Bioremediation with fungal mycelia

Roland  Treu  

   

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  

Fungal mycelium (400 x)

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

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Isolation of fungal cultures

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

Simulation of aquatic pollution using wood based fungal inocula

Inoculation units

We started our experiments with aquatic systems, measuring toxicity over 10-14 days.

Microtox instrument

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  

Example 1: Pleurotus

Example 2: Bjerkandera

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  

Thank  you!      

Any  ques2ons?