MICR 306 Advanced Applications of Fungi

1. MICR 306 Advanced Applications of Fungi Prof. J. Lin University of KwaZulu-Natal Westville campus Microbiology Discipline 2013 School of Life Sciences

2. Theory Test 1 • Venue: T2 • Time: 2:10 pm – 4:40 pm

3. Fungi • Molds consist of long, branching filaments of cells called hyphae (singular, hypha). A tangled mass of hyphae visible to the unaided eye is a mycelium (plural, mycelia). Avascular -- no specialized respiratory, digestive or transport systems. • Yeasts are microscopic, unicellular fungi with a single nucleus and eukaryotic organelles. They reproduce asexually by a process of budding. (some are dimorphic.)

4. Replication Cycle

5. Unique Characters • Fungi grow best where there is a rich supply of organic matter.(lack photosynthsis) • Usually found as opportunistic saprophytes(living on dead organic matter) or in some parasitic or symbiotic relationship with plants or other autotroph. (chemoheterotrophic)

6. Major Roles in Ecology • Along with bacteria, fungi are the major decomposers in most terrestrial (and some aquatic) ecosystems, and therefore play a critical role in biogeochemical cyclesand in many food webs. • Fungi are versatile organisms that feed on a variety of substrates. • Digest food outside their bodies release enzymes into the surrounding environment (exoenzymes).

8. Biotechnology • Organisms (bacteria, fungi, viruses) with desirable properties • Environments – Conditions • Enrichment process • Competitive exclusive principle

9. Applications • Agricultural Biotechnology • Industrial Applications • Biotechnological Processes Using Fungi • Use of Fungi as Expression Hosts • Agaric farming (pharming) for production of human proteins • Environmental Biotechnology • Use of fungi for insect biological control • Food and Feeds Industry

10. Agricultural Biotechnology • A Biotechnological Approach to Plant Protection with Fungi  Increase the biomass production, better health and better resistance • Fungi as Plant Growth Promoter and Disease Suppressor • Challenges and Strategies for Development of Mycoherbicides • The Biological Control Agent Trichoderma: Fundamentals to Applications • Control of Nematodes by Fungi • Insectside

11. SYMBIOTIC RELATIONSHIP WITH PLANTS

12. Plant growth mechanisms can be grouped as follows: • Direct-like asymbiotic fixation of atmospheric nitrogen, solubilization of mineralssuch as phosphates; • Production of plant growth regulatorslike auxins, gibberellins, cytokinin and ethylene; • Indirect- like HCN production, antibiotics, siderophores, synthesis of cell wall enzymes, and • Competitions with detrimental microorganisms for sites on plant roots.

13. Organic Matters • Filamentous fungi in soil  degrade organic matter and help in soil aggregation  Humic substances. (species of Alternaria, Aspergillus, Cladosporium, Dematium, Gliocladium, Helminthosporium, Humicola and Metarhizium)  maintenance of soil organic matter. • Producers of organic acids particularly black Aspergilli and some species of Penicillium.

14. Acquisition of phosphates • Fungi capable  forming ectotrophic associations on the root system of tree (Boletus and Lactarius) mobilization of soil phosphorus and nitrogen into plants • Fungi have been reported to possess greater ability to solubilize insoluble phosphate than bacteria. (Aspergillus spp., Penicillium spp., and Fusarium spp.)

15. Acquisition of Iron • High affinity iron transport system termed as Siderophore • The majority of fungal siderophores are hydroxamates, apart from the carboxylate-type siderophore rhizoferrin produced by Zygomycetes. Most species of the genus Aspergillusare known to produce several hydroxamate type siderophores.

16. Role of Fungi as PGP • Some rhizosphere fungi able to promote plant growth upon root colonization are functionally designated as 'plant-growth-promoting-fungi’ (PGPF). PGPF belong to genera Penicillium, Trichoderma, Fusarium and Phoma.

17. Phytohormones • Phytohormones such as indole-3-acetic acid (IAA), cytokines, gibberellins (GA) and other plant growth promoting substances  enhance the hosts' uptake of nutritional elements such as nitrogen and phosphorous. • Some to trigger systemic resistance against various pathogens in cucumber plants.

18. Biofertilizers • Microbes involved in these formulations not only mobilize N and P but also secrete various growth promoting and health promoting substances. In broad sense, the term 'biofertilizer' may be used to include all organic resources for plant growth, which are rendered in an available form for absorption through microorganisms or plant association or interactions. • Arbuscular mycorrhizal fungi (AMF) -- alleviate some nutrient deficiencies, improve drought tolerance, overcome the detrimental effects of salinity and enhance tolerance to pollution.

19. Industrial Applications (1) • Antibiotics • Aspergillus, Penicillium and actinomycetes • Cephalosporin from Cephalosporium spp. Penicillin and Cephalosporin are antibacterial against Gram (-) bacteria • Griseofulvin from Penicilliumgriseofulvum antifungal antibiotic useful in treating dermatophyte infections • Lentinan from Lentinus sp. against Mycobacterium tuberculosis, Listeria sp. and Herpes Simplex Virus • Schizophyllan from Schizophyllum commune. antibacterial and antifungal activity  controlling Candida albicans and Staphylococcus aureus.

20. -Lactams biosynthetic pathway Secondary Metabolic Products

21. Non-antibiotic Therapeutics • Cyclosporin is an important immunosuppressant drug that is used in organ transplantation surgery • Lovastatin from Aspergillus terreus & Pravastatin from Penicillium citrinum are cholesterol biosynthesis inhibitors. • Vitamin B12 (Saccharomyces cerevisiae) and other vitamins (S. cerevisiae, Ashbya sp., Blakeslea sp.), hallucinogens (Psylocybe sp.), and steroids useful in fertility regulation (Rhizopus spp.).

22. Industrial Applications (2) • Enzymes Use of Enzymes for different purposes Food 45 % Detergent 34 % Textile 11 % Leather 3 % Pulp/paper 1 % Others 6 % Bio-fuel productions • ENZYME SOURCE • Acid, alkaline & neutral proteases Aspergillusoryzae; A. niger; A. flavus; A. sojae • CellulaseTrichodermakoningi • Diastase Aspergillusoryzae • GlucoamylaseAspergillusniger; A. oryzae • InvertaseSaccharomycescerevisiae • Lactase S. lactis; Rhizopusoryzae • LigninasePhanerochaetechrysosporium • Lipase Rhizopus spp. • Pectinase (flavours, clear) A. niger; Sclerotinialibertina

23. High-fructose corn syrup • Cornstarchis treated with α-amylase to produce shorter chains of sugars called oligosaccharides. • Glucoamylase - which is produced by Aspergillus, in a fermentation vat — breaks the sugar chains down even further to yield the simple sugar glucose.(Starch  glucose) • Xylose isomerase (aka glucose isomerase) converts glucose to a mixture of about 42% fructose and 50–52% glucose with some other sugars mixed in. • Glucose  insulin release; Fructose  X(sweetness)

24. Organic acids from Fungi Organic acid Source Citric acid Aspergillus niger Fumaric acid Rhizopus nigricans Gluconic acid Aspergillus niger - Food additive, acidity regulator, cleaning products Itaconic acid A. terreus - Paper and architectural coating industry Kojic acid A. oryzae - food and cosmetics (preserve color; anti-bacteria)

25. Biotechnological Processes Applications • Fungi are chemo-organotrophs and therefore require fixed organic compounds for their carbon and energy supply. Carbohydrates  Glucose ( metabolic pathways) most abundant renewable  Biomolecules energy source  Fermentation  Sugar (fructose, syrups) Alkane  acetyl-coenzyme A (Acetyl-coA)  TCA  Energy/bio-molecules

26. Plant Tissues

28. Cellulose  Glucose • Endoglucanases hydrolyze internal bonds of amorphous cellulose, cellodextrins and cellulose derivatives. This releases new terminal ends and cellobiose which consists of two glucose units. • Cellobiohydrolases are exo-1,4--glucanases that act on both amorphous and crystalline cellulose as well as endoglucanase-generated chain ends to release cellobiose. • -Glucosidasebreaks down cellobiose into two glucose molecules that serve as easily metabolisable carbon source for fungi.

29. Xylan  Xylose • Endoxylanases are endo-acting enzymes that randomly hydrolyze the xylan backbone to produce a mixture of xylooligosaccharides. • -Xylosidases are exozymes that liberate single xylose units from xylooligosaccharides. Xylose is then catabolized through the xylitol pathway in filamentous fungi and through the phosphoketolase pathway in yeasts. • -l-arabinofuranosidase, -d-glucuronidases, Acetyl xylanesterases, Feruloyl and coumaroylesterases. Thermomyceslanuginosus

30. Mannans • The mannans consisting mainly of the hexose mannose are similarly hydrolyzed by endo-1,4--mannanases, -mannosidase and accessory enzymes such as -galactosidase and -glucosidase, and esterases.

31. Ligninase • The chemical structure of lignin makes it resistant to attack by most microorganisms; however, several groups of fungi can utilize it as a source of carbon. • The major groups of lignicolous fungi are the white-rot and litter-decomposing Basidiomycetes. In addition, the brown-rotBasidiomycetes and certain soft-rot Ascomycetes and Deuteromycetes can partially degraded lignin.

32. Sources • Ruminomyces • White ants ……

33. Biofuel - Yeast

34. Leather productions • the heavy use of polluting chemicals in the tanning process (chromium) • air pollution due to the transformation process (dehairing H2S and deliming  NH4+). • Leather biodegrades slowly; it takes 25–40 years Solutions • Proteases hydrolyze non-structural proteins (casein, elastin, albumin and globulin) (bating). • Lipases  degreasing operation to hydrolyze fat particles. • Amylases  to soften skin (strength and flexibility)

35. Environmental Biotechnology • Cellulose Degradation by Fungi & Lignocellulose biodegradation by White Rot Fungi • The Importance of Wood Decay Fungi in Forest Ecosystems • Biomineralization of Heavy Metals  Bioleaching • Decolouration of Industrial Waste and Degradation of Dye Water, Azo Dyes & Bioconversion of Distillery Waste By Fungi • Degradation of Hydrocarbons by Yeasts and Filamentous Fungi • Fungal Degradation of Explosives • Restoration of Mycorrhizae in Arid Ecosystems

36. Azo yellow dye Permethrin polychlorinated biphenyls (PCB) TNT

37. Examples • Biodegradation of Azo dye and Hydrocarbons Peroxidase of Penicillium crysosporium & Streptomyces sp. The filamentous fungi are also having role in degradation of toxic hydrocarbons. • Fungi in Hazardous waste remediation by its lignin degrading Enzymes of Pleurotus ostreatus • Biomineralization of Heavy Metals in the removal & recovery of heavy metals from wastewater and industrial effluents. Hg, Cu, Ni, Pb, Cd are extracted at pH 2-5 by myceliar beads of Penicillium. (Bioflocculants, biosorption…)

38. Metabolism-independent accumulation The positively charged ions in the solution are attracted to negatively charged ligands in cell materials. Biosorption of metal ion occurs on microbial cell surface. (bioflocculants) But composition of biomass and other factors affect biosorption. For example, in Rhizopus arrhizus adsorption depends on ionic radius of Li3+, Mn2+, Cu2+, Zn2+, Cd2+, Ba2+, Hg2+ and Pb2+. However, binding of Hg2+, Ag2+, Cd2+, A13+, Ni2+, Cu2+ and Pb2+ strongly depends on concentration of yeast cells.

39. Metabolism-dependent accumulation In fungi and yeast, heavy metal ions are transported into the cells through cell membrane. However, as a result of metabolic processes ions are precipitated around the cells, and synthesized intracellularly as metal-binding proteins. Energy-dependent uptake of Cu2+, Cd2+, Co2+, Ni2+, Zn2+ by fungi has been demonstrated. Moreover, intracellular uptake is influenced by certain external factors such as pH, anions, cations and organic materials, growth phase, etc. Metal uptake by growing batch culture was found maximum during lag phase and early log phase in Aspergillus niger, Penicillium spinulosum and Trichoderma viride.

40. Limitations and technical challenges for application • Sensitivity to biological process operations • The fungus does not grow well in a suspended cell system. • Enzyme induction is negatively affected by mixing actions and the ability of the fungus to effectively attach itself to a fixed medium is poor. • The majority of the research on fungal performance has been conducted on autoclaved soil or on synthetic media.  In situ? low competitive capabilities in the environment

41. Biological Control Agents • Biological control is the use of biological organisms, or their by-products, to control pests. Biocontrol is popular in theory, because of its potential to be host-specific virtually without non-target effects.  predators, parasitoids, and pathogens

42. Understanding and Preventing Disease • Holistic

43. Classical biological control • Classical biological control is the use of natural enemies against a host which is exotic in an area and has established without its full guild of natural enemies. This approach has been used successfully to control a wide variety of pests including larvae of the gypsy moth, aphids and rabbits.

44. Augmentation • Augmentation is based on the knowledge or assumption that in some situations there are not adequate numbers or species of natural enemies to provide optimal biological control, but that the numbers can be increased (and control improved) by releases. This method involves the addition of in vitro-produced mycelia or conidia, (genetic modified) viruses in aqueous suspensions to a field or glass house crop often in combination with synthetic materials which are formulation components to enhance persistence and/or infectivity.

45. Augmentation • Augmentation involves the addition of in vitro-produced mycelia or conidia in aqueous suspensions to a field or glass house crop often in combination with synthetic materials which are formulation components to enhance persistence and/or infectivity.  Inundation  Inoculation

46. Inundation • Inundation involves the application of a biocontrol agent in large amounts, so that the pest can be rapidly removed. However, the biocontrol agent (fungus) is only active within a short period and no secondary infection is expected. • Hyphomycetes have great potential as inunadative biocontrol agents since they are relatively easy to mass produce and formulate for use with conventional spray application equipment .

47. Example • Verticillium lecaniiis used as an inundative mycoinsecticide in glasshouses in Europe for the control of aphids and related insects. • V. lecanii blastospores are produced and formulated with a nutrient source in a wettable powder which can be applied with a carrier such as aphid alarm pheromone or an adjuvant based on emulsifiable vegetable oil. The nutrient formulations allow satellite colonies to grow on leaf surfaces, increasing effective coverage 40-fold. The pheromone serves to attract the aphids and therefore facilitates widespread infections while oil based adjuvants promote adherence of the spores to the cuticle and enhance activity at low humidity.

48. Inoculation • Inoculative augmentation is the introduction of the biocontrol agent in small amounts early in the season of the crop with the expectation that it will repeatedly cycle (i.e. establish epizootics) in pest populations and spread over a period of time thereby maintaining the pest populations below the economic threshold.

49. Examples using viruses • Weeds control: Araujia mosaic virus vs Moth plant? • Pathogenic fungi control: Totivirusesagainst Fusarium • Rabbit population control: calicivirus, myxoma virus • Insect population control: baculoviruses • Rat control: synthetic pox viruses Advantages: specificity; natural present Disadvantages: Ecology?

50. Examples using fungi • Insect population control: Advantages: specificity; natural present Disadvantages: Ecology?