Fungal Collection, Culturing and Identification

Fungi are Eukaryotic cells that are able to synthesize enzymes when required to break down complex mono and polysaccharides when in contact with it. There is much to understand with this fascinating Kingdom that the Fungi belong to as Biotechnology within these organisms may play a fundamental role within Industrial waste. There is much to study and learn from these organisms and possible outcomes of collecting, culturing and Identifying these complexed organisms as they may possibly resolve and revolutionize the waste industry to prevent damage and ecologically reverse damage to the planet.
Chapter 1: Introduction
Overview
1. Fungi
Are a large group of eukaryotic organisms that are classified as having its’ own recognized kingdom. This kingdom has an enormous variety that includes unicellular microorganism and multicellular organisms that are separate from the other kingdoms of life. It is Estimated anywhere between 1.5 to 5 million species exist. These unique organisms contain properties that produce fungal compounds that provides many possible applications within industry. General characteristics of fungi include but not limited to:

Cell wall consists of Chitin.
Fungi are heterotrophs meaning they are not able to produce their own food, but obtain Its’ nutrients by the absorption of its surroundings by the secretion of digestive enzymes.
In an ecological system, fungi are described as decomposer organisms.
It is now known that the DNA of fungi are more related to animals than plants.

These organisms are abundant worldwide, A majority of fungi are not clearly visible due to their small size, structure, and perplexed biological processes in soil or on dead matter.
2. Classification / Taxonomy
Taxonomy is a scientific method to group biological organisms on the grounds of characteristics and naming the organisms into groups or categories. Formally only 5% of Kingdom Fungi has been formally classified, methods of arrangement have been based on biological taxonomy based upon different characteristics such as type of hypha, spore, and reproduction process. This provides a template of key features to identify and class organisms in the form of a list. All concepts are man-made and to a certain degree are arbitrary.
3. Morphology
General:
Fungi displays itself in a wide variety of size and shape, from unicellular, microscopic organisms to more advanced multicellular forms that are able to be seen with the naked eye. Fungi cell size ranges from 1″ to 30″. Fungi that are microscopic are classified as molds, yeasts or both.
Molds:
Are larger multicellular organisms that contains a network of branching filaments. This is called hyphae. The two known types of hyphae are vegetative and reproductive hyphae. The reproductive hyphae contain spores (fungal spores are different from bacterial spores as bacterial spores are for the fundamental purpose of survival in harsh conditions as opposed to reproductive purposes for fungi). The spores produced from fungi are used for the role of classification and identification of fungi. The hyphae are described to contain a tube-like appearance, the hyphae and other structures combine to form an elaborate network called a mycelium.
Yeasts:
These single-celled organisms are large (5 to 8″) that rarely form filaments. Most yeasts undergo an asexual process referred to as budding. Colonies of yeast are usually described as having a surface considered to be smooth similar to many bacteria.
4. Physiology
Is the scientific observation of living systems in organisms, focusing on nutrition, reproduction, and growth.
Nutrition for most fungi contain enzymes that are complex with other chemical substances that diffuse from the fungi to break down complex substances that are available, e.g. wood, vegetation, bread etc. into simpler substances that is digested by fungi. The products of digestion are formed outside of the organism and the fungus absorbs the end products.
Reproduction for fungi is unique as organisms can reproduce sexually or asexually, or both depending upon various factors e.g. the species, the environmental factors etc. Sexual reproduction undergoes when two spores are compatible to reproduce with one another.
Growth for fungi are similar as to the growth of bacteria, favourable conditions involve warmth and moisture. As the temperature lowers, fungal activity also lowers. However, spores are very resistant to cold temperatures for long periods of time. At high temperatures, fungi are easily killed.

Figure 1: Typical mycelium structure of a fungus

Figure 2: Morphology and General Properties of Fungi.
Figure 2 describes the morphology of an individual colony of fungi growing in a Petri dish. Figure 2 can be used to establish and identify organisms in the fungi Kingdom.Each type of fungus will produce different colonies. Specific terminology is established to describe the different types of common colony forming organisms.

Size – The size of a colony is measured by the diameter.
Form – This establishes the basic shape of the colony in question e.g. if its circular, filamentous, etc.
Elevation – This establishes how to describe a colony from observing the colony at an angle.
Surface – This establishes how to describe the appearance of the colony whether if it is rough, wrinkled, smooth, etc.
Opacity- This describes how translucent the colony in question is e.g. transparent (clear), opaque etc.
Colour – Also referred to as pigmentation of the colony of whether if the colony has a distinguishable colour associated with the organism.

Identifying similarities between different fungal organisms allowing the organisms to be catalogued in a unique way for different Phyla to be established and recognizing different organisms to be closely related to one another depicted in figure 3 creating a cluster of analysis.

Figure 3 is a cluster analysis.
In Figure 3. The 26 sites cluster into four major groups from A to D. These sites have been clustered based on similarities of the fungal community that the organisms belong to.
4. Species
To Examine a group of fungi that would have similar traits. The filamentous fungi contain cells that grow as tubular, elongated, and thread-like structures. These structures are called hyphae, which may contain multiple nuclei and extend by growing at their tips. Each tip contains a set of aggregated vesicles – cellular structures consisting of proteins, lipids, and other organic molecules associated with fungi chemical compound production.
Penicillium
is a genus of ascomycetous fungi. This genus is of major importance in the food and drug industry. Some members of this genus produce the antibiotic penicillin, a molecule that is used to kill or inhibit the growth of certain kinds of bacteria inside the body. Other species are used in cheese making. The characteristics to expect typically consists of a highly-branched network of multinucleate, septate, usually colourless hyphae. The mycelia contain many-branched conidiophores, the conidiospores are the main route of dispersal of the fungi, and often are green in colour.
Aspergillus
Is a genus a genus that is recognized as a mould species found in various climates worldwide. This genus is known as a group of conidial fungi, which in turn cause this group to be in an asexual state. Members of the genus possess the ability to grow where a high osmotic concentration (high sugar, salt, etc. exists. The species are highly aerobic and are widely found where oxygen-rich environments on the surface of substrates. Commonly grown on carbon-rich substrates like monosaccharides (e.g. glucose) and polysaccharides (e.g. amylose). Starchy foods are generally contaminated by this genus.
Talaromyces
Is a genus of fungi, described as species in the genus form soft, cottony fruit bodies with cell walls with an intricate network of hyphae. The fruit bodies are often described with yellowish characteristics or are surrounded by yellowish granules.
Rhizomucor
Is a genus of fungi that are described to be thermophilic, with a minimum growth temperature of 20° C with a maximum growth temperature of 60° C.
Trichoderma
Is a genus of fungi that is present in all soils, they are most prevalent culturable fungi as they are fast growing at 25 – 30° C, colonies are transparent at first on media such as cornmeal dextrose agar (CMD) or white on richer media such as potato dextrose agar (PDA). Conidia typically form within a week in shades of green or yellow pigment. A yellow pigment maybe secreted into the agar, especially on PDA. Industrial applications involve enzyme production for cellulose, xylanase and chitinase.
Fusarium
Is a genus of filamentous fungi. Its’ industrial application is used in human consumption. This genus is produced and marketed under the name Quorn.
5. Culturing
Organisms of interest will be cultured in the lab and Identified accordingly on the characteristics they exhibit by Classical Methods that rely on direct observation of fungi, including microscopy as well as direct sampling of fungal fruiting bodies, incubation of substrata in most chambers, culturing of endophytes and particle plating.
6. Laboratory Methods
Are used to collect, culture, and identify organisms in the lab with the following processes of Aseptic Technique, Microscopy, and biochemical analysis.
7. Aseptic Technique
Is a critical method to successfully observe fungal growth and distinct characteristics with the prevention of microbial contamination. This technique is carried out in a variety of ways. The use of a Bunsen burner when dealing with petri dishes to keep the laboratory environment as clean as possible and to prevent possible contamination, as well as sterilising inoculation loops when inoculating fungal organisms. A disinfectant such as Distil is utilized to ensure that the work surfaces and equipment are free from possible contamination. It is also encouraged to flame any containers where appropriate before and after use. This is to prevent any unwanted organisms being introduced in the lab when observing the interested fungal organisms with the interest to preserve fungal purity.
8. Microscopy
Classical Methods of identification of fungal organisms are examined under a microscope to identify specific characteristics within fungi. Specific families of fungi are able to by morphologically identified by colourless hyphae that would be presented on organisms. Others are able to be characterized by dark colonies and pigmented fungal elements seen on microscopic examination of fungal material that was prepared by biopsy. Fungi that has the ablity to infect hair and nails; often displays fluffy or fine texture and are pale coloured with distinct hyphae.
Biochemical Tests
Are laboratory methods to Identify fungal organisms. These include carbohydrate fermentation, this tests whether a certain yeast ferments different carbohydrates. Carbohydate assimilation studies: this tests whether a yeast can aerobically utize various carbon and nitrogen compounds with oxygen. API 20C system involves freeze-dried sugars are placed into wells on a plstic strip, yeast medium is pipetted into wells, incubated and turbidity determines growth. Uni-yeast tek system Identifies yeasts based on their ablity to ferment certain sugars.
Media
Selecting the correct media for optimal fungal growth in the lab is essential. Sabouraud’s Dextrose agar (SDA) is sufficient for culturing fungi as it’s rich in nutrients to support fungal growth and is a preferred medium.
Enzymes
Enzymes play a fundamental role in fungal extracellular activity. Enzymes are synthesized inside the cell and are secreted outside the cell. The main function of these enzymes involve the breakdown of complex macromolecules into smaller units to be digested by the cell for growth and assimilation. Organic matter such as cellulose are degraded into simple sugars that enzyme-producing organisms use as a source of carbon, energy, and nutrition. Enzymes are categorized in the following: hydrolases, lyases, oxidoreductases and transferases. These enzymes breakdown complexed compounds in specific ways for the cell to take up nutrients. Fungi are widely diverse giving this kingdom the
Application in Industry
Microorganisms produce enzymes, with current understandings of cell knowledge and recombinant protein, it is possible to modify the cell to produce enzymes with more efficient proderties e.g. better yield and purity. For saccharification of plant polysaccharides and biofuel production, fungi are valuable sources for enzyme production.
Enzymatic degradation of wastes.
Waste material is a fundamental factor in Industry. A majority of pharmaceutical, industrial, agricultural and forestation waste costs more to dispose of properly than sometimes the production. This results in several problems leading to environmental, health and safety issues that prevent sustainable development that could be dealt with in alternative ways for the aim to reduce waste. Current technology is investigating if Enzymes are able to play a fundamental role in waste degradation to prevent, reduce, reuse, and recycle pharmaceutical waste that would be considered costly to properly dispose of. Fungi is recognized to be a complexed eukaryotic cell that may hold the answers industry is looking for to improve these matters.
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