Three-point inoculation

Historically, the study of fungi, mycology, was based on morphology. The color, the shape, the size and the spores of the fungus were used for the first categorizations of fungal species. Macroscopic morphology observations are still widely used for characterization of fungi, but as the morphology of a fungus varies depending on the physiological state and environment, a fungus is usually grown on several different growth media.

Inoculation of a fungus on agar plates is done using the technique called three-point inoculation, where a toothpick or inoculation loop containing fungal spores is poked into the agar in three different spots on the plate. Growth and sporulation were observed after 5 days of incubation at 30°C.

ATCC 1015 and the nine A. niger mutant strains were inoculated and grown on four different media.

Yeast Extract Peptone Dextrose (YPD)
YPD is a complex medium. The carbon source for the medium is glucose. It has a neutral pH. Complex media are preferred in the industry as they are cheaper than defined media as minimal media.

Transformation media (TM)
The medium is normally used for plating after transformation of A. niger. Its a minimal medium where the carbon source is sucrose. It has a very high concentration of 34.2%w/v, compared to other media types. This makes the media ideal for transformations. It has a neutral pH.

Creatine Sucrose agar (CREA)
The medium is a differential media used to characterize fungi. Its carbon source is sucrose and the nitrogen source is creatine. The medium contains bromocresol purple that serves as a pH indicator. It can therefor be used to test acid production. If acid is produced a yellow halo appears around the colonies.

Czapek Yeast Extract Agar (CYA)
The medium is used for cultivation of saprophytic fungi such as Aspergillus niger. Its carbon source is sucrose and contains both yeast extract and nitrate as nitrogen source. It has a neutral pH.

Microscopy

Morphological observations made with the bare eye can provide a wide palette of information about a fungus, but it only tells half the story. The macroscopic growth pattern of a fungus is highly interlinked and affected by the microscopic growth pattern. Microscopy is therefore an important and informative tool when analyzing morphology. It can be used to observe changes to the hyphal growth pattern, such as dispersed or pelleted hyphae, branching and tip growth/extension. The interconnected hyphae system is a way for the fungus to transport resources such as water and nutrients between its cells. The process of exocytosis further occurs at the apex of the hyphae. The ability to make predictions and gather information about the hyphal growth pattern, might provide information about the fungus' wellbeing as well as its ability to produce and secrete proteins. This would provide indications about its industrial relevance.

Spores from the reference A. niger strain, ATCC 1015, and from the nine mutants were grown for 24 hours on microscope slides in imaging spacer wells (Grace Bio-Labs SecureSeal imaging spacer) containing liquid YPD media with uridine at 30°C. Before microscope pictures were taken, calcofluor white stain, which binds to the chitin in the cell wall, was added. Pictures of the strains were taken with a confocal microscope (Leica SP8 confocal microscope).

Glucoamylase assay

This assay was conducted in order to determine the glucoamylase activity.

Glucoamylase is an enzyme (exoglucohydrolase) which can degrade starch and soluble oligosaccharides to beta-glucose. It is naturally produced by Aspergillus niger and therefore was a protein of interest quantifiable through this assay. Conducting this assay would therefore show how the secretion was affected in the new strains compared to the reference strain.

The assay was performed on the bioreactor samples from different time-points. It works by adding starch to the supernatant of the sample, which can be degraded by glucoamylase produced by the fungus. After a 10 minutes incubation at 37°C, an iodine reagent which can bind to starch polymers was added. When this reaction occurs, color will develop. This means if glucoamylase has been produced, it will degrade the starch, and there will thereby be less starch that the iodine reagent can bind to and thereby less of a color change will develop. The absorbance was measured at 580nm.

This assay was also performed for the signal peptide mutants. These mutants were grown on agar plates with starch added as carbon source. After 72 hours of growth, iodine reagent was added to the plates for 30 seconds. Yellow coloration on the plate indicates that starch has been degraded.

Bicinchoninic acid assay (BCA)

The morphology of a fungus can greatly impact the efficiency of protein production, positively as well as negatively. It has been hypothesized that hyperbranching might result in increased production. This can be explained on the basis of exocytosis occurring at the apex, so if the amount of apexes increases, this might also increase the possibility of increased protein secretion. The morphology mutants all have altered morphology which might have modified their protein profile and activity, which made this analysis relevant.

BCA is a protein assay based on bicinchoninic acid (BCA) which can determine the concentration of protein in a solution. In an alkaline environment, proteins are able to perform a reduction of Cu⁺² into Cu⁺. Cu⁺ cations are able to chelate two BCA molecules which results in the formation of a purple color that can then be measured at 562 nm. The more proteins which are present in the solution, the more intense the color will thus become.

Growth Measurements

We performed two types of measurements to characterize growth profiles and parameters. These where done using a BioLector and multiple bioreactors.

BioLector

The BioLector is a microbioreactor which can be used for high-throughput fermentations. It is possible to control the temperature, shaking speed and the humidity of the cultivation chamber while at the same time measuring absorbance at 620 nm. The experiment was set up with a 48-well flowerplate containing liquid YPD media with uridine and inoculated with the same concentration of spores in all the wells. It ran for 72 hours. The data provided an indication of the growth profile of the mutants compared to the reference strain (ATCC 1015). These profiles could be used to predict which of the morphological mutants would be the most interesting strains to grow in the bioreactors.

Bioreactors

Filamentous fungi, including Aspergillus niger, are widely used as cell factories as they are able to produce a broad spectrum of secondary metabolites, proteins, enzymes and organic acids. These compounds are produced by growing the fungi in large-scale fermentation tanks, where the environmental conditions can be manipulated and optimized in order to promote production of specific compounds.

The mutants created for this project were cultured in 1L bioreactors as a batch culture in order to try to predict how they would behave for large-scale industrial processes. The bioreactors were run with liquid YPD media with uridine with the following conditions:

Temperature: Set to 30°C
pH: Set to pH 3 for germination and changed to pH 5 after 16-18 hours
Stirring: Started at 100 rpm and increased gradually to 800 rpm after 12 hours
Airflow: Started at 0.01 vvm and increased exponentially to 1 vvm over 10 hours

The strains were cultured in the bioreactors until they reached the stationary phase. During the fermentation, CO₂ data was measured continuously, which can be used to define the growth profile. From the collected data it was possible to measure and characterize the growth kinetics, therein the lag phase, the exponential phase, and stationary phase, which is highly useful information considering that different compounds are produced in different growth phases. Around 8 samples were taken during growth for HPLC analysis, glucoamylase and BCA assays, and for microscopic pictures.

Team:DTU-Denmark/Measurement