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Outline • Introduction:
Pollution, a worldwide problem, Canadian context Bioremediation with fungi (mycoremediation) Examples of pollutants degraded by fungi, mechanisms Mycelial organization White rot fungi
• Methods: Fungal inocula, wood substrates and Microtox • Results: Reduction in toxicity • Conclusions, further work
Photo credit: Julia Kilpatrick © The Pembina Institute; www.pembina.org
Bitumen operation in Alberta, Canada
Mycoremediation
• In bioremediation we use the natural processes of biodegradation for toxin removal
• Apart from fungi, bacteria, plants and algae are used for remediation
• Mycoremediation is bioremediation with fungi
Why fungi? • There is an excessive amount of data in the literature that show the biodegrada8on capaci8es of fungi*.
• Fungal nutri8on depends on the release of enzymes by the mycelium into the substrate. Those fungal enzymes are able to degrade many pollutants.
• Fungi are organized as mycelia which provide flexibility and resilience not found in any other organism group.
* Reviewed in Singh H (2012) Mycoremedia8on. Wiley & Sons, Hoboken, USA
Fungi degrade numerous hazardous substances • DDT • PCBs (polychlorinated biphenyls) • PAH (polycyclic aromatic hydrocarbons) • pesticides • dioxin • chlorophenols • explosives
Mechanisms of toxin biodegradation • Various mechanisms of detoxication exist:
Hydrolysis, dehalogenation, ether cleavage, methylation, hydroxylation, deamination and others
• Many basidiomycetes use enzyme systems such as phenoloxidases. There is no uniform chemical mechanism of degradation for these enzymes.
• It is suggested that free radical ions are formed* which attack various
chemical bonds *Harvey PJ and Thurston CF. 2001. The biochemistry of ligninoly8c fungi. In Fungi in bioremedia/on, Gadd GM, ed. Pp. 27-‐51. Cambridge
University Press, Cambridge, UK.
Complex chain of degradation reactions • Ideally bioremediation would be a complete mineralization.
E.g. all carbon would be released as CO2
• In reality bioremediation is usually a detoxication
• Bioremediation needs to avoid activation
Image source: Wikimedia Commons
Ø Mycelia are mul8cellular Ø Mycelia are able to grow around barriers Ø Mycelia avoid areas with adverse condi8on Ø Mycelia can quickly reallocate resources (i.e. more mycelium) to areas
with preferen8al condi8ons (e.g. high amount of nutrients) Ø Mycelia are able to recycle dead hyphae
Mycelia
Positive enzyme test with syringaldazine confirms the presence of laccase
Enzyme produc2on by fungal mycelia
Basidiomycetes: white rot fungi White rot fungi are a group of basidiomycetes that are able to degrade lignin with phenoloxidases. The unspecific nature of the degrada8on mechanism explains their ability to degrade pollutants in addi8on to lignin.
Structure of the lignin molecule
Image source: © Karol Głąb / Wikimedia Commons / CC-‐BY-‐SA-‐3.0 / GFDL hap://commons.wikimedia.org/wiki/File:Lignin_structure.svg
Basidiomycetes: white rot fungi
There are few thousand species of fungi in Alberta. Each fungal strain has specific and unique enzyme systems.
We use pure cultures, isolated directly from fruitbodies
Methods
• Isolation of fungal cultures • AU Bioresource collection • Choice of wood substrates • Microtox method for measuring pollution
Fungal cultures are stored in slants and refrigerated. Currently we have more than 100 strains of fungi, most of them are white rot basidiomycetes.
AU Bioresource collec2on
Wood dowels
Choice of wood substrates
Toothpicks
Popsickle s8cks
Wood substrates are the most promising choice for in situ applica8on
Methods
• We measured toxicity of crude oil, phenol and naphtol over time in aquatic systems using wood based fungal inocula
• Various basidiomycete strains (e.g. Ganoderma, Pleurotus, Trametes, Bjerkandera) were used
• Toxicity was measured with the Microtox method
Inoculation units
We started our experiments with aquatic systems, measuring toxicity over 10-14 days.
Microtox method • Microtox u8lizes the bioluminescent bacterium Vibrio fischeri • The bacteria are very sensi8ve to environmental pollu8on • Toxins impact bacterial metabolism and immediately result in a reduc8on of light emission
• Toxicity is measured as IC50 which represents a 50% reduc8on of bacterial light emission
• Drawback: the nature of the pollutants is not known • Advantage: sensi8vity to a large number of pollutants, therefore useful in situa8ons where a mix of various toxins occurs
Results
• Typical examples for the effect of fungal mycelia on polluted aqua8c systems (phenol)
• IC50 was converted to toxicity units: 1TU = 100/IC50
• Toxicity was consistently reduced aher 10-‐14 days for all tested strains but in some cases there was an ini8al increase in toxicity
Summary of results
• Toxicity was consistently reduced aher 10-‐14 days of exposure in all fungal strains
• A few fungal strains caused an ini8al, temporary increase in toxicity
• There are considerable differences in efficiency between fungal taxa
• Efficiency of toxin degrada8on depends on the age of the fungal inocula
Conclusions and further work • Microtox is a sensitive method for application in
mycoremediation
• Due to the considerable differences between fungal strains it will be crucial to screen as many species of fungi as possible
• We are currently moving into soil based systems using the same inocula
• Wood based systems are promising for in situ applications
Acknowledgements All the work was done by:
Funding was provided by the Government of Denmark Mitacs, Canada CAPES, Brazil
Anni F. Holm Michele Berreta