SEPA
                           United States
                           Environmental Protection
                           Agency
                         Environmental Research
                         Laboratory
                         Athens, GA 30613-7799
 Research and Development   EPA/600/M-90/004 July 1990	
ENVIRONMENTAL
RESEARCH   BRIEF
  Sorption of Heavy Metals by Intact Microorganisms, Cell Walls, and Clay-
                                     Wall Composites

            M. D. Mullen1, T.J Beveridge2, F. G. Ferris2, D. C. Wolf3, and G. W. Bailey4
Abstract
Sorption of Ag + , Cd2', Cu2*. and La?~ from solution by
four bacteria, Bacillus cereus, B. subtilis, Escherichia coli,
and Pseudomonas aeruginosa, and two fungi, Aspergillus
mger and Mucor rouxii, was examined.  Metal sorption was
assessed using  Freundlich adsorption isotherms  to
partitioning of these metals between the solution and
microbial biomass phases. Precipitation of Ag and La by the
bacteria precluded the use of the Freundlich isotherm for
these metals.  Freundlich K values for bacterial sorption of
Cd and Cu ranged from 0.389 to 1.067 and 2.188 to 4.150,
respectively.  The affinity series for bacterial  sorption of
these metals decreased in the order Ag > La >  Cu > Cd.
Mean K values for fungal  metal sorption were 2.235, 0.098,
0.818, and 4.290 for  Ag, Cd, Cu, and La, respectively. The
fungal affinity series was La > Ag > Cu > Cd.
    To further define toxic heavy metal sorption by bacterial
surfaces, walls from representative gram-negative  (E. coli)
(E) and gram-positive (B.  subtilis} (B) bacteria were isolated
and purified, and compared to smectite  (S) and kaolinite (K)
    Department of Agriculture and Natural Resources.  University of
    Tennessee-Martin, Martin. TN 38238

    Department of Microbiology,  CBS, University of Guelph.Guelph,
    Canada NIG 2W1

    Department of Agronomy, University of Arkansas. Fayetteville. AR
    72701
    Environmental Research Laboratory. U.S. Environmental Protection
    Agency,Athens. GA 30613-7799
                       with regards to  metal binding  capacity.  Metal binding
                       decreased in the order B>E>S>Kfora group of
                       metals consisting of Ag, Cu. Ni. Cd, Pb, Zn, and Cr.  High
                       levels of metal immobilization tn B and E were the result of
                       surface-associated  heavy metal  precipitates.  Adsorption
                       isotherms were  constructed for  wall-clay interactions and
                       clearly showed that there were strong interactions between
                       each clay and each wall type to form composite aggregates.
                       The composites  utilized a variable proportion of the innate
                       reactive sites available to heavy metal ions and resulted in
                       lower concentrations of immobilized metals
                           Binding capacity was in the order  B + S  > B + K >
                       E + S  > E + K  and it was apparent that  the biological
                       constituents  dominated the  immobilization process.
                       Experiments were designed to remobilize three  bound
                       metals (Ag, Cu and Cr) and relied on several parameters -
                       pH fluctuation, metal chelation  by outside agents (EDTA),
                       metal  complexation by  natural  organic  acids (fulvic acid),
                       competition for binding sites by non-toxic metal ions (Ca2 + ),
                       and enzyme hydrolysis of the wall fabric (lysozyme).  The
                       results of these  experiments suggested that there was no
                       easily observed trend for remobilization; each particulate
                       component, each composite, and each metal had a distinct
                       influence on the ease of  heavy metal  remobifization.
                       Increased knowledge of the metal sorption  capacity of
                       microbial cells and their cell walls should enable us to better
                       predict the fate  of metals introduced  into the environment,
                       and may also  be of value for enhanced utilization of
                        microbial cells  in renovation  and  metal  recovery  from
                        municipal and industrial wastewaters.

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  Background

      Adsorption-desorption processes regulate the fxtent of
  binding that exists betyveen a given solid  surface  and a
  chemical species in solution. Adsorption refers to a'process
  whereby solutes such  as  metal ions adhere  tola  solid
  surface such as that  presented  by microbial cells.   In the
  environment, mineral and  organic  surfaces in  soils  and
  sediments  are  the principal sites of metal  ion  adsorption.
  One important  organic surface category that has riot been
  extensively  studied is that possessed  by microorganisms.
  The cell surfaces of bacteria and  fungi  have chemical
  properties that  could play important roles in the adsorption
  and mass  partitioning of ,metal  ions.  , To conduqt  a risk
  assessment of  potential heavy  metal contamination of
  terrestrial  and  aquatic   ecosystems,  it  is  necessary to
  quantitate  the magnitude of metal adsorption to biological
  surfaces.                                       \

     The information  available,  on metal  adsorption by
  microbial surfaces is rather  limited, and most of it has been
  published in the last 20  years. Bacteria behave as colloidal
  particles in  aqueous systems and have a pH-dependent net
 negative surface charge1. Metal binding studies of Bacterial
 cell  walls  have demonstrated  that these surfaces  are
 capable of  removing appreciable quantities of a  variety of
 metals 2- 3.  Isolated cell walls of the gram-positive bacteria
 B. subtilis and B. licheniformis bound "larger quantities than
 cell envelopes  of th'e gram-negative bacterium  E.\ coli 2.
 Metal ion  uptake by  the fungus Rhizopus arrhiziis  was
 found to be'directly related  to the Ionic radii of  some ten
 metals tested, and it was concluded that adsorption  of the
 metals occurred at sites  in  and on the cell that  contained
 phosphate and carboxylate groups 4.

     Information  on partitioning of  heavy metals in tejrestrial
 and  aquatic  ecosystems is not  complete.   Increased
 knowledge of heavy  metal   partition  coefficients between
 aqueous solutions and solid  surfaces will enable us to  more
 accurately predict heavy  metal  behavior in the environment
 and, thus, more  carefully assess potential toxic heav^ metal
 exposure.  Specifically,  this information will aid Jin the
 development of  risk assessment models.  It will  aid in our
 understanding of the mobility and  fate of metals in  both
 surface and  groundwaters.   Land application of municipal
 sewage  sludge  and disposal of heavy metal-containing
 hazardous  wastes are  also  areas where  information
 regarding metal partition coefficients Is  crucial    An
 additional area where  heavy metal adsorption to  bacteria
 and other  biomass is  particularly important is [in  the
 understanding of metal  partitioning  in  municipal  and
 industrial wastewater treatment facilities and their evaluation
 relative to metal  removal efficiency.                I

 Laboratory Procedures                       j
 Intact Microbial Cell Studies                  \
    Bacterial cells of  B.  cereus  strain ATCC  117|78  P
 aeruginosa strain ATCC 14886, B. subtilis 168 and £  coli
 K-12 strain  AB264 were maintained  and  cultured  as
 previously described s.  The  fungi examined were A\  niger
 ATCC 34467 and M. rouxii ATCC 24905. Fungal celte were
 harvested by vacuum filtration and washed with 3 volumes
of cold, 10 mM Ca(N03)2 solution.  Portions of moist'fungal
biomass  were weighed,  placed  in  10-mL  polypropylene
tubes, and 8  ml. of Ca(NO3)2 were added.  The cells were  "
stored at 5°C for approximately 2  h prior to use.   Mpisture
  determinations were done on six subsamples to determine
  the dry weight added to the tubes.

      Nitrate salts of Ag*, Cd2*, Cu2*, and La3* were used.
  All metal solutions were made in  "pH 4, 10TnM  Ca(N03)2
  solutions to minimize precipitation of metals and differences
  in ionic strength across metal concentrations.  Initial metal
  concentrations for the  bacterial experiments  were:  1  0 1
  0.01, and 0.001 mM Cd2 + ; 1, 0.1, 0.01 and 0.005 mM Cu2 + ;
  and  10, 1, 0.1, and 0.01  mM  for Ag + and La3*. Equilibrium
  concentrations of  both  Ag  and La were typically below
  detection limits at  initial concentrations of 0.01 mM or less.
  For fungal sorption experiments, initial concentrations of all
  metals were 1, 0.1, 0,01, and 0.005 mM.

     Bacterial  metal   sorption   was   determined   by
  equilibrating cells in the metal solutions at a concentration of
  2  to  3  mg  dry wt mL-1.   The  cell suspensions  for  all
  microorganisms  were equilibrated  for 2  h  at 5 °C on  a
  rotating shaker.  Timed  equilibration experiments  indicated
  that  metal sorption was  relatively  constant within the 2  h
  time.  After equilibration, bacterial  cells were removed from
  solution by centnfugation and fungal cells were removed  by
  filtration through 0.45-nm membrane filters.  Concentrations
  of metal in solution were determined by inductively coupled
 argon plasma spectroscopy.  Sorption  is defined  as  the
  removal of metal from solution by microorganisms ,by one or
 more processes,  such  as  adsorption,  precipitation,  or
 uptake.   Where  applicable, sorption  isotherms  were
 evaluated using  the logarithmic form of the  Freundlich
 adsorption equation.
             logS = logK + nlogC
(1)
 where S is the metal  sorbed  in iimol  g dry wt-1,  C is the
 equilibrium solution concentration in nmol L-i, and K and n
 are  constants  6.   isotherms  were constructed using the
 methods outlined by Dao et al.7

 Isolated   Bacterial Wall,  Clay,  and  Wall-Clay
 Composite Studies
     Walls from B. subtilis  168 and from E. coli K-12 strain
 AB264 were isolated and purified according to Walker et al.s
 Na-smectite (montmorillonite,  SWY-1  Crook County, WY)
 and  Na-kaolinite (KGA-1,   Washington  County,  GA) were
 obtained from  the Source Clays  Repository of  the  Clay
 Minerals Society.

     Adsorption isotherms of the  wall-clay composites were
 determined by reacting 1 mg mL-1 of clays in distilled water
 with  0, 0.05, 0.1, 0.2, 0:4, 0.6,  0.8 or 1.0 mg  dry weight mg
 mL-1 of walls at circumneutral  pH for 10 min at 22°C. The
 experiments relied on the natural buffering capacity of the
 particulate  material.  Centnfugation at 12000  x g for 30 min
 into  a  60% (w/v)  sucrose  cushion separated unabsorbed
 walls from the clay and the  clay-wall composite and allowed
 adsorption efficiency to be estimated.

     Sorption of Ag, Cu, Ni, Cd, Zn, Pb and Cr nitrate salts
 was  accomplished  in 5 mM  metal solutions at a clay, wall or
 clay-wall concentration of 1. mg dry  weight mL-1 for 10 min
 at 22°C.   The  particulates were  washed  five times  in
distilled water to remove unbound metal.  The metals were
analyzed by atomic absorption  spectrophotometry.   In
addition,  location of metal concentration was monitored by
transmission electron microscopy and energy dispersive X-

-------
ray speetroscopy.
in Walker et al.s
More experimental details can be found
Remobilization Experiments
    Ag ~, Cu,"  or Cr" loaded B. subtilis and E.  coli walls,
clays, and  wall-clay composites  (as outlined in  section 2)
were used  in this study.   Fulvic acid  (10  to 120 mg L-1),
Ca2+ (0 to 160 mg L-1), EDTA (0 to 500 M), H+ (pH 3 to
9).  and  lysozyme  (40 to  160 mg  L-1)  were  used  as
remobilization agents and  were  interacted  with  the  heavy
metal-loaded particulates for 48 h at 22°C. Particulates were
separated from the fluid phase by centrifugation (18000 x g
for 30  min)  and  the supernatant  was  analyzed  for
remobilized metal.  The amount of  cell  wall hydrolysis by
lysozyme was estimated by the acid  ninhydrin test 9.

Results and Discussion

Intact Microbial Cell Studies
    Constants  for sorption of Cd and  Cu  from solution by
the four bacteria are given in Table 1.  B.  subtilis was the
least efficient bacterium for  sorption of  Cd  and  B. cereus
was the least efficient for Cu  sorption, with K values of 0.147
and 2.188, respectively.   £ coli and  B. subtilis were the
most efficient for Cd and Cu  sorption with K values of 1.067
and 4.150, respectively. The slopes of the  isotherms were
all  less  than one  and were generally  different  among
bacteria within metals.  Because the slopes were not equal,
the  differences in affinities for the  metals by the bacteria
predicted by K at an equilibrium  concentration of 1 iiM  may
not hold  at  higher concentrations.   For example,  P.
aeruginosa removed  the  most  Cd and Cu from  solution
 when the initial concentration was 1  mM. Figure 1 presents
 the actual Cd  sorption isotherms  for  B.  cereus  and  P.
 aeruginosa.
     Bacterial sorption of Ag and La did not conform to the
 Freundlich equation because of precipitation of these metals
 by the bacteria.  On average, 99% of  the total Ag+  and
 89% of the total  La3+ were removed from the 0.1  mM
 solutions by the bacteria.  Electron  microscopy  and  energy
 dispersive X-ray analysis indicated that Ag precipitation was
 likely a reductive process with the formation of colloidal Ag
aggregates, whereas La precipitates were crystalline  and
probably La-oxides or -hydroxides 5.  The affinity series for
bacterial sorption of these metals decreased in the order Ag
> La > Cu > Cd.
    Freundlich constants for sorption of all four metals by
the filamentous fungi are given  in Table 2.  The isotherms
adequately  described the removal of  the  metals by  the
fungi,  although  some  precipitation  of Ag may  still  be
occurring as Ag  sorption was much greater than La sorption
(188.3 versus 48.6 nmol g-1  at  an initial concentration  of 1
mM).  Based on K values, M. rouxii was more efficient at Ag
and La sorption, whereas A. niger removed the most Cd and
Cu from solution.  Because  of  the differences  in  isotherm
slopes, however, A. niger was more efficient than M. rouxii
for sorption of  Ag and  La from  1-mM solutions.  The K
values indicated  that fungal  affinity  for  these  metals
decreased in the order La  >  Ag > Cu  > Cd. Although not
statistically comparable, the bacteria in this study generally

Figure 1. Freundlich isotherms for cadmium sorption by B.
cereus and P.  aeruginosa. The dotted lines represent 95%
confidence intervals about the isotherms. Reproduced from
Applied and Environmental Microbiology 55: (in press), 1989 by
permission of the American Society for Microbiology and the
authors.
                                                100.0
                                             f


                                             •a
                                             (D
                                             _a
                                             O
                                                 10.0
                                                  1.0
                                                  0.1
                                                    0.1       1-0       10.0      100.0

                                                              Equilibrium Concentration (\iM)
                                                                                          1000.0
                          Table 1. Freundlich isotherms for sorption of Cd2+ and Cu2+ by bacteria3
                           Metal   Bacterium
                               log K ± SE
                                                                   n± SE
Cd



Cu



B.cereus
B. subtilis
E. coli
P. aeruginosa
B. cereus
B. subtilis
E. coli
P. aeruginosa
-0.673+ 0.083
-0.833 ±0.027
0.028 ±0.066
-0.410 + 0.043
0.340 ±0.064
0.61 8 ±0.032
0.411 +0.049
0.399 + 0.140
0.657 ±0.043
0.857 + 0.014
0.497 + 0.033
0.770 + 0.023
0.482 + 0.036
0.521 ±0.019
0.574 + 0.029
0.677 ±0.091
0.212
0.147
1.067
0.389
2.188
4.150
2.576
2.506
0.962
0.998
0.966
0.992
0.952
0.988
0.977
0.860
                           a Log K is the intercept and n is the slope of the regression line. The constant K
                           represents the amount of metal sorbed in umol g-1 at an equilibrium concentration
                           of 1 uM (logC=0).
                           Reproduced from Applied and Environmental Microbiology 55: (in press), 1989  by
                           permission of the American Society for Microbiology and the authors.

-------
 appeared to bind more metal than the fungi on a iimol g
 wt-1 basis.
dry
     The disparity between predicted efficiencies of different
 microorganisms at low and high concentrations for a given
 metal may be indicative of differences in total binding sites
 per gram  dry weight and/or  affinities  of individual binding
 sites on the bacteria for the metals.  For example, B. subtilis
 may have sites with a higher affinity for  Cu than does;  P.
 aeruginosa, with P.  aeruginosa  having more total sites
 available on  a dry  weight basis. This  may explain!  P.
 aeruginosa binding more Cu  at high concentrations and  B.
 sublilis binding more at low concentrations.

     These data  indicate  that  microorganism-metal
 interactions may be  amenable  to  equilibrium  modelijng,
 particularly when precipitation of metal is not a major factor.
 Such  modeling  may  allow for the  eventual  inclusionj  of
 biological surfaces in equilibrium solution chemistry codes,
 (or example MINTEQ.                                '

 Isolated  Bacterial  Wall,  Clay,  and  Wall-Clay
 Composite Studies                                I
     Of all  bacterial  structures,  cell  walls  are  the  most
 resilient and  are  virtually indestructible  unless  either
 degraded  by  specific  enzymes  (muramidases) lor
 hydrolyzed by extreme pH conditions. 1° Frequently, once a
 bacterial cell  dies, uncontrolled autolysis  ensues,  the cell
 lyses, and the wall is degraded into small fragments, YLet,
 the  very  wall  enzymes  that are responsible  for this
 phenomenon are easily inactivated by dilute heavy  metals,
 such as Fe,  Cu, Cr  and Ag, within  the cell's  aqueous
 environment   Because  bacteria are ubiquitous  to  natiiral
 soils,  sediments, and  groundwater systems, it  is  very
 possible that  they  can affect  the  migration of toxic  he^vy
 metals throughout these natural environments; indeed, these
 metals may actually increase the residence time of bactej-ial
 walls within waters, soils  and sediments  thereby making
 them  a  major force  in  the determination of  toxic  metal
 mobility.  For  this reason, it was important to study exactly
 how representative bacterial  wall  types modified hea'vy
 metal migration patterns in simple soil simulations, such las
 clay suspensions in the laboratory.                     j
    The metallic ion adsorption capacity of soils is control-
 led  by  aluminosilicate clay  minerals,  metal  oxides/-
 hydroxides and organic  matter.  For this  reason, our

 Table 2. Freundlich constants for metal sorption by filamentous
        fungi"                                       I
 Molal       Fungus            K          n          r2 I
Ag

Cd


Cu

La

A. niger
M. rouxii
A. niger

M. rouxii
A. niger
M. rouxii
A. niger
M. rouxii
1.096
3.373
0.156

0.039
0.889
0.746
2.877
5.702
0.892
0.641
0.679

0.875
0.495
0.551
0.426
0.314
0.953I
0.806
0.861


i
0.994
0.921
0.963


0.971 ! •
0.968
8 The constant K represents the amount of metal sorbed in jimo! g-1 [at
 an equilibrium concentration of 1 yM and n is the slope of the log i
 transformed isotherm.
 simulation  experiments  used  two  clays,  smectite
 (montmorillonite)  and kaolinite, that at circumneutral  pH
 carry a  net negative  charge  and  function  as cation
 exchangers.  Cell walls of B. subtilis  (representative of a
 gram-positive bacterium) and £  coli  (representative of a
 gram-negative  bacterium)  were  used as  the  biological
 component of  the system.  At neutral  pH,  the  metal ion
 sorption capacity of these walls  is dominated by  ionized
 carboxyl and phosphoryl groups.2  Previous experimentation
 has shown that these walls can immobilize large quantities
 of soluble metaf cations and act as nucleation sites for the
 production of various minerals.2
     Before laboratory simulations  using clay-bacterial wall
 composites  could  be  performed, it  was  necessary  to
 establish  the heavy metal sorption capacity  of each of the
 single components (Table  3); it was already apparent that
 the order of reactivity was B. subtilis (B)  >  £ coli (E) >
 smectite  (S)  >  kaolinite  (K).   Further  experimentation
 revealed that each of the clays also was capable  of  binding
 to the  bacterial walls,  presumably  through  polynuclear
 aluminohydroxide  bridging, 11  to  make   organo-clay
 composites.8  Kinetic analysis of these adsorption isotherms
 revealed  that saturation occurred  at approximately a 1:1
 stoichiometry of clay-to-wall  masses (Figure 2).    Metal
 immobilization experiments revealed that a proportion of the
 reactive  sites of  each component  (wall  and clay) were
 apparently used in composite production and, consequently,
 were  not available for metal binding yielding a reduced
 capacity for  heavy  metal  immobilization  (see  calculated
 versus observed binding values in Table  4  and following
 discussion).  Yet, it  was  apparent that the  biological
 components of  the  clay-wall  composites  continued  to
 dominate  the system  (Table 5).  The order of reactivity for
 the composites was B + S>B + K>E + S>E + K.
    This study was conducted to determine the net effect of
 clay  sorption  on the  metal  binding  capacity of bacterial
 walls.   The results  indicated that metal  binding  was
 substantially  reduced in  wall-clay  aggregates, a  reduction

Table 3. Metal bound by native Bacillus subtilis walls, Escherichia
        coli envelopes, kaol;inite and smectite.3
                        nMole Metal Bound/Gram Dry Weight
       Metal
                  Wall
                             Envelope
                                    Kaolinite
                                                 Smectite
Ag
Cu
Ni
Cd
Pb
Zn
Cr
423 ±15
530±13
654 ±25
683 + 19
543 ± 1 1
973 ±13
435 + 37
176+ 3
172+ 9
190+ 3
221 ± 6
254+ 5
529 + 32
102+ 2
0.46 ±0.02
5 ± 0.03
4 +0.2
6 ±0.2
3 + 0.2
37 ±1
8 ±0.5
43 ±0.3
197±4
173 + 10
1 +0.02
118 + 6
65 ±2
39 ±5
      a  Each component was suspended for 10 min. at 22° C in a 5 mM metal
        nitrate solution and washed 5 times by centrifugation to remove
        unbound metal. Metals were analyzed by atomic absorption
        spectrophotometry. The data represents the average of 3-5
        determinations for each sample  + standard error.

      Reproduced from Applied and Environmental Microbiology 55: (in press),
      ,1989 by permission of the American Society for Microbiology and the
      authors.

-------
attributed to a physical blocking of negatively charged sites
in the cell walls and envelopes by the sorbed clay particles.
The  contribution of the clays to heavy  metal  binding was
small in comparison to that of the organic constituents of
these composites  (Table 5).  These  results suggest that
small remnants  of bacterial  walls in  soils or  sediments,
adsorbed to clay particles, would substantially  increase the
metal binding capacity of the soil., An  important question
Figure 2. Adsorption of Bacillus subtilis walls (A) and
        Escherichia coli envelopes (B) to smectite (*) and
        kaolinite (o) clays. Reproduced  from Applied and
        Environmental Microbiology 55:  (in press), 1989 by
        permission of the American Society for Microbiology
        and the authors.
          1.25
          1.00
     CB
     €
     O
          0.75
     =    0.50
     g
     O)


          0.25
                      0.5       1.0      1.5

                        mg walls reacted/mL
                                                2.0
         1.25
     •§    1.00
     O>
     -Q
     O
         0.75
     §   0.50

     CD
     O>
          0.25
                      0.5      1.0      1.5
                       mg envelopes reacted/mL
                                                2.0
which should be addressed  in future research regards the
stability  and immobility  of  metals bound  by wall-clay
complexes compared to metals bound to the walls or clays
alone.
Remobilization Experiments
    Once toxic heavy  metals have been concentrated  by
walls, clays,  and composites in  a natural  environmental
setting,  they would  be  subject to  a range of chemical and
enzymatic  agents that  could  leach the particulates of their
bound  metal.   For instance,  low pH  frequently has  the
capability of remobilizing metal precipitates.12  To test this
reaction, four different pHs (pH 9,  7, 5 and 3) were used  on
our single and multicomponent systems (Figure 3); Ag was
remobilized best, whereas Cr was  little affected.
    It is also possible that competing,  non-toxic, naturally
occurring counter-ions  such  as Ca2 + could prove effective
at displacing  bound  heavy metals.   In this  instance,,
laboratory  tests showed  that  Cu was  remobilized  best
(Figure  4)  followed by Ag and Cr.  Fulvic acid, a  natural
complexing agent,  proved  to  be  not  as effective  at
remobilization as Ca2 + (cf. Figure 4 and 5), whereas EDTA,
which has a high binding constant  for  these metals and
which forms true chemical chelates,  had a profound effect
on Cu but not on Ag or Cr (data not shown).
    Lysozyme  is a muramidase  that cleaves  the covalent
bonds  holding  the glycan chains of  a major bacterial wall
constituent, peptidoglycan, together10  and, consequently,
should solubilize the wall fabric away from the bound metal.
Interestingly, there  was a highly  variable response to this
enzyme and Cr was barely remoblized at all. Previous work
suggested that heavy  metal ions can inactivate  native
muramidases (autolysins) within the wall13, and it  is possible
that a variable proportion of the lysozyme used in our study
was denatured by the heavy metals liberated during  initial
wall digestion.  We  currently  are conducting  studies to
confirm this hypothesis.
Summary and Future Research
    It is clear  from the studies outlined in this  report that
microorganisms and  their surfaces  can have  a profound
effect  on  the  immobilization  of toxic heavy  metals in
aqueous solution.   Furthermore, they  are capable of
chemically interacting with  aluminosilicate  minerals to
produce composites  in  which the  microbial  component
dominates the aggregate binding of metals.  Frequently,  the
remobilization of these bound metals is difficult and does
not follow a set pattern.  Clearly, microbial complexation
with metals in soils, sediments, and pore waters is important
and must be taken  into account when  modeling  the
transport patterns and  ultimate fate of toxic heavy metals in
natural  systems.
     Future research should  include the evaluation of other
environmentally relevant  metals  and  microorganisms, and
their  physiological  processes related  to metal  dynamics.
For example,  microorganisms produce  extracellular
compounds  that  assist  in  Fe  transport14,  and  these
compounds also may  act as complexing agents for  a  full
range of metals.15  The role  of microbial metal complexing
agents  in metal transport is poorly understood and requires
careful  assessment  if  we  are to  develop  complete
mathematical  models  that accurately predict  metal
movement in the soil, surface, and ground water.

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    Table 4. Comparison of the metal binding capacities of clay-wall and clay-envelope mixtures with predicted values as
             calculated from Table 3                 '
      Metals
                      Wall* Smectite
pMole Metal Bound/Gram Dry Weight

Wall + Kaolinite         Envelope + Smectite
                                                                                                Envelope + Kaolinite
Calculated3
Ag
Cu
Ni
Cd
Pb
Zn
Cr
233 ±
364 ±
414 ±
342 ±
331 ±
519 ±
237 ±
8
3
18
10
8
7
21
Observed13
115 ± 2
263 ± 4
148 ± 2
689 ± 13
148 ± 5
464 ± 15
122 ± 22
Calculated | Observed
212± 8 107 ±
268 ± 7
329± 13
181 ±
176 ±
345 ± 10 299 ±
273 ± 6
505 ± 7
222 ± 19
271 ±
367 ±
122 ±
2
10
0.3
8
5
7
16
Calculated
110 ± 8
364 ± 9
414 ± 18
342 ± 10
331 ± 9
519 ± 8
237 ± 21
Observed
19 ±
100 ±
37 ±
141 ±
134 ±
92 ±
19 ±
0.2
2
2
5
6
6
1
Calculated
87 ± 2
89 ± 6
97 ± 2
114 ± 3
129 ± 3
283 ±17
55 ±13
Observed
30 ± 2
49+2
28 ± 0.6
57 ± 1
27 ± 2
82 ± 2
28 ± 4
                                                     I
    a Calculated from average uptake for each component of the mixture in Table 3.
    b The same metal binding conditions as outlined in Table 3 were used and the data represents averages from 3-5 determinations ±
     standard error.

     Reproduced from Applied and Environmental Microbiology 55: (in press), 1989 by permission of the American Society for
     Microbiology and the authors.
Table 5. Metal Proportion (%) associated with the cellular
         constituents in the clay-wall/envelope        |
         components"                               |
Metal
Ag
Cu
Ni
Cd
Pb
Zn
Cr
Wall-
Kaolinite
100
99
99
99
99
99
99
Wall-
Smectite
91
73
79
100
82
94
92
Envelope-
Kaolinite
100
97
98
97
99
99
93
Envejope-
Smeptite
80
4J7
5(2
100
68
89
^
"Calculated from the data in Table 3.

Reproduced from Applied and Environmental Microbiology 5J5: (in
press), 1989 by permission of the American Society for     l
Microbiology and the authors.
    55: (in

-------
Figure 3.   Effect of decreasing pH on the remobilization of bound metal. B = B. subtilis walls, E = E. coli envelopes, S =
           smectite, and K =  kaolinite. The pHs are arranged in groups of four and, from left to right, are pH = 9,7, 5 and
           3.
                          Ag
100
50
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o
N
S
o n


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                     100
                          Cr
                      50
                                  IL
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                                                   S       K     B + K

                                                      Decreasing pH

                                                      (pH .9,  7, 5, 3)
                                                                           B + S     E + K    E + S

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  Figure 4.  Effect of increasing Ca concentrations on the remobilization of Cu. The Ca concentrations are arranged in
            groups of four and are Ca = 0,40, 80, and 160 mg I/1.
                         100
                    I

                    §     50
                              Cu
                                                        S      K      B + K     B + S


                                                        Increasing Ca concentrations

                                                           (0, 40, 80, 160 mg/L)
                                      E + K    E + S
Figure 5.  Effect of fulvic acid on Cu remobilization. Ipulvic acid concentrations = 10, 30, 60, and 120 mg I/1.
                          100
                               Cu
                     13     50
                     CD





                     I
                     CU
                     Q.
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     (10, 30, 60, 120 mg/L)
                                                                                          E+K    E + S

-------
Acknowledgments
    This  research  was  funded  through  EPA
Cooperative. Agreements  CR813605-01-1   and
CR813609-01-2. The  technical  assistance of  S.G.
Walker, L. Smith and C.A. Flemming is acknowledged.

References
1. Marshall,  K.C. 1980.   Microorganisms  and
interfaces.  Bioscience 30: 246-249.
2. Beveridge, T.J. and W.S. Fyfe. 1985. Metal fixation
by bacterial cell walls.  Can. J. Earth. Sci. 22:  1893-
1898.

3. Beverfdge, T.J.  and R.G.E.  Murray. 1976.  Uptake
and  retention of metals by  cell  walls  of Bacillus
subtilis. J. Bacteriol. 127:  1502-1518.

4. Tobin, J.M.,  D.G. Cooper, and R.J. Neufeld.  1984.
Uptake  of  metal ions  by Rhizopus  arrhizus.  Appl.
Environ. Microbiol.  47:  821-824.

5. Mullen, M.D., D.C. Wolf, F.G. Ferris, T.J. Beveridge,
C.A. Flemming, and G.W.  Bailey.   1989.   Bacterial
sorption of heavy metals. Appl. Environ. Microbiol. 55:
(in press).
6. Travis, C.C. and E.L, Etnier.  1981.  A  survey of
sorption relationships  for reactive solutes in soil.  J.
Environ. Qual. 10:  8-17.

7. Dao, T.H., D.B. Marx,  T.L Lavy, and J. Dragun.
1982.   Effect and    statistical  evaluation  of  soil
sterilization  on  aniline  and  diuron   adsorption
isotherms.  Soil Sci. Soc. Am. J. 46:  963-969.
8. Walker, S.G., C.A. Flemming, F.G.  Ferris,  T.J.
Beveridge and  G.W. Bailey. 1989.  Physiocochemical
interaction of Escherichia coli envelopes and Bacillus
subtilis walls  with two clays  and the ability of  the
composite  to immobilize heavy metals from solution.
Appl. Environ. Microbiol.55: (in press).
9. Work,  E.  1957.   Reaction of  ninhydrin in  avid
solution with   straight-chain amino acids  containing
two amino groups and its application to the estimation
of meso-diaminopimelic acid.   Biochem. J.  67:   416-
423.
10. Beveridge, T.J. 1981.  Ultrastructure, chemistry,
and function of the bacterial wall.  Int. Rev. Cytol. 72:
229-317.
11.  Hsu,  P.H.  1977.  Aluminum hydroxides  and
oxyhydroxides.  In: Minerals in the Soil Environment.
J.B.  Dixon and S.W.  Weed  (eds.).   Soil Science
Society of America, Madison, Wisconsin, pp. 99-143.
12. Cotton, F.A. and  G. Wilkinson.  1962.   Advanced
inorganic  chemistry:   a  comprehensive text.  John
Wiley and Sons, Inc., N.Y.
13. Ferris, F.G., W.S. Fyfe,  and T.J. Beveridge. 1988.
Metallic ion binding by Bacillus subtilis:  Implications
for the  fossilization of  microorganisms. Geology 16:
149-152.
14. Byers, B.R. and J.E.L. Arceneaux. 1977. Microbial
transport and  utilization of iron.  In, E.D.  Weinberg
(ed.) Microorganisms and metals, Marcel Dekker, Inc.,
New York. pp. 215-249.
15. Huyer,  M.  and W.J. Page. 1988. Zr\2+  increases
siderophore production  in   Azotobacter  vinelandii.
Appl. Environ. Microbiol. 54:  2625-2631.

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