WATER POLLUTION CONTROL RESEARCH SERIES
14010 DKN 11/70
           Microbial Factor
                   in
   Acid Mine Drainage  Formation
U.S. DEPARTMENT OF THE INTERIOR  FEDERAL WATER QUALITY ADMINISTRATION

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       WATER POLLUTION CONTROL RESEARCH SERIES
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    Microbial Factor  in Acid Mine  Drainage Formation
        MICROBIOLOGICAL FACTOR IN ACID MINE DRAINAGE FORMATION
Microbiological Factor in Acid Mine Drainage Formation:   II.   Further
                Observations from a Pilot Plant Study
                                 by
                          Mellon Institute
                     Carnegie-Mellon University
                          4400 Fifth Avenue
                   Pittsburgh, Pennsylvania  15213
                               for the

                 FEDERAL WATER QUALITY ADMINISTRATION
                     DEPARTMENT OF THE INTERIOR
                           Program Number
                      FWQA Grant No.  14010 DKN
                             July, 1970

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This  report has been  reviewed by the  Federal
Water Quality Administration and approved for
publication.  Approval  does not signify that
the contents necessarily reflect the  views and
policies  of the Federal Water Quality Admin-
istration,  nor does mention of trade  names or
commercial  products constitute endorsement or
recommendation for use.
    For gale by the Superintendent of Documents, U.S. Government Printing Office
               Washington, D.C. 20402 - Trice 70 cents

                         ii

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                               ABSTRACT

        MICROBIOLOGICAL FACTOR IN ACID MINE DRAINAGE FORMATION

               by Robert A. Baker and Albert G. Wilshire


           The role of chemoautotrophic organisms (Ferrobaciltus fevrooxi-
dans, Ferrobacillus sulfooxidans and Thiobacillus thiooxidans) in the for-
mation of acid mine drainage from pyritic materials associated with coal
mining has been investigated by pilot plant techniques.  Dynamic flow, con-
trolled environment units which served as models of mines were used.

           It was demonstrated that the concentration of acidity, ferrous
and total iron and sulfate in effluent from aerobic, biologically-seeded
or unseeded pyritic beds is zero order with respect to flow, except at low
flow rates where mass transport is diffusion limited.  Algal growth occurred
in the acidic, aerobic environment but did not affect acid production.

           Nonaerobic systems produce acidity consisting only of ferrous
iron.  Total acidity is lower from biologically-seeded than -nonseeded, aero-
bic systems at retention times exceeding three hours because microorganisms
consume acid.  At higher hydraulic rates, lower retention time, total acidity
is greater in the aerobic-nonseeded effluents.  Under aerobic-nonseeded
conditions acid mine drainage release reaches a maximum rate at a specific
flow.  Further increase in flow only dilutes the concentration with total
mass discharge remaining constant.  Under aerobic-seeded conditions, a
dynamic equilibrium exists between the rates at which pyrite dissolves,
organisms reproduce and are flushed from the system.  Maximum release
occurs at a specific hydraulic rate.  Higher or lower flows result in de-
creased discharges.

           Acid mine drainage release (a) was not significantly affected
by seeding with the individual or a mixture of the three organisms; (b)
increased directly with recycle ratios up to Ail of forward flow rate;
(c) increased appreciably under forced aeration; and, (d) is directly
related to available pyritic surface area.

           This report was submitted in fulfillment of Research Grant No.
14010 DKN between the Federal Water Quality Administration and the Mellon
Institute.
Key Words:  Mine Drainage/Chemoautotrophic Microorganisms/FerrofcaeiZZttS/
            Tfr0Z>acZZtts/Pyrite/Pilot Plant/Dynamic Equilibria

                                  iii

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                           TABLE OF CONTENTS

Section                                                            Page

   1.     Conclusions                                                1

   2.     Recommendations                                            4

   3.     Introduction                                               5

   4.     Experimental Units                                         9
          A.  Horizontal* Flooded-Bed Reactors                       9
          B.  Horizontal, Flooded-Bed Reactor with Recycle
              Chamber                                               12
          C.  Vertical, Flooded-Bed Reactors                        12

   5.     Experimental                                              16
          A.  Feedwater                                             16
          B.  Pyrite                                                18
          C.  Chemoautotrophic Organisms                            18
          D.  Operating Conditions                                  19
          E.  Analytical                                            20
              1.  Chemical Analyses                                 20
              2.  Biological Analyses                               20

   6.     Results                                                   23
          A.  Horizontal, Flooded-Bed Reactors                      23
              1.  Effect of Environment and Seeding                 23
              2.  Effect of Nature of Seed                          46
              3.  Effect of Recycle                                 46
          B.  Vertical, Flooded-Bed Reactors                        52
              1.  Effect of Supplemental Carbon Dioxide             52
              2.  Effect of Aeration                                56
              3.  Effect of Flow                                    57
              4.  Effect of Surface Area                            57

   7.     Acknowledgments                                           61

   8.     References                                                62

   9.     List of Publications                                      65

  10.     Appendix I  Pyrite Electronmicrographs                    66

          Abstract Cards
                                  iv

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                               FIGURES

Number                                                           Page
  1      Schematic of Horizontal Reactor Pilot Plant              10

  2      Horizontal Reactor Pilot Plant                           11

  3      Schematic of Horizontal Reactor Pilot Plant with         13
         Recycle

  4      Horizontal Reactor Pilot Plant with Recycle Chamber      14

  5      Vertical Reactor Pilot Unit                              15

  6      Effluent pH from Aerobic-Seeded Horizontal Reactor       24

  7      Acidity of Effluent from Aerobic-Seeded Horizontal
         Reactor                                                  25

  8      Iron Content of Effluent from Aerobic-Seeded Horizontal
         Reactor                                                  26

  9      Sulfate Content of Effluent from Aerobic-Seeded Hori-
         zontal Reactor                                           27

 10      Effluent Characteristics of Nonaerobic-Seeded Horizontal
         Reactor                                                  28

 11      Effluent Characteristics of Aerobic-Nonseeded Horizontal
         Reactor                                                  29

 12      Effluent Characteristics of Aerobic-Seeded Horizontal
         Reactor                                                  30

 13      Mass Release of Acidity from Horizontal Reactors Under
         Varying Operating Modes                                  38

 14      Mass Release of Iron from Horizontal Reactors Under
         Varying Operating Modes                                  39

 15      Mass Release of Sulfate from Horizontal Reactors Under
         Varying Operating Modes                                  ^

 16      Effluent Concentration of Acidity Corrected for Ferrous
         Iron Content                                             ^

 17      Effluent Concentration of Sulfate in Excess of Sulfuric
         Acid Equivalent of Acidity                               45

 18      Effluent Characteristics of Acid Mine Drainage with
         Recycle                                                  50

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                         FIGURES (Continued)
Number                                                            Page

  19      Mass Release of Acid Mine Drainage with Varying
          Recycle Ratio                                            51

  20      Effluent Concentration of Acidity Corrected for
          Ferrous Iron Content and of Sulfate in Excess of
          Sulfuric Acid Equivalent of Acidity                      53

  21      Photomicrograph of Unreacted and Reacted Pyrite
          at 1000 Magnifications                                   67

  22      Photomicrograph of Unreacted and Reacted Pyrite
          at 5000 Magnifications                                   68
                                  vi

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                               TABLES

Number                                                           Page

  1      Feedwater Composition                                    17

  2      Chemical Analytical Procedures                           21

  3      Effect of Biological Seed on Horizontal, Flooded-
         Bed Reactor Effluents Under Anaerobic Conditions         31

  4      Characteristics of Horizontal, Flooded-Bed Reactor
         Effluents Under Varying Operating Modes                  32

  5      Least Squares Fit of Horizontal Flooded-Bed
         Reactor Effluent Characteristics                         34

  6      Presence of Viable Sulfide- and Ferrous-Utilizing
         Organisms in Horizontal, Flooded-Bed Reactor
         Effluents                                                35

  7      Effect of Biological Seed on Effluent Characteristics
         of Aerobic Horizontal, Flooded-Bed Reactors              47

  8      Effect of Recycling on Effluent Characteristics          49

  9      Effect of Gasification by Carbon Dioxide or Air on
         Effluent Composition from Aerobic-Seeded, Flooded
         Vertical Columns of Pyrite                               55

 10      Effect of Flow Rate on Effluent Composition from
         Aerobic-Seeded, Flooded Vertical Columns of Pyrite       58

 11      Relative Surface Area of Pyrite Partiples Charged
         to Vertical Reactors                                     59

 12      Effect of Varying Surface Area on Effluent Composition
         from Aerobic-Seeded, Flooded Vertical Reactors Con-
         taining Pyrite                                           59
                                vii

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                                Section 1

                               CONCLUSIONS

           The role of chemoautotrophic microorganisms (Ferrobaoil'Liis
ferrooxidans, Ferrobaeillus sulfooxidans, and Thiobaoillus thiooxidans)
in the formation of acid mine drainage from pyritic materials associated
with coal mining has been studied using pilot plant reactors.  The units
were charged with crushed pyrite from actual mining sites.  Most of the
tests were conducted with horizontal, flooded-bed reactors through which
water of fixed mineralization was passed at a controlled rate of flow.

           Dissolution of pyrite, FeS2 or iron sulfide, leads to ferrous
(Fe II) ion and the sulfur species, S2"11.  These are subsequently oxidized
to ferric and sulfate reaction products.  When the microorganisms were added
to the system the unit is described as being seeded.  Nonseeded is used to
described the condition when the organisms were not added.

           The conclusions are:

           1.  The concentration of acidity, ferrous ion and total iron and
sulfate in effluents from pyritic beds is zero order with respect to flow if
the flow is expressed in reciprocal time units, Day"-'-, (total volume of water
per day * volume of pyrite bed) over the range of 4.6 to 13.8, Days"  or
1.5 to 4.3 hours retention time.  At low flow rates of water flow the reaction
products are mass transport limited and increase in concentration as flow
decreases.

           2.  There is continuous release of Fe (II) and 82"JI from non-
aerobic pyritic systems.  The mass per unit time discharged is constant
if the flow of water is not so low as to be diffusion rate limited.  Fyrite
dissolution takes place in the absence of air (oxygen) although at a lower
rate than under aerobic conditions.  Since the Fe (II)- and S2~II-utilizing
chemoautotrophic microorganisms are aerobes they are not active and do not
affect release of these ions under nonaerobic conditions.

           3.  Under aerobic conditions a mycelial growth may develop in
the pyritic beds despite acidic pH 3 to 4.5.  There was no evidence that
the presence of the heterotrophic organisms affected chemoautotrophic or-
ganism activity or altered the mass of acid mine drainage released per unit
time.  Acid mine drainage discharge was similar in periods of high or low
algal growth.

           4.  The effect of flow rate on effluent characteristics was
examined for nonaerobic,aerobic-nonsceded and aerobic-seeded operating
conditions.  Flow was varied to provide retention times through the pyritic
beds of 1.5 to 10 hours.

           a.  The effluent pH under nonaerobic conditions increased slightly
over feedwater pH of 4.5 because of dissolution of alkaline materials in the
pyritic mineral.  The pH of the seeded and nonseeded aerobic effluents were
3.8 to 4.1 over the entire hydraulic flow range.

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           b.  Effluent acidity under nonaerobic conditions was solely that
contributed by ferrous ion.  For aerobic effluents the total acidity is
lower for seeded than nonseeded systems at flows providing retention times
greater than approximately three hours.  This is attributed to consumption
of acid by the chemoautotrophic microorganisms.  At higher flow rates or
lower retention times the total acidity is greater in the aerobic-nonseeded
effluents.  Acidity, corrected for ferrous ion and expressed as l^SO,, de-
creases as flow increases and retention time decreases from 7 to 1.5 hours
for the aerobic-seeded system.  The corrected acidity is much greater in
aerobic-nonseeded systems at 7 hours retention but decreases to concen-
trations equivalent to aerobic-seeded systems at retention time of 1.5 hours.

           c.  Ferrous ion and total iron release is greater from the aerobic-
seeded than from the aerobic-nonseeded systems at all retention times
greater than 1.5 hours.   Most of the total iron content of the effluent
is Fe (II).  Ferric compounds precipitate in the bed.  Mass release of
iron reaches a maximum at approximately 2 hours retention time in the
aerobic-nonseeded system then remains constant as flow rate increases.  For
the aerobic-seeded system the iron release reaches a higher maximum value
than for the aerobic-nonseeded system at approximately the same hydraulic
rate.  With increasing flow, lower retention times, there is a drop in mass
release of iron until the mass release from the aerobic-seeded and aerobic-
nonseeded systems is comparable at <1.5 hours retention time.

           d.  There is no sulfate in nonaerobic system effluents.  For
aerobic systems, the sulfate mass release is greater for the seeded than
for the nonseeded condition over most of the hydraulic range studied.  Max-
imum release is at approximately 2 hours retention time in each case.  For
the nonseeded system, increasing flow does not alter the mass release of
sulfate.  For the seeded system, the sulfate release drops as retention
time decreases to <2 hours and approaches the nonseeded system mass release
at retention times <1.5 hours.  Sulfate required to balance nonferrous
acidity, expressed as sulfuric acid, is in slight excess and increases
with flow rate for aerobic-nonseeded systems but decreases from a great
excess at low flow rates to values comparable to the aerated-nonseeded
system at retention times of 1.5 hours.

           e.  In aerobic systems the mass release of sulfur as sulfate
and of total iron approximates the stoichiometric ratio expected from
pyritic dissolution; however, because of reaction product accumulation within
the pyritic beds, the effluent concentrations are not indicative of the pyrite
dissolution rate.

           f.  The effect of chemoautotrophic organisms on formation of
acid mine drainage is the result of a dynamic equilibrium between the rate
at which their cells reproduce, their flushout rate within the effluent
and the dissolution of pyrite.  It is not possible from these results to
establish whether these microorganisms directly affect pyrite surface re-
actions. They enhance oxidation of Fe(H) but the increased Fe (II) in the
effluent of seeded systems may be an indirect result of higner ferric:ferrous
ion ratios at the mineral surface rather than a direct microbial action.

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           5.  The standard chemoautdtrophic microorganism mixture used to
seed the pilot plant systems contained equal numbers of fhidbausi'iluB thiooxi-
danSy Ferrobaeillus ferrooxidans and Femobacillus sulfooxidan&.  In tests
made at flow rates which provided 4.3 hours retention it was found that the
effluent showed little difference in composition whether the seed was the
individual organism or the ternary mixture.

           6.  When effluent is recycled at 1:1 and 4:1 times the forward
flow rate, the mass release of acidity, ferrous ion and total iron and sul-
fate increases directly with the total flow through the pyritic bed.

           Supplemental studies made with flooded, vertical, packed-bed
columns of pyrites operated as aerobic-seeded systems demonstrated that:

           7.  Forced aeration of the pyritic bed raised acidity 35-fold.
The corresponding pH changed from 5.2 to 2.8 in comparison to a nonaerated
system.  Ferrous ion, total iron and sulfate discharge increased by approxi-
mately 30-, 70-, and 120-fold respectively with aeration.  Passing gaseous
carbon dioxide through the pyritic bed raised the acidity 12-fold.  Although
pH remained comparable, iron concentration increased 3- to 4-fold and sui-
fate only 50%.

           8.  If reactant  (chemical or biological) depletion or reaction
product concentration is not rate limiting, the release of acid mine drainage
is proportional to the available pyritic surface area for a given hydraulic
flow rate.  This relationship does not apply if flow is so low that transport
is diffusion rate limited.

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                                Section 2

                             RECOMMENDATIONS

           Despite recent significant contributions from investigators en-
gaged in biological and chemical research on the subject of pyritic oxidation
there is still a lack of knowledge of the nature of the reaction mechanism
at the mineral surface.  The need for defining this oxidation mechanism is
of practical, as well as academic consequence.  For example, application of
bacteriocidal agents to mineral surfaces is predicated on the proposition
that the chemoautotrophic organisms will be destroyed, thus retarding or
preventing ferrous iron and sulfide release.  The validity of this premise
is in doubt.  Research should be undertaken to firmly establish the nature
of the pyritic oxidation at the mineral surface in the presence and/or
absence of chemoautotrophic microorganisms.

           There is a serious problem in interpreting the results obtained
in fundamental chemical and biological investigations for actual field use.
Basic studies are usually made in small-volume, batch systems.  In coal
mining operations which release acid mine drainage, a dynamic equilibrium
exists between the dissolution of pyrite and the transport of the released
mineralization as a function of water flow.  Thus, small pilot plants, such
as those employed in this study, which serve as models of mines should be
put to greater use for testing the postulates derived from fundamental studies.
Typical is the need to clarify the interrelationship between the limiting
waterrpyrite ratio, ferric:ferrous ion concentration, and microbial density.

           The major emphasis was placed on establishing the effect of en-
vironmental conditions and hydraulic rate on acid formation and release in
this study.  The effect of surface area; differences in pyrite source;
supplemental COo (by a partial pressure increase of the gaseous environ-
ment rather than by forced gasification); biocides to arrest chemoautotrophic
activity; and dark reactions (eliminate sunlight or artificial light which
promotes algal activity); and the effect of varying feedwater composition
merit further pilot plant investigation.

           This study demonstrated a need for development of accurate and
reliable miniature probes which could be operated remotely in closed systems
to measure oxidation-reduction potential and dissolved oxygen.  At the present
time it is not possible to make an accurate oxygen balance on the reactor
or to determine the Eh at which pyrite dissolution takes place, particularly
under nonaerobic conditions.

           The nature of the sulfur species which is released on dissolution
of pyrite and its fate in subsequent oxidation reactions is unknown and should
be elucidated if the pyritic oxidation processes are to be understood.

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                                Section 3

                              INTRODUCTION

           The objective of this research was to advance the understanding
of the effect of microbiological factors in the formation of acid mine drain-
age from pyritic materials associated with coal mining.  The study was made
in pilot plant reactors amenable to environmental and hydraulic control
which were charged with pyritic material taken from actual mining locations.
Thus, the pilot plants served as models of mines.  Results obtained under
dynamic equilibrium conditions are directly relevant to the particular mining
locations from which the pyritic samples were obtained.  Specific factors
which were investigated included the effect of:  aerobic versus anaerobic
environments; liquid flow rate; biological seeding; nature of chemoauto-
trophic microorganism seed; supplemental gasification by carbon dioxide or
air; surface area; and recycle of effluent.

           Although significant advances in the basic knowledge of chemical
and biological factors have been made in recent years, these results have
not been readily applicable to acid mine drainage problems in the field.
The pilot plant concept was advocated to bridge the gap between fundamental
studies and field needs.  It is suggested that this concept, once demon-
strated, could be applied to other laboratory and field locations as needed
in the study of regional problems.  The technique could be used by operators
to determine acid-producing potential in advance of mining.  This should
enhance pollution abatement activities by indicating the most readily con-
trolled factor(s) for a specific site.  Since major operators presently
take core samples ahead of operation, this screening step could be a com-
plimentary activity.

           Table-top and batch systems used in basic studies are often
rate limiting because of consumption of reactants, depletion of vital nu-
trient or accumulation of reaction products.  A continuous pilot plant permits
attainment of dynamic equilibrium and better represents field conditions.
However, there are disadvantages.  In this study, the effect of the variables
tested was determined by monitoring the composition of the effluent from
the reactors.  It was not possible to make material balances on specific
ions or compounds.  Simultaneous dissolution, oxidation and precipitation
processes occur.  Thus, information obtained from batch and continuous
pilot plant experimentation is necessary.

           'The oxidation of coal mine pyrite, FeS2J in the presence of
moisture leads to acidity.  Half of the acidity is attributable to S,"11
oxidation to sulfate and half to Fe (II) oxidation to ferric iron and its
hydrolysis.  The overall reaction suggested by Leathen, et.al. (1953a) was
These authors further postulated that the reaction may be biologically
promoted to increase the rate of oxidation and subsequent hydrolysis of
FeSO, in the presence of sulfuric acid to yield ferric sulfate, ferric hydroxy-
sulfate and ferric hydroxide.  In the colloquial language of the coal in-
dustry, these resulting yellow-brown reaction products are called "yellowboy"
(Girard, 1965).  Elucidation of the interrelationship between the chemically-
and biologically-promoted oxidation reactions has been the basis of con-
siderable effort in recent years.  Stumm and Lee (1961) found the ferrous

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                                        _ 2
oxidation rate to be proportional to [OH ]  above pH 4.5 for bicarbonate
solutions comparable to natural waters.  A transition occurs at pH 3.
At lower pH the oxidation rate is independent of pH.  Hem and Cropper (1959)
and Hem (1960) defined the solubility field diagrams for ferrous ion
release from pyrite.  At pH $6, Fe (II) solubility is high and its con-
centration may change by several orders of magnitude over a narrow Eh range.
Singer and Stumm (1968) found that sulfate retards Fe (II) oxidation.
Though the oxidation rate continues to be first order with respect to Fe (II)
it is slower than in the absence of sulfate.  The rate of ferric hydrolysis
is linear second-order with respect  to Fe (III) and faster in the presence
of sulfate. They proposed that in the absence of bacteria the rate deter-
mining step is the Fe (II) oxidation.  Schematically:
                               2       -2
                       FeS0   r^ -  SO, * + Fe (II)
                          2   slow     4
                      Fe (II) + 02  slow-  Fe (III)
and
                   Fe (III) + FeS2       > Fe(II)+ S04

The Fe  (II) oxidation rate is dependent upon the anionic species under
acidic  conditions  (Huffman and Davidson, 1956) .  The rate increases in the
same order as the  complexing affinity for Fe (III):  perchlorate, sulfate,
chloride, phosphate and pyrophosphate.  Since the hydroxyl ligand has a
strong  Fe (III) affinity it was postulated to be a factor in Fe (II) oxi-
dation.  The hydrolysis of free Fe (III) is therefore involved in Fe (II)
dissolution from the pyrite.  Garrels and Thompson (1960) studied pyrite
oxidation by iron  sulfate and showed that the rate is independent of total
iron content and is controlled by differential adsorption of Fe (III) and
Fe (II) ions on the pyritic surface.  Oxidation of pyrites to release
ferrous and sulfate ions was observed only at sites occupied by ferric
ions.   This oxidation rate is slow relative to the adsorption process
hence the latter controls.  They postulated that pyrite oxidation may in-
volve release of molecular sulfur.

           The specific role of acidophilic chemoautotrophic bacteria in
pyritic conversion to acid mine drainage is undefined.  These Fe (II)- and
82"** -utilizing organisms are active at pH 2 to 4.5 and use carbon dioxide
as their carbon source.  Since oxidation of FeS2 may proceed solely by chemical
routes  the microorganisms are not essential to acid formation.  Their role
may be  (1) as a direct catalyst to alter the overall chemical reaction
rates or (2) as specific catalytic agents which alter the rate of inter-
mediate reactions  and the nature of the resulting byproducts but not the
overall rate.  The microorganisms may remove electrons from surface pyritic
iron to start a reaction chain and/or catalyze sulfur oxidation or they
may simply increase Fe (III) concentration and hence the Fe (III) to Fe (II)
ionic ratio.  The  Fe (III) is reduced by the pyrite and releases Fe (II).  '

           CoLner, Temple and Hinkle (1950) reported an organism which
oxidized Fe (II) to Fe (III) and thiosulfate to sulfuric acid.  Later
Temple  and Colmer  (1951) named this organism Thiobacfilus ferrooxidans.
Leathen, et.al. (1953a) could not confirm that, a single organism was capable
of oxidizing both  the Fe (II) and thiosulfate energy sources.  Leathen, et.al.
(1953b) demonstrated that Thiobaoillus thiooxidans increased acidity and

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sulfate formation when elemental sulfur was present in the medium.   Leathen
and Braley (1954) subsequently described an organism capable of oxidizing
ferrous to ferric sulfate and named it Ferrobaoillus ferrooxidans.   Leathen,
et.al. (1956) further characterized this organism showing that neither
sulfur nor thiosulfate are oxidized by it, thus distinguishing it from
the genus Thiobaaillus.

           Unz and Lundgren (1961) examined the nutritional requirements
of Thiobaeillus thiooxidans3 Thiobaeillus ferrooxidans, and Ferrobaoillus
ferrooxidans and found them to be similar.  Morphologically, these or-
ganisms are indistinguishable and these authors suggest that they cannot
be classified by their nutritional differences.  Kinsel (1960) reported
a sulfur-oxidizing, iron chemoautotroph, Ferrobaoillus sulfooxidans.
The dispute about the classification of these organisms remains unresolved.
Lundgren and Schnaitman (1965) examined cultural characteristics and kinetics
of biological iron oxidation. They showed that Fe (II) oxidation was directly
proportional to cell count of Ferrobaoillus ferrooxidans.

           The role of bacteria in the oxidation of pyritic materials has
been the focus of much debate.  Silverman and Ehrlich  (1964) outlined two
alternate mechanisms for bacterial conversion of 89      One involves Fe (III)
oxidation of pyrites to obtain Fe (II) which the organism oxidizes back to
Fe (III).  The alternate mechanism is independent of Fe (III) and requires
only contact between the bacteria and 82     It is likely that both mechan-
isms could take place in a given system depending on the nature of the
pyritic material.  Silverman  (1967) further elaborated on the mechanism
of the bacterial action and proposed that two mechanisms of bacterial pyrite
oxidation operate concurrently.  These were termed the direct contact and
indirect contact mechanisms.  The direct contact mechanism requires physical
contact between the bacteria  and the pyrite particles.  The indirect con-
tact mechanism requires that  the bacteria oxidize Fe (II) to the Fe (III) state
thereby regenerating the Fe (III) ions required for chemical oxidation of
pyrite.  During this investigation it was reported that Fe (III) oxidizes
pyrite in the absence of bacteria and oxygen.  Smith and Shumate (1970)
claim that direct oxygen oxidation and Fe (III) oxidation of pyrite are in-
dependent processes and that  the latter is a chemical  analogy of the micro-
bially-enhanced pyrite oxidation process.

           The role of S2~i:t  and the ferrous ion is two fold in the meta-
bolism of these bacteria.  They supply both the energy and reducing power for
carbon dioxide fixation.  Dugan and Lungren (1965) have proposed, in the
case of the Fe (II), a model  to explain the coupling of the energy released
from Fe (II) in the form of an elefctron to the carbon  reduction mechanism
within the cell.  In this model, an initial iron and oxygen complex is
oxygenated but not oxidized in the absence of electron transport.  Subsequently
this complex reacts with iron oxidase or oxygenase to  affect the electron
transport.  It is not evident from this study whether  the complex is formed
in solution or on the pyrite  surface.

           These often unreconcilable reports prompted the initial pilot
plant study.  In the first phase of this research  (Baker and Wilshire,
1968) it was demonstrated that:

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           1.  Chemoautotrophic microorganisms significantly accelerate
the oxidation of Fe (II) and Sj"11 released from the pyrites but apparently
do not directly alter the rate of pyrite dissolution in an aerobic environ-
ment.

           2.  Excluding oxygen does not prevent dissolution of pyrites.
Under dynamic conditions a steady-state release of Fe (II) and S2~IJ takes
place; however, the rate is very slow.  The practice of sealing mines to
exclude air may reduce but will not eliminate acid formation.  Since the
autotrophic organisms are aerobes they are not active and have no effect
in an anaerobic environment of Fe (II) or 82   -  Fe (II) emitting from
sealed mines must ultimately be oxidized in the receiving body of water
with the production of acidity and "yellowboy" precipitate.

           3.  In the absence of biological organisms, effluent Fe (II)
content is greater under an aerobic than anaerobic environment.  Effluent
Fe (III) content is negligible under highly acidic, aerobic conditions since
it is precipitated.

           4.  Organic carbon if available from coal or its associated hy-
drocarbons will supply energy for heterotrophic microorganisms.  Certain
heterotrophs can exist in the highly acidic environment of an aerobic
system.  A mycelial growth of the species Pen-iei/llium developed in the
aerated reactors of the pilot plant.  These heterotrophs may affect the
autotrophic oxidation processes through a succession reaction mechanism.
They release carbon dioxide and enzymes.  Since autotrophs utilize inor-
ganic carbon this supplemental carbon source could be available to increase
autotrophic metabolism in cases where C02 availability is rate limiting.

           5.  Total acidity released by aerobic-nonseeded systems exceeds
that from aerobic-seeded systems since Fe (II) content is greater.  However,
the acidity corrected for Fe (II) equivalent is greater from the seeded
systems.  The acidity released from nonaerobic systems consists almost en-
tirely of Fe (II).

           6.  Sulfate release in the nonaerobic reactor effluents was
essentially nonexistent.  Under aerobic environments the biologically seeded
systems release lower sulfate effluent concentrations than nonseeded systems.

           7.  Viable autotrophic organisms capable of utilizing Fe (II)
were native to each of the pyritic materials tested.  These have been found
to survive extended periods of relative inactivity pending establishment
of favorable environmental and other conditons.

           These conclusions were reached from pilot plant studies at a
specific hydraulic rate.  It was recognized that more extensive testing
was required to determine the validity of these findings over a range of
hydraulic rates.  The heterogeneous reaction of pyrite oxidation could be
limited by mass transport or reaction kinetics depending upon the particular
limitations imposed on the experimental system.
                                     8

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                                Section 4

                           EXPERIMENTAL UNITS

           The microbiological factor in acid mine drainage formation was
studied with  continuously operating pilot plants.  These units served as
models of mining sites.  They provided for controlled environment, hydraulic
flow of preestablished composition and rate over pyritic minerals of known
composition.  Microbiological seeding provisions were made.  Three versions
of packed bed reactor units were employed:  horizontal, flooded-bed; horizon-
tal, flooded-bed with recycle chamber; and, vertical, flooded-bed.

           A.  Horizontal, Flooded-Bed Reactors

           The unit used to make most of the tests provided four identical
horizontal, flooded, packed bed reactors in controlled environmental chambers.
The reactors  and associated control systems are depicted schematically in
Fig. 1.  This drawing shows two reactors each under  aerobic and nonaerobic
conditions.

           All four may be operated under identical  environments by making
the appropriate line adjustments.  Feedwater flow to the aerated and non-
aerated reactors is from 45-liter pyrex carboys (C)  each under 3 psig Cono-
flow regulated (R) air or nitrogen pressure (P).  Liquid flow control is
adjusted by Manostat teflon needle valves.  Liquid-(aerated or nonaerated,
(L ) or (L )) containing lines are 7 mm glass tubing connected by Tygon
tuf>ing sections.  Filtered air (A) or nitrogen  (N) is delivered via a
Gelman model  1235 filter holder containing 47 mm, O.ly millipore filters
which are  changed each time feedwater is prepared.   Filtered, house compressed
air is used.  Nitrogen is obtained from tank supplies.  The carboy tops
are securely  wired during operation but are removed  during feedwater pre-
paration.  A  fritted-glass sparger permits air or nitrogen as desired to
be bubbled through the vented carboys during the liquid transfer and chemi-
cal addition  steps.  Once the carboys are filled and the flow to the spargers
terminated, the caps are repositioned and the uniform pressure head main-
tained.  The  aerobic and nonaerobic reactors are kept under a slight positive
(approximately 2 to 4 cm water) pressure of air or nitrogen respectively
measured by U-tube manometers  (U) filled with water.  Effluent liquid (E)
passes through a loop which serves as a seal to maintain environmental
control.  Biological organisms are added by syringe  through septa mounted
in holders, on the inlet end of small glass tubes leading into the reactors
at inoculation points  (I).  Inoculations are made without disrupting the
atmosphere.   An outer shell serves' as an environmental chamber and an
internal reactor contains the mineral and feedwater.  This chamber consists
of a 4-inch I.D., 4.25-inch O.D. plexiglass section  36 inches long.  It is
fitted with 0.5 inch thick plexiglass end caps machined to accept standard
pipe thread fittings.  The liquid and environmental  gas service lines, mano-
meter and inoculation septa are mounted on these end caps.  It is supported
on a spring-loaded mount for leveling adjustment.  The reactor is a 30-inch
long plexiglass tray cut from a 2.25-inch I.D., 2.5-inch O.D. tube.  A
longitudinal  section has been cut away to create a tray 1 13/16-inches
high.  Overflow and underflow weirs are used to reduce short circuiting of
liquid through reactor.  These are cut from 1/32-inch cellulose acetate and
are cemented  into the reactor.  Overflow weirs at the inlet, center and
exit and underflow weirs at the 25 and 75% points between the inlet and exit
weirs are provided.  The beds are filled with 625 ml or 1350 g of mineral

-------
   N
V  R   P

   a  T
         9
                   I ,
                                              Ln
                                            La

                                                u
Figure 1.  Schematic of Horizontal Reactor Pilot Plant
A     filtered air
C     carboy
E     effluent
I     inoculation septum
L     aerated feedwater
                         1^    nonaerated  feedwater
                         N    filtered nitrogen
                         P    pressure guage
                         R    regulator
                         V    valve
                           10

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Eigure 2.  Horizontal Reactor Pilot Plant
                           11

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and have a liquid retention volume of 520 ml.  When the reactors are opera-
ting the liquid just covers the surface.  The bed is flooded.  Fig. 2 is a
photograph of this unit.

           B.  Horizontal, Flooded-Bed Reactor with Recycle Chamber

           The effect of recycling effluent was determined with a horizontal,
flooded, packed bed reactor unit similar, except for size, to the afore-
mentioned units.  Feedwater was added to the reactor by a peristaltic pump
(P) from a carboy (C).  Fig. 3.  A short section of Tygon tubing was used
in the line at the point of pump operation.  The remaining lines were
glass tubing.  Feedwater (La) and recycle (Le) entered the reactor inlet
by separate lines.  The reactor is a 11.7-inch long glass tray cut from a
2.2-inch I.D., 2.4-inch O.D. glass tube.  A longitudinal section has been
cut away to create a tray 1.75-inches high.  An underflow weir at midpoint
is used to reduce short circuiting of liquid through the reactor.  These
are cut from 1/32-inch cellulose acetate and are cemented into place.  The
flooded beds contain 230 ml of pyritic mineral when filled so that liquid
just covers the surface.  All tests with this unit used an air environ-
ment.  The exterior environmental chamber consists of a 4-inch I.D., 4.25-
inch O.D. plexiglass section 14 inches long.  End plates containing the tubing
and pipe threaded fittings were made of 0.5 inch thick plexiglass.  The
endplates were held against the environmental chamber by four stainless
steel tie rods threaded at the ends to take wing nuts.  The reactor base was
a spring-loaded mounting which provided leveling adjustment.  The recycle
chamber was a plexiglass, two-chambered box (3.25 x 3.25 x 1.44 inches)
with a total retention volume of 370 ml.  The inlet end provides the recycle
source.  An underflow weir is located 1.125 inches from the effluent end.
Effluent (E) is discharged over an overflow weir.  Figure 4 is a photograph
of this unit.

           C.  Vertical, Flooded-Bed Reactors

           Three to four small vertical, flooded packed-bed reactors were
used to examine the effect of pyritic surface area and supplemental C02
(Fig. 5).  The units were made of acrylic tubing with overall height of 51 cm
and internal diameter of 6.3 cm.  The columns were mounted on a plexiglass
base.  They were first packed with 4 cm polystyrene and glass beads. This
layer was arranged around a polyethylene gas dispersion tube and a glass
feedwater inlet tube coming down the center of the column.  The inert
beads serve to promote uniform cross sectional flow distribution.  Pyrites
were then added to a depth of approximately 44 cm (925 ml of pyrite per
unit).  Feedwater is added by a peristaltic pump from a carboy.  Effluent
is discharged via a sidearm.  Microrotameters were calibrated and used to
measure tank supplied gases, either carbon dioxide or air.
                                  12

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                              a. Schemotic
                        Constructed of -y Plexiglas
             Effluent
                              .
                                     U
                                                   l  Top
                                                   10  view
                                                   . '  Section
                                                   1s  A-A

Recycle
                        b. Recycle Chamber Size
Figure 3.  Schematic of Horizontal Reactor Pilot  Plant with  Recycle
C       carboy
E       effluent
I       inoculation septum
     La    aerated  feedwater
     P     pump
     U     manometer
                                13

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Figure 4.   Horizontal Reactor Pilot Plant with Recycle Chamber
                               14

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Figure 5.  Vertical Reactor Pilot Unit
                   15

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                               Section 5

                             EXPERIMENTAL

           The rate of acid formation from pyritic material of given com-
position and surface area will vary as a function of (1) temperature, (2)
hydraulic rate, (3) dissolved oxygen content of the water, (4) nature and
concentration of the microbiological population, (5) nature and composi-
tion of the inorganic species present in aqueous solution, (6) pH, and (7)
the oxidation-reduction potential of the system.

           Deep coal mines approximate 15C in temperature.  Simulation of
this temperature would have required a system beyond the resources of this
project.  Operation was at ambient laboratory temperature of 23 to 26C.

           A.  Feedwater

           The flow rate of water over or through the pyritic mineral
material determines the immediate reaction contact time, the rate at which
nutrients and trace elements are supplied and the rate at which reaction
byproducts are removed.

           Various considerations were involved in devising a feedwater
formulation.  It should be of constant composition and contain typical
mineralization present in aqueous soil solutions, provide the ions necessary
for bacterial growth and reproduction but not contain the primary energy
sources of ferrous ion and 82"** for the bacteria.  Typical mine waters
are characterized by minimal quantities of the biological nutrients nitrogen
and phosphorous.  These nutrients should not be rate limiting factors for
the biological activity of the test organisms in this research.  Higher
levels were therefore needed.  Previous studies, Baker and Wilshire (1968),
resulted in formulation of a feedwater which contained sulfate.  This for-
mulation was modified for this study by substitution of chloride as the
anion for those salts which were originally added as sulfate (Table 1).
The pH was adjusted by phosphoric acid, H^P04, to a final value of 4.5
to 4.7.  This produced a final phosphate content of approximately 30 mg/1.
The final pH represents the upper limit of environmental suitability for
reproduction of the microorganisms.  It also represents an important point
in the pH-ferrous oxidation relationship (Stumm & Lee, 1961).  It was demon-
strated that:  (a) no deleterious osmotic effects were induced by organism
(Thiobaeillus thiooxidans, Ferrobaeillus sulfooxidansj and Ferrobacillus
ferrooxidans) transfer from culture media to feedwater or vice versa; (b)
biological growth was maintained in the feedwater; and (c) the feedwater
need not be sterilized since no iron- or sulfur-utilizing organisms are
present.  Despite the last result, every care was exercised to avoid con-
tamination of the feedwater supplies.

           The deionized water used to prepare the feedwater was either
aerated by air or deaerated by tank nitrogen.  Final dissolved oxygen con-
tent was approximately 7 and 0 mg/1 respectively.  This was done in
separate 13-gallon Nalgene polyethylene tanks.  The deionized conditioned
water was transferred to the feedwater carboys under their respective gas
heads (air or nitrogen).  The chemicals were added to the carboys imme-
diately and the carboys were sealed.  Feedwater characteristics were routinely
monitored to assure constancy of composition and oxygen level.


                                   16

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                     TABLE  1

             Feed Water Composition
Component
Ca
Mg
K
Mn
Cl
P4
P42
pH?
Specific Conductance
(timhos/cm)
Composition, mg/1
76
17
13
6.5
6.8
10
199.5
16
30
^6.7
-V4.5
690
After adjustment with HaPOi*; with  a resulting acidity of approx-
imately 20 to 25 mg/1 as H2SOit.
                       17

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           B.  Pyrite

           The iron sulfide associated with coal seams is chiefly pyritic.
Another mineralogical form, marcasite, is usually less abundant at locations
where acid mine drainage is a problem.  The oxidation rate of pyrite is
affected by such factors as the crystal size, nature and concentration of
impurities.  The pyrite used in these studies was obtained from Shawville,
Pennsylvania.  This mineral was also used in preliminary investigations,
Baker and Wilshire (1968).  It contains 45% iron, 0.05% magnesium, 0.26%
maganese and 0.08% calcium.  Loss on ignition is 27.8%.  This loss includes
entrained coal and conversion of carbonates or other anionic forms to
oxides.  X-ray analysis indicates pyrite content of 90% or more with minor
quantities of siderite, marcasite and quartz.  If all the iron were assumed
to be present as FeS2 then the material would be 98% pyrite.

           Surface area and pore size characteristics were measured by
a BET apparatus using nitrogen as sorbate.  The sample was first reduced
to 60/80 mesh screen size and degassed for four hours at 200C.  Sorption
capacity is low.  Sorption and desorption loops do not close and the sorption
isotherm is classified as Type I in the Brunaer-Deming-Deming-Teller classi-
fication.  Type I characterizes nonporous and microporous solids with cap-
illaries which have a width not exceeding a few molecular diameters.  Data
between 0.02 and.0.20 relative pressure where linearity exists were used
to calculate a specific surface of 1.09 m /gm.  For a cubic particle with
edge length of the average size screened the specific surface is 0.006 m /gm.
The BET value is 184 times as great which appears excessive for the triple
product of shape factor, size distribution factor and surface roughness
factor.  The density was measured by helium displacement and by mercury
displacement.  Helium which penetrates all voids with access from the surface
gave a density of 4.12 gin/ml.  Mercury at a pressure equivalent to pene-
tration into pores 9 microns in radius gave a density of 3.588 gm/ml.  From
these densities the calculated pore volume is 0.0358 ml/gm or on a volume
basis 0.128 ml/ml.  In summary, the pyritic material is a microporous solid
with at least 0.036 ml/gm internal voids.  These voids have an internal
specific surface of approximately 1 m^/gm.

           The sulfur ball and similar pyritic material as received was
not washed with acid, solvents or water.  The outer surfaces were rejected
during crushing operations and the freshly-exposed, ground mineral was
screened to the desired size range.  Most studies were made with 3.5 to 7
mesh particle size.  The freshly-ground pyrite was sterilized to eliminate
native organisms by exposing the particles to an atmosphere of carboxide
(10% ethylene oxide and 90% carbon dioxide) for at least 24 hours prior to
charging reactors (Allison, 1951).  Studies were made which demonstrated
that the gaseous sterilization did not affect activity of the pyrite in
acid formation reactions.  Sterilization effectiveness was monitored by
placing representative mineral in sulfur and iron culture media and checking
for autotrophic activity over a period of at least ten days.

           C.  Chemoautotrophic Organisms

           Certain autotrophic organisms have long been known to occur
in mine drainage.  The specific role of these bacteria in the acid formation
reactions has not been adequately defined.  Three specific organisms were


                                   18

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used:  Ferrobacillus fevrooxidans3 FF3 JferrobaoU'Lus eulfooxidans3 FS,  and
Thiobaaillus thiooxidansf TT.  They do not use the same energy sources.  FF
oxidizes only ferrous ion.  FS oxidizes ferrous ion and 82"  .  TT oxidizes
sulfur and thiosulfate but not ferrous ion.  All three species are chemo-
autotrophic.  They obtain carbon from carbon dioxide and carry on metabolic
processes at pH 2 to 4.5.

           The original FF and FS organisms were obtained from cultures
maintained at the Mellon Institute by Dr. W. Leathen.  TT was culture number
8085 obtained from American Type Culture Collection.  Subcultures were pre-
pared every seven days and maintained at 26C.  Waksman's media (Waksman,
1922) was used for the TT.  Leathen1s media (Leathen, 1951) was used for
the FF and FS.  An inoculum of 1 ml was transferred aseptically to 99 ml
of appropriate medium in a 250 ml Erlenmyer flask.  It was determined by
dilution and culturing tests that one week old cultures each contained at
least 10' with maxima of 10^ organisms per ml.  These counts were essentially
constant for the next few weeks so cultures 7 to 14 days old were used to
seed the biological reactors.

           D.  Operating Conditions

           The effect of biological seeding on acid formation was the
primary factor to be examined in this research.  Preliminary studies
(Baker and Wilshire, 1970) had established that in the absence of oxygen,
biological seeding was without effect since the autotrophic organisms are
aerobic.  Consequently, major emphasis was placed on assessing acid pro-
duction under the following operating modes:  aerobic biologically-seeded;
aerobic-nonseeded; and, nonaerobic-nonseeded.  These operating modes were
tested in the horizontal flooded-bed  reactors.  The secondary variables of
recycle, surface area, C0~ supplement and nature of seed were tested in
the horizontal-recycle and vertical units.  In each case, pyrite was
charged to the units, the atmosphere  adjusted and feedwater added.  Seeding,
except for the specific study of the  nature of biological seed, was by a
ternary mixture of Thiobadllus thiooxidans3 Ferrobaoillus ferrooxidans
and Ferrobaeillns sutfooxidans.  Equal volumes of stock culture containing
10 ^ to 10H organisms/ml were first settled to remove excessive sediment
and energy sources.  The clarified supernatant was withdrawn by syringe
for inoculation into the units through septa-covered ports.  One ml of
seed was added then a 5 ml rinse of deionized water to flush the  organisms
into the reactors.  Seeding was practiced for 14 days then the organisms
were allowed to adapt to the particular experimental conditions.  Equili-
brium, judged by steady-state effluent characteristics, generally required
10 to 90 days.  The primary  operating modes were evaluated in the four-
reactor, horizontal, flooded-bed,'unit at flows from 1.44 to 8.64  liters
per day or 8.7 to 1.5 hours  retention time.  The effect of recycle at 0:1,
1:1 and 4:1 ratio of recycle to forward flow rates, was determined in
the smaller, flooded-bed reactor with recycle chamber.  The effect of
varying pyrite surface area  and adding supplemental C02 or air was tested
in the vertical packed-bed columns.
                                   19

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           E.  Analytical

               1.  Chemical Analyses

           The chemical analytical procedures used to monitor the reactions
were chiefly those described by &STM (1968).  Table 2.  Acidity was measured
to an end point of 7.3 pH after oxidizing Fe (II) with hydrogen peroxide
by the procedure described by Salotto, Earth, Ettinger and Tolliver (1967).
Their studies demonstrated that hydrolysis of Fe (III) and Al (III) was
complete at pH 7.3 before formation of aluminate ion.  Carbon dioxide
acidity is removed by aeration and sodium hydroxide is the titrant.  The
alkalinity was measured to an end point of 4.5 pH.  Dissolved oxygen cali-
bration of the Yellow Springs instrument was by the Alsterberg-Azide
modification of the Winkler test.  Sulfate in preliminary studies was
measured indirectly by measurement of excess barium after addition of barium
chloride, formation of barium sulfate, 24-hour growth of precipitate, fil-
tration, and atomic absorption analyses of barium to measure the extent
of barium consumed.  However, the feedwater formulation was adjusted to
eliminate the sulfate anion for this study and the effluents often contained
<20 mg/1 sulfate.  It was necessary to increase sample size prolonging
sampling time at the low flow rates which was considered undesirable.
Therefore, an alternate turbidimetric analytical procedure was used for
sulfate at <20 mg/1 as described in Standard Methods (1965), pp. 291-293.
Ferric ion was occasionally measured by D-1068-62T as an analytical check
but generally by atomic absorption analyses from an acidified (HC1) solution.
Additional cations present in the pyritic samples were measured by atomic
absorption analyses after solution of the material.  A pyritic sample of
known weight was digested with phosphoric and nitric acids'.  The residue
was removed by filtration, ashed, and treated with concentrated hydrofloric
acid to remove silica.  The filtrates were combined and diluted with de-
ionized water.  An equivalent acid blank was also prepared to serve as a
reference in atomic absorption analyses.  Carbon dioxide content of the
water could not be routinely monitored.  Phosphates were measured as an
indicator of biological activity but significant depletion only occurred
at the beginning of biologically seeded, aerobic test series (Baker and
Wilshire, 1968).

               2.  Biological Analyses

           The presence of viable chemoautotrophic organisms in the effluent
from the reactors was determined by inoculating tubes containing 10 ml of
either Waksman's or Leathen's media with 1 ml of effluent.  The tubes
were incubated in the dark at 26C.  They were examined after 7 and 10 days
incubation.  A drop of more than one pH unit from 4.5 for the .Waksman
medium was considered positive evidence of the presence of ThiobaeHlus
thiooxidans or Fevrobaoillus sulfooxidans.  Thiobaeillus thiooxidans do
not oxidize Fe (II) and Ferrobaeillue fexvooxidane do not use elemental
sulfur as an energy source.  A positive potassium thiocyanate reaction indi-
cating oxidation of ferrous to ferric ion in the Leathen medium was con-
sidered evidence of Ferrobaeillue fervooxidans, Ferrobaaillits sulfooxiddne
or both.                                                                   i
                                  20

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 Component


   PH

   Fe II


   Specific Conductance


   NH* Nitrogen


   N0~ Nitrogen


   POA

   Dissolved Oxygen


   so4


   Fe, Total
                               TABLE 2


                   Chemical Analytical Procedures


                            ASTM* Designation


                              D-1293-62T


                              D-1068-62T


                              D-1125-64


                              D-1426-58


                              D-992-52


                              D-515-62T


                              D-1589-60

                                           **
                              Turbidimetric


                              Atomic Absorption
fc*
ASTM Book of Standards, Part 23, 1968.

Standard Methods, 12th Edition, APHA, 1965.
    Instrument
Beckman Zeromatic
 Yellow Springs
Perkin Elmer 303
                                   21

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           Supplemental analyses were made of the biological growth present
in the water and on the pyritic surface at the end of some of the test
series.  A microscopic examination and a plate culturing procedure were
used.  Plates of Leathen's and Waksman's media solidified with #2 ion
agar were streaked with sample to isolate the chemoautotrophic organisms.
Heterotrophic organisms were isolated by streaking plates of Sabouraud
dextrose agar and subsequently making transfers to specific media including:
lead acetate agar; phenol red broths containing sucrose or dextrose or
lactose; gelatin; and EMB agar.                '
                                  22

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                               Section 6

                                RESULTS

           A.  Horizontal, Flooded-Bed Reactors

               1.  Effect of Environment and Seeding

           The reactors were operated over intervals varying from 60 to
140 days at fixed forward flow rates during which time the effluent charac-
teristics were monitored.  Once equilibrium was achieved, as indicated by
constancy of all analytical measurements, the tests were terminated.
Typical effluent measurements are shown for an aerobic-seeded horizontal
reactor test:  pH, Fig. 6; acidity, Fig. 7; total and ferrous ion, Fig. 8;
and sulfate, Fig. 9.  The units generally required approximately 30 days
to approach equilibrium.  Initially high values of effluent components are
the result of flushout of pyritic fines of greater surface area and hence
greater immediate oxidation potential.  The acidity values presented in
Fig. 7 are uncorrected for feedwater acidity which varied slightly from
batch to batch, Table 1,  These data were obtained at feedwater flow rates
corresponding to 2.3 D   volume ratio or 8.7 hours retention.  In the
presentation of results which follows, only the equilibrium characteris-
tics are given although each point is the result of a comparable time-
analyses series.

           Effluent characteristics (pH, acidity, sulfate, total and
ferrous ion) were measured as a function of water flow rate and environ-
mental operating modes.  The latter include:  nonaerobic-seeded, Fig. 10;
aerobic-nonseeded, Fig. 11; and, aerobic-seeded, Fig. 12.  Preliminary
tests, Table 3, demonstrated that since the chemoautotrophic organisms
are aerobic, there is no difference in acid mine drainage emitted from
pyrite under nonaerobic-seeded and nonaerobic-nonseeded modes.  Hence,
only the combination of nonaerobic-seeded was examined in this study.
The pyrite used in the preliminary test differs from the mineral used in
the rest of the experiments.  Therefore, effluent characteristics differ
from those obtained at comparable conditions.  Furthermore, the preliminary
test was made at a flow rate which was diffusion limited with respect to
transport as determined in subsequent studies.

           The effluent characteristics are essentially linear functions of
flow rate (expressed as the ratio, Q * V = D"1, where Q = volume of water
per day and V = volume of pyrite) except fpr the very lowest flows.  Since
only two values are available at low flow  (high retention time) operation,
the regression in this zone is depicted by a dashed line.  Figs. 10-12.  The
entire regressions of sulfate and acidity concentration vs. flow under non-
aerobic conditions are dashed to indicate limits of analytical reliability
as values approach zero.  A summary of the average effluent characteristics
for each flow rate tested under each operating mode is given in Table 4.
The concentration, C, of components of the effluent fit the general equation

                            C = a - b (D"1)
                                   23

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              10      20      30      40
                           Time, Days
50
60
Figure 6<  Effluent pH from Aerobic-Seeded Horizontal Reactor

Flow,  2.3 D~\ volume water per day * pyrite volume.  Shawville pyrite,
625 ml, 3.5 to 7 mesh size.  Retention time, 8.7 hours.  Influent pH, 4.5.
                                24

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          5000
          2000
          1000


        E  500
           100


            50



            20
Vv
   ^^.....
                    10     20      30     40
                                Time, Days
                        50
60
Figure 7.  Acidity of Effluent from Aerobic-Seeded Horizontal Reactor

Flow,  2.3 D  , volume water per day * pyrite volume.   Shawville pyrite,
625 ml,  3.5 to 7 mesh size.  Retention time, 8.7 hours.  Influent acidity,
20-25  mg/1.
                                25

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      e
      o
        500
        200
        100
         50
         20
         10
                              I    I   I    I
A Total
 Ferrous
                   **
                  10     20      30     40
                             Time, Days
          50
60
Figure 8.   Iron Content of Effluent  from Aerobic-Seeded Horizontal Reactor

Flow, 2.3  D  ,  volume water per day  * pyrite volume.   Shawville pyrite, 625
ml, 3.5 to 7  mesh size.  Retention time, 8.7 hours.
                                26

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       2000
       1000
        500
     f  200
     5  100
     V)
         50
         20
         10
                                      i    i    I    I
                                        
                  10      20      30      40
                               Time, Days
50
60
Figure 9.  Sulfate Content of Effluent from Aerobic-Seeded Horizontal
           Reactor

Flow, 2.3 D"1, volume water per day * pyrite volume.  Shawville pyrite,
625 ml, 3.5 to 7 mesh size.  Retention time, 8.7 hours.
                                  27

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                 <0
                 <  of-
                    I
                                           Totol Fe
                           \
                              \

                                J	.	.	1	,	L
                                             10

                       Flow,Day'1, Volume Water Per Day-r Volume Pyrlte
                              J	1_
                                 15
10   5  4   3        2

       Retention  Time,Hour*
                                                      1.5
Figure  10.   Effluent  Characteristics  of Nonaerobic-Seeded Horizontal Reactor


Shawville pyrite, 625 ml,  3.5 to 7 mesh size.  Dashed lines and arrows indi-
cate analytical limit as  concentrations approached zero.
                                        28

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                 =5 10
                                       FeU
                    0            5            10            (5
                       Flow.Day"',Volume Water Per Day-^ Volume Pyrite
                         10     5  4   3        2
                                Retention Time,Hours
1.5
Figure 11.  Effluent Characteristics of Aerobic-Nonseeded Horizontal Reactor

           Shawville pyrite, 625 ml, 3.5 to 7 mesh size.
                                       29

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                  f 4
                  I 2
                    30-
                    20-
                    to-
                    0-
                    30-
                    20
                    10
                    0


                           *
Totol Fe
                                  5            1O           15
                        Flow.Doy"1,Volume Water Per Doy-t-Volume Pyrite
                          10    5 4    3         2
                                 Retention Time,Hours
        1.5
Figure 12.  Effluent  Characteristics of Aerobic-Seeded Horizontal Reactor
           Shawville pyrite, 625 ml, 3.5 to 7 mesh size.
                                        30

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                                TABLE  3



Effect of Biological Seed on Horizontal, Flooded-Bed Reactor Effluents

                      Under Anaerobic Conditions3
                                        Seeded          Nonseeded



               pHb                       5.5               5.7



               Acidity, mg/1            18                18



               Ferrous iron, mg/1        2.9               2.9



               Total iron, mg/1          2.9               2.9



               sulfate, mg/1            10                10
a                                                                     -1
 Shawville pyrite, 625 ml, 3.5 to 7 mesh size; feedwater flow at 2.3 D  ,

 volume water per day * volume pyrite or 8.7 hours retention


 Feedwater pH 4.5
                                  31

-------
                                                          TABLE  4

                Characteristics of Horizontal, Flooded-Bed Reactor Effluents Under Varying Operating Modes2
Flow
L/Df
1.44
2.16
2.88
5.76
8.64
D~18
2.3
3.15
4.6
9.2
13.8
PH
N-Sb
4.68

4.76
4.76
4.76
A-NC
3.51

3.77
3.96
4 .07
A-Sd
3.73
3.81

3.99
4 .04
Acidity, mg/1
N-Sb
11.7

^2e
<2e
<26
A-NC
24.7

13.7
9.9
6.1
A-Sd
21.1
13.5

9.8
7.4
Total Iron, mg/1
N-Sb
3.1

2.1
1.3
0.9e
A-NC
4.5

2.4
1.9
1.4
A-Sd
5.0
3.9

2.5
1.7
Ferrous Iron,
mg/1
N-Sb
2.7

1.2
0.6
0.4
A-NC
3.7

1.6
1.2
0.7
A-Sd
4.2
3.5

1.9
0.8
Sulfate, mg/1
N-Sb
5.0

'v-l6
<16
<16
A-NC
22.6

11.3
8.8
6.2
A-Sd
21 6
14.6

9.8
6.9
LO
N9
      a
       Shawville pyrite, 625 ml, 3.5 to 7 mesh size; feedwater pH 4.5.

       nonaerobic-seeded, N-S
      Q
       aerobic-nonseeded, A-N

       aerobic-seeded, A-S

       approximations, limit of analytical procedures.

       liters per day

      ^volume water per day * volume of pyrite

-------
which differentiates to indicate that concentration is zero order with
respect to (D"1).  Least squares analyses of the effluent characteristics
over the linear range are summarized in Table 5.

           All effluents were monitored every 14 days for presence of viable
Fe (II)- and S2~II-utilizing organisms.  Table 6.  The effectiveness of
gaseous sterilization of the pyrite is readily apparent.  Preliminary
tests showed that native Fe (II)- and S2~II-utilizing organisms were present
in the Shawville pyrite but none were found after 84 days of operation
of the aerobic-nonseeded reactor.  Chemoautotrophs were present in the
reactor with a nonaerobic (nitrogen) atmosphere.  Tests for both Fe (II)-
and S2~II-utilizing organisms were positive at 28 days.  S2~II-oxidizing
organisms continued to persist but Fe (II)-oxidizing organisms were not
found after 42 days.  The latter did not survive in the nonaerobic en-
vironment and the seeded Fe (II)-utilizing organisms were either flushed
or destroyed.  Remaining S^"  -utilizing organisms were evidently in an
inactive or resting stage based on the effluent characteristics and visual
appearance of the pyritic beds.

           Biological growth appeared in the aerobic reactors despite a
pH 4.  The source of seed for this growth is the unsterilized deionized
water used to prepare the feedwater.  Sterilization of the large quantities
of water being used was not practical and biological culturing studies
demonstrated that no organisms were present which affected Fe (II) or
82"^ oxidation.  A greyish fungal growth began to appear at the exit end
of the reactor after approximately one week.  The growth intensified and
gradually progressed toward the inlet end until the entire surface was
covered.  Maximum growth was achieved within 30 to 70 days and then began
to diminish in most cases.  Phosphate depletion from 30 to 3 mg/1 was
maximum at approximately 30 days then rapidly decreased to 1 to 2 mg/1
after 60 days.  At no time did phosphate drop to zero concentration.  The
diminution of growth in the extended runs is attributed to depletion of the
carbon source necessary for the heterotrophic organisms.  (Baker and Wilshire,
1968).  Oxidation products, ferric hydroxides and sulfates, tended to
accumulate in the mycelial mat so that it gradually changed from a grey
to yellow-brown color.  The "yellowboy" appearance was a function of flow.
It was first noticed after 11 days at a flow corresponding to 4 hours
residence time but took 34 days to be evident at half this residence time
in the aerobic  reactor.  The mat undoubtedly affected the transport of
colloidal-sized reaction products in aerobic operation and reduced their
concentration in the effluent.  On the other hand, release of C02 by
the mycelia would be beneficial to the chemoautotrophic organisms if that
carbon source were limited.

           Microscopic examination and culturing techniques of the mycelial
mat indicated a fungus.  The mycelia were branched, septate, had fruiting
bodies and spores.  A Penieilliwrn was indicated by growth on Sabouraud
dextrose agar.  The heterotrophic bacteria isolated were tentatively
identified as species of the genus Pseudomonas or Aevobaeter. The presence
of Chemoautotrophs was confirmed on agar containing Waksman's or Leathen's
media.

           No mycelial growth developed at any time in the nonaerobic
reactors.  No sulfuritic or any other characteristic odor was detected
                                   33

-------
                                                       TABLE 5
                      Least Squares Fit of Horizontal, Flooded-Bed Reactor Effluent Characteristics
                              nonaerobic-seedede
                                                    aerobic~nonseeded
                                     aerobic-seeded
CO
*
PH
acidity, mg/1
total iron, mg/1
ferrous iron, mg/1
sulfate, mg/1
                          (4.72*0.07)-(0.000.01)D
                            d
                                                  -1
                          <2
                          (2.59*0.22)-(0.13*0.02)D
                                                  -1
                          (1.51*0.22)-(0.08*0.02)D
                                                  -1
 (3.640.04)+(0.03*0.00)D
(17.46*1.26)-(0.83*0.13)D
 (2.87*0.27)-(0.11*0.03)D
 (2.09*0.22)-(0.10*0.02)0
                                                                        -1
-1
-1
-1
(13.81*0.08)-(0.55*0.08)D
                                                                              -1
 (3.750.04)+(0.02*0.00)D
(15.28*0.18)-(0.58*0.02)D
 (4.550.24)-(0.210.02)D
 (4.300.13)-(0.250.01)D
(16.86*0.62)-(0.73*0.07)D
                                                       -1
-1
-1
-1
                                                       -1
      a                                                                                           -1
       nonaerobic-seeded and nonaerobic-nonseeded results identical; data apply from 2.3 to 13.8 D
       data apply from 4.6 to 13.8 D" .
      Cdata apply from 2.3 to 13.8 D~ .
       lower limit set by analytical procedure.
     " basic equation of general form c = a  b (D"1)

-------
                                                TABLE 6




Presence of Viable Sulfide- and Ferrous-Utilizing Organisms in Horizontal, Flooded-Bed Reactor Effluents
Days
Environment
nitrogen
air
air
Seeding
yes
no r;_
a
yes
0
Fe S
- -
- -
-
14
Fe S
+ +
-
+ +
28
Fe S
+ +
-
+ +
42
Fe S
+
-
+ +
56
Fe S
+
-
+ +
70
Fe S
+
-
+ +
84
Fe S

-
+ +
140
Fe S


+ +

-------
in the exit gases from the reactors or from the effluents .

           Samples of the pyrite remaining in aerobic-seeded reactor after
140 days of pilot plant operation were submitted to x-ray diffraction
analysis.  Debye-Scherrer patterns of the exposed and fresh mineral did
not differ and showed only the diffraction lines of pyrite.  Thus, surface
reaction product accumulation, if any, was too slight to be measured by
this spectrographic procedure.  There were only small quantities of fines
in the reactors after extended operating periods.  The individual pyrite
particles were essentially unaltered in appearance to the naked eye.
Electronmicrographs of the pyrite before and after oxidation show differ-
ences.  Appendix I.  Clusters of unidentified material developed on the
pyrite surface during oxidation.

           To facilitate comparison of the acid mine drainage release under
varying operating modes, the effluent concentrations at each flow rate
predicted from the least squares regressions, Table 4, were used to cal-
culate the total mass release of each component.

           An equation for relating the mass release in milligrams per
day, mg/d, as related to flow expressed as (D  ), may readily be obtained
from the aforementioned concentration-flow equation.  The mass release is
CQ and hence
                           CQ = aQ -
                                        - bVCD"1)2
Maximum mass release occurs when
                              - aV - bVUD'1) =0                   or
                                         fb
Maximum mass release occurs when
Fig. 13 depicts the pH and acidity (I^SO^ in mg/d, for each of the three
operating modes.  There is a slight but uniform increase in pH from a feed-
water value of 4.5 to 4.7 in the effluent over the entire hydraulic range
for the nonaerobic system.  The increase in pH is attributed to release of
basic minerals from the pyrite and the absence of oxidative, acid-producing
reactions.  Correspondingly, acidity approaches that of the feedwater, Fig.
10, over the entire hydraulic range except for a higher value at the very
lowest flow rate.  This acidity value reflects Fe (II) content.  The
aerobic-nonseeded reactor effluent characteristics are the result of       i
chemically-promoted oxidative reactions.  Fig. 11.  The pH increases only  j_
slightly from approximately 3.8 to 4.1 over the flow range of 2.3 to 13.8 D  .
Fig. 13.  This pH is the result of increased acidity released by pyrite
oxidation compared to the nonaerobic dissolution process.  The mass release
of acidity reaches a maximum at a flow corresponding to approximately 9
to 10 D"1, or 2 hours residence time.  Fig. 13.  Further increase in flow
produces no increase in acidity discharged per unit time suggesting that


                                   36

-------
the oxidation reaction is rate limited with respect to time.  Seeding the
aerobic system results in only minor differences in effluent pH from that
obtained with a nonseeded aerobic system.  Fig. 12.  The acidity does vary.
With seeding, mass release of acidity is lower at flows <10 D"1 and higher
at flows >10 D'1 than in the case without seeding.  Fig. 13.  At lower
flows, greater residence time, the organisms have an increased opportunity
to affect ferrous and sulfide oxidation in the seeded system.  Chemoauto-
trophic metabolic processes utilize acid, Silverman and Lundgren (1959),
and Fe (II) which is included in the acidity, is also consumed.  These
factors would account for the decreased mass release of acidity under
seeded compared to nonseeded aerobic operation.  At higher flow rates, the
rate limiting factors are the rate of pyritic dissolution to release Fe (II)
and 82  * ions and the limited contact time necessary for biologically
catalyzed oxidation of these ions.

           The mass release of iron under an anaerobic environment is con-
stant at 3.5 mg/d of ferrous and 7.5 mg/d of total iron at flows >4.6 D~^.
Fig. 14.  The ferrous and total iron concentration under nonaerobic con-
ditions should be identical.  Differences, Fig. 10, at a given flow rate
are attributed to oxidation during the lengthy sampling period required.
For anaerobic operation under equilibrium dynamic conditions a steady-
state release of Fe (II) and S2~** ions occurs even though oxygen is ex-
cluded from the system.  As noted in the report of the first phase of this
research, Baker and Wilshire (1968), mine sealing is practiced to keep
air from promoting pyritic oxidation.  However, pyritic dissolution takes
place without air being present although at a much lower level.  Ultimately,
the Fe (II) and S2"11 discharged with the effluent into the receiving
water body will exert an oxygen demand.

           Aerobic systems release significantly greater quantities of
Fe (II) and Sy~   ions an<^ t^6*1 oxidation products than do nonaerobic
systems.  Biologically promoted aerobic systems further accelerate Fe (II)
oxidation over levels obtained by strictly chemical means.  The aerobic-non-
seeded mass release of total iron approaches a maximum of 12 mg/d at 10 D"1
flow but then decreases  slightly to 14 mg/d at 14 D~^ flow for the aerobic-
seeded system.  Fig. 14.  Fe (II) release reaches a maximum of 6.8 mg/d
at approximately 10 D~^ and remains constant at higher flow rates for the
aerobic-nonseeded system.  For the aerobic-seeded system the maximum of
11.3 mg/d at approximately 9 D~^ is followed by a significant decrease
at higher flow rates.  At approximately 14 D"1 the mass release of Fe (II)
from the aerobic units is comparable whether or not chemoautotrophic or-
ganisms were seeded.  The Fe (II) I and total iron mass releases from the
seeded and nonseeded aerobic units are expected to correspond at an identical
high flow rate.  In this case, the failure of correspondence is an ex-
perimental limitation.  At the highest hydraulic rates applied to the
reactors, the effluent concentrations of the constituents approached the
lower limits of analytical reliability.  Very slight deviations in the
average effluent concentrations when multiplied by the large volumes would
alter the mass release values sufficiently for correspondence.  Definition
of the exact hydraulic rate for correspondence is of less importance than
in the theoretical basis for suggesting that a difference in mass release
occurs between the seeded and nonseeded aerobic systems.  The number of
organisms per unit volume of liquid in the continuously operating system
is a function of the availability of Fe  (II) and S2"i:t ions, their reproduction
                                   37

-------
                 80
                 60
              r  40
              TO
              'o
              <
                 20
                                    N-S
                                                  N-S
                        10     5  4    3         2
                                 Retention Time,Hours
1.5
                  "0               5              10              15
                      Flow,Day'.Volume Water Per Doy-rVolume Pyrite
Figure 13.  Mass Release of Acidity from Horizontal Reactors  Under Varying
            Operating Modes.

Charged with 625 ml  Shawville pyrite, 3.5 to 7 mesh size.  Operating mode:
Nonaerobic-seeded, N-S;  Aerobic-seeded, A-S; Aerobic-nonseeded, A-N. Influ-
ent pH 4.5 and acidity  20 to 25 mg/1.
                                   38

-------
                    15


                  T>
                  ^

                  6 10
                  c
                  o
                  o  5
                  p
                  1 10
A-S
                                           N-S
                      0           5           1O           15
                         Flow,Doy1,Volume Water Per Dayi-Volume PyrHe
                           10    5 4   3        2
                                  Retention Time,Hours
                      1.5
Figure  14.   Mass Release of Iron from Horizontal Reactors  Under Varying
             Operating Modes.

Charged with 625 ml Shawville pyrite, 3.5  to 7 mesh size.   Operating mode:
Nonaerobic-seeded, N-S;  Aerobic-seeded, A-S; Aerobic-nonseeded, A-N.
                                     39

-------
rate and their removal from the system.  At lower flow rates the reproduction
rate is sufficiently great relative to their residence time in the unit
so that a dynamic equilibrium is achieved.  At higher flow rates residence
time becomes limiting and flushout exceeds reproduction.  Eventually,
residence time is too short for the chemoautotrophs to affect the rate of
oxidation and the seeded and nonseeded mass release rates correspond.

           An examination of the effluent characteristics from the units
operated under various environmental and seeding modes shows agreement with
the strictly chemical model of ferrous iron oxidation described by Singer
and Stumm (1968).  They proposed a cyclical reaction model involving the
slow oxidation of Fe (II) to Fe (III) followed immediately by the rapid
reduction of Fe (III) by pyrite, which in turn generates more Fe (II) and
acidity.  The rate determing step is the oxidation of Fe (II).  Oxygen is
involved only indirectly in pyritic oxidation in their model.  Oxygen
serves to regenerate Fe (III) which is itself the specific pyritic oxidant.
In the biologically seeded aerobic systems the organisms promote Fe (II)
oxidation.  This increases the Fe (III) concentration leading to greater
ferric:ferrous ion ratios and increased acid formation.  In the report
of the first phase of this study, Baker and Wilshire (1968), it was suggested
that chemoautotrophic microorganisms significantly accelerate oxidation of
Fe (II) and S2-I1 ions released from the pyrite but apparently do not
directly alter the rate of pyrite dissolution in an aerobic environment.
The present results tend to support this contention.  The effect of the
microorganisms raising the ferric:ferrous ion ratio and hence, indirectly
affecting the rate of pyritic oxidation is in accord with the original
proposition.  Further research is necessary to specifically define whether
the microorganisms actually take part in the reactions at the pyrite
surface.

           Sulfate release from nonaerobic systems is essentially nonexis-
tent, Fig. 15, with oxidation during sampling accounting for the minimal
quantities measured.  Aerobic-nonseeded system mass release of sulfate
reached a maximum of approximately 53 mg/d at 13 D~^ flow.  Further in-
crease in flow does not affect sulfate release.  The aerobic-seeded system
released more sulfate than the aerobic-nonseeded system over the entire
hydraulic range and reached a maximum sulfate release of approximately
61 mg/d near 11 D   flow then decreased to 56 mg/d at 14 D""-*- flow.  For
the particular system being studied, maximum acid mine drainage occurs at
a flow of approximately 10 D~  or 2 hours retention time.

           One limitation of the pilot plant system being used to make
these studies is that an exact material balance cannot be calculated for
individual chemical characteristics.  The process is being monitored ex-
clusively on the basis of effluent characteristics.  Ferric reaction pro-
ducts formed are relatively insoluble and precipitate within the reactor
or are incorporated within the mycelial mat.  Only those products carried
over in the effluent are measured.  Carryover is obviously enhanced as
flow rate increases.  Sulfate often does not account for all of the S2"11
released from the pyrite.  Elemental sulfur has been suggested as a reaction
product by Silverman (1967).  Pyrite, FeS2, dissolution should lead to a
Fe (II) to S2"11 molar ratio of 1.0.
                                  40

-------
                80
                60
              *  40
              o
             *


             v>
                                      A-S
                                                  N-S
                 "05              10              15


                     Flow,Day"1,Volume Water Per Day-f Volume Pyrite
                        10     5  4    3          2

                                Retention  Time,Hours
1.5
Figure 15.  Mass  Release of Sulfate from Horizontal Reactors Under Varying

            Operating Modes.



Charged with  625  ml Shawville pyrite, 3.5  to 7 mesh size.  Operating mode:

Nonaerobic-seeded,  N-S; Aerobic-seeded, A-S; Aerobic-nonseeded, A-N.
                                     41

-------
                                 Fe (II)

and the possible sulfur species of
           For the nonaerobic system the total iron to sulfate sulfur molar
ratios in the effluent are >7, >4 and >3 at 4.6, 9.2 and 13.8 D'1 flow
rates.  Thus, mass release of total iron is in great excess of the mass
release of sulfur measured as the sulfate discharged per unit time.
For the aerobic-nonseeded system the Fe (II) to S2"11 molar ratio changes
only slightly from 0.67 to 0.77 over the flow range 2.3 to 13.8 D  .
The biologically-seeded, aerobic system produces ratios of 0.79, 0.92, 0.90
and 0.84 for flows of 2.3, 3.15, 9.2 and 13.8 D"1.  In the aerobic systems
the iron content approximates roughly the stoichiometric quantity needed
to balance the sulfur.  Neither total iron nor sulfur is a measure of the
pyrite dissolved.  Reaction products precipitated are retained in the
aerobic reactors so that effluent concentrations represent minimum rates
of pyritic dissolution.

           The concentration-flow relationships depicted in Figs. 10 to 12
for the effluents under varying operating modes show a distinct break in
slope at approximately 4.6 D~  flow or 4.3 hours retention time.  Concen-
trations increase at a greater rate as flow decreases (retention time in-
creases) over that predicted by extrapolation of the zero order relation-
ships at higher flow rates.  The point at which this linearity ceases
represents a mass transport limitation.  At lower flow rates reaction
products accumulate in the system because their removal rate is slower
than their formation rate.  Oxygen transport was by diffusion through
a film of water which floods the pyrite surface.  The gaseous diffusion
processes are slow.  It was not possible to devise an internal monitoring
probe to be inserted into the closed system to measure dissolved oxygen
in the liquid phase.  Measurements taken of the dissolved oxygen in the
effluent leaving the system were inadequate since reaeration occurred
while the sample was collecting at the relatively slow flow rates.  Oxygen
for the pyritic oxidation was supplied by the feedwater and by diffusion
into the liquid phase from the gaseous phase over the reactor surface.
Oxygen transfer to the flooded pyrite surface more nearly approximates
actual mine conditions than would be the case if forced aeration were
practiced to accelerate the oxidative reactions.  Mine pyrite surfaces
which are alternately flooded then drained will yield higher concentrations
of acid mine drainage on a spot basis simply because reaction product
accumulation may occur.  Smith and Shumate (1970) reported oxygen consumed
per mass of pyrite as a function of the waterrpyrite ratio in batch ex-
periments.  Their results also fall in two distinct regions represented
by a common point of transition between zones.  The point dividing
the mass transport limited regime from that not so restricted is analogous
in these studies.  However, experimental differences preclude direct com-
parison.

           In all these tests, the effluents from aerobic operation re-
mained in the range pH 3.5 to 4.1 at equilibrium.  In no case did pH
drop to levels which would inhibit chemoautotrophic activity or signifi-
cantly raise the solubility of Fe (III), thereby decreasing the potential
for Fe (II) oxidation in the sequential model of Singer and Stunm (1969).

                                  42

-------
           Further clarification of the role of the chemoautotrophic micro-
organisms and the effect of environment is obtained by examining the acidity-
ferrous ion interrelationship.  Each mole of ferrous ion is equivalent
to a mole of sulfuric acid.  Conversion of the effluent concentrations of
total acidity and ferrous ion to a molar basis permits subtraction to de-
termine the sulfuric acid equivalent of the remaining acidity.  For the
nonaerobic reactor this difference is negligible confirming that the total
acidity consists of the ferrous ion released.  For the aerobic systems the
effluent acidity concentration corrected for ferrous ion is expressed as
a function of flow over the range 3.15 to 14 D"1 for aerobic-seeded,

           Acidity (H2S04), m moles/I = [(7.890.05)-(0.140.00)D~1]10~2

For aerobic-nonseeded,

           Acidity (H2S04) m moles/I = [(14.080.90)-(0.670.00)D~1]10~2

           These least squares regressions are depicted in Fig. 16.  The
aforementioned claim that the microorganisms consume acid is evident in
these results.  Since the chemoautotrophic organisms must have a source of
hydrogen as well as the carbon to reproduce new cells, it is not unexpected
that the hydrogen ion concentration is depleted.  It is also apparent
that at a flow approximating 14 D~l that the seeded and nonseeded aerobic
units approach comparable acid release.  (The crossover of the regressions
at approximately 12 D~* is well within experimental error at m molar con-
centrations. )  As flow rates decrease the concentration of nonferrous
acidity markedly increases in the nonseeded over the seeded system.

           The effluent sulfate content may be considered to consist of
sulfuric acid and various sulfates.  A calculation was made of the sulfate
concentration in the effluent in excess of that necessary to balance the
sulfuric acid content depicted in Fig. 16 as a function of flow.  For the
nonaerobic reactor the quantity is negligible.  For the aerobic systems
the sulfate in excess of sulfuric acid equivalency is expressed as a
function of flow from 3.15 to 14 D"1.  For aerobic-seeded,

           Sulfate, m moles/1 = [(9.670.60)-(0.620.07)D~1]10"2

For aerobic-nonseeded,

           Sulfate, m moles/1  [(O.SliO.OO+CO.HO.OOD"1]!*)"2

           These least squares regressions are depicted in Fig. 17.
Sulfate concentration in the effluent in excess of sulfuric acid equi-
valency decreased rapidly as flow rate increases (retention time de-
creases) for the seeded system.  At approximately 13 D"-*- it is comparable to
the nonseeded system.  The nonseeded system sulfate in excess of sulfuric
acid equivalency decreases somewhat as flow decreases.  This modest in-
crease in effluent sulfate content of the nonseeded system with increased
flow may simply be a flushout of ferric or other sulfates.

           The results of this analysis of the corrected acidity expressed
as sulfuric acid and sulfate equivalency support the mechanism proposed for
role of microorganisms in acid mine drainage formation.  They promote
                                   43

-------
           _  0.12

           I
           |  0.10

           %
           c^Q.08

           >>
           ^  0.06
             0.04
                          i    i   i   i   I   i
                                                I	I
                 0              5               10              15
                    Flow,Day"1,Volume Water Per Day * Volume  Pyrite
                       10    5  4     3         2
                               Retention Time, Hours
1.5
Figure 16.   Effluent Concentration of Acidity Corrected for  Ferrous Iron
             Content.

Horizontal  reactors charged with 625 ml Shawville pyrite,  3.5  to 7 mesh.
size.  Operating mode:  Aerobic-nonseeded,  A-N; Aerobic-seeded,  A-S.
                                    44

-------
             0.08
             0.06
            0.04
            E
            0.02
            3
           V)
             0.00
                       1	1	1	1	1	1	1	T
                                                         ir
                       J	L
J	1	'   '
                                                     J	L
                 0              5              10              15
                    Flow,Day~1,Volume  Water Per Day + Volume  Pyrlte
                             J	L.
                                               _L
                       10     5  4    3          2
                               Retention Time, Hours
                          1.5
Figure 17.  Effluent Concentration of Sulfate in Excess of Sulfuric Acid
            Equivalent of Acidity.

Horizontal  reactors charged with 625 ml  Shawville pyrite, 3.5 to 7 mesh
size.  Operating mode:  Aerobic-nonseeded,  A-N; Aerobic-seeded, A-S.
                                    45

-------
Fe (II) and S2    oxidation.  The extent to which they affect oxidation is
a function of the hydraulic flow rate since microbial regeneration, or-
ganism flushout and pyritic dissolution are involved in the dynamic equili-
brium.

               2.  Effect of Varying Biological Seed

           In the aforementioned sections the effect of biological seeding
with a ternary mixture of Fe (II)- and $2"  -utilizing organisms was des-
cribed.  It was deemed expedient to test the effect on effluent charac-
teristics of seeding with the individual organisms and to compare the
results with those for the ternary seeding.  The four horizontal, flooded-
bed reactors were used.  These were charged with 625 ml of 3.5 to 7 mesh
Shawville pyrite.  Normally, fresh pyritic charge was used for each test.
In this case, the four units were pressed into this service immediately
after completion of the aerobic-nonseeded test series to save several
months of operating time.  Biological culturing tests confirmed that no
Fe (II)- or S2~**-utilizing organisms were present in the system prior to
seeding.  Flow through all four reactors was adjusted to 4.6 D   or 4.3
hours retention time.  The effluent characteristics after several weeks
equilibration were comparable in concentration and closely approximated
values predicted by the least squares equations of Table 4 for aerobic-
nonseeded operation.

           The reactors were then seeded for 14 days as follows:  (1)
Thiobaeillus thiooxidans^ TTf  (2) Ferrobaaillus sulfooxidanss FS, (3)
Ferrobaeillus ferrooxidanSj FF,  and (4) a ternary mixture of all three.
After 21 days the four effluent characteristics were not too different.
Table 7.  Sulfate tended to be slightly higher in the effluent of the
Thiobaaillus-seeded unit as might be expected.  The iron content of the
effluent from all four reactors was comparable and was less than that pre-
dicted from the least squares equations obtained from the analyses of
results using fresh pyrite, seeded immediately on start-up of the test
series.  All other effluent characteristics correspond closely to least
squares prediction values for aerobic-seeded operation.  The important
finding is that the nature of the biological seed made little difference
in the effluent composition from the four reactors.

               3.  Effect of Recycle

           The tests series in which the environmental and seeding factors
were examined utilized once-through flow systems.  Reaction products
which formed were chiefly precipitated with only soluble products and fine
particulates discharged with the effluent.  In an actual mining situation
the water arriving at a given pyritic site will probably contain previously-
formed reaction products.  How the chemical and microbiological materials
in the water might affect acid mine drainage formation was determined in
the smaller horizontal, flooded-bed pilot unit.  The unit was charged with
230 ml of 3.5 to 7 mesh Shawville pyrite.  (Additional details of the system
are given in Section 4, Part B.)  Feedwater flow was at 1.06 L/D or 4.6 D~^-.
Liquid volume in the bed was 103 ml for a retention time of 2.34 hours on
a once-through basis.  Operation was under an aerobic-seeded mode with
seeding of the standard ternary mixture of'chemoautotrophic microorganisms
for the first fourteen days.  The presence of viable Fe (II)- and S2"11-
utilizing microorganisms was confirmed by making inoculations in Waksman's
and Leathen's media every 14 days.  Equilibrium was reached in 53 days

                                  46

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                                TABLE 7


Effect of Biological Seed on Effluent Characteristics of Aerobic Horizontal,
                         Flooded-Bed Reactors3

     Reactor                       i           _3      _4

organism                          TT     FS    FF     Mix

pH                                3.8    4.0   4.0    4.0

acidity (H2SO,), mg/1            16     16    16     16

ferrous iron, mg/1                1.4    1.3   1.3    1.1

total iron, mg/1                  2.3    2.0   2.4    2.0

sulfate, mg/1                    15     11    12     12
aseeded after 84 days by Thiobaoillue thiooxidane, TT;  Ferrobacillus  eulf-
         f FS; Ferrobaeillue ferrooxidans, FF; or a  ternary mixture of  all
 three, Mix.  Flow at 4.6 D'1 or 4.3 hours  retention.
                                   47

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without the recycle chamber being on-stream.  The recycle chamber was then
added to determine the effect of eflfuent composition of the additional
retention volume of 190 ml or retention of 260 minutes at the forward flow
rate of 1.06 liters per day or 4.6 D  .  A slight decrease in Fe (II) and
total iron concentration and a slight increase in sulfate was measured.
The units were also operated at 1:1 and 4:1 recycle to forward flow ratios,
corresponding to hydraulic rates through the pyrite packed bed of two-
and five-fold over the nonrecycled operation.  The unit was operated for
approximately five weeks at each recycle ratio.  Table 8.  Effluent charac-
teristics as a function of recycleratio are presented in Fig. 18.  For
a fixed forward flow rate of 4.6 D  , the effluent composition for various
recycle rates from 0:1 to 4:1 is predicted by the following relationships
expressed in reciprocal time units:

         acidity, mg/1 -                  (10.921.21)+(0.880.07)D~1

         sulfate, mg/l =                  (8.031.40)+(1.070.08)D~1

         ferrous ion, mg/1 =              (-0.0510.02)+(0.3210.01)D"1

         total iron, mg/1 =               (Q.210.17)+^0.360.01)lT1

The mass release of these components per day as a function of recycle
rate is presented in Fig. 19.  The mass release of acidity, Fe (II), total
iron, and sulfate increase directly with increased hydraulic flow through
the reactor.  This is in marked contrast with the mass release of these
components when recycle is not used.  Figs. 13 to 15.  On a once-through
basis the mass release reaches a limiting value at a critical flow.  Further
increase in flow serves only to dilute the maximum concentration.  With
recycle, the effluent concentration continues to increase proportionally
with flow through the bed.  Of particular Interest is the increased Fe (II)
content of the effluent.  The higher flow rates through the bed facilitate
pyrite dissolution and transport of the Fe (II) from the pyrite surface.
The difference between total iron and Fe (II) concentrations is 0.4, 0.6
and 1.1 at 0:1, 1:1 and 4:1 recycle rates.  Thus, these effluent Fe (III)
concentrations cannot be used to judge the feasibility of increased pyrite
dissolution because of increased ferric:ferrous ion ratios.  The rate of
change of Fe (III) concentration is not proportional to the Fe (II) con-
centration increase.  The effective ferric:ferrous ion ratio has to be that
at the mineral surface.  Because of Fe (III) reaction product precipitation,
it is not possible from these results to estimate the actual surface con-
centration.  Chemically- and biologically-promoted reactions in the liquid
phase at some finite distance from the mineral surface most likely dominate
the oxidation process.

           An examination of the acidity-iron interrelationship facilitates
interpretation of these results.  The effluent concentrations were converted
to a molar basis and the contribution of the ferrous ion subtracted from
the total acidity.  The relationship of the acidity remaining, expressed as
sulfuric acid equivalents, to recycle ratio, expressed as flow through the1
pyrite bed, is                                                19
      Acidity (H2S04), m moles/1 -  [(11.230.88)+(0.320.05)D"i]10
                                  48

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                    TABLE 8

Effect of Recycling on Effluent Characteristics3
recycle ratio                0:1     1:1     4:1

pH                           3.8     3.8     3.6

acidity , mg/1       16      18      31

ferrous iron, mg/1           1.6     2.8     7.5

total iron, mg/1             2.0     3.4     8.6

sulfate, mg/1               15      15      33
aaerobic-seeded reactor charged with 3.5 to 7 mesh Shaw-
 ville pyrite; forward flow rate 1.06 L/D (4.6 D"1).
                       49

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                             8-
                             0     S     10     15     20    25
                              Flow,Day;1 Volume Water Per Day*Volume Pyrlte
                                  0:1    t:i                4M
                              Recycle Ratio,Volume Recycled* Forward Flow
Figure 18.   Effluent  Characteristics  of Acid Mine Drainage with Recycle

Shawville pyrite, 230 ml, 3.5 to 7 mesh size.  Forward flow  1.06 L/D
(4.6
                                         50

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                         40-
                                   Total Fe
                                                Fn
                          0

                      ^200
                      9

                        iooH
                      5 200
                      9

                      >
                      i  too-
                           0      9     10     15     20     29
                            Flow,Day? Volume Water Per Day! Volume Pyrlte

                                0:1    1:1                4:1
                            Recycle Ratio,Volume Recycled? Forward Flow
Figure 19.   Mass Release of Acid Mine Drainage with Varying Recycle Ratio

Shawville pyrite,  230 ml,  3.5 to 7  mesh size.  Forward flow 1.06 L/D
(4.6  D'1).
                                       51

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The results are presented in Fig. 20 which shows that for the fixed for-
ward flow of 4.6 D~l that recycling to increase flow through the pyritic
bed serves to increase effluent concentration of acidity corrected for
ferrous ion content.  This contrasts with a decrease of nonferrous acidity
with increasing flow rates on a once-through basis.  Fig. 16.

           The sulfate concentration in the effluent increases with recycle,
Fig. 18.  When the sulfate equivalent to the corrected acidity expressed
as sulfuric acid is subtracted from the total sulfate concentration, the
remainder as a function of total flow through the pyrite bed is

           Sulfate, m moles/1 = [(-2.8710.58)+(0.800.04)D"1] x 10~2

The results are presented in Fig. 20.  The accumulation of sulfate with
increased recycle is in contrast to the decrease experienced with increased
flow on a once-through basis.  Fig. 17.  On a once-through basis, the sul-
fate in excess of sulfuric acid equivalence is not significant.  As flow
(recycle) increases, the sulfate excess increases.  With the recycle system,
the chemoautotrophic microorganisms can achieve a higher count per unit
volume than would be the case for a once-through operation at the same hy-
draulic rate through the pyrite bed.  Flushout is solely a function of
the forward flow rate.  Microorganism count represents an equilibrium
between regeneration rate and flushout.  Hydraulic factors affecting
ferric concentration at the surface and similar mass transport considera-
tions also influence the acid mine drainage formation rates.

           B.  Vertical, Flooded-Bed Reactors

           Supplementary studies of the effect of adding carbon dioxide,
forced aeration and varying particle size were examined in vertical, packed-
bed, flooded reactors.  These units are described in Section 4B.

               1.  Effect of Supplemental Carbon Dioxide

           Examination of the effect of environment and seeding took place
in clear plastic reactors exposed to light.  Algal and heterotrophic as
well as chemoautotrophic microorganism growth was favored under aerobic
conditions.  Mines would not be equally appropriate locations for such
symbiotic processes.  The proliferation of mycelial growth has already
been suggested as a supplemental source of carbon dioxide for the chemo-
autotrophic microorganisms (Baker and Wilshire, 1968).  This supplemental
supply could be important if the dissolved carbon dioxide content of the
water were rate limiting.  This is unlikely.  In some tests of many months
duration, the effluent characteristics were comparable at the same flow
for the periods when mycelial growth was at its peak and after depletion of
the available carbon source reduced such growth.  A quantification of the
symbiotic relationship was beyond the scope of this study.  Normally,
natural waters in equilibrium with the atmosphere have carbon dioxide
concentrations of 0.4 to <1.0 mg/1.  Fixation of carbon dioxide by the
chemoautotrophic microorganisms might be expected to raise the pH except that
their metabolic end products from Fe (II)- and S2~II-utilization are acidic.
                                  52

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                   0.20
                  0.15
                 2 0.10
                 o
                 V
                 o

                 o 0.05
Figure  20.
          0       5       10      15      20       25
           Flow,Day"*,Volume Water Per Day*Volume Pyrlte
           	i	|	i	
                 0:1     1.1                    4:1
             Recycle Ratio, Volume  Recycled + Inward Flow


Effluent Concentration of Acidity Corrected for Ferrous Iron
Content and of Sulfate in Excess of Sulfuric Acid Equivalent
of Acidity.
Aerobic-seeded operating mode.  Horizontal, flooded-bed  reactor charged
with  230 ml of 3.5 to 7 mesh  Shawville pyrite.
                                    53

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           Whitesell, et.al. (1969) and  Borichenski (1967) demonstrated
that high levels of carbon dioxide were inhibitory to chemoautotrophic
organism cell growth and to iron oxidation.  Earlier, Schnaitman and Lundgren
(1965) found Ferrobaaillus ferrooxidans growth to be stimulated when carbon
dioxide supplement was added to carefully controlled cultures.  The maximum
pyruvic acid level of spent media from (^-supplemented and nonsupplemented
cultures was the same but the maximum acid level was reached earlier in
the (X^-supplemented culture.  These references suggest that although carbon
dioxide may be a limiting factor in pure culture experiments, high cell
counts and limited carbon dioxide solubility are more likely to affect
such systems.  Spot checks of Fe (II)- and S2~-^-utilizing microorganism
counts in the effluent from the horizontal, aerobic-seeded reactors of this
study indicated 10^ to 10  cells per ml under dynamic equilibrium condi-
tions.  The cell concentration of 10^ per ml is identical to that reported
by Smith and Shumate (1970) as the population required to supply ferric ions
at sufficient rate to support a detectable oxidation of pyrite by ferric
ions.  Their study of the microbiological effect on oxidation utilized
a batch respirometer with recirculation.

           The effect of bubbling carbon dioxide through a flooded, vertical
bed of pyrite has no relevance to the aforementioned horizontal reactor
studies.  The experiment only demonstrates the general effect on pyritic
dissolution and oxidation of pyrite under aerobic-seeded operating condi-
tions supplemented by carbon dioxide.  Two reactors were used.  One served
as a control while the other was subjected to carbon dioxide gasification
at 10 ml/min.  Both units contained 925 ml of 3.5 to 7 mesh Shawville
pyrite.  Feedwater flow corresponded to a rate of 2.3 D~^ providing 5.6
hours retention time.  The units were seeded with the standard ternary
mixture of chemoautotrophic microorganisms for the first 14 days.  Pre-
sence of viable Fe (II)- and S2~**-utilizing organisms in the effluent
was confirmed over the entire experimental period of 56 days.  The effluent
characteristics at equilibrium are presented in Table 9.

           Although the effluent pH of the carbonated system differs only
slightly, 5.1 versus 5.2 for the control, the increase in acidity is from
6 to 74 mg/1.  Fe (II) and total iron concentrations increase three- to
four-fold.  Sulfate increases only moderately from 7 to 10 mg/1.  The molar
ratio of Fe (II) to S2"11 in the effluents is 1.48 and 3.54 respectively
for the control and the carbonated systems.  Both show significant excess
of total iron to sulfur when compared to the molar ratio of 1.0 in pyrite.

           At pH 5.1 the hydroxyl and carbonate equilibrium concentrations
are calculated to be 0.8 x 10~9 and 5 x 10"11 moles/i respectively.  These
values and the solubility products

                    CaC03                    4.5 x 10~9

                    Fe(OH)2                  1.6 x 10~14

                    Fe(OH)3                  1.1 x 10~36

                    FeO>3                    3.0 x 10~14


                                  54

-------
                                TABLE 9

Effect of Gasification by Carbon Dioxide or Air on Effluent Composition
       from Aerobic-Seeded, Flooded Vertical Columns of Pyrite3
Characteristic

  pH

  acidity, mg/1

  ferrous iron, mg/1   2.5

  total iron, mg/1

  sulfate, mg/1
Control
5.2
6
2.5
3.2
7
C02 Addedb
5.1
74
9.1
10.5
10
Air Added0
2.8
208
97
224
866
aShawville pyrite, 925 ml of 3.5 to 7 mesh particle size; hydraulic rate
 2.3 D""^- (volume water per day * volume pyrite); retention time 5.6 hours.

 carbon dioxide rate, 10 ml/min
Caeration rate, 10 ml/min
                                  55

-------
permit further characterization of the system.  Ferrous hydroxide,
and ferrous carbonate. FeCO.,, cannot exist since their equilibrium concen-
trations are 1.4 x 10" and 33.5 g/1 respectively.  Feedwater calcium con-
centration is 76 mg/1 (1.9 x 10"-* moles/I) and it, too, will remain in
solution rather than precipitate as its carbonate (3600 g/1 required).
Also, Fe (II) content would have to be approximately 150 times greater
than calcium concentration for ferrous carbonate to form according to

                    CaC03 + Fe"""  ^^  Ca*"1" + FeCOg

so that its formation is not favored.  Singer and Stumm (1970) demonstrated
that ferrous bicarbonate concentration relative to ferrous carbonate con-
centration is insignificant for natural waters of normal alkalinities.
Only ferric hydroxide, Fe(OH)o, formation is likely at 1.2 x 10~5 mg/1.
These results explain why Fe (II) released from the pyrite tends to remain
in solution until oxidized to the Fe (III) state.  Biologically-promoted
oxidation is not favored at pH 5.1 since the chemoautotrophic organisms
are most active at pH 2 to 4.5.  Chemical oxidation processes are expected
to dominate but are limited since the dissolved oxygen in the aqueous
phase is being stripped by the carbon dioxide flow through the bed.
Chemical oxidation depends on the dissolved oxygen content of the water
in the absence of forced aeration.  The bubbling action of the carbon
dioxide promotes mixing and transport which also contributes to the in-
crease in effluent concentration of constituents over that in the quiescent
control pyrite column.  Fe (II) accounts for 72% of the total acidity of
the control reaction, (0.061 - 0.045 m moles/I).  If the remainder
of the acidity is assumed to be sulfuric acid, then the sulfate balance is
negative.  This occurs because carbon dioxide is oversaturated in the
system.  Total acidity is consequently artificially increased over that
attributable to the Fe (II) and sulfuric acid contributions.

               2.  Effect of Aeration

           In the horizontal and vertical reactor studies described to
this point, only the oxygen dissolved in the aqueous phase was available
to chemically- or biologically-promoted oxidation of pyrite.  This is the
predominant condition existing in mines.  A test was also made to determine
the dissolution and oxidation of pyrite when forced aeration was practiced.
The experiment was made in the same vertical reactors and under identical
conditions described in the carbonation study.  Air was bubbled through
the reactors at 10 ml/min.  The effluent characteristics are given in
Table 9.

           There is a decrease of pH from 5.2 for the control to 2.8.
Acidity, sulfate and. iron content of the effluent is appreciably increased.
At pH 2.8, hydroxyl concentration is 6.3 x 10~^.  At these conditions,
precipitation of ferrous hydroxide, Fe(OH)2, would require 2.24 x 1010 g/1
but ferric hydroxide, Fe(OH)3, only 245 mg/1.  The ferrous iron to S2~1^ molar
ratio is 0.88 in the aerated reactor effluent which approximates the stoi-
chiometrically necessary quantity of 1.0 for pyrite dissolution.  However,
the total iron of 224 mg/1 includes 97 mg/1 ferrous ion.  Hence, either
S2""11 oxidation is favored over Fe (II) oxidation; or, more likely, ferric
hydroxide, Fe (OH)3, rapid formation and precipitation within the bed
accounts for the difference.  Fe (II) accounts for 28% of the total acidity
(6.2-1.7 m moles/1) leaving a great excess which may be attributed to
sulfuric acid.  There is also an excess of effluent sulfate of 4.6 m moles/I

                                  56

-------
over that necessary to balance sulfuric acid acidity of 4.5 m moles/I.
All chemical and biological factors favor increased pyrite dissolution
and oxidation under forced aerated conditions.   These results demonstrate
that the reactions in the horizontal-flooded bed reactors were oxygen
rate limited.

               3.  Effect of Flow

           The effect on effluent composition and mass release of acid
mine drainage of varying hydraulic flow rate through the vertical columns
of pyrite was determined.  The vertical columns, pyrite and operating
conditions were identical to those described in 6 B.I.  The flow rate was
adjusted to 2.15, 4.3, 8.6 and 12.9 liters/day.  These flow rates corres-
pond to 2.3, 4.6, 9.2 and 13.8 D~^ (volume of water per day T volume of
pyrite) or 5.6, 2.8, 1.4 and 0.9 hours retention time respectively.  The
retention time versus D"1 relationship in the case of the vertical units
differs from that in the horizontal units described in 6A.  Although the
depth of liquid film over the pyrite was approximately the same in each
case, volume of water over the surface was greater with the horizontal
reactors.  The surface area for the vertical reactor was very small relative
to that of the horizontal bed although the former contained approximately
50% more pyrite.  The ratio of volume of liquid to volume of pyrite is
smaller for the vertical columns.  Actual liquid volumes in the pyrite-
containing zones of the reactors were measured.  Retention time is based
on this volume.  Effluent characteristics for the horizontal and vertical
units are therefore not comparable at the same volumetric ratio, D 1.

           The effluent composition and corresponding mass release per day
of various constituents is tabulated as a function of flow in Table 10.
The effluent characteristics at 2.3 D~l agree very closely with those for
the control unit operated under identical conditions in the carbon dioxide
study, Table 9.  This illustrates the reproducibility of the tests.  The
mass release rates suggest that flow rate was limiting total acid mine
drainage production probably because available oxygen was limited.  Pyrite
dissolution and chemical-biological oxidation rates were sufficiently
rapid so that at lower flow rates, <9.2 D~* or <1.4 hours retention time,
there is poor correlation of mass release with flow.  Precipitation of
reaction products is also enhanced at low flow rates.  At higher flow
rates insoluble (particulate) carryover is more likely.

               4.  Effect of Surface Area

           It is expected that a direct relationship should exist between
available pyritic surface area and release of acid mine drainage.  This
is a resonable assumption if reaction product accumulation or depletion of
reactant are not factors under aerobic conditions.  An estimate  of the
effect of varying surface area on effluent composition under aerobic-
seeded conditions was obtained with the vertical reactors.  Operating
procedures were identical to those described in 6 B.I except that three
reactors were filled with Shawville pyrite of three different sizes.
Table 11.  The crushed pyrite was first screened for size range.  The
mean diameter based on a spherical shape and the number of particles making
up the reactor volume of 925 ml provided an estimate of mean area for the
pyrite surface.  These mean area values are uncorrected for contact area.
The original particles are so irregular that only a rough estimate of relative


                                   57

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                               TABLE 10

Effect of Flow Rate on Effluent Composition from Aerobic-Seeded, Flooded
                      Vertical Columns of Pyritea
Effluent
Concentration
pH
acidity, mg/1
ferrous iron, mg/1
total iron, mg/1
sulfate, mg/1
Mass Release
acidity, mg/d
ferrous iron, mg/d
total iron, mg/d
sulfate, mg/d
c
Flow Rate, Volume Water Per Day * Volume Pyrite
2.3
5.2
6
2.5
3.0
8

13
5.4
6.5
17
4.6
4.2
6
1.3
1.6
2

26
5.6
6.9
9
9.2
4.2
7
0.7
1.0
4

61
6.1
8.6
35
13.8
4.1
7
0.8
1.3
7

90
10.3
16.8
90
aShawville pyrite,  925 ml of 3.5 to 7 mesh particle size
 mass released, milligrams per day

 retention times:   5.6,  2.8,  1.4 and 0.9 hours,  respectively
                                 58

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                               TABLE 11

Relative Surface Area of Pyrite Particles Charged to Vertical Reactors3

Size
0.5 - 0,75
incn
3.5 mesh -
0.5 inch
3.5-7 mesh

Mean
Dia. , mm

15.9

9.18
4.25

Mean
Area, mm2

795

266
56.7

Number
Particles

139

760
11,137

Mean Area
10 3 mm2

107

202
632
Relative
Surface
Area

0.17

0.32
1.00
 Shawville pyrite, 925 ml volume
                               TABLE 12

Effect of Varying Surface Area on Effluent Composition from Aerobic-Seeded,
             Flooded Vertical Reactors Containing Pyrite3
     Effluent
Relative Surface Area
uoncentracion
pH
acidity, mg/1
ferrous iron, mg/1
total iron, mg/1
sulfate, mg/1
Al
4.1
7
0.8
1.3 ,
7
0.32A1
4.1
6
0.7
1.2
6
0.17A1
4.2
4
0.5
1.0
5
aShawille pyrite, 925 ml volume; flow at 13.8 D"1, 0.9 hours retention
                                  59

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surface area could be expected.  The 3.5 to 7 mesh particles were used in
most of these experiments.  The surface areas of the two beds containing
larger particles are 0.32 and 0.17 relative to the standard particle size.
The void space in the bed also varied with particle size and is 6% and
16% greater for the larger particles than for the 3.5 to 7 mesh size
pyrite.  The results of varying flow through the vertical reactors, Table 10,
suggested that the study of the effect of surface area be made at 13.8 D~*
or 0.9 hours retention time for the 3.5 to 7 mesh particles.  Retention
time in the other units is 1.0 and 1.1 hours respectively.  The reactors
were seeded for the first 14 days with the standard ternary mixture of
chemoautotrophic microorganisms.  The effluent continued to show the
presence of viable Fe (II)- and S2~II-utilizing organisms through the
duration of the study, 95 days.  The gray mycelial growth observed in
the horizontal, flooded-bed reactors also developed in these vertical
reactors.  In time, it assumed the yellow-brown color typical of pyrite
oxidation products.

           Effluent characteristics at equilibrium are summarized in
Table 12.  Effluent pH varies only slightly, increasing from 4.1 to 4.2
as particle size increases.  Acidity, Fe (II) and total iron and sulfate
decrease as particle size increases (surface area decreases). Although
the relative surface area of the pyrite bed with the largest particles is
only 17% of that with the smallest particles, the mass release of each of
the acid mine contaminants decreases only about 30 to 40%.  The increased
mass release is not directly proportional to the increased surface areas of
1:2:6.  The difference is chiefly attributable to the variances involved
in estimating surface area, variation in residence time and the probability
that the choice of operating conditions may have been rate limited with
respect to oxidation rate and/or transport relationships.  A more exact
experiment would have to be performed to isolate the surface area effect.
However, this study suggests that if reactant (chemical or biological)
depletion or reaction product concentration is not rate limiting, the
release of acid mine drainage will be proportional to the available pyritic
surface area for a given hydraulic rate under flow conditions which are
not so slow as to be diffusion rate limited.
                                  60

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                               Section 7

                            ACKNOWLEDGMENTS

           During the period of this investigation, the authors were privi-
leged to have had the advice and constructive criticism of colleagues at
the Mellon Institute.  Dr. Ming-Dean Luh of the Environmental Sciences
Fellowship was especially helpful with the calculations and critical review
of the draft of this final report.  Dr. William R. Samples, Water Resources
Fellowship head, cheerfully acted as sounding-board during conceptual
discussions with the senior author and also reviewed the draft of this
report.

           The research was supported financially by the Federal Water
Quality Administration through Research Grant 14010 DKN.  Technical liaison
was provided by Ronald Hill, Chief, Mine Drainage Pollution Control Activities,
of FWQA.
                                   61

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                                Section 8

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 2.   ASTM Book of Standards, Part 23,  1968.

 3.   Baker,  R. A. and Wilshire, A. G.  (1968)   Acid mine drainage-pilot
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 4.   Baker,  R. A. and Wilshire, A. G.  (1970)   Microbiological factor in
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 5.   Borichenski, R.  M. (1967)   Keto acids as growth-limiting factors in
     autotrophic growth of Thiobaaillus thiooxidans.   J. Bacter., 93,
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 6.   Colmer, A. R.,  Temple,  K.  L., and Hinkle, M. E.  (1950)  An iron
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 7.   Dugan,  P. R. and Lundgren, D. G.  (1965)   Energy supply for the chemo-
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 8.   Garrels,  R.  M.  and Thompson, M. E. (1960)  Oxidation of pyrite by
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 9.   Girard, L. (1965)  Operation yellowboy.   Proceedings, Second Symposium
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10.   Hem, J. D. (1960)  Some chemical relationships among sulfur species
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11.   Hem, J. D. and Cropper, W. H. (1959)  Survey of ferrous-ferric chemi-
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     Paper 1459 A, 31 pp.

12.   Huffman, R.  E.  and Davidson, N. (1956)  Kinetics of ferrous iron-oxygen
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13.   Kinsel, N.  (1960)  New sulfur oxidizing iron bacterium:  Ferrobaoillus
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14.   Leathen, W. W.  and Braley, S. A.  (1954)  A new iron-oxidizing bacterium:
     Ferrobaoillus ferrooxidans.  Bact. Proc., 44.
                                   62

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15.   Leathen, W.  W.,  Braley, S.  A., and Mclntyre, L.  D.  (1953a)   The role
     of bacteria in the formation of acid from certain sulfuritic consti-
     tuents associated with bituminous coal.  I.  Thiobaoillus thiooxidans.
     Applied Microbiology, ^, 61-64.

16.   Leathen, W.  W.,  Braley, S.  A. and Mclntyre, L. D. (1953b)  The role of
     bacteria in the formation of acid from certain sulfuritic constitu-
     ents associated with bituminous coal.  II.  Ferrous iron oxidizing
     bacteria.  Applied Microbiology, _!, 65-68.

17.   Leathen, W.  W.,  Kinsel, N.  A. and Braley, S. A.  (1956)  Ferrobaaillus
     ferrooxidans:  a chemosynthetic autotrophic bacterium.  J.  Bacter.,
     72, 700-704.

18.   Leathen, W.  W., Mclntyre, L. D. and Braley, S. A. (1951)  A medium
     for the study of the bacterial oxidation of ferrous iron.  Science,
     1141, 280-281.

19.   Lundgren, D. G.  and Schnaitman, C. A.  (1965)  The iron oxidizing
     bacteria-culture and iron oxidation.  Proceedings, Second Symposium
     on Coal Mine Drainage Research, Mellon Institute, Pittsburgh, Penn-
     sylvania, 14-22.

20.   Salotto, B. V., Earth, E. F., Ettinger, M. G., and Tolliver, W. E.
     (1967)  Determination of mine waste acidity.  Private communication.

21.   Schnaitman, C. and Lundgren, D. G.  (1965)  Organic compounds in the
     spent medium of Ferrobaoillus ferrooxidans.   Can. J. Micro., 11, 23-
     27.

22.   Silverman, M. P. (1967)  Mechanism of bacterial pyritic  oxidation.
     J. Bacter., 94^ 1046-1051.

23.   Silverman, M. P. and Ehrlich, H. L.  (1964)  Microbial formation and
     degradation of minerals in Advances in Appl.  Microbiol.  (W. W. Um-
     breit, Ed.), , 153-206.

24.   Silverman, M. P. and Lundgren, D. G.  (1959)   Studies on  the chemoauto-
     trophic iron bacterium Femobaoillus ferrooxidans.  J. Bacter., 77,
     642-647.

25.   Singer, P. C. and Stumm, W.  (1968)  Kinetics  of  the oxidation  of
     ferrous ion.  Proceedings, Second Symposium on Coal Mine Drainage
     Research, Mellon Institute,  Pittsburgh, Pennsylvania.

26.   Singer, P. C. and Stumm, W.  (1969)  Oxygenation  of ferrous iron.
     Final Report to Federal Water Quality  Administration, Program  Number
     14010 by Harvard University.

27.  Smith, E. E. and Shumate, K. S.  (1970)  The sulfide to sulfate reaction.
     Final Report to Federal Water Quality  Administration, Program  Number
     14010 FRS by Ohio State University  Research Foundation.
                                    63

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28.  Standard Methods for the Examination of Water and Wastewater, Twelfth
     Edition (1965) APHA, AWWA and WPCF, New York.

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     & Eng. Chem., 53, 143-146.

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     iron by a new bacterium:  Thiobacillus ferrooxidans.  J. Bacter.,
     62., 605-611.

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     of three chemoautotrophic bacteria.  Soil Science, 92, 302-313.

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     Presented at ACS 157th National Meeting, Minneapolis, Minn., April
     13-18.
                                   64

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                                Section 9

                          LIST OF PUBLICATIONS

Baker, R. A. and Wilshire, A. G. (1970)  Microbiological Factor in Acid
Mine Drainage Formation:  A Pilot Plant Study, Environmental Science &
Technology, 4^ No. 5, 401-407.

Baker, R. A. and Wilshire, A. 6. (1970)  Evaluation of Potential Acid
Mine Drainage, Water & Sewage Works, 117, No. 6, IW/10-16.

Baker, R. A. and Wilshire, A. G. (1970)  Microbiological Factor in Acid
Mine Drainage Formation:  II.  Further Observations from a Pilot Plant
Study, Paper to 160th National Meeting of American Chemical Society,
Chicago, Illinois, September 13-18.
                                   65

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                               Section 10

                               APPENDIX I

                        PYRITE  ELECTRONMICROGRAPHS

            The surface characteristics of selected 3.5 to 7 mesh particles
of Shawville pyrite were examined by means of a Jeolco scanning electron
microscope before and after oxidation.  The reacted pyrite has been exposed
to an aerobic-seeded environment for 50 days.  It was taken from the reactor
and air dried.  The unreacted pyrite was not freshly-ground but had been
stored in a closed container prior to this examination so that some oxi-
dation may have occurred on the mineral surface.  The surface appearance
at 1000 and 5000 magnifications for the unreacted and reactor pyrite is
shown in Figs. 21 and 22, respectively.  Although x-ray diffraction spec-
troscopy failed to detect differences between the unreacted and reactor
pyrite, the photomicrographs indicate a significant surface alteration.
Clusters which appear as lighter-colored areas developed on the pyrite
surface during oxidation.  Further elaboration on the nature of these
crystal clusters is not possible at this time.
                                   66

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          Unreacted
1000X
Reacted     1000X
                                                   HQu I
Figure 21.   Photomicrograph of Unreacted and Reacted Pyrite at 1000 Magnifications

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00
                 Unreacted     5000 X
                                                          2y
Reacted     5000X
     Figure 22.  Photomicrograph of Unreacted and Reacted Pyrite  at  5000  Magnifications,

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BIBLIOGRAPHIC:  Carnegie-Mellon University,- Mellon
Institute
Microbiological Factor in Acid Mine Drainage Form-
ation.  FWQA Publication No. 14010 DKN

   The role of chemoautotrophic organisms in the
formation of acid mine drainage from pyritic ma-
terials associated with coal mining has been inves-
tigated by pilot plant techniques.  Dynamic flow,
controlled environment  units which served as
models of mines were used.
   It was demonstrated that the concentration of
acidity, ferrous and total iron and sulfate in
effluent from aerobic-seeded or -unseeded pyritic
beds is zero order with respect to flow, except
where mass transport is diffusion limited.
   Nonaerobic systems produce acidity consisting
only of ferrous iron.  Total acidity was lower
from seeded- than nonseeded-aerobic systems at
retention times exceeding three hours because micro-
organisms consume acid.  For lower retention time,
ACCESSION NO.

KEY WORDS:

Mine Drainage
Chemoautotrophic
 Microorganisms
Ferrobacillus
Thiobacillus
Pyrite
Pilot Plant
Dynamic Equilibria
BIBLIOGRAPHIC:  Carnegie-Mellon University, Mellon
Institute
Microbiological Factor in Acid Mine Drainage Form-
ation. FWQA  Publication No.  14010 DKN

   The role of chemoautotrophic organisms in the
formation of acid mine drainage from pyritic ma-
terials associated with coal mining has been inves-
tigated by pilot plant techniques.  Dynamic flow,
controlled environment units which served as
models of mines were used.
   It was demonstrated that the concentration of
acidity, ferrous and total iron and sulfate in
effluent from aerobic-seeded or -unseeded pyritic
beds is zero order with respect to flow, except
where mass transport is diffusion limited.
   Nonaerobic systems produce acidity  consisting
only of ferrous iron.  Total acidity was lower
from seeded- than nonseeded-aerobic systems at
retention times exceeding three hours because micro-
organisms consume acid.  For lower retention time,
ACCESSION NO.

KEY WORDS:

Mine Drainage
Chemoautotrophic
 Microorganisms
Ferrobacillus
Thiobacillus
Pyrite
Pilot Plant
Dynamic Equilibria
BIBLIOGRAPHIC:   Carnegie-Mellon  University, Mellon
Institute
Microbiological  Factor  in Acid Mine  Drainage  Form-
ation.  FWQA Publication No.  14010 DKN

   The role of chemoautotrophic  organisms in  the
formation of acid mine  drainage  from pyritic  ma-
terials associated with coal mining  has been  inves-
tigated by pilot plant  techniques.   Dynamic flow,
controlled environment  units which served as
models of mines were used.
   It was demonstrated  that the  concentration of
acidity, ferrous and total iron  and  sulfate in
effluent from aerobic-seeded or  -unseeded pyritic
beds is zero order with respect  to flow, except
where mass transport is diffusion limited.
   Nonaerobic systems produce acidity consisting
only of ferrous iron.   Total acidity was lower
from seeded- than nonseeded-aerobic  systems at
retention times exceeding three hours because micro-
organisms consume acid.  For lower retention  time,
 ACCESSION NO.

 KEY WORDS:

 Mine Drainage
 Chemoautotrophic
  Microorganisms
 Ferrobacillus
 Thiobacillus
 Pyrite
 Pilot Plant
 Dynamic Equilibria

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total acidity is greater in the aerobic-nonseeded effluents.
Under aerobic-nonseeded conditions acid discharge reaches a
maximum rate at a specific flow.  Further increase in flow
only dilutes the concentration.  Under aerobic-seeded con-
ditions, a dynamic equilibrium exists between the rates at
which pyrite dissolves, organisms reproduce and are removed
from the system, and a maximum discharge occurs at a specific
hydraulic rate.
total acidity is greater in the aerobic-nonseeded effluents.
Under aerobic-nonseeded conditions acid discharge reaches a
maximum rate at a specific flow.  Further increase in flow
only dilutes the concentration.  Under aerobic-seeded con-
ditions, a dynamic equilibrium exists between the rates at
which pyrite dissolves, organisms reproduce and are removed
from the system, and a maximumodlscharge occurs at a specific
hydraulic rate.
total acidity is greater in the aerobic-nonseeded effluents.
Under aerobic-nonseeded conditions acid discharge reaches a
maximum rate at a specific flow.  Further increase in flow
only dilutes the concentration.  Under aerobic-seeded con-
ditions, a dynamic equilibrium exists between the rates at
which pyrite dissolves, organisms reproduce and are removed
from the system, and a maximum discharge occurs at a specific
hydraulic rate.

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    Accession Number
                            Subject Field &, Group
                                                SELECTED  WATER RESOURCES  ABSTRACTS
                                                      INPUT TRANSACTION FORM
    Organization
    Carnegie-Mellon University
    Title
     Microbial Factor in Mine Drainage Formation
| Q Authors)
Wilshire, Albert G.
16

21
Project Designation
U010DKN (Federal
Water Quality Administration^
Note
 22
    Citation
 23
   Descriptors (Starred First)

   *Mine Drainage
   *Mine Acid
   *Mine Wastes
   *Mine Water
                                        Bacteria
                                        Ferrobacillus
                                        Thiobacillus
                                        Mine Models
 95 I Identifiers (Starred First)
 27
   Abstract
   The role of chemoaujirophic organisms in the formation of acid mine drainage
   from  pyritic materials associated with coal mining has been investigated by
   pilot plant techniques. Dynamic  flow, controlled environment units which
   served as models of mines were used.

   It was demonstrated that the  concentration of acidity, ferrous  and total
   iron  and sulfate in the effluent from aerobic, biologically-seeded or
   unseeded pyritic beds is zero order with respect to flow, except at low
   flow  rates where mass transport  is diffusion limited. Algal growth occurred
   in the acidic, aerobic environment but did not affect acid production.

   Nonaerobic systems produce acidity consisting only of ferrous iron. Total
   acidity is lower from biologically-seeded than -nonseeded aerobic systems.
   Acid  mine drainage was not significantly affected by seeding with the
   individual or a mixture of three different organisms but was increased by
   recycle of the flow and increased appreciably under forced aeration. The
   rate  is directly related to the  available pyrite surface area.
Abstractor
      T-M.
                             Institution
                             	Federal Watfti- Quality  Adtni ni at.ra-M on
WR;102 (REV. JULY 188BI
WRSIC
                                               SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
                                                      U.S. DEPARTMENT OF THE INTERIOR
                                                      WASHINGTON. D. C. SOZ4O

                                                              * U. S. GOVERNMENT PRINTING OFFICE : 1TO O - 4M-M

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