Environmental

 Microbiology  (BI  304):  Lecture  4  

Biogeochemical  Cycling  
Instructor:  Dr.  Nalina  Nadarajah  
Email:  nnadarajah@centennialcollege.ca  
Biogeochemical  Cycles  (Chapter  16)  
ObjecEve:  To  demonstrate  and  understand  the  carbon  and  
nitrogen  cycles,  including  their  significance  in  terms  of  the  
environment,  and  the  roles  of  microbes  in  each  cycle  

Agenda:  
–  IntroducEon  to  Biogeochemical  Cycles  
–  The  Carbon  Cycle  
–  The  Nitrogen  Cycle  

2  
Introduc:on  to  Biogeochemical  Cycles  
•  Biogeochemical  Cycle:  a  circuit  or  pathway  by  which  a  
chemical  element  or  molecule  moves  through  both  bioEc  
("bio-­‐")  and  abioEc  ("geo-­‐")  compartments  of  an  
ecosystem    

•  All  major  elements  found  in  biological  organisms  are  

cycled  

•  Understanding  these  cycles  allows  us  to  understand  and  

predict  the  development  of  microbial  communiEes  in  the  
environment  

 
3  
Chemical  Composi:on  of  an  E.  coli  Cell  
Elemental   %  dry  mass  of  an   E.g.  Func:on  
Breakdown   E.coli  cell  
Major  Elements  
Carbon   50   Building   blocks   of  
Oxygen   20   all  macromolecules  
Hydrogen   8  
Nitrogen   14   Proteins,  nucleic  acids  
Sulfur   1   Amino  acids,  vitamins  
Phosphorus   3   Nucleic  acids,  ATP  
Minor  Elements  
Potassium   2   OsmoEc  control  
Calcium   0.05   Cell  wall  stability  
Magnesium   0.05   Enzyme  cofactor  
Sodium   1   OsmoEc  control  
4  
 The  Gaia  Hypothesis  
•  Originated  by  James  Lovelock  in  1970’s  (co-­‐developed  by  
the  microbiologist  Lynn  Margulis)  

•  The  earth  is  a  super-­‐organism  and  can  respond  to  drasEc  

environmental  changes  

•  “Living  organisms  and  their  material  environment  are  

Eghtly  coupled.  The  coupled  system  is  a  super-­‐organism,  
and  as  it  evolves  there  emerges  a  new  property,  the  
ability  to  self-­‐regulate  climate  and  chemistry”  

5  
 Comparing  Planetary  Atmospheres  
Current  
Earth  w/o  
Gas   Venus   Mars   Earth  with  
life  
Life  
CO2   96.5%   95%   98%   0.03%  
N2   3.5%   2.7%   1.9%   78%  
O2   trace   0.13%   0.0%   21%  
Ar   70  ppm   1.6%   0.1%   1%  
CH4   0.0   0.0   0.0   1.7  ppm  
Surface  
459   -­‐53   290   14.8  
Temp.  (°C)  

•  According  to  the  Gaia  hypothesis,  development  and  

conEnued  presence  of  life  on  earth  is  responsible  for  the  
drasEc  changes  in  our  atmosphere  
6  
 How  Did  This  Change  Happen?  
•  Microbial  and  plant  acEvity  changed  the  heat-­‐trapping  CO2-­‐
rich  atmosphere  to  the  present:  O2  rich,  CO2  poor  one  

•  Allowed  the  earth’s  average  surface  temperature  to  

decrease  from  290oC  to  14.8oC    

7  
 Development  of  the  Carbon  Cycle  
•  ~  3.8  billion  years  ago,  organic  carbon  was  formed  by  large  
amounts  of  UV  light  reacEon  with  the  CO2  rich  atmosphere  
•  Organic  maeer  used  by  early  heterotrophs  
•  Followed  by  the  ability  of  microbes  to  fix  CO2  
photosyntheEcally  (~3.5  billion  years  ago)  
–  evidence  from  Stromatolites  
–  provided  a  mechanism  for  carbon  recycling  
•  ~  2.8  billion  years  ago  photosyntheEc  microbes  developed  
ability  to  produce  O2  
–  led  to  the  change  in  atmosphere  (accumulaEon  of  O2)  
–  the  development  of  the  ozone  layer    
–  the  development  of  higher  forms  of  life   8  
How  Do  Biogeochemical  Cycles  Relate  To  
The  Gaia  Hypothesis?  
•  Consider  the  basic  Carbon  cycle:  

Autotrophs

Organic
CO2
Carbon

Heterotrophs 9  
 If  Earth  is  a  Superorganism  
•  it  should  be  able  to  respond  to  the  environmental  
changes  
•  why  do  we  see  increasing  environmental  disasters  all  
over  the  world?  

10  
Global  Warming  and  Greenhouse  Gases  
•  The  troposphere  (Earth’s  lower  atmosphere,  up  to  15  km  
thick)  consists  of  a  blanket-­‐like  layer  of  gases  keeping  Earth  
warm  
•  TradiEonally  major  gases  contribuEng  to  heat  storage  were  
H2O  (67%)  and  CO2  (33%)  
•  But  during  last  century,  several  natural  and  syntheEc  gases  
in  troposphere  have  increased  –  resulEng  in  increased  heat  
trapping  
–  natural:  CO2,  methane  (CH4),  nitrous  oxide  (N2O)  
–  syntheEc:  chlorofluorocarbon  (CFC),  trichlorofluoromethane  
(CFC-­‐11;  freon;  CCl3F)  
11  
 Greenhouse  Effect  

Animation 12  
www.combatclimatechange.ie
Why  Biogeochemical  Cycles  Go  Wild?  
•  Remained  stable  for  millions  of  years  
•  Growing  need  for  food  and  energy  has  interfered  
–  formaEon  of  GH  gases    

•  Global  Warming  à  Climate  change  

13  
Why  Biogeochemical  Cycles  Go  Wild?  
•  Remained  stable  for  millions  of  years  
•  Growing  need  for  food  and  energy  has  interfered  
–  formaEon  of  GH  gases    
•  Major  greenhouse  gases  are  the  subject  to  the  Kyoto  Protocol  
–  CO2,  CH4,  N2O  and  three  groups  of  fluorinated  gases    
•  sulfur  hexafluoride  (SF6),  Hydrofluorocarbons  (HFC)  and  
perfluorocarbons  (PFC)  

30  
 Canada  and  the  Kyoto  Protocol  
•  Kyoto  Protocol  established  in  1997  
•  Canada’s  target:  6%  reducEon  in  GHG  by  2012  (compared  to  1990  
levels)  
•  between  1990  and  2008  –  Canada’s  GHG  emission  increased  by  24%  
•  change  of  government  a  factor  (Liberal  Govt.  in  1997;  ConservaEve  
Govt.  from  2006  -­‐  2015)  
•  dramaEc  rise  in  GHG  in  2007  

•  2011  UN  Climate  Change  Conference  in  Durban,  S.A.  –  Canada  

announced  its  formal  withdrawal  from  Kyoto  Accord  as  of  2012  
•  Minister  of  Environment  –  Peter  Kent  (2011):  "The  Kyoto  Protocol  
does  not  cover  the  world's  largest  two  emieers,  United  States  
and  China,  and  therefore  cannot  work"    
 Canada  and  the  Kyoto  Protocol  
•  Canada  has  the  world's  third-­‐largest  oil  reserves  
–  ConservaEve  government  is  reluctant  to  hurt  Canada's  
booming  oil  sands  sector  
•  Canada's  overall  greenhouse  gas  (GHG)  emissions  
–   oil  &  gas  and  transportaEon  
•  Canada  is  one  of  the  largest  per  capita  greenhouse  gas  polluter  
 Latest:  Paris  Agreement  
•  December  2015  in  Paris,  France  
•  195  countries  signed  –  including  USA,  China,  Canada  &  India  
(13  countries  (incl.  Russia,  Iran,  Iraq,  Turkey  remain  yet  to  
raEfy)  
•  Former  US  President  Donald  Trump  announced  U.S.  would  
cease  all  parEcipaEon  in  the  Paris  Agreement  
•  Current  US  President  Joe  Biden  has  requested  UN  on  
rejoining  the  Paris  Agreement  
 Latest:  Paris  Accord  
•  December  2015  in  Paris,  France  
•  195  countries  signed  –  including  USA,  China,  Canada  &  India  
–  On  June  1,  2017,  USA  announced  its  withdrawal  by  2020  

•  Canada  commieed  to  emissions  targets  of  17%  reducEon  

from  2005  levels  by  2020  and  30%  by  2030  
•  How  do  we  achieve  this  goal?  
–  carbon  tax  
•  Canada  will  impose  a  tax  on  carbon  emissions  starEng  in  2018    

–  cap-­‐and-­‐trade  
Why  Biogeochemical  Cycles  Go  Wild?  
•  Remained  stable  for  millions  of  years  
•  Growing  need  for  food  and  energy  has  interfered  
–  formaEon  of  GH  gases    
•  Major  greenhouse  gases  are  the  subject  to  the  Kyoto  Protocol  
–  CO2,  CH4,  N2O  and  three  groups  of  fluorinated  gases    
•  sulfur  hexafluoride  (SF6),  Hydrofluorocarbons  (HFC)  and  
perfluorocarbons  (PFC)  
•  AcEviEes  releasing  greenhouse  gasses:  
–  burning  of  fossil  fuels  and  deforestaEon  –  CO2    
–  manure  management,  paddy  rice  farming,  wetland  changes,  and  
covered  vented  landfill  emissions  –  CH4  
–  over  use  of  ferElizers  –  N2O   39  
Global  Atmospheric  Concentra:on  of  
Selected  GH  gasses  
Microbially  mediated  &  Anthropogenic  
Anthropogenic  Only    (ppt)  
(ppm)  

Sulfur  
CO2   CH4   N2O   CFC  
hexaflouride  (SF6)  

Pre-­‐industrial   278   0.700   0.275   0   0  

2004   377   1.789   0.319   5.22   794  

Atmospheric  
Life  Time     50-­‐200   12   114   3200   45-­‐100  
(in  years)  
40  
Source: Table 31.1 from Text
 Comparison  of  Various  Gases  on  

Source: Environmental Microbiology By Masden, E. Box 7.1, p.291

Greenhouse  Effect  
Type  of  Gas   Contribu:on  Rela:ve  to  CO2  to  GH  Effect  

CO2   1  

CH4   25  

N2O   200  

Ozone   2  000  

CFC-­‐11  (freon)   12  000  

CFC-­‐12   15  000  

41  
 Carbon  Cycle  
•  Reservoir:  sink  or  source  of  an  element  
•  Global  carbon  reservoirs  include    
–  carbonate  rock  in  the  earth’s  crust  (1.2  x  1017  metric  tons)  
–  DOM  &  POM  in  oceans  (2.1  x  1012  metric  tons)  
–  CO2  in  the  atmosphere  (6.7  x  1011  metric  tons)  
•  The  atmospheric  reservoir  is  most  accessible  and  most  
ac:vely  cycled  
•  This  cycle  is  parEcularly  sensiEve  to  human  acEvity;  the  
last  100  years  has  seen  a  28%  increase  in  atmospheric  CO2  
•  This  increase  is  responsible,  in  part,  for  the  Greenhouse  
Effect  and  global  warming  
43  
 •  Global  Carbon  Cycle  
Carbon  Cycle  AnimaEon  

44  

Photo Source: Windows to the Universe; Animation: Environmental Science, Toward a Sustainable Future, 9th ed. by Richard T. Wright
 Carbon  RespiraEon  
•  CO2  fixed  into  organic  compounds  is  consumed  by  
animals  &  heterotrophic  microbes    
–  end  products  of  respiraEon  are  CO2  and  new  cell  mass  
•  More  complex  carbon  cycle  includes  anaerobic  acEvity  
–  fermentaEon  &  methanogenesis  
Aerobic Anaerobic
Fossil fuels
Photosynthesis Fermentation

CO2 + H2O O2 + CH2O CH2O Alcohols, acids,

H2 + CO2

Respiration Methanogenesis

CH4 45  
Source: Figure 14.3 from text
 Organic  Polymers  
•  Most  common  organic  carbon  in  the  environment  are  
plant  polymers,  polymers  used  in  bacterial  and  fungal  
cell  walls,  arthropod  exoskeletons  
•  Form  the  basic  food  supply  that  supports  heterotrophs  
•  3  most  abundant  are  cellulose,  hemicellulose  and  lignin  

Plant  component   %  dry  mass  of  plant  

Cellulose   15-­‐60  
Hemicellulose   10-­‐30  
Lignin   5-­‐30  
Protein  and  nucleic  acids   2-­‐16  
46  
Table 16.6 from Text
 Cellulose  
•  Most  abundant  polymer  found  on  Earth  
–  make  up  the  woody  structures  of  plants  
•  Consists  of  linear,  β-­‐1,4  linked  glucose  subunits  (1000  –  
10,000;  MW:  1.8  x  106  g/mol)  
•  Large  and  insoluble  molecule,  must  be  par:ally  degraded  by  
extracellular  microbial  enzymes  (β-­‐1,4-­‐endoglucanase  and  
β-­‐1,4  –exoglucanase  aka  cellulases)  before  it  can  be  taken  
up  and  used  by  bacteria  

47  
 Hemicellulose  
•  Second  most  common  plant  polymer  
•  Branched  and  more  heterogeneous  
–  made  from  a  mixture  of  several  monosaccharides  including  
various  hexoses,  pentoses  (~  200  monomers)  &  uronic  acids  
–  E.g.  pecEn  
•  DegradaEon  similar  to  cellulose;  more  enzymes  involved  
 

Fig. 16.6 from Text 48  

 Lignin  
•  Third  most  common  plant  polymer  
•  Building  blocks  are  randomly  polymerized  tyrosine  &  phenylalanine  
•  Strengthens  cell  wall  and  improves  pathogen  resistance  
•  BiodegradaEon  is  slower  and  less  complete  than  for  other  polymers  
•  Extreme  heterogeneity  makes  evoluEon  of  specific  degradaEve  
enzymes  difficult  
–  broken  down  somewhat  
into  smaller  subunits  by  
H2O2-­‐dependent  lignin  
peroxidase  
–  free-­‐radicals  generated  
help  break-­‐down  
–  subunits  taken  up  by  
microbes  (white  rot  fungi)  
and  degraded  
49  
 Methane  
•  Formed  mostly  by  microbes  in  an  anaerobic  process  called  
methanogenesis    
–  By  methanogens  (obligatory  anaerobic  archae)  
–  e.g.  Methanobacterium,  Methanobrevibacter,  Methanococcus  
4H2  +  CO2                    CH4  +  2H2O        auto  or  heterotrophic?  
•  Occurs  in  specialized  environments  like  wetlands  and  paddy  
fields,  landfills  and  in  the  rumen  gut  
•  ContribuEon  to  global  carbon  cycle,  though  emission  is  of  
environmental  concern  
–  Greenhouse  gas;  25  Emes  more  effecEve  than  CO2  at  trapping  heat  
–  Explosive;  generaEon  in  landfill  sites  must  be  managed  (i.e.  vented)  
50  
Estimates of methane released into the atmosphere
Source Methane emission
(106 metric tons/year)
Biogenic
Ruminants 80 - 100 ü 
Termites 25 - 150
Paddy fields 70 - 120 ü 
Natural wetlands 120 - 200
Landfills 5 - 70 ü 
Oceans and lakes 1 - 20
Tundra 1-5

Abiogenic
Coal mining 10 - 35 ü 
Natural gas flaring and venting 10 - 35
ü 
Industrial and pipeline losses 15 - 45 ü 
Biomass burning 10 - 40
Methane hydrates 2-4
Volcanoes 0.5
Automobiles 0.5 ü 

Total 349 - 820

Total biogenic 302 - 665 81 - 86% of total
Total abiogenic 48 - 155 13 - 19% of total
Anthropogenic 190 – 405 54 - 49% of total
 Methane  Oxida:on  
•  Methanotrophs  (a.k.a  methanophiles)  are  a  group  of  bacteria  
that  have  developed  the  ability  to  use  methane  as  a  source  of  
carbon  and  energy  
•  Chemoheterotrophic  and  aerobic  
–  Methylococcaceae  and  Methylocystaceae  
methane
monooxygenase
CH4 + O2 CH3OH HCHO HCOOH CO2 + H2O
methanol formaldehyde formic
  acid

•  A  new  bacterium  Methylomirabilis  oxifera  was  idenEfied  that  

can  couple  anaerobic  oxida:on  of  methane  to  nitrite  reducEon  
 
Nature. 2006 Apr 13;440:918-21. A microbial consortium couples anaerobic methane oxidation to denitrification.
Raghoebarsing AA, Pol A, van de Pas-Schoonen KT, Smolders AJ, Ettwig KF, Rijpstra WI, Schouten S, Damsté JS, Op den Camp HJ, Jetten MS, Strous M.
Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, Netherlands.
52  
 Methane  Monooxygenase  
•  First  enzyme  idenEfied  as  able  to  
cometabolize  highly  chlorinate  
solvents  like  trichloroethylene  (TCE)  

•  Nonspecific  enzyme  able  to  oxidize  

both  methane  and  TCE  

•  Subsequent  steps  in  degradaEon  are  

either  spontaneous  or  catalyzed  by  
other  bacteria  

•  Working  on  strategy  to  use  

methanotrophs  in  bioremediaEon  of  
contaminated  groundwater   53  
 The  Nitrogen  Cycle  
•  Best  studied  and  most  complex  of  the  mineral  cycles  
•  Nitrogen  is  the  mineral  nutrient  in  most  demand  by  
microbes  and  plants    
•  4th  most  common  element  in  cells  (12-­‐14%  of  dry  weight)  
•  Stable  valences  from  -­‐3  (NH3+)  to  +5  (NO3-­‐)  
–  occurs  in  numerous  oxidaEon  states  

•  Cycle  includes  microbially-­‐catalyzed  processes  of  

nitrogen  fixaEon,  ammonificaEon,  nitrificaEon  and  
denitrificaEon  

57  
Development  of  the  Nitrogen  Cycle  
•  Nitrogen  cycle  emerged  because  nitrogen  was  a  limiEng  
element  for  microbial  growth  

•  N2  was  abundant  in  the  atmosphere,  but  inaccessible  

–  cells  need  organic  N  or  reduced  inorganic  N  

•  Some  microbes  developed  the  ability  to  fix  N2  into  

organic  nitrogen  needed  for  growth  of  other  microbes  

–  using  the  enzyme  nitrogenase  

–  using  the  reducing  atmosphere  found  in  early  Earth  

56  
 •  Nitrogen  Cycle  
Nitrogen  Cycle  

58  

Photo: http://en.wikipedia.org/wiki/File:Nitrogen_Cycle.svg; Animation: Environmental Science, Toward a Sustainable Future, 9th ed. by R. T. Wright
 Nitrogen  Reservoirs  
•  Large  Reservoirs    
–  largely  inaccessible  and  not  acEvely  cycled  
•  N2(g)  in  atmosphere  (78%)  
–  conEnually  released  from  volcanic  and  hydrothermal  erupEons  
•  bound  ammonium  in  the  Earth’s  crust  
 

•  Small  Reservoirs  
–  acEvely  cycled  
•  organic  nitrogen  found  in  living  biomass  and  dead  maeer  
•  inorganic  N  ions  (ammonium,  nitrite  and  nitrate)  are  highly  
water  soluble  and  are  distributed  throughout  the  ecosphere  
1.  Nitrogen  FixaEon  
2.  Ammonium  AssimilaEon/  AmmonificaEon  
3.  NitrificaEon  
4.  Nitrate  ReducEon  
–  assimilatory/  dissimilatory  nitrate  reducEon,  denitrificaEon,  anamox   59  
THE NITROGEN CYCLE
 Nitrogen  Fixa:on  
•  Plants,  animals  &  most  microbes  require  combined  forms  
of  nitrogen  for  growth  
–  ability  to  fix  nitrogen  is  limited  to  a  small  number  of  
bacteria,  archae  and  symbioEc  associaEons  

–  energy  intensive  process  

•  N  fixaEon  –  67%  by  microbial  fixaEon;  30%  by  ferElizer  

producEon  –  Haber-­‐Bosch  Process;  3%  by  atmospheric  
fixaEon  -­‐  lightening    

•  FerElizer  producEon  is  expensive;  alternaEves  such  as  

rotaEon  of  crops  between  nitrogen-­‐fixers  (soybeans)  &  
non-­‐fixers  (corn)  are  becoming  popular  
61  
 Nitrogen  Fixa:on:  methods  
•  Atmospheric  fixa:on  by  lightning  (3%)  
–  high  energy  of  lightning  breaks  nitrogen  molecules;  combine  
with  oxygen  in  the  air  forming  nitrogen  oxides;  dissolve  in  
rain,  forming  nitrates    
•  Biological  fixa:on  by  certain  microbes  (67%)  
–  alone  or  in  a  symbioEc  relaEonship  with  some  plants  and  
animals    
–  e.g.  Cyanobacteria  (Anabaena,  Nostoc),  Azotobacteraceae,  
Rhizobia,  Frankia  
•  Industrial  fixa:on  (Haber-­‐Bosch  process)  (30%)  
–  atmospheric  N2  and  H2  (usually  from  natural  gas  or  petroleum)  
can  be  combined  to  form  NH3  under  high  P,  high  T  and  catalyst   62  
 Nitrogen  Fixa:on:  the  chemistry  
•  ReacEon  is  catalyzed  by  the  nitrogenase  enzyme  complex;  
requires  ATP  and  cytochromes  
N2  +  8  H+  +  6  e−  →  2  NH3  +  H2                          ΔG  =  +150  kcal/mol  
•  Ammonia  is  incorporated  into  amino  acids,  nucleic  acids  etc.    
•  ReacEon  is  under  strict  regulaEon;  expression  of  required  
genes  (nif  )  is  inhibited  by  NH3  -­‐-­‐>  Feedback  Inhibi:on  
•  Enzyme  is  extremely  oxygen  sensi:ve  (requires  low  O2  
tensions  to  funcEon)  

63  
 Nitrogen  Fixa:on:  who?  
•  Free-­‐living  soil  bacteria  
–  Azotobacter  (aerobic),  Beijerinckia  (aerobic),  Clostridium  
(anaerobic)  
–  Azotobacter  and  Beijerinckia  can  fix  at  normal  O2  tension  –  
mechanism  to  protect  nitrogenase  enzyme  
•  Rhizobia-­‐legume  symbioEc  relaEonship  
–  rate  of  fixaEon  is  2-­‐3  orders  of  magnitude  higher  than  free-­‐living  
•  Cyanobacteria  
–  predominant  in  aquaEc  environments  
–  fixaEon  rate  1-­‐2  orders  of  magnitude  higher  than  free-­‐living  
terrestrial  microbes  because  they  are  photosyntheEc  
–  have  specialized  heterocysts  with  thick  walls  impermeable  to  O2  
64  
–  e.g.  Anabaena,  Nostoc  
 Rates  of  Nitrogen  Fixa:on  
N2-­‐fixing  system   Nitrogen  fixa:on    
(kg  N/hectare/year)  

Rhizobium-­‐legume   200-­‐300  

Anabaena-­‐Azolla   100-­‐120  

Cyanobacteria-­‐moss   30-­‐40  

Rhizosphere  associaEons   2-­‐25  

Free-­‐living   1-­‐2  

Source: Table 16.13 from Text

Summary for Nitrogen Fixation:

energy intensive

end-product is ammonia

inhibited by ammonia à feed back regulation

occurs in aerobic and anaerobic environments

nitrogenase is O2 sensitive
Ammonium  Assimila:on  (Immobiliza:on)  
•  IncorporaEon  of  NH4+  into  amino  acids  (proteins),  purines  &  
pyrimidines  (nucleic  acids)  and  N-­‐acetylmuramic  acid  (cell  wall)  

 
Ammonifica:on  (Mineraliza:on)  
•  SequenEal  degradaEon  of  nitrogenous  organic  compounds  with  
the  release  of  ammonia  
           Proteins        à            Amino  acid        à        Organic  acid    +    Ammonia  
 
•  Under  N  limi:ng  condi:ons:  immobiliza:on  predominant  
•  Under  N  non-­‐limi:ng  condi:ons:  mineraliza:on  predominant  
•  Fate  of  ammonium  released  into  the  environment:  
-  taken  up  by  plants/  microbes  (incorporated  into  biomass)  
-  bound  to  soil/  humus  
-  adds  to  caEon  exchange  capacity  (CEC),  trapped  in  clay,  escape  to  
67  
atmosphere,  nitrificaEon    
Summary for ammonia assimilation and ammonification
Assimilation and ammonification cycles ammonia between its organic and
inorganic forms

Assimilation predominates at C:N ratios > 20

Ammonification predominates at C:N ratios < 20

68  
 Nitrifica:on  
•  Biological  oxidaEon  of  ammonia  to  nitrite  followed  by  the  
oxidaEon  of  nitrites  to  nitrates  
•  Carried  out  by  a  limited  number  of  autotrophic  bacteria;  2  
steps  carried  out  by  different  populaEons  of  bacteria  
–  closely  coupled,  hence  build-­‐up  of  nitrite  does  not  occur  
–  pH  sensi:ve:  op:mal  6.6  –  8;  completely  inhibited  <  4.5  
•  OxidaEon  of  ammonia  to  nitrite  carried  out  by  
Nitrosomonas;  nitrite  to  nitrate  by  Nitrobacter  or  Nitrospira  
•  Both  reacEons  are  energy-­‐yielding;  nitrifying  bacteria  use  
the  energy  derived  from  nitrificaEon  to  assimilate  CO2  
ammonium monooxygenase
NH3  +  CO2  +  1.5  O2  +  Nitrosomonas  -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐→  NO2-­‐  +  H2O  +  H+    
NO2-­‐  +  CO2  +  0.5  O2  +  Nitrobacter          -­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐-­‐→  NO3-­‐   69  
 Importance  of  Nitrifica:on  to  Soil  
Chemistry  
•  TransformaEon  of  ammonium  ions  to  nitrite  and  nitrate  ions  
results  in  a  change  in  charge  from  ‘+’  to  ‘–’  

•  PosiEvely  charged  ions  are  generally  bound  by  negaEvely  

charged  clay  parEcles  in  soil  

•  NegaEvely  charged  ions  migrate  freely  in  soil  

•  NitrificaEon  is  therefore  ‘nitrogen  mobilizaEon’  

•  Ammonia  in  soil  is  rapidly  oxidized  

–  nitrate  is  taken  up  by  plants  but  can  also  be  leached  from  the  soil  
into  the  groundwater;  eutrophicaEon  in  lakes  
–  health  concerns:  methemoglobinemia,  nitrosamine  (carcinogen)  
70  
Summary for nitrification

Nitrification is an chemoautotrophic, aerobic process

Nitrification is sensitive to a variety of chemical inhibitors and is inhibited at

low pH. (There are a variety of nitrification inhibitors on the market)

Nitrification in managed systems can result in nitrate leaching and

groundwater contamination

71  
 Denitrifica:on  
•  Biologically  mediated,  more  complete  reducEon  of  
nitrate  to  nitrogen  gas  
•  Primary  denitrifying  genera  in  soil  are  Pseudomonas  and  
Alcaligenes  
NO3−  →  NO2−  →  NO  +  N2O  →  N2  (g)  
•  Usually  produce  a  mixture  of  nitrous  oxide  and  nitrogen  
•  Most  oyen  occurs  under  strictly  anaerobic  condi:ons    
–  more  common  in  standing  waters  than  in  running  streams  
•  Problems:  
–  removal  of  limiEng  nutrient,  N2O  causes  depleEon  of  
ozone,  greenhouse  gas  (N2O)  
72  
Summary for Denitrification

Anaerobic respiration using nitrate as TEA

Inhibited by oxygen

Produces a mix of N2 and N2O

Many heterotrophs denitrify

ANaerobic  AMMonium  Oxida:on  
(Anammox)  
•  A  biological  process,  where  ammonium  oxida:on  occurs  
under  anaerobic  condi:ons  using  nitrite  as  TEA  

NH4+  +  NO2−  →  N2  +  2H2O  

•  Responsible  for  50%  of  the  N2  gas  produced  in  the  oceans  

•  e.g.  Brocadia,  Kuenenia,  Anammoxoglobus  

–  produces  hydrazine  (rocket  fuel;  highly  toxic)  as  an  intermediate  

•  Used  in  the  removal  of  ammonium  from  wastewater  

treatment  (full-­‐scale  plants  in  the  Netherlands)  
74  
Interrup:on  of  N  Cycle  –  Release  of  N2O  
•  Agricultural  pracEces  responsible  for  large  proporEon  of  
N2O  released  by  human  acEvity:  
–  ammonia  is  primary  source  of  nitrogen  in  ferElizers  
–  only  50%  of  applied  nitrogen  is  assimilated  by  crops  
–  rest  is  lost  through  leaching,  erosion  &  gaseous  emission  and  
may  ulEmately  be  released  as  N2O  
 
•  Other  sources  of  N2O  include    
–  burning  of  biomass  
–  combusEon  of  fossil  fuel  
–  chemical  manufacturing  of  nylon  
75  
Nitrous  Oxide  &  Earth’s  Atmosphere  

•  N2O  is  released  to  the  atmosphere  from  industrial  and  

biological  sources  

•  Contributes  to  global  warming  (Greenhouse  Effect)  &  ozone  

depleEon  

–  long  residence  Eme  (114  years)  &  efficient  at  radiaEon  

absorpEon  (200  X  more  than  CO2)  

–  solar  radiaEon  convert  N2O  to  NO;  factor  in  O3  depleEon  

76  
Photo-­‐dissocia:on  of  N2O  and  
Deple:on  of  Ozone  
N2O  +  hν                              N2  +  O*    
N2O  +  O*                        2  NO  
 
NO  +  O3                          NO2  +  O2  
O3  +  hν                                  O  +  O2  
NO2  +  O                            NO  +  O2  
2O3  +  hν                                            3  O2  

77  
Nitrous  Oxide  &  Earth’s  Atmosphere  
Cont.  
•  N2O  is  produced  by  mulEple  phases  of  the  N  cycle  
–  intermediate  in  denitrificaEon    (wet  soil  with  restricted  O2)  
 

               NO3-­‐                  NO2-­‐                    NO  +  N2O                    N2  (g)  

 
–  by-­‐product  of  nitrificaEon  (aerated,  moist  soils  with  low  O2)  
 

     NH3  +  O2                        NO2-­‐                        N2O                

78  
Nitrate  Contamina:on  of  Groundwater  
•  Use  of  ferElizers  and  large  amounts  of  animal  waste  
cause  excess  ammonia  in  soil  &  groundwater  
•  Nitrifying  bacteria  convert  NH4  to  NO3-­‐;  accumulaEon  in  
soil  
•  Nitrate  is  mobile  in  soil;  transported  to  groundwater  
•  Excess  nitrate  causes  methemoglobinemia  in  infants  
–  nitrate  is  transformed  to  nitrite  in  the  digesEve  system;  nitrite  
oxidizes  iron  in  the  hemoglobin  to  form  methemoglobin,  which  lacks  
the  oxygen-­‐carrying  ability.  This  creates  the  condiEon  known  as  
methemoglobinemia  ("blue  baby  syndrome”)  

•  FormaEon  of  highly  carcinogenic  nitrosamines  in  adults  

79  
Preven:on  of  Nitrate  Contamina:on  

•  Best  Management  PracEces  (BMPs)  for  amount  and  

Eme  of  ferElizer  applicaEon  and  irrigaEon  of  crops  
–  region-­‐specific  since  climate  and  soil  types  vary  by  region  

•  Use  of  slow-­‐release  ferElizers  

–  more  controlled  release  of  ammonia  into  environment  

•  ApplicaEon  of  nitrificaEon  inhibitors  

80  
 Summary  
•  GH  gases  à  global  warming  à  climate  change  
•  UN  acEviEes  to  combat  climate  change  –  Kyoto  Protocol  
&  Paris  Agreement  -­‐  what  is  your  contribuEon?  
•  Carbon  cycle  
–  carbon  respiraEon  
–  organic  carbon  polymers  
–  methane  generaEon  &  oxidaEon  
•  Nitrogen  cycle  
–  Nitrogen  FixaEon  
–  Ammonium  AssimilaEon/  AmmonificaEon  
–  NitrificaEon  
–  DenitrificaEon  
–  Anamox   81  
 Environmental  Microbiology  (BI  304):  Lecture  5  
 
Beneficial  and  Pathogenic  Microbes  in  
Agriculture  
Instructor:  Dr.  Nalina  Nadarajah  
Email:  nnadarajah@centennialcollege.ca  
 Overview  (Chapter  19)  
•  ExaminaLon  of  plant-­‐microbe  interacLons  in  the  
phyllosphere  and  soil-­‐plant-­‐  microbe  interacLons  in  the  
rhizosphere.  
•  Agenda  
–  Categories  of  ecological  relaLonships  
–  Plant  Microbial  Habitats  
(i)  Phyllosphere    (ii)  Rhizosphere  
–  Beneficial  Root-­‐Microbe  InteracLons  
•  Nitrogen  FixaLon  
•  Mycorrhizal  AssociaLons  
–  Pathogenic  Microbes  in  Agriculture  
2  
 Bdellovibrio

3  

Source: Box 8.1 from Environmental Microbiology by Eugene Masden

Back
4  
 Plant  Microbial  Habitats  
•  Plants  consist  of  two  primary  microbial  habitats:  the  
phyllosphere  and  the  rhizosphere  
•  Phyllosphere:  the  aerial  porLons  of  plants  (trunk,  
branches,  stems,  buds,  flowers,  leaves)  
–  phyllosphere  inhabitants  are  termed  epiphytes  
•  most  important  is  the  leaf  habitat  (106  –  107  microbes/  cm2)  

Microorganisms found on the Bacteria on the surface of an Electron microscope image of

leaves of a bean plant Arabidopsis leaf a leaf surface 5  
 Plant  Phyllosphere  
•  Most  work  on  phyllosphere  has  focused  on  leaves  à  more  
dominant  aerial  plant  structure  
•  What  condiLons  and  resources  prevail  in  the  phyllosphere?  
–  Physical  condiLons:  fluctuaLons  in  temperature,  humidity,  
free-­‐  standing  rainwater,  ultraviolet  light  
–  Plant  surfaces:  waxy  surface,  intercellular  spaces  within  plant  
stomata  
–  Carbon  sources:  waxes,  sloughed  cells,  sugars    
–  SpaLally  heterogeneous  (micro-­‐scale)  distribuLons  of  
nutrients  and  micronutrients,  such  as  iron  
6  
•  What  organisms  occur?  
–  The  microbial  communiLes  include  many  different  genera  of  
bacteria,  filamentous  fungi,  yeasts,  algae  &  less  frequently,  
protozoa  and  nematodes  
–  Patchy,  spaLally  heterogeneous  distribuLons  of  heterotrophic  
microorganisms  and  plant  pathogens  
•  What  processes  occur?  
–  CommensalisLc  relaLonships  between  plant  and  epiphyLc  MO  
–  CompeLLon  among  heterotrophs  for  limited  nutrients    
–  Plant  immune-­‐like  defenses  against  pathogenic  bacteria  include  
localized  cell  death  (the  hypersensiLve  response)  
–  Plant  pathogens  may  release  virulence  factors  that  assist  in  their  
colonizaLon  and  infecLon  
–  Microorganisms  may  also  release  surfactants  and  auxins  that  assist  
them  colonizing  the  plant   7  
 Why  Study  Plant  Phyllosphere  
– Understanding  the  ecology  of  an  important  biosphere
habitat
– To  use  that  understanding  to  control  microbial  plant
disease,  food-­‐transmifed  human  disease  &  crop  damage
– Microbial  ecological  strategies  can  establish  microbes  that
are  antagonisLc  to  plant  pathogens  (biological  control)
– Microbial  ecological  strategies  can  use  compeLLve
exclusion  to:
(i)  curtail  establishment  by  plant  pathogens  

(ii)  curtail  frost  damage  insLgated  by  phyllosphere  bacteria  

that  catalyze  ice  nucleaLon  
8  
 Ice  nuclea:on  à  Frost  damage  
• Some  bacteria  normally  found  on  plant  surfaces
iniLate  formaLon  of  frost,  which  damages  crops
–  e.g.  Pseudomonas  syringae  and  Erwinia  herbicola  
– ice  nucleaLng  bacteria  iniLates  frost  damage  at  -­‐2°C;  in  the
absence  bacteria,  damage  does  not  begin  unLl  -­‐8  to  -­‐10°C
• Ice-­‐minus  bacteria:  a  variant  of  P.  syringae  that  lacks  the
ability  to  produce  INA  (ice  nucleaLon-­‐acLve)  protein
• Could  there  be  any  benefits  of  ice  nucleaLng  bacteria?

9  
Use  of  Bt  genes  in  GeneLc  Engineering  
of  Plants  for  Pest  Control  
• Bt  toxins  present  in  peanut  
leaves  (right  image)  protect  it  
from  extensive  damage  
caused  by  larvae  

• Bt  cofon  and  cofon  from  

untreated  control  plant        
Source  California  Agriculture  

• Problem:  pink  bollworms  

have  become  resistant   10  
 Plant  Rhizosphere  
• The  rhizosphere  is  the  narrow  region  of  soil  (5  mm)  directly  

http://en.wikipedia.org/wiki/Rhizosphere
influenced  by  root  secreLons  and  associated  soil  MO  

• It  has  abundant  nutrients  

Biofilm of fluorescently labeled bacteria on the root of a plant

Structure  of  a  Root  &  its  Rhizosphere  

Fig. 18.3
12  
 The  Rhizosphere  Environment  
• Term  coined  in  1904  by  Hiltner;  describes  part  of  soil  
influenced  by  plant  roots  
• Generally  extends  at  least  5mm  
– microscale  biogeochemical  gradients  (pH,  organic  substrate,  
O2,  CO2,  H2O)  
• Consists  of  two  main  regions:  
– Rhizosphere  soil  
– Rhizoplane  –  soil  in  direct  contact  with  plant  root  
• endophytes:  microorganisms  that  inhabit  the  root  itself  
– microflora  (bacteria,  acLnomycetes,  fungi  &  algae)  and  micro  
and  mesofauna  (protozoa,  nematodes,  mites  &  insects)  
 Rhizosphere  Cont’d  
•  Rhizosphere  effect  caused  by  release  of  compounds  (organic  
&  inorganic)  from  the  plant  roots  
•  Rhizosphere  soil  very  different  from  bulk  soil  but  difficult  to  
study  &  therefore  define  
•  Challenge  of  sampling  methods,  huge  number  of  possible  
‘environments’  (specific  plants  in  specific  soil  in  specific  
environment)  
•  CharacterisLcs  compared  to  bulk  soil:    
–  pH  of  1  unit  higher  or  lower  depending  on  N  concentraLon  
–  drier  due  to  plant  transpiraLon  
–  richer  in  organics  
14  
PotenLal  Influences  of  the  Rhizosphere  
Microbial  Community  on  Plant  Growth  

(Fig 18.2)
15  
Key  Influences  in  Plant  Rhizosphere  
•  Physical  change  of  habitat  
–  Growing  root  surface  extending  through  soil    
–  Moisture  flux  toward  root  
•  Chemical  change  of  habitat  
–  Nutrient  (e.g.,  N,  P,  K)  depleLon  near  root  surface    
–  Root  products:    
•  diffusates  (sugars,  amino  acids,  organic  acids,  inorganic  ions,  
oxygen,  growth  factors,  water)  
•  excreLons:  CO2,  protons,  bicarbonate,  ethylene    
•  secreLons:  mucilage,  enzymes,  iron-­‐binding  siderophores,  
allelochemicals  that  inhibit  other  organisms    
•  debris:  root  cap  cells,  sloughed  Lssues  
16  
Key  Processes  in  Plant  Rhizosphere  
• Microbiological  processes  in  habitat  
– Mycorrhizal  infecLon,  nitrogen  fixaLon,  pathogen  infecLon,  
compeLLon  for  nutrients  among  heterotrophs,  interacLons  with  
soil-­‐fauna  (e.g.,  nematodes,  insects)  
• Plant  processes  in  habitat  
– AcquisiLon  of  nutrients  (e.g.,  Fe  uptake,  P  solubilizaLon)  
– AcquisiLon  of  water  via  transpiraLon  and  as  modulated  by  
mucilage  release  
– ProtecLon  against  toxic  agents  (e.g.,  complexaLon  of  Al3+)    
– ProtecLon  against  compeLLon  and  plant  pathogens  (e.g.,  
allelochemicals  that  inhibit  other  organisms)    
– Establishment  of  symbioLc  relaLonships  (by  Rhizobia  and  
mycorrhizae)   17  
 The  Soil-­‐Plant-­‐Microbe  System  
• Rhizosphere  exists  because  of  soil-­‐plant-­‐microbe  
interacLons  
– interacLons  control  microbial  gene  expression  in  
rhizosphere  
– interacLons  are  influenced  by  environmental  factors  
– microbial  populaLons  affect  plant  growth  (+/-­‐)  

18  
Organic  Compounds  Released  by  Plants  
• Exudates  
– low  MW,  soluble  compounds  that  leak  non-­‐metabolically  
from  intact  plant  cells  via  simple  diffusion  
– can  change  pH,  structure  of  rhizosphere  soil,  availability  of  
inorganic  nutrients  &  induce  toxic/sLmulatory  effects  
• SecreLons  
– compounds  metabolically  released  from  acLve  plant  cells  
– can  occur  against  concentraLon  gradients,  but  require  
metabolic  energy  
– include  carbohydrates,  amino  acids,  organic  acids,  lipids,  
growth  factors,  enzymes  
19  
• Lysates  
– Compounds  released  by  the  autolysis  of  older  cells  
• Plant  mucilages  
– polysaccharides  from  the  root  cap,  root  cap  cells,  primary  
cell  wall  &  other  cells  
• Mucigel  
– gelaLnous  material  of  plant  and  microbial  origin  
– contains  excellent  substrates  for  microbial  growth  
– may  protect  root  Lp  from  injury/dessicaLon  &  have  role  in  
nutrient  uptake  
– Bacteria  consume  mucigel;  also  contribute  to  
polysaccharide  that  makes  mucigel  
20  
 Factors  AffecLng  the  Release  of  
Compounds  
• Plant  species  
• Age  
• Stage  of  development  
• Light  intensity  
• Temperature  
• Soil  factors  
• Plant  injury  
• Soil  microbes  (microflora,  microfauna  &  mesofauna)  
21  
 Rhizosphere  PopulaLons  

• plant  
– crop  plant  roots  >  tree  roots  
• physical  environment  
– light,  moisture,  temperature  change  plant  metabolism  &  
affect  type/amount  of  organic  mafer  released  into  the  soil  

22  
Rhizosphere  PopulaLons  and  R/S  raLo  

• Rhizosphere  
effect  described  
as  R/S  raLo  
– R  =  #MOs  in  
rhizosphere  
– S  =  #MOs  in  bulk  
soil  
– greater  R/S  raLo,  
more  
pronounced  
rhizosphere  
effect  
23  
Beneficial  Root-­‐Microbe  InteracLons  
• Demonstrated  by  growing  plants  in  sterilized  soil  versus  
non-­‐sterilized  soil  
• Predominant  mechanisms  involve  N  and  P  
– prokaryoLc  bacteria  enhance  plant  N  uptake  through  N  
fixaLon  
– eukaryoLc  fungi  enhance  plant  P  and  other  micronutrient  
uptake  through  mycorrhizal  associaLon  

24  
 Biological  Dinitrogen  FixaLon  
•  Conversion  of  nitrogen  gas  to  ammonia  
•  Mediated  only  by  prokaryotes  (bacteria,  cyanobacteria  &  
acLnomycete  Frankia)  
•  Exist  as  free-­‐living  organisms  or  in  complex  with  other  
microbes,  plants  &  animals  
•  Diazotrophs:  bacteria  and  archaea  that  fix  atmospheric  
nitrogen  gas  into  a  more  usable  form  such  as  ammonia  
–  can  uLlize  N2  gas  as  sole  source  of  nitrogen  for  growth  
(e.g.  Rhizobia,  Frankia  and  Azospirillum)  

25  
 Process  of  Nitrogen  FixaLon  
• N2  +  3H2                              2NH3  
• requires  large  energy  input  (226  kcal/  
mol)  to  break  triple  bonded  N2    
• energy  derived  from  oxidaLon  of  
carbon  sources  (heterotrophs)  or  light  
(photosyntheLc  diazotrophs)  
• reacLon  accomplished  by  nitrogenase  enzyme  complex
– dinitrogenase  reductase  (iron  protein);  reduces  dinitrogenase  
– dinitrogenase  (molybdenum-­‐iron  protein);  reduces  N2  to  NH3  

26  
http://www.cliffsnotes.com/WileyCDA/CliffsReviewTopic/Bacterial-Nitrogenase.topicArticleId-24594,articleId-24526.html
 Process  of  Nitrogen  FixaLon  
•  reacLon  requires  at  least  16  ATPs  (and  
osen  up  to  30)  
•  dinitrogenase  reductase  accepts  
electrons  from  low-­‐redox  donor  
(reduced  ferredoxin)  and  binds  2  Mg-­‐
ATPs  
Dinitrogenase  
•  complex  forms,  electrons  are   reductase  
transferred  to  dinitrogenase  &  2  Mg-­‐
ATPs  are  hydrolyzed,  complex  
dissociates  
•  process  repeated  unLl  dinitrogenase   N-fixing bacteria constitute 10-40%
has  enough  e-­‐  to  bind  and  reduce  one   proteins as nitrogenase enzyme
molecule  of  nitrogen  gas  
25% energy on H2 production –
•  reducLon  of  one  N2  molecule  requires   some diazotrophs contain
that  protein  complex  form  and   hydrogenase enzyme to reoxidize
dissociate  8  Lmes!   and generate energy 27  
 Process  of  Nitrogen  FixaLon  Cont.  
• N  fixaLon  is  an  anaerobic  process  because  dinitrogenase  
reductase  is  irreversibly  inacLvated  by  oxygen  
• Aerobic  organisms  have  developed  novel  mechanisms  to  
prevent  interacLons  between  nitrogenease  enzyme  and  O2    
– Clostridium  fixes  nitrogen  in  anaerobic  environments  
– Azotobacter  uses  very  fast  aerobic  respiraLon  (oxygen  uptake  
and  reducLon)  to  do  it  in  aerobic  environments  
– Anabaena  uses  thick  walled  heterocysts  (micro  anaerobic  env)  
to  restrict  oxygen  access  to  dinitrogenase  reductase  
– Rhizobium  forms  nodules  in  plant  roots  and  oxygen  access  is  
restricted  by  leghemoglobin  
– Frankia  also  forms  nodules  in  plant  roots  with  thick  vesicles  and  
envelopes   28  
Free-­‐living  and  AssociaLve  N2  FixaLon  
•  Free-­‐living  
–  amount  of  nitrogen  fixed  by  free-­‐living  diazotrophs  is  small  (2-­‐25  kg  per  hectare  /  yr)  
–  due  to  high  energy  requirements  &  O2  inhibiLon  
–  examples:  Xanthobacter,  Azospirillum,  Beijerinickia  and  others  

•  AssociaLve  
–  associaLve  organisms  are  established  on/in  plant  cells  and  use  carbon  reserves  
–  in  return  they  fix  nitrogen,  which  can  be  used  by  the  plant  
–  casual/associaLve  symbioses  do  not  require  geneLc  interacLons  between  plant  &  
microbe,  and  no  morphological  modificaLons  occur  in  either  partner  
•  e.g  include  Azotobacter  paspali  associaLng  with  Paspalum  notatum  (tropical  grass)  and  
Acetobacter  diazotrophicus  with  tropical  sugarcane  
•  small  amount  of  nitrogen  fixed  (~20  kg/hectare/yr)  

29  
SymbioLc  N  FixaLon  -­‐  Legume-­‐Rhizobia  
• Formal  symbiosis  in  which  both  
partners  benefit  
• Rhizobium  sp  interact  with  
leguminous  plants  causing  
physiological  &  geneLc  changes  
(nod  genes)  in  both  (forms  
bacteroids)  
• Bacteria  fix  nitrogen  in  
exchange  for  carbon  source  
(supplied  by  plant  by  
photosynthesis)  
• Occurs  in  root  nodules;  develop  
in  response  to  soil  borne  
rhizobia   http://www.bio.usyd.edu.au/DavidDay/root_nodules.jpg
30  
 Bacteroid  FormaLon  in  the  
Rhizobium-­‐Legume  Symbiosos  

31  
 Legume-­‐Rhizobia  Symbioses  
• Leguminous  plants  undergo  physiological  change  as  the  root  
produces  root  nodules  
• Rhizobia  also  undergo  physiological  changes,  become  
bacteroids  which  actually  conduct  N-­‐fixaLon  
• When  plant  host  matures,  root  nodules  lyse  and  Rhizobia  are  
released  back  into  the  soil  
• Many  legumes  (peas,  beans,  soybeans)  can  fix  50%  of  total  
nitrogen  requirements  and  can  be  grown  commercially  with  
reduced  N  ferLlizer  

The pink color is the The lightest areas contain

compound leghaemoglobin high levels of Rhizobium 32  
 Azolla  –  Anabaena  Symbiosis  
• The  small  floaLng  fern  (Azolla)  provides  caviLes  within  its  
leaves  that  serve  as  a  habitat  for  the  cyanobacteria  
(Anabaena)    
• The  cyanobacterium  fixes  nitrogen  in  this  intracellular  
associaLon  
• Exploited  by  rice  farmers  for  centuries  as  a  way  to  boost  
the  nitrogen  status  of  rice  paddies  

Azolla Anabaena 33  
 Mycorrhiza  
• Mycorrhizae  is  a  symbioLc  (generally  mutualisLc)  associaLon  
between  a  fungus  and  the  roots  of  a  vascular  plant;    
• Commonly  divided  into  (i)  endomycorrhizas  and  (ii)  
ectomycorrhizas    
• Hyphae  of  endomycorrhizal  fungi  penetrate  the  cell  wall  and  
invaginate  the  cell  membrane  
–  e.g.  Arbuscular  mycorrhizas  –  formed  by  obligately  symbioLc  fungi  
from  the  division  Glomeromycota  -­‐  85%  of  all  plant  families  
• Ectomycorrhizal  fungi  do  not  penetrate  individual  cells  within  
the  root;  root  is  enveloped  in  a  fungal  network  
–  e.g.  fungi  belonging  to  the  Basidiomycota,  Ascomycota,  and  
Zygomycota  -­‐  ~  10%  of  plant  families,  mostly  woody  plants  
including  the  birch,  eucalyptus,  oak,  pine,  and  rose   34  
Images  of  Endomycorrhizae    

Source: http://ifgtb.icfre.gov.in/
1) vesicles
2) arbuscules

35  
 Images  of  Ectomycorrhizae  
Ectomycorrhizal Fungus

Root Cortical
Cells

http://www.ktsa.com/ 36  
http://www.apsnet.org/education/illustratedglossary/PhotosI-M/mycorrhiza.htm
 Mycorrhizal  AssociaLons  
• Mycorrhiza  assist  plants  in  obtaining  water,  phosphorus  and  
other  micronutrients  (e.g.,  Zn  and  Cu)  from  the  soil  
– phosphates  typically  have  low  solubility  and  exist  at  low  
concentraLons  
– fungi  assist  in  uptake  by  scavenging  nutrients  &  by  using  acLve  
transport  to  concentrate  nutrients  against  concentraLon  
gradients  
• Nutrients  are  released  from  fungal  hyphae  and  taken  up  by  
plant  roots;  also  fungi  act  as  reservoirs  
• Plant  supplies  fungi  with  carbon  compounds  
–  both  endo  and  ectomycorrhiza  can  demand  up  to  20-­‐40%  of  
the  total  photosyntheLcally  fixed  carbon  the  plant  produces  
37  
Pathogenic  Microbes  in  Agriculture  
• Plant  pathology:  study  of  causes,  mechanisms,  
environmental  factors  and  control  of  diseases  caused  by  
microbes  
• Plant  pathogens:  fungi,  bacteria,  virus,  protozoa,  
nematodes  

38  
 Plant  Disease  Caused  by  Fungi  
• Most  plant  pathogenic  fungi  
are  filamentous  (mycelium  
&  hyphae)  
• Survival  and  effects  of  
fungal  pathogens  controlled  
mostly  by  bioLc  (microbial)  
and  abioLc  (temperature,  
moisture)  soil  
environmental  factors  
• Cause  billions  in  crop  
damage  to  seeds,  roots,  
stems,  leaves,  fruit  

Leaf mildew Root rot

 Fungus–Bacterium  in  Rice-­‐Blight  
Disease  
• Rhizopus,  a  plant  pathogenic  fungus,  long-­‐thought  to  be  
responsible  for  the  plant  disease  rice  blight  disease    
• Rhizopus  is  pathogenic  because  of  endosymbioLc  
bacteria  Burkholderia  rhizoxinica  that  synthesize  rhizoxin  
 hfps://cen.acs.org/content/cen/arLcles/83/i41/Symbiosis-­‐Behind-­‐Rice-­‐Blight.html  

• New  strategies  for  controlling  the  plant  disease:    

– inhibit  the  bacterium  that  inhabits  the  fungus  

Source: https://doi.org/10.7554/eLife.03007.003
Rice plants affected by rice blight disease Microscopic image of Burkholderia rhizoxinica (green)
residing in the cytosol of Rhizopus microsporus
 Diseases  Caused  by  Bacteria  
• Pathogenic  bacteria  can  occur  
within  the  host  plant  as  
parasites,  on  the  leaves  as  
epiphytes  or  in  the  soil  or  
plant  debris  as  saprophytes  
• Best  studied  is  Agrobacterium
tumefaciens  because  its  
mode  of  afack  involves  
nucleic  acids  
• Crown  gall  disease:  uncontrolled  
cell  division  in  host  –  tumor  or  gall  
• Ti  plasmid  (vir  genes)  induced  

Leaf spots Crown gall in Rose plant

 Plant  Diseases  Caused  by  Viruses  
• Typically  enter  cells  through  wounds  or  infected  pollen;  result  
in  leaf  lesions  
• Viral  diseases  tend  to  occur  on  crops  year  aser  year,  causing  
small  to  moderate  losses  
• Historically  been  hard  to  study;  more  research  recently  by  
PCR/RT-­‐PCR  

42  
Soil  Biological  Control  of  Plant  Diseases  
• Uses  microbes  instead  of  chemicals  to  control  plant  pathogens  
– Antagonists:  biological  agents  that  reduce  numbers  or  acLviLes  
of  pathogens  through  anLbiosis,  compeLLon  or  
hyperparasiLsm  
– anLbiosis  
• occurs  when  pathogen  is  inhibited  or  killed  by  metabolic  products  of  
antagonist  (enzymes,  acidic  agents,  anLbioLcs)  
– compeLLon  
• for  nutrients,  growth  factors,  oxygen  
– hyperparasiLsm  
• a  hyperparasite  is  a  parasite  whose  host  is  a  parasite  
• result  of  invasion  of  parasite  by  secreLon  of  lyLc  enzymes  
• (-­‐)  can  take  longer  to  act,  and  effect  can  be  hard  to  predict  
• (+)  when  successful,  can  last  longer  than  chemical  control   43  
 Pathogen  Suppressive  Soils  
• Soils  in  which  a  parLcular  pathogen  doesn’t  establish  
itself  or  persist  
• Controlled  by  soil  management  processes  
– crop  rotaLons  
– soil  Lllage  (reducing  specific  crop  residues  that  harbor  pathogens)  
– addiLon  of  organics  to  enhance  populaLons  of  antagonists  
relaLve  to  pathogens  
• example:  ‘take-­‐all  disease’  in  wheat  
– P.  fluorescens  produces  phenazine  anLbioLc  that  inhibits  
the  fungal  pathogen  
44  
 Biological  Control  of  Crown  Gall  
Disease  
• Inoculated  planLng  stock  with  non-­‐pathogenic  
Agrobacterium  tumefaciens    strain  K84  
• Produces  bacteriocin  that  inhibits  closely-­‐related  bacteria;  
pathogenic  strains  don’t  grow  
• bacteriocin  is  known  as  Agrocin  84  
– mode  of  acLon:  adenine  analog  that  inhibits  DNA  synthesis  
 

45  
 Environmental  Microbiology  (BI  304):  Lecture  6  
 
Microorganisms  and  Metal  Pollutants    
Instructor:  Dr.  Nalina  Nadarajah  
Email:  nnadarajah@centennialcollege.ca  

Shewanella metabolizes Uranium

Photo Source: http://dsc.discovery.com/; http://soilchem.usu.edu/CurrentResearch.html; South African Journal of Science

Microorganisms  and  Metal  Pollutants  
•  Agenda:  
–  IntroducFon  
–  Important  DefiniFons  
–  Sources  of  Metal  ContaminaFon  
–  Bioavailability  
–  Toxicity  
–  Mechanisms  of  MO  Resistance  &  DetoxificaFon  
–  Methods  for  Studying  Metal-­‐MO  InteracFons  
–  Adverse  Effects  of  MO-­‐Metal  InteracFons  
–  Metal  RemediaFon  
2  
Microorganisms  and  Metal  Pollutants  
(Chapter  18)  
Objec5ves:  

•  To  understand  the  problem  of  metal  contaminaFon  in  

the  environment  and  the  factors  that  contribute  to  it  

•  To  explain  methods  of  microbial  resistance  to  metal  

contaminaFon,  metal-­‐microbe  interacFons  and  the  
applicaFons  of  MOs  to  metal  remediaFon  

3  
 The  Overview  
•  Several  metals  are  required  by  living  organisms  in  trace  
amounts  (Mg,  Mn,  Cu,  Se)  
•  Heavy  metals  play  no  metabolic  role  and  are  highly  toxic  
even  at  low  concentraFons  
•  Main  sources  of  heavy  metals  in  the  environment  are  
smelters,  power  plants,  waste  incinerators  and  vehicle  
exhaust  -­‐  Anthropogenic  emissions  up  to  100  fold  than  that  
from  natural  sources  e.g.  for  Pb,  Cd,  Va,  and  Zn  
•  It  poses  serious  health  and  ecological  risks  -­‐  major  problem  
is  accumulaFon  in  the  food  chain  

4  
 The  Overview  -­‐  II  
•  Metal  contaminants    are    not    'biodegradable’-­‐  
persistent  in  environment  

•  Microbes  have  developed  unique  and  someFmes  bizarre  

ways  of  dealing  with  unwanted  metals,  including:  
–  sequestraFon  and  immobilizaFon  of  metals  
–  enhancement  of  metal  solubility  in  the  environment  
•  Other  strategies  include  bioaccumulaFon  within  the  cell  
in  an  inaccessible  form  so  as  not  to  cause  damage  to  the  
cell  and  chemical  transformaFon  of  a  toxic  compound  
into  a  less  toxic  one      
5  
 Metals  Defined  
•  3  classes  of  metals:  
–  Metals:  elements  that  form  lustrous  solids  and  
are  good  conductors  of  heat  and  electricity  
(excepFons  include  Hg)  
–  Metalloids:  semi-­‐metals;  intermediate  properFes  
between  metals  and  non-­‐metals  (e.g.  arsenic,  
boron,  germanium,  tellurium)  
–  Heavy  metals:  defined  in  many  ways;  specific  
gravity  >  5g  /  mL,  complex  formaFon;  toxicity  
e.g.  Hg,  Cd,  As,  Pb,  Cr  
6  
 Essen5al  &  Toxic  Metals  
•  EssenFal  metals;  known  biological  funcFon  
–  Na,  K,  Mg,  Ca,  V,  Mn,  Fe,  Co,  Ni,  Cu,  Zn,  Mo,  W  
–  generally  funcFon  in  enzyme  catalysis,  transport,  protein  structure,  
charge  neutralizaFon  
–  can  be  toxic  @  high  concentraFon  (e.g.  Cu)  
•  Toxic  metals;  no  known  biological  funcFon  
–  Ag,  Cd,  Sn,  Au,  Hg,  Tl,  Pb,  Al  –  predominantly  caFonic  
–  Metalloids:  Ge,  As,  Sb  –  predominantly  anionic      
–  toxicity  caused  by  displacing  essenFal  metals  from  normal  binding  
sites  (AsO43-­‐  &  Cd  vs  PO43-­‐  &  Zn),  inhibiFng  enzyme  funcFon  &  
disrupFon  of  nucleic  acid  structure  
–  toxicity  depends  on  speciaFon  (form);  influences  bioavailability  
–  someFmes  bejer  tolerated  by  MOs  at  high  concentraFons  
•  Non-­‐toxic,  non-­‐essenFal  metals;  no  known  biological  effects  
–  Rb,  Cs,  Sr,  Ti   7  
 Top 20 Hazardous Substances List
ATSDR/EPA 2017
1.  Arsenic 11. Chloroform
2.  Lead 12. Arochlor 1260
3.  Mercury 13. DDT
4. Vinyl Chloride 14. Arochlor 1254
5. Polychlorinated Biphenyls (PCBs) 15. Dibenz[a,h]anthracene
6. Benzene 16. Trichloroethylene
7.  Cadmium 17. Chromium (+6)
8. Benzo(a)pyrene 18. Dieldrin
9. Polycyclic Aromatic Hydrocarbons 19. Phosphorus, white
10. Benzo(b)fluoranthene 20. Hexachlorobutadiene

ATSDR’s Substance Priority List

Source: https://www.atsdr.cdc.gov/spl/#2017spl
Toxic metals Essential metals 9  
http://www.elementsdatabase.com/
 Sources  of  Metals  
•  Metal  polluFon  generally  results  from  human  ac5vity  
–  mining  
–  ore  refinement  
–  nuclear  processing  
–  industrial  manufacturing  of  bajeries,  metal  alloys,  
electrical  components,  paints  and  insecFcides  
–  weathering  of  parent  materials    
–  metal-­‐containing  sewage  sludge  
PolluFon  tends  to  accumulate  in  soil  à  ‘metal  sink’ à  
contaminates  surface  water,  groundwater  (leaching)  and  
the  vadose  (sub-­‐surface)  zone   10  
 Sources  of  Metal  Pollu5on  
•  Common  causes  of  metal  polluFon:  
–  mining  

11  
12  
Mount  Polley  Mine  in  BC,  Canada    
• open  pit  copper/gold  mine  with  an  underground
component
• owned  by  Imperial  Metals
• On  Aug.  4,  2014,  a  breach  of  the  copper  and  gold  mine
tailings  pond  released  mining  waste  into  Polley  Lake
• ContaminaFon  spread  to  HazelFne  Creek,  and  conFnued
into  nearby  Quesnel  Lake  and  Cariboo  River  of  the
Secwepemc  NaFon  (First  NaFon  community)
• Mine  reopened  parFally  aner  2  years
• Environmentalists  are  concerned  more  tailing  ponds
could  collapse
14  
Metals and Metalloids of Concern - Quantities Produced and
Uses
• Arsenic- As 43,000 tons/yr (1995)
used in: insecticides, herbicides, seed additives, wood
preservatives, desiccants, ceramics, glass (0.2-1%) additives

• Cadmium- Cd 14,500 tons/yr (1995)

used in: battery-powered cellular telephones, camcorders,
personal computers, pigments, stabilizers, coatings, alloys, cheap jewelry
• A CBC Marketplace investigation: some Ardene and Aldo jewelry
manufactured in China contains 1000 X more Cd than levels safe
for children

• Cobalt- Co 18,500 tons/yr (1994)

used in: alloys, nuclear industry, pigment in glazes, UV
protectant in eye protective equipment, paint additive, catalyst
in the petroleum industry.
• Lead- Pb 1,510,000 metric tons/yr in the US (2002)
(a large portion is recycled) over half of lead is used by the
auto industry in batteries. Other uses include manufacture of
cable sheathings, sheet, pipe foil and tubes, solders, alloys,
ammunition, and paints.

• Mercury- Hg 10,000 tons/yr (1980)

major uses include electrical apparatus, the electrolytic
preparation of chlorine and caustic soda, the manufacture of
mildew-proof paint and in industrial and control instruments.

• Nickel- Ni 875,00 tons/yr (1995)

used in alloys, plating, batteries, magnets, electrical
contacts, electrodes, spark plugs, machinery parts, and as a\
catalyst.
 Metal  Bioavailability  in  the  
Environment  
• bioavailable  metals  
– soluble,  non-­‐sorbed,  mobile  
– taken  up  by,  and  toxic  to  biological  
systems  

• non-­‐bioavailable  metals  
– precipitated,  complexed,  sorbed,  
non-­‐mobile  
Fig 18.2
• total  conc  of  metals  in  soil  is  not  
enough  to  predict  toxicity  to   Factors affecting bioavailability:
biological  systems;  toxicity  is   metal chemistry, sorption to soil,
determined  by  bioavailability   pH, redox potential, MO present
17  
 Metal  Chemistry  
• Most  metals  are  caFonic  &  react  with  negaFvely  charged  surfaces  
• in  soil  these  surfaces  include:  
– clay    
– anionic  salts  (phosphates,  sulfates)  
– humic  funcFonal  groups  
– cell  surfaces  
• tendency  to  sorb  to  soil  &  cell  surfaces  called  adsorpFon  affinity;  
determined  by  size  and  charge
Al3+    >    Ca2+    =    Mg2+    >    K+    >    Na+  
• high  adsorp5on  affinity  means  low  bioavailability
• anionic  metals/metalloids  can  be  sorbed  to  negaFvely  charged  
surfaces  through  ca5on  bridging  using  Ca2+  Mg2+  
18  
 Ca5on  Exchange  Capacity  (CEC)  
• Dependent  on  both  organic  majer  and  clay  content  of  the  
soil    
• CEC  reflects  ability  of  soil  to  sorb  metals  
• In  general  CEC  is  inversely  related  to  bioavailability  &  toxicity  
– toxicity  of  metals  within  soils  with  high  CEC  (organic  and  clay  
soil)  is  low  even  at  high  total  metal  concentraFons  
– sandy  soil  with  low  CEC  have  low  metal  binding  capacity,  thus  
high  metal  toxicity  

19  
 Redox  Poten5al  
• Metal  bioavailability  changes  in  response  to  changing  
redox  condiFons    
• Oxidizing/aerobic  condiFons  
– surface  soils  
– metals  found  as  soluble  caFons  (Cu2+,  Pb2+)  
• Reducing/anaerobic  condiFons  
– sediments  /  saturated  soils  
– metals  found  as  insoluble  precipitates  (e.g.  accumulaFon  
of  CuS  /  PbS  in  areas  rich  in  sulfur  and  SRB)  

20  
 pH  
•  High  pH:  

–  metals  found  as  insoluble  metal  mineral  phosphates/  

carbonates  

•  Low  pH:  

–  metals  found  as  free  ions  or  soluble  organometals  

•  pH  also  affects  metal  sorpFon  to  soil  surfaces,  owing  to  

the  changes  in  the  net  charge  on  soil  and  organic  
parFcles  

21  
 pH  
• As  soil  pH  increases  

– the  electrostaFc  ajracFon  between  a  metal  and  soil  

consFtuents  is  enhanced  by  increased  pH-­‐dependent  CEC  

– metal  solubility  decreases  with  increase  in  pH,  thus  

decreasing  metal  bioavailability  

• As  soil  pH  decreases  

– metal  solubility  increases,  thus  enhancing  metal  

bioavailability  

22  
Metal  Toxicity  Effects  on  Microbial  Cell  
• The  toxic  nature  of  metals  results  from  their  strong  ionic  nature  
• Bind  cellular  ligands  &  displace  essenFal  metals  (As  replaces  
PO43-­‐;  Cd  vs.  Zn)  
• Disrupt  proteins  by  binding  sulrydryl  groups  
• Disrupt  nucleic  acid  structure  by  binding  phosphate  /  hydroxyl  
groups  
• Alter  membrane  permeability  
• Decreased  growth,  morphology  change,  inhibiFon  of  
biochemical  processes  

23  
Metal  Toxicity  Effects  on  Microbial  Cell  

Fig 18.4 from Text

24  
 Metal  Entry  Into  Cells  
• Cells  use  specific  transport  mechanisms  
to  get  essenFal  metals  inside  
• Toxic  metals  can  also  use  these  
pathways  to  enter  cells  
• Ionophores:  special  carrier  molecules  that  wrap  
around  metal  ions  so  they  can  pass  through  the  
membrane  by  diffusion  (passive)  

• Ion  channels:  large,  membrane-­‐spanning  molecule  

that  form  a  hydrophilic  path  for  diffusion  (passive)  

• Ion  pumps:  molecules  using  energy  to  transport  ions  

in  one  direcFon  through  a  membrane  (acFve)  
Fig 18.5
25  
 Metal-­‐Microbe  Interac5ons  
• Decreased  growth  
• Abnormal  morphological  changes  
• InhibiFon  of  biochemical  processes  
• ReducFon  in  overall  community  richness  (number  of  
microbes)  
• ReducFon  in  overall  community  diversity  (different  types  
of  microbes)  

26  
 Mechanisms  of  Microbial  Metal  
Resistance  &  Detoxifica5on  
•  Some  MOs  evolved  metal  resistance  because  of  exposure  
to  metals  shortly  aner  life  began  
•  Others  have  evolved  resistance  in  response  to  recent  
exposure  to  polluFon    
•  Some  resistance  mechanisms  in  MOs  are  plasmid  encoded  
and  tend  to  be  specific  for  a  parFcular  metal  
•  Others  are  general  conferring  resistance  to  a  variety  of  
metals.  These  include  resistance  mechanisms  that  are:  
–  general  and  do  not  require  metal  stress  
–  specific  that  dependent  on  a  specific  metal  for  acFvaFon  
27  
 1.  General  Mechanisms  
• Do  not  require  metal  stress  &  onen  serve  other  func5ons    
• Slime  layers  (exopolymers;  carbohydrates  &  proteins)  
– facilitates  adhesion  to  various  surfaces  and  protecFon  against  desiccaFon  and  
predaFon  
– bind  heavy  metals  &  prevent  entry  into  cell  (Pb,  Cd,  U)  
• Siderophores  (iron-­‐complexing,  low  MW  organic  complexes)  
– Natural  iron  binding  compounds  that  chelate  ferric  ions  (which  form  insoluble  
colloidal  hydroxides  at  neutral  pH  and  are  then  inaccessible  
– concentrate  iron  in  environments  where  concentraFon  is  low  &  facilitate  
transport  into  cell  
– can  bind  other,  similar  metals  and  reduce  their  bioavailability/  toxicity  
• Biosurfactants
– produced  and  excreted  by  some  MOs  
– able  to  bind  Cd,  Pb  and  Zn  
– increase  apparent  solubility  of  metals,  but  bound  form  is  not  toxic  to  cells   28  
 Siderophore  Mediated  Iron  
Bioavailability  

Back
29  
 2.  Metal-­‐Dependent  Mechanisms  

Source: http://www.pathconsultddx.com/pathCon/diagnosis?pii=S1559-8675(06)70948-2
• Not  clearly  understood;  depend  on  a  specific  metal  for  
acFvaFon  
• Metal-­‐binding  by  metallothioneins    
– low  MW,  cysFne-­‐rich  proteins  with  high  affinity  for  Cd,  Zn,  Cu,  Ag,  Hg  

– producFon  induced  by  presence  of  specific  metals;  primary  funcFon  

is  metal  detoxificaFon  

• Metal-­‐efflux  system  
– plasmid-­‐encoded,  energy-­‐dependent  
Copper metallothioneins

– pump  toxic  ions  out  of  the  cell  via  acFve  transport  or  diffusion  

– systems  observed  in  many  bacteria  for  As,  Cr  and  Cd  
30  
 Examples  of  Efflux  
Mechanisms  
Cd

H+
Fig 18.7
• Arsenate  enters  cell  via  
phosphate-­‐specific  transport  
Fig 18.8
system  
• Arsenate  reduced  to  arsenite   n  Cadmium  enters  cell  via  
inside  cell   manganese  transport  
pathway  
• Arsenite  pumped  out  via  efflux  
mechanism  (plasmid;  ATP)   n  Cadmium  is  excreted  by  
• As  not  detoxified   a  Cd-­‐proton  pump   31  
2.  Metal-­‐Dependent  Mechanisms  Cont.  
• MethylaFon  
– only  certain  metals  are  involved  
– increases  liphophilicity  -­‐-­‐-­‐-­‐-­‐>  increases  permeaFon/  toxicity  
– increases  metal  volaFlizaFon    -­‐-­‐-­‐-­‐-­‐>  decreasing  toxicity  to  MO  
– removes  significant  amounts  of  metals  from  aquaFc  systems,  
sewage  and  soil    
•  e.g.  methymercury  is  more  toxic  than  mercury  (Hg2+),  but  also  
more  volaFle  
– adverse  effects:    
• methylmercury  poisoning  
• dimethyl  mercury  poisoning  
•  methylarsenes  by  fungi         32  
Methyl  Mercury  Poisoning  –  Minimata  
Bay,  Japan  
•  Minimata  is  a  small  town  in  Japan    
•  It  is  a  Bay  (inlet  of  the  sea)  –  community  mainly  fishing  
•  Chisso  CorporaFon  –  ferFlizer,  petrochemical  and  plasFc-­‐
maker  company  
•  1932  -­‐  1968,  Chisso  Corp.  dumped  ~  27  tons  of  mercury  
compounds  into  the  Bay  
•  Mercury  poisoning  –  “Minimata  Disease”  

33  
Methyl  Mercury  Poisoning  -­‐  Canada  
• Grassy  Narrows,  Ont.  

• Reed  Paper  in  Dryden,  Ont.,  dumped  

chemicals  in  the  river  in  ‘60s  -­‐’70s  

• Way  of  life:  fishing,  hunFng  &  

trapping  

• High  concentraFon  of  mercury  in  fish  

• Community  was  not  informed  about  

mercury  contaminaFon  in  fish  
34  
Microbial  Resistance/  De-­‐toxificaFon  
Mechanisms  

Fig 18.6 36  
 Adverse  Effects  of  Microbial-­‐Metal  
TransformaFons  
•  Acid  mine  drainage    
–  FeS2  exposed  to  oxygen  and  
water  
–  microbial  oxidaFon  by  
Acidithiobacillus  ferrooxidans  
results  in  large  producFon  of  acid  
•  Metal  pipe  corrosion  
–  pipelines  corroded  by  acFons  of  
sulphate-­‐reducing  bacterium  
(SRB)     37  
• Metal  methyla5on  

− methylaFon  makes  metals  more  lipophilic  

− potenFal  for  bioaccumulaFon  and  bio-­‐magnificaFon  in  food  
webs  
− primary  organism  involved  in  mercury  methylaFon  sulphate-­‐
reducing  bacteria  (SRB)  
− Hg  resistance  may  involve  the  reducFon  of  Hg2+  to  elemental  
mercury  (Hg0)  by  mercuric  reductase  in  both  G+ve  and  G-­‐ve  
bacteria  

38  
Fig 17.12 from 2nd Edition Text
Methods  for  Studying  Metal-­‐Microbe  
Interac5ons  
• Challenges:    

– total  metal  concentra5ons  do  not  accurately  predict  

biologically  toxic  concentra5on  

– metals  are  not  biologically  degradable;  difficult  to  

determine  if  and  how  a  metal  is  being  detoxified  when  
total  concentraFon  does  not  change  

• Primary  method  involves  culturing  metal-­‐resistant  

microbes  in  the  lab  followed  by  measurement/
calculaFon  of  total  and  soluble  metal  concentraFon  
– any  precauFons  in  interpretaFon  of  results?   39  
 Culturing  Metal-­‐Resistant  MOs  
• Metal  resistance  can  be  affected  by  contents  of  media  
(yeast  extract,  phosphate  buffers,  amino  acids)  which  
may  bind  to  metals  and  by  pH    
• Choose  defined  media  that  minimizes  metal  binding  
– phosphate-­‐free  buffers  (MOPS,  MES,  PIPES)  
• phosphate  buffers  strongly  precipitate  metals  
– slightly  acidic  pH  (~6.0)  to  avoid  metal  precipitaFon  

40  
 Determining  Metal  Concentra5on  
• Total:  
– dissolve  soil  parFcles  by  digesFon  of  sample  with  hot  acid  (nitric,  
percholoric);  releases  all  metals  

• Soluble:  
– sequester  metal  via  interacFon  with  weak  acid  or  extracFon  with  
deionized  water  

• For  Both:  
– determine  metal  concentraFons  in  extract  using  AA  or  inducFvely  coupled  
plasma  atomic  emission  (ICP-­‐AE)  spectroscopy  

• Caveats:  
– metals  may  precipitate  with  culture  medium  components  and/or  may  bind  
to  walls  of  glassware;  must  perform  metal  controls  with  no  inoculum  to  
disFnguish  between  bio  and  chem  metal  removal   41  
 Determining  Metal  Concentra5on  
• Ion-­‐selecFve  electrodes  
– only  available  for  some  metals  (Cd,  Pb,  As)  
– soluble  metal  concentraFon  as  low  as  1  μM  
– measures  only  free  (bioavailable)  metal  ions    
– advantages:  ease  of  use,  inexpensive,  colour/  turbidity  
doesn’t  interfere  
– disadvantage:  other  ions  in  soluFon  interfere  
• Ion  exchange  columns  
– chromatographic  separaFon  
– metals  bound  to  ion  exchange  resin  are  eluted  with  metal-­‐
complexing  soluFon  
– can  be  analyzed  via  conducFvity,  AA,  ICP-­‐AE,  GC  or  HPLC      
42  
Visualiza5on  of  Metal  Deposits  inside  
Bacteria  
• Transmission  electron  microscopy  (TEM)  
– spaFal  visualizaFon  (sequestered  metal  inside  or  outside  cell)  

• Energy  dispersive  x-­‐ray  spectroscopy  (EDS)  

– metal  element  can  be  idenFfied  based  on  signature  spectra  
– Used  in  combinaFon  with  TEM  

Copper metallothioneins Intracellular accumulation of Pb 43  

Methods  for  Preven5ng  Movement  of  
Metals  to  the  Surroundings  
• immobilizaFon  –  Reduces  metal  solubility  and  
bioavailability  

• pH  alteraFon  (metal  solubility  decreases  with  increasing  

pH)  

• addiFon  of  organic  majer  –  electrostaFc  ajracFon  

between  metals  and  organic  majer  

• Problem:  impossible  to  know  if  the  metals  will  remain  

immobilized  indefinitely  
44  
 Methods  Aimed  at  Physically  
Removing  Metal  Contamina5on  
•  Physical  ExcavaFon  –  e.g.  of  sediments  (dredging)  

•  Soil  washing  techniques  –  washing  with  acidic  soluFons  

or  chelaFng  agents  (e.g.  EDTA)  solubilizes  metals  

•  IncineraFon  of  soils  –  thermal  treatment  involving  

combusFon  at  high  temperature  

•  Problem:  expensive  and  moves  the  problem  to  another  

locaFon  
45  
 Microbial  Approaches  for  Metal  
Pollu5on  Remedia5on  in  Soils  
Metals  can  not  be  degraded,  making  remedia5on  difficult  
• Metal  solubilizaFon  /  leaching  
– removal  from  soils/sludge  using  methods  developed  for  valuable  metals  
• Microbial  surfactants  and  polysaccharides  
– bind  to  metals  and  increase  solubility  
• Microbially  induced  Metal  VolaFlizaFon  (e.g.  methylaFon)  
– increases  bioavailability  &  toxicity;  MOs  volaFlize  to  remove  metals  from  
immediate  environment  
– used  in  bioremediaFon  of  selenium-­‐contaminated  soil  in  San  Joaquin,  
California  by  selenium-­‐volaFlizing  MOs  (Case  Study  pg  432)  
• Metal  sequestraFon    
– relies  on  the  ability  of  some  microorganisms  to  produce  metal-­‐complexing  
polymers,  such  as  siderophore  
46  
Microbial  Metal  Remedia5on  

47  
Fig 18.15
Microbial  Approaches  for  RemediaFon  of  
Metal-­‐Contaminated  AquaFc  Systems  
• Based  on  ability  of  MOs  to  complex  and  precipitate  
metals  

– binding  to  cell  surface/  EPS,  intracellular  uptakes,  

volatlizaFon,  precipitaFon    

• Result  is  detoxificaFon  and  removal  

• Most  common  is  treatment  of  metal-­‐contaminated  

waters  with  microbial  biofilms  

– metals  have  high  affinity  for  exopolymers  that  make  up  

biofilm   48  
 Microbial  Metal  RemediaFon  
Approaches  for  Metal-­‐Contaminated  
Waters  

49  
Fig. 18.17 from Text
 Biofilms  in  Removal  of  Metals  from  
Contaminated  Waters  

•  biofilm  immobilized  on  support  

•  contaminated  water  passed  through  &  metals  are  removed  
•  can  have  biofilm-­‐producing  MOs  growing  on  support  to  provide  constant,  fresh  
supply  of  biofilm  
•  or  commercially-­‐produced,  non-­‐living  microbial  preparaFons  e.g.  BIOCLAIM  
(Bacillus  biomass)  ,  AlgaSORB  (Chlorella  vulgaris)   50  
 What  about  True  BioremediaFon?  
• Complete  microbe-­‐mediated  transforma5on  of  toxic  metal  
compounds  to  non-­‐toxic  compounds  like  CO2  and  H2O  is  NOT  
possible  

• Microbial  processes  that  contribute  to  conversion  of  toxic  to  

less-­‐toxic  compounds  are  being  acFvely  studied  

• Specific  bacterial  genes  have  been  idenFfied  that  funcFon  in  

bacterial  resistance  to  many  heavy  metals  

• Have  succeeded  at  creaFng  geneFcally-­‐modified  MOs  that  

consFtuFvely  express  operons  for  heavy-­‐metal  resistance;  
being  looked  at  for  roles  in  bioremediaFon  efforts  
51