Het microbiologisch leven in de bodem by xiangpeng

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									Microbial Life in Soil

         Prof. dr. ir. Willy Vestraete
          Dr. ir. Tom Van de Wiele


Laboratory of Microbial Ecology and Technology
                   (LabMET)
           Faculty of Bioengineering
               Ghent University
              LabMET.Ugent.be

                       1                            Laboratory of
                                Microbial Ecology and Technology
           Topics of Discussion
The microbial ecosystem in the soil

The most common bacterial soil processes

The microbial growth

The simulation of the microbial transport in the soil

The bioavailability of contaminants

                           2                              Laboratory of
                                      Microbial Ecology and Technology
           Topics of Discussion
The microbial ecosystem in the soil

The most common bacterial soil processes

The microbial growth

The simulation of the microbial transport in the soil

The bioavailability of contaminants

                           3                              Laboratory of
                                      Microbial Ecology and Technology
       1. The Microbial Ecosystem
   Ecological importance of soil:
    – The production of biomass (food,…)
    – The natural biotope for:
       • Micro-organisms
       • The plant-communities
       • The animal world
    – To filter or to buffer soil contaminants:
       • By retaining, transforming, neutralizing…




                                 4                              Laboratory of
                                            Microbial Ecology and Technology
       1. The Microbial Ecosystem
   The interactions between soil and soil-biotic
    communities


                              geological substrate;
             climate            mother material          topography




              vegetation                              soil properties
             and soil biota                           and soil profile

                                      time




                                        5                                   Laboratory of
                                                        Microbial Ecology and Technology
      1. The Microbial Ecosystem
 “The soil represents a set of physical-
  chemical conditions in which life
  develops in all diversity.”
 Life: complex communities with ten
  thousand different species of micro-
  organisms:
    – Bacteria
    – Fungi      Micro-aggregates
    – Protozoa
    and macro-organisms

                      6                          Laboratory of
                             Microbial Ecology and Technology
            1. The Microbial Ecosystem
   The soil biodiversity:
    Group             Number of species            Density
    Micro-organisms        35.000                 105-108/g
    Nematodes               7.000                 104-105/g
    Protozoa                5.000                       -
    Insects                60.000                       -
    Mites                  30.000                       -
    Grubs                   3.500                       -




                             7                                Laboratory of
                                          Microbial Ecology and Technology
       1. The Microbial Ecosystem
   The microbial biodiversity:
    – 35.000 different species
    – 105-108 per gram soil
    – Great diversity of „genetic capacity‟ and
      „biological know-how‟
    – Participant of a „food-web‟ in the soil, that
      develops and grows in complexity until a
      maximally efficient filling in of the soil
      functions is obtained


                          8                            Laboratory of
                                   Microbial Ecology and Technology
         1. The Microbial Ecosystem
   Soil-profile and micro-organisms:
    micro-organisms contribute to the profile-development by
    increasing the solubility of the organic and inorganic material
       A0                                    Deposition of organic material
       A1: much humus                 Elution of anorganic and organic compounds
       A2: less humus                             from the upper layer
       B1: humus                               Depositon of compounds
       B2: iron
       Mother-material


       cm depth                   Horizon                    Bacteria                   Fungi
          3-8                          A1                       7800                      119
         20-25                         A2                       1800                      50
         65-75                         B1                        10                        6
       135-145                         B2                         1                        3
       Podzol: number of propagules x 103/g


                                                      9                                       Laboratory of
                                                                          Microbial Ecology and Technology
           Topics of Discussion
The microbial ecosystem in the soil

The most common bacterial soil processes

The microbial growth

The simulation of the microbial transport in the soil

The bioavailability of contaminants

                          10                              Laboratory of
                                      Microbial Ecology and Technology
         2. Bacterial Soil Processes
   Soil bacteria are nutritionally exigent, more
    than one half of the bacteria requires one or
    more growth factors
        Requirements                  % of the soil bacteria
a. Minerals + Organic C-Source                    15

b. a + Amino-acids                                15

c. a + b + Vitamins                               30

d. a + b + c + Soil-extract                       40



                                 11                            Laboratory of
                                           Microbial Ecology and Technology
          2. Bacterial Soil Processes
   Organo-heterotrophic bacteria:
    building organic cell-compounds out of
    organic material
    Bacillus: amino-acids
    Clostridium: carbohydrates + amino-acids


   Chemo-lithotrophic bacteria (autotrophic):
    building organic cell-compounds out of
    chemical reactions with anorganic material
    Nitrosomonas: NH4+ + 3/2 O2  NO2- + 2H+ + H2O
    Nitrobacter: NO2- + 1/2 O2  NO3-


                             12                            Laboratory of
                                       Microbial Ecology and Technology
       2. Bacterial Soil Processes
   Microbial respiration: oxygen or other
    compounds act as hydrogen(=electron)-
    acceptor
    – Aerobic: O2
    – Facultative aerobic: O2, NO3-
    – Facultative anaerobic: O2, NO3-, organic
      acceptors
    – Anaerobic: Fe3+, Mn4+, SO42-, CO2, organic
      acceptors
   Aerobic conditions: Eh > 0, anaerobic
    or anoxic conditions: Eh < 0
                         13                          Laboratory of
                                 Microbial Ecology and Technology
         STANDARD REDUCTION POTENTIALS

                 substrate              product

                                H+ e-
Aerobic
conditions O2                0.82 V
                                                        H2O

Anaerobic Fe3+               0.77 V
                                                        Fe2+
conditions                   0.74 V
           NO3-                                         N2




                             -0.23 V
         SO42-                                          H2S
         CO2                 -0.24 V
                                                        CH4
                                   14                         Laboratory of
                                          Microbial Ecology and Technology
       2. Bacterial Soil Processes
   The degradation of organic compounds:
    – Happens through selective enzymes and
      delivers energy for the microbial
      metabolism: metabolic degradation
    – Happens fortuitously by non selective
      enzymes and delivers no energy for the
      metabolism: cometabolic degradation

   Reaction kinetics:
    metabolic > cometabolic

                       15                          Laboratory of
                               Microbial Ecology and Technology
        2. Bacterial Soil Processes
   Degradation of biotic organic material:
    If favorable conditions are present, every
    compound will be degraded by the micro-
    organisms, in a quick (DT50: hours-days) or
    slow way (DT50: months-years), e.g.

    – Cellulose (Cellovibrio, Aspergillus, Streptomyces)
      (DT50-aerobically: 3-4-5 months)
    – Lignin (Basidiomycetes)
      (DT50-aerobically: 0,5-1y)
    – Hydrocarbons e.g. aromatic compounds (Bacillus)
      (DT50-aerobically-monomers: 0,5-1 month)
      (DT50-anaerobically-polymers: months-years)

                            16                             Laboratory of
                                       Microbial Ecology and Technology
        2. Bacterial Soil Processes
   Example: Aerobic cleavage of the aromatic ring of
    catechol by oxygenase enzymes




                            17                            Laboratory of
                                      Microbial Ecology and Technology
      2. Bacterial Soil Processes
 Degradation of xenobiotic organic material:
  If favorable conditions are present, some
  compounds will be degraded, other ones are
  recalcitrant.
 The more a xenobiotic compound resembles
  a biotic one, so much the more it will be
  recognized by microbial enzymes and be
  transformed




                      18                          Laboratory of
                              Microbial Ecology and Technology
        2. Bacterial Soil Processes
   Degradation pathways
    for the pesticide
    parathion




                           19                       Laboratory of
                                Microbial Ecology and Technology
         2. Bacterial Soil Processes
   Rules of thumb to judge the biodegradability of an
    unknown aliphatic chemical compound
    – The C2-C18 chain length is optimal
    – CC > C=C > C-C
    – The more branched, the less the biodegradability

              >         >


    – Substitution with –OH or –COOH is positive
    – Substitution with –Cl, –NO2, –SO3H is negative
    – The more substituents, the stronger the positive or
      negative effect
    – The closer the substituents towards the active group, the
      greater its influence            O


                            Cl
                                           OH


                                  Cl


                                 20                                 Laboratory of
                                                Microbial Ecology and Technology
         2. Bacterial Soil Processes
   Rules of thumb to judge the biodegradability of an
    unknown aromatic chemical compound
    – Substitution: see aliphatic compounds
    – Para isomers are more biodegradable than ortho, resp. meta
      isomers.     OH           OH        OH


                                                  Cl


                               >                      >
                                                                           Cl

                    Cl

    – Poly aromatic compounds are difficult to degrade, e.g.
      benzopyrenes



                  Naphtalene       Pyrene                 Benzo[a]pyrene
                                      Recalcitrance


                                            21                                           Laboratory of
                                                                     Microbial Ecology and Technology
       2. Bacterial Soil Processes
   Environmental factors:
    – A higher microbial diversity increases the
      degradation-capacity by proto-coöperation
    – Water-content: optimal ca. 20%
    – Temperature: factor 1,5-2 for 10°C
    – Sorption: through sorption processes,
      compounds are no longer bio-available
      (see below), e.g. straws slows down the
      degradation of atrazin.


                        22                          Laboratory of
                                Microbial Ecology and Technology
           Topics of Discussion
The microbial ecosystem in the soil

The most common bacterial soil processes

The microbial growth

The simulation of the microbial transport in the soil

The bioavailability of contaminants

                          23                              Laboratory of
                                      Microbial Ecology and Technology
            3. Microbial Growth
 Growth: increase in the number of cells
 Essential: any given cell has finite life span in
  nature  species maintains only as result of
  continued growth of the population
 Useful in designing methods to control microbial
  growth




                          24                           Laboratory of
                                   Microbial Ecology and Technology
           3. Microbial growth
☞ Time required for complete growth cycle is highly
 variable and dependent on nutritional,
 environmental and genetic factors
                                         Time         Total number
                              20                      of E. coli cells
                                          0u00                  1
                              21          0u20                  2
                                          0u40                  4
                                          1u00                  8
                              22          1u20                 16
                                          1u40                 32
                                          2u00                 64
                              23
                                          2u20                128
                                          2u40                256
                              24          3u00                512
                                          3u20               1024
                              2n          3u40               2048
                                          4u00               4096
                                           …                   …
                                          7u00             2097152

                         25                            Laboratory of
                                   Microbial Ecology and Technology
              3. Microbial growth
   Bacterial growth: cells divide into two new cells by
    binary fission




                         Bacillus subtilis




                         Dividing streptococci

                              26                                 Laboratory of
                                             Microbial Ecology and Technology
                 3. Microbial growth
☞ Bacterial population growth: typical growth curve

           Log10 viable organisms/ml                                       600

                                                                           500




                                                                                 Substrate (mg/l)
                                                                           400

                                                                           300

                                                                           200

                                                                           100
                                                                           0




☞ Growth rate: change in cell number or cell
mass per unit time
                                       27                       Laboratory of
                                            Microbial Ecology and Technology
              3. Microbial Growth
   Most information available resulting from
    controlled laboratory studies using pure cultures
    of micro-organisms




    ☞ Compare the complexity of growth in a flask and
    growth in a soil environment. Although we understand
    growth in a flask quite well, we stil cannot always
    predict growth in the environment!
                              28                            Laboratory of
                                        Microbial Ecology and Technology
           Topics of Discussion
The microbial ecosystem in the soil

The most common bacterial soil processes

The microbial growth

The simulation of the microbial transport in the soil

The bioavailability of contaminants

                          29                              Laboratory of
                                      Microbial Ecology and Technology
    4. Microbial transport in the soil
   The knowledge about bacterial
    transport in soil is required:
    – To protect groundwater sources from
      microbial contamination
    – To estimate the influence of rainfall on
      microbial transport in soil
    – To design sustainable and safe in situ
      bioremediation techniques
      (Can the contact between micro-organisms
      and the contaminants be realized?)

                       30                          Laboratory of
                               Microbial Ecology and Technology
     4. Microbial transport in the soil
   Determined by:
     – Dispersion (no straight path by diffusion (concentration
       gradient and Brownian movement) and mechanical mixing)
    – Advection (transport of non-reactive components at a rate equal
       to the average velocity of the percolating water)
    – Sorption (a part of the bacteria will be sorbed onto the soil
       particles)
    – Retention (a part of the bacteria will be retained in the pores in
       the soil)
    – Microbial die-off

   Modeling this transport requires interdisciplinary
    research (microbiology + hydrogeology)
                                     31                                Laboratory of
                                                   Microbial Ecology and Technology
     4. Microbial transport in the soil
   Example: The modelling of the evolution of the concentration
    of the anaerobic micro-organism Desulfitobacterium
    dichloroeliminans strain DCA1 and the contaminant 1,2-
    dichloroethane in an in situ bioaugmentation strategy by
    MOCBAC-3D (Prof. L. Lebbe and K. Smith, UGent)
          Concentration of Desulfitobacterium dichloroeliminans strain DCA1




                                         32                                  Laboratory of
                                                         Microbial Ecology and Technology
     4. Microbial transport in the soil
   Example: The modelling of the evolution of the concentration
    of the anaerobic micro-organism Desulfitobacterium
    dichloroeliminans strain DCA1 and the contaminant 1,2-
    dichloroethane in an in situ bioaugmentation strategy by
    MOCBAC-3D (Prof. L. Lebbe and K. Smith, UGent)
                  Concentration of the contaminant 1,2-DCA




                                     33                                 Laboratory of
                                                    Microbial Ecology and Technology
           Topics of Discussion
The microbial ecosystem in the soil

The most common bacterial soil processes

The microbial growth

The simulation of the microbial transport in the soil

The bioavailability of contaminants

                          34                              Laboratory of
                                      Microbial Ecology and Technology
                5. Bio-availability
   Definition: the fraction of the total concentration of a
    contaminant that will be taken up by the micro-
    organisms out of the environment
   Generally: the bio-availability to the micro-organisms
    is directly dependent on the solubility of the
    contaminant in the aqueous phase
   Affecting processes: diffusion of the contaminant in
    the boundary layer, the macro-pores and the micro-
    pores, physico-chemical interactions with the particle
    surface and the desorption velocity of the
    contaminant out of the sediment which is strongly
    dependent on the particle size and particle density
   Consequence: The degradation efficiency of a
    contaminant will be reduced as much as
    the mass transfer is limited to the micro-organism


                              35                            Laboratory of
                                        Microbial Ecology and Technology
                            5. Bio-availability
   Processes of bio-availability
                                                 Biological membrane


                    Bound
                  Contaminant


                                                                       Absorbed                 Place of
        Dissociation        Association                              contaminant in             biological
                                                                     micro-organism             response


                     Free
                  Contaminant
      Partitioning and interaction       Passive or facilitated         Assimilation, dissimilation and
       of the contaminant with       diffusion or active transport      accumulation of the contaminant
            different phases              of the contaminant            with specific reaction kinetics
                                        through the membrane
                                        to the micro-organism




                                                            36                                      Laboratory of
                                                                                Microbial Ecology and Technology
3. Bio-availability




         37                         Laboratory of
                Microbial Ecology and Technology
              5. Bio-availability
   Significance of bio-availability:
    – The mass transfer limits the bio-availability
    – The endpoint of bioremediation must be
      related to the matrix
    – The concentration of a contaminant in a
      specific soil must be recalculated to the
      concentration in a „standard soil‟ to
      evaluate the contamination extent
    – Important for legislation: the line must be
      drawn, but where? (high „grey-value‟)

                         38                           Laboratory of
                                  Microbial Ecology and Technology
            Take-home message
   Great diversity in the ecosystem of the soil
   Micro-organisms participate in
    biogeochemical processes and are able to
    biodegrade a variety of biotic and xenobiotic
    compounds
   Knowledge about the transport of micro-
    organisms in soil is required for safely
    designing clean-up strategies
   Bio-availability is determined by the mass-
    transfer of compounds to the micro-
    organisms, so the endpoint of bioremediation
    is not absolute
                          39                          Laboratory of
                                  Microbial Ecology and Technology

								
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