitative precise and holistic morphological description is targeted.
Thus, we present a method to generate and characterize micro particle dependent morphological structures and to correlate fungal morphology with productivity (Figure 1) which possibly contributes to a better understanding of the morphogenesis of filamentous microorganisms.
The recombinant strain A. niger SKAn1015 is cultivated for 72 h in a 3 L stirred tank bioreactor. By addition of talc micro particles in concentrations of 1 g/L, 3 g/L and 10 g/L prior to inoculation a variety of morphological structures is reproducibly generated. Sterile samples are taken after 24, 48 and 72 hours for determination of growth progress and activity of the produced enzyme. The formed product is the high-value enzyme &beta;-fructofuranosidase, an important biocatalyst for neo-sugar formation in food or pharmaceutical industry, which catalyzes among others the reaction of sucrose to glucose 7-9. Therefore, the quantification of glucose after adding sucrose implies the amount of produced &beta;-fructofuranosidase. Glucose quantification is made by a GOD/POD-Assay 10, which is modified for high-throughput analysis in 96-well micro titer plates.
Fungal morphology after 72 hours is examined by microscope and characterized by digital image analysis. In doing so, particle shape factors for fungal macro morphology like Feret's diameter, projected area, perimeter, circularity, aspect ratio, roundness und solidity are calculated with the open source image processing program ImageJ. Relevant parameters are combined to a dimensionless Morphology number (Mn) 11, which enables a comprehensive characterization of fungal morphology. The close correlation of the Morphology number and productivity are highlighted by mathematical regression.

A method to precisely generate and to comprehensively characterize morphology of filamentous fungus Aspergillus niger is described, which allows the mathematical correlation of morphological appearance and productivity.

The filamentous fungus A. niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most ...

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.

customization-aspergillus-niger-morphology-through-addition-talc

Research

JoVE Journal

Immunology and Infection

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.  Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Here, we describe a simple and inexpensive technique for inoculating and observing arbuscular mycorrhizal fungi in superabsorbent polymer-based autotrophic systems.

Inoculating and Observing Arbuscular Mycorrhizal Cultures on Superabsorbent Polymer-Based Autotrophic Systems

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Learn to customize Aspergillus niger morphology using talc microparticles in bioreactor cultivation. This method enables precise correlation between fungal morphology and productivity through image analysis.

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

Explore More Videos