WATER POLLUTION CONTROL RESEARCH SERIES ,g ^ 14Q10 ENW gg/71
     Microbiological  Treatment of
      Acid Mine  Drainage Waters
ENVIRONMENTAL PROTECTION AGENCY •

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       WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control  Research Series describes
the results and progress in the control  and abatement
of pollution in our Nation's waters.  They provide a
central source of information on the research,  develop-
ment, and demonstration activities in the Environmental
Protection Agency, through inhouse research and grants
and contracts with Federal, State, and local  agencies,
research institutions, and industrial  organizations.

Inquires pertaining to Water Pollution Control  Research
Reports should be directed to the Head,  Project Reports
System, Office of Research and Monitoring, Environmental
Protection Agency, Washington, D. C.  20242

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Microbiological Treatment of Acid Mine Drainage Waters
                      Continental Oil Company
                Research and Development Department
                    Ponca City,  Oklahoma  74601
                              for the

                  EWIRONMEWTAL PROTECTION AGENCY
                         Grant # 14010 ENW
                          September 1971
  For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1

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         EPA Review Notice

This report has been reviewed by the Office
of Research & Monitoring, EPA, and approved
for publication.  Approval does not signify
that the contents necessarily reflect the
views and policies of the Environmental
Protection Agency, nor does mention of
trade names or commerical products
consitute endorsement or recommendation
for use.
                   11

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                               ABSTRACT
Laboratory studies demonstrated that both pure cultures and fresh field
cultures of acidophilic iron bacteria could readily oxidize ferrous iron
in both synthetic and natural acid mine drainage waters.  In the presence
of adequate forms and amounts of oxygen, carbon dioxide, nitrogen and
phosphorus, ferrous iron was oxidized at rates up to 600 mg/l/hr.  Average
oxidation rate was found to vary with the ferrous iron concentration in
the water; i.e., the higher the ferrous iron content, the greater the
oxidation rate.  Approximate requirements of oxygen, carbon dioxide,
nitrogen and phosphorus by the iron bacteria were established.

Multistaging of oxidation vessels in series was found to produce a more
effective microbial oxidation system than use of a single oxidation
reservoir.

Limestone neutralizations of partially oxidized acid mine waters showed
that such waters containing up to 9° mg/1 ferrous iron could be success-
fully neutralized and result in discharge waters containing < 7 mg/1
total iron.

For reasons as yet undetermined, attempts to duplicate laboratory findings
with a 2,000-gallon pilot plant were not completely successful.

Although sulfate-reducing bacteria were isolated from all of nine acid
mine discharges examined, attempts to grow the cultures or produce hydrogen
sulfide at pH values below 5-5 were unsuccessful.

This report was submitted in fulfillment of Grant 1^010 EWW between the
Environmental Protection Agency and Continental Oil Company.
Key Words:  Acid mine water, bacteria, iron bacteria, sulfate-reducing
            bacteria, limestone, oxidation of ferrous iron, oxidation
            rate, neutralization, pilot plant
                                  111

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                               CONTENTS




Section                                                           Page




  I       Conclusions                                               1




  II      Recommendations                                           3




  III     Introduction                                              5




  IV      Methods                                                   9




  V       Experimental                                             19




  VI      Discussion                                               73




  VII     Acknowledgments                                          75




  VIII    References                                               77

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                       FIGURES



                                                         PAGE
1
2

3
4
5
6

7
8
9
10

11
12
13
14
15
16
17
CARBON CONTENT OF GROWING IRON BACTERIAL CULTURES
CARBON PRODUCTION AND FERROUS IRON OXIDATION BY
SOUTHWEST- 1 CULTURE
SCHEMATIC OF CONTINUOUS FEED BIO-OXIDATION SYSTEM
Fe++ OXIDATION RATES IN 9K MEDIUM SHAKE FLASKS
Je++ OXIDATION AT VARIOUS AMMONIUM ION CONCENTRATIONS
Fe++ OXIDATION USING NOg OR NO^ AS NITROGEN SOURCE IN
9K MEDIUM SHAKE FLASKS
Fe++ OXIDATION AT VAROUS P0| CONCENTRATIONS
EFFECT OF PHOSPHATE CONCENTRATION ON FERROUS IRON
OXIDATION BY THIOBACILLUS FERROOXIDANS (BECK)
EFFECT OF PHOSPHATE CONCENTRATION ON FERROUS IRON
OXIDATION BY T. FERROOXIDANS (BECK) - SUBCULTURES
EFFECT OF INITIAL FERROUS IRON CONCENTRATION ON EARLY
IRON OXIDATION IN BATCH CULTURE
FERROUS IRON OXIDATION AT AN INITIAL IRON CONCENTRATION
OF 9,000 mg/liter
IRON OXIDATION AT AN INITIAL FERROUS IRON CONCENTRATION
OF ABOUT 10,000 mg/liter
EFFECT OF TEMPERATURE ON FERROUS IRON OXIDATION BY
SOUTHWEST- 1 CULTURE
EFFECT OF TEMPERATURE ON FERROUS IRON OXIDATION BY
THIOBACILLUS FERROOXIDANS (BECK)
EFFECT OF TEMPERATURE ON FERROUS IRON OXIDATION BY
FERROBACILLUS FERROOXIDANS (SYRACUSE)
EFFECT OF TEMPERATURE ON FERROUS IRON OXIDATION BY
FERROBACILLUS FERROOXIDANS (DUGAN)
FERROUS IRON OXIDATION AT VARIED INITIAL Fe++
14
15

16
20
22
24

25
26
27
29

30
31
32
33
3^
35
36
CONCENTRATIONS BY BECK CULTURE  -  SHAKE FLASKS 10 °C

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                          FIGURES (CONTINUED)

                                                                 PAGE

18    FERROUS IRON OXIDATION AT VARIED INITIAL Fe++
        CONCENTRATIONS BY BECK CULTURE - SHAKE FLASKS 20°C

19    FERROUS IRON OXIDATION AT VARIED INITIAL Fe++                38
        CONCENTRATIONS BY BECK CULTURE - SHAKE FLASKS 35 °C

20    FERROUS IRON OXIDATION AT VARIED INITIAL Fe++                39
        .CONCENTRATIONS BY SOUTHWEST-1 CULTURE  - SHAKE
        FLASKS 10°C

21    FERROUS IRON OXIDATION AT VARIED INITIAL Fe++                40
        CONCENTRATIONS BY SOUTHWEST-1 CULTURE  - SHAKE
        FLASKS 20°C

22    FERROUS IRON OXIDATION AT VARIED INITIAL Fe++                4l
        CONCENTRATIONS BY SOUTHWEST-1 CULTURE  - SHAKE
        FLASKS 35 °C

23    EFFECT OF INITIAL pH ON FERROUS OXIDATION IN SHAKE            45
        FLASKS - 25°C

24    EFFECT OF UNLEACHED REDWOOP ON FERROUS OXIDATION             48

25    IRON PRECIPITATION FOLLOWING LIMESTONE NEUTRALIZATION        6l
        OF PARTIALLY OXIDIZED MEDIUM

26    -EFFECT OF ADDED SOLIDS ON LIMESTONE NEUTRALIZATION            63
        AND SETTLING OF STAGE II EFFLUENT

27    BIO-OXIDATION PILOT PLANT                                    65
                                  Vll

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                                TABLES

No.                                                               Page

1     Composition of 9K Iron Bacterial Medium                      10

2     Acidophilic Iron Microbial Cultures and Their Sources        11

3     Chemical Compositions of Several Mine Drainage Waters        21

4     Ferrous Iron Oxidation Rates at Varied Initial Fe++          42
        Concentrations and Incubation Temperatures in Shake
        Flasks

5     Air Oxidation of Ferrous Iron at Several Initial pH          44
        Values at 25 °C

6     Effect of Sodium Polyvinylsulfonate on the Bio-Oxidation     4?
        of Ferrous Iron in Shake Flasks

7     Oxidation of 9K Medium by Continuous Culture                 49

8     Oxidation of 1,000 mg/1 and 500 mg/1 Ferrous Iron by         51
        Continuous Culture

9     Effect of Aeration on the Microbial Oxidation of             52
        Ferrous Iron

10    Effect of Carbon Dioxide Content of Sparger Gas on           53
        Iron Oxidation

11    Multistage Continuous Oxidation of Ferrous Iron              54

12    Chemical Analysis of Mine Drainage Waters                    56

13    Continuous Feed Studies vith Natural Mine Waters             57

14    Effect of Non-Aeration on Ferrous Oxidation During           58
        Continuous Feed

15    Effect of Residual Ferrous Iron on Limestone                 60
        Neutralization

16    Neutralization and Settling Studies of Stage II              62
        Oxidized Liquor

17    Effect of Ferrous Feed Variation on Oxidation Rate           67

18    Effect of Residence Time on Ferrous Oxidation                68

19    Effect of Temperature on Ferrous Oxidation                   70
                                 Vlll

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                          TABLES  (COMTNUED)



No.                                                              Page



20    Effect of Feed Water on Oxidation Rate                       71



21    Pilot Plant and Bench Unit  Surface-to-Volume Ratios          72
                                  IX

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                               SECTION I

                              CONCLUSIONS

1.  Acidophilic  iron bacteria can be used  successfully to oxidize ferrous
iron  in acid mine drainage waters to prepare such waters for relatively
inexpensive limestone neutralization.
2.  At ferrous  iron  concentrations between 10 and 9^000 mg/lj the rate of
iron bio-oxidation is  significantly dependent on ferrous iron concentra-
tion.  High oxidation  rates occur at high iron concentrations and low rates
at low iron concentrations.

3«  Series multistaging of microbial oxidation vessels offers operational
efficiency over a single oxidation vessel.

4.  To achieve  a ferrous iron level of about 100 mg/1 the following oxida-
tion treatment  periods are required:

            Influent Fe++             Total Residence Time
                (mg/l)        Stages   _ (hours) _

                9,000           1              87
                9,000           2              18
                1,000           1               4
                  500           1               k

5.  Bacterial oxidation of ferrous iron is most rapid at 20 - 30°C with
decreased rates occuring at higher or lower temperatures.  An exception
is a species of Thiobacillus which oxidizes iron equally well at ^0°C-

6.  Aeration rates of  1/100 WM appear to be adequate for microbial oxidation
of mine water containing 500 mg/1 ferrous iron.

T.  Between 1 and 10 mg/1 ammonium ion is required to support microbial
oxidation of 800 mg/1  ferrous iron.  Nitrate or nitrite can serve as the
nitrogen source.

8.  Between 0.55 and 5-5 mg/1 of phosphate is needed to support microbial
oxidation of 9; 000 mg/1 ferrous iron.

9.  Significant scale-up problems were encountered in going from a 1 1/2-
gallon bench-size microbial oxidation system to a 1,000-gallon pilot plant
oxidation vessel.

10. Although sulf ate -reducing bacteria are present in acid mine drainage
water, they will not grow or produce H2S at pH values below 5-5-
                                   1

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                              SECTION II

                            BECOMMEKDATIONS

Microbial Oxidation of Ferrous Iron

1.  The problems encountered in scale-up from laboratory-size systems to
pilot plant units should be more thoroughly investigated.

2.  Other types of new waste treatment concepts, in particular those having
large surface contact areas, should be examined as microbial oxidation
systems for mine drainage water.

3-  Pilot plant microbial treatment studies of mine drainage waters should
include limestone neutralization and settling steps so that a complete
economic analysis of the process can be ascertained.

Removal of Iron with Mlcrobially Produced H2S

1.  Since sulfate-reducing bacteria will not grow at pH values 'below 5-5^
use of these organisms downstream of the neutralization step should be
investigated.

2.  A search should be made for a suitable, inexpensive carbon source
that will allow the rapid propagation of sulfate-reducing bacteria in
neutralized mine discharge waters.

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                              SECTION III

                             INTRODUCTION

The purpose of this study was to determine if the abilities of certain
bacteria to oxidize ferrous iron or to convert sulfate to hydrogen
sulf ide could be applied to the neutralization and subsequent removal of
iron from difficult- to- treat mine drainage waters.  If one or both of
these concepts could be successfully utilized, the expense of adequately
treating these types of problem waters might be significantly reduced.

In certain sections of the country mine drainage waters are one of the
principal contributors to water pollution.  These discharge waters are
characterized by a high dissolved iron content, and many are quite acidic.
The problem is particularly acute in the coal mining industry even though
modern mining practices have greatly reduced such discharges from
operating mines.  Recent estimates indicate that from 60 to 90 percent
of the total mine drainage water is emitted from worked out or abandoned
mines (1,2).  The quantity of mine drainage in the state of Pennsylvania
alone is 5 "to 10 billion gallons per day (3)-

The acidity and iron content of mine drainage waters originate from
pyrites present in the coal seams and surrounding strata.  In the presence
of water ( surface water and underground aquifers) and oxygen, pyrite is
oxidized to sulfuric acid and ferrous sulfate which are carried away by
the water.

                 FeS + H20 + 3.5 02 - >H2S04 + FeS04

Although some success in preventing this reaction from occurring can be
achieved by water diversion and maintenance of a reducing environment,
the complexities of mining and the abandoned mine problem make it seem
likely that treatment of acid mine drainage waters will remain a
significant problem for the foreseeable future.

Numerous concepts for removing the undesirable iron and acidity from
mine discharge waters have been investigated, and some are listed below:
          Ion Exchange
          Reverse Osmosis ( 5)
          Activated Carbon Catalysis ( 6)
          Foaming (Addition of a Surfactant) (?)
          Freezing (8)
          Addition of Hydrogen Sulf ide Gas (9)
          Production of Microbial Polymer Adsorbents (10)
          Neutralization Followed by Aeration ( ll)

Except for special circumstances, poor efficiencies or adverse economics
appear to rule out the present use of most of these techniques.

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The most widely used treatment for acid mine water at the present time
is hydrated lime neutralization.  This process typically Involves the
addition of lime followed by vigorous aeration to oxidize the ferrpus
hydroxide to the ferric state.  The ferric hydroxide is then allowed to
settle in a series of holding ponds.  The precipitate thus formed has a
very slow settling rate and a low compaction factor.  Not only are large
ponds required to provide -ample settling time but also are necessary to
accommodate the voluminous amount of iron sludge produced.  Although the
final effluent from such a system is acceptable for surface stream
discharge, the problem of sludge recovery, dewatering and disposal is a
formidable one (12).

While much less expensive limestone can be successfully used to treat
acid discharge waters containing ferric iron or waters having low concen
trations of ferrous iron, the more prevalent acid mine discharges that
contain relatively high concentrations of ferrous iron are not directly
amenable to limestone treatment.

When limestone (calcium carbonate) is added to water bearing an acidic
pH, the following reactions occur:
Carbon dioxide is released and acidity is neutralized.  While these
reactions proceed readily at acidic pH values, calcium carbonate neutral-
ization cannot efficiently take the pH to much above 6.

Ferric hydroxide is formed by ferric iron salts in the presence of water.

                   Fe+++ + 3 HgOv    NFe(OH)3 + 3 H+

Ferric hydroxide becomes water insoluble at about pH 5-5; thus limestone
neutralization of acid mine water bearing ferric iron will result in the
precipitation of ferric hydroxide.  Ferric hydroxide precipitates rapidly
and produces a relatively small amount of sludge which has a relatively
high solids content.

Ferrous salts in water also hydrolyze.

                   Fe^* + 2 H20^=^Fe(OH)2 + 2 E+

However, ferrous hydroxide is soluble in water up to pH 9-10.  Since
calcium carbonate cannot elevate the pH to this region, limestone
neutralization will not allow the precipitation of ferrous hydroxide (13).

Thus,  in order to realize the obvious economic benefits from using lime-
stone  instead of hydrated lime to neutralize acid ferrous mine drainage

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waters (limestone costs about one third as much as hydrated lime),  it  is
necessary that the iron present he converted to the ferric state  prior
to neutralization.  Even in the presence of oxygen, this oxidation
process is extremely slow at low pH values.  For example, at pH 3-5 the
half time of this reaction in water saturated with oxygen is 2,000 days

The existence of certain bacteria, commonly known as the acidophilic
iron bacteria, that specialize in the oxidation of ferrous to ferric
iron at low pH values has been known for many years (15)•  In fact,
these microorganisms have been isolated from many mine discharge  waters.
Acidophilic iron bacteria are autotrophic (do not require organic sub-
stances for growth) and are classified as the Thiobacillus-Ferrobacillus
group.  These bacteria utilize carbon dioxide as their source of  carbon
and oxidize ferrous iron to the ferric state in order to obtain energy
to drive their cell machinery.  In addition to_ carbon dioxide, oxygen  and
ferrous iron, these organisms also require lesser quantities of nitrogen
and phosphorus and trace amounts of other minerals.  It has not been
demonstrated, however, that this microbial catalytic activity can be
economically utilized to satisfactorily prepare acid ferrous mine drainage
water for limestone treatment.

While the use of hydrogen sulfide gas has been examined as a technique
for the precipitation and removal of iron in mine drainage water  as iron
sulfide, the value of commercial hydrogen sulfide has made this concept
relatively unattractive (9)-  However, mine discharge waters are  not
lacking in sulfate and certain bacteria, in particular Desulfovibrio
desulfuricans, are capable of prolific hydrogen sulfide production from
sulfate.  If this process could be shown to operate in mine drainage
waters, using a cheap carbon source to satisfy the carbon requirements
of the organisms, an inexpensive technique for iron removal from  these
discharges would result.
                                    7

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                              SECTION  IV

                                METHODS


                      MICROBIAL OXIDATION OF IRON

                         Iron Bacterial Cultures

Acidophilic  autotrophic  iron bacteria  were  cultured from  six mine drainage
water  outfalls  in  southwestern Pennsylvania and northwestern West Virginia.
Waters were  selected  that  differed  in  chemical composition in an attempt
to  cover the "broad spectrum of water types  associated with mine drainage.
Culture  of the  iron bacteria from these sources was initiated on location
by  inoculating  10  ml  samples of the mine waters into 90 ml volumes of
sterile  9K medium  (Table l) in 4-ounce screw cap bottles.  Following
transport to the laboratory and incubation  at room temperature, 10 ml
aliquots from the  bottles  were used to inoculate 90 ml volumes of sterile
9K  medium in 250 ml erlenmeyer flasks.  These flasks were incubated at
25 °C on  a gyrotory shaker  operating at 200  rpm and culture ability to
oxidize  ferrous to ferric  iron verified.  Acidophilic autotrophic iron
bacterial cultures were  obtained in this manner from all  of the mine
drainage outfalls  samples  (see top  section  of Table 2).  Viable stock
cultures were maintained throughout the study by transferring every four
days into fresh 9K medium.

In  addition  to  these  field cultures of acidophilic iron bacteria, pure
cultures of  Thiobacillus ferrooxidans, Ferrobacillus ferrooxidans and
Ferrobacillus sulfooxidans were obtained through the courtesies of Drs.
J.  V.  Beck,  D.  G.  Lundgren, W- W. Leathen and P. R. Dugan.  These cultures
were maintained in the same manner as  described for the field cultures
and are  also listed in Table 2.

                             Iron Analysis

Ferrous  iron and total iron analyses were performed using the
p-diphenylamine sulfonic acid - dichromate  titration technique (l6).

                       Media and Culture Methods

The basic synthetic medium used throughout  the study was  the 9K medium
of  Silverman and Lundgren  (l?)-  The synthetic medium was used for most
of  the investigations to provide reproducibility and to eliminate the
problems of  transportation and instability  associated with natural mine
drainage  waters.  For studies that required the utilization of mine
discharge waters,  the waters were collected and maintained at ice bath
temperature  until used.

All of the bacterial growth and oxidation studies were carried out in
250 ml or 2,000 ml  erlenmeyer flasks containing 100 ml or 1,000 ml of

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                       TABLE 1




        COMPOSITION OF 9K IRON BACTERIAL MEDIUM







(NH4)2S04                                      3 g




KC1                                            0.1 g




K2HP04                                         0.5 g




MgS04 • T H20                                  0.5 g




Ca(N03)e                                       0.01 g




H2S04(lO l)                                    1 ml




FeS04 « 7 H20 (lij-.?^ wt/vol solution)         300 ml









Distilled Water                                TOO ml









Final pH                                       2.0-2-5
                         10

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                                TABLE 2

        ACIDOPHILIC IRON MICROBIAL CULTURES AND THEIR SOURCES
     IRON BACTERIAL CULTURES

FIELD CULTURES
   Southwest-1
   Southwest-2
   Arden-1
   Arden-2
   Marcliand-1
   Marchand-2
   Indian Creek
   Main ¥est
   Lynch Shaft

PURE CULTURES
   Thiobacillus ferrooxidans
      (Beck)
   Ferrobacillus ferrooxidans
      (Syracuse)
   Ferrobacillus ferrooxidans
      (Leathen)
   Ferrobacillus sulfooxidans
      (Leathen)
   Ferrobacillus ferrooxidans
      (Dugan)
              SOURCES
Southwest Mine Drainage Outfall*
Southwest Mine Drainage Outfall*
Arden Mine Drainage Outfall*
Arden Mine Drainage Outfall*
Mar.chand Mine Drainage Outfall*
Marchand Mine Drainage Outfall*
Indian Creek Mine Drainage Outfall*
Main ¥est Mine Drainage Outfall*
Lynch Shaft Mine Drainage Outfall*
J. V. Beck, Brigham Young University

D. G. Lundgren, Syracuse University

¥• ¥. Leathen, Mellon Institute

¥. ¥. Leathen, Mellon Institute

P. R. Dugan, Ohio State University
          * Mine outfalls located in southwestern Pennsylvania and
            northwestern ¥est Virginia
SULFATE-REDUCING BACTERIAL CULTURES

   Southwest*
   Arden*
   Indian Creek*
   •Core*
   Brewer*
   Marchand
   Main ¥est
   Lynch Shaft
   Fetty

   Mid-Continent Strain*

          * Test cultures
              SOURCES

        Mine Drainage ¥ater
        Mine Drainage ¥ater
        Mine Drainage ¥ater
        Mine Drainage ¥ater
        Mine Drainage ¥ater
        Mine Drainage ¥ater
        Mine Drainage ¥ater
        Mine Drainage ¥ater
        Mine Drainage ¥ater

        API Stock Culture
                                  11

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medium or in a continuous culture system that will be described in a
subsequent section of this report.  Culture flasks vere agitated on a
gyrotory shaker operating at 200 rpm at a controlled temperature.

Inoculum for flasks or the continuous culture system consisted of a
lj-8-72 hour old shake flask culture either used directly from the flask
or as a washed cell suspension.  Cell suspensions were prepared by
centrifuging the cells from the culture medium, washing two times with
pH 2-5 water (distilled water acidified with sulfuric acid) and resus-
pending the cells in pH 2-5 water.  The suspension was used immediately
on a 10 percent basis; i.e., cells collected from a 100 ml growth flask
were used as inoculum for 1,000 ml of fresh medium.

                            Cell Population

Direct microscopic counts using a Petroff-Hausser counting chamber were
found to be exceedingly difficult to perform because of the presence of
inanimate solids (primarily precipitated iron) in the preparations which
obscured many of the very small cells.  These solids also interferred with
the direct microscopic counting technique when the organisms were collected
on a membrane filter.

The use of solid 9K medium agar plates for dilution counting was also
found to be unacceptable.  The principal problem encountered with this
method was the very long incubation period required for colonial
development.

It was found that serial dilution in tubes of 9K medium could be success-
fully used to determine the approximate iron bacterial population of a
given system.  Regardless of whether the tubes were agitated or left
undisturbed, high dilutions required seven to eight days of incubation
before they could even be read microscopically.  Upon further incubation,
growth was evidenced by the precipitation of iron from solution in the
tubes and development of turbidity.  The reciprocal of the highest
dilution tube exhibiting these characteristics was taken to represent
the number of organisms in the original sample.  Although this technique
required more than a desirable length of time to determine populations,
it was selected for use in studies requiring iron bacterial counts.

                               Cell Mass

A Hewlett-Packard FM Model 185 CHEF analyzer was used to quantitate the
amount of carbon present in known volumes of iron bacterial culture.
Since 9K medium is inorganic, the carbon present in the very acid culture
should represent cellular carbon.

Samples were withdrawn at intervals from growing shake flask cultures and
subjected to one of two preparatory procedures for carbon analyses.  One
technique consisted of collecting the solids present in a 10 ml sample on
a tared 0.^5 micron membrane filter which was then dried in a vacuum at
65°C to a constant weight.  Following careful recovery of the solids
                                  12

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present on the filter, they were subjected to CHN analysis.  The other
method entailed centrifugal sedimentation of the solids present in a
10 ml sample which were then recovered, dried, weighed and analyzed for
carbon content.

The data shown in Figures 1 and 2 illustrate the utility of this concept
in following iron bacterial cell mass by organic carbon content of the
medium.  Although cell mass determinations were not carried out in ensuing
studies, it was felt that the- success of the technique should be reported.

An attempt to directly measure the organic content of growing culture
samples was made utilizing a total organic carbon analyzer.  Serious
positive interferences by medium constituents were encountered, and no
attempts were made to identify or circumvent the interferences.

                       Continuous Culture System

For continuous culture studies a continuous feed system was constructed
using a FABCO-BUSH apparatus (Houston Glass Fabricating Company, Houston,
Texas) as the central component.  A schematic of the apparatus is shown
in Figure 3-

This system was used to more thoroughly investigate the nutritional and
environmental factors found to be significant in shake flask studies as
influencing microbial oxidation of ferrous iron.  Substrate (9K medium
or field water) was stored in a reservoir from which it was pumped to the
first oxidation stage.  Average retention time in this vessel was varied
by changing the feed pump rate.  The first stage contained a working volume
of six liters and was aerated via three fritted glass dispersion tubes
located at the bottom of the vessel.  The inner cone and center tube were
designed primarily to permit control over the solids content of the vessel
contents; but since the iron bacteria being used tend to exist as single
discrete cells rather than clumps, these system components served only to
insure complete mixing of the vessel contents.  The second oxidation stage
was aerated via a single, coarse-grade gas dispersion tube; and mixing was
insured by use of a magnetic stirrer insulated to prevent significant heat
transfer-  A five-gallon carboy served to collect the final effluent.
Reduced pressure on this vessel was used to draw liquid from the first
stage into the second stage and then into the effluent collector.

                       Sulfate-Reducing Bacteria

Wine different mine drainage water outfalls in Pennsylvania and West
Virginia were sampled and examined for the presence of sulfate-reducing
bacteria.  D. desulfuricans (identification based on morphology and
hydrogen sulfide production) was found in all nine waters.

Isolation of I), desulfuricans was carried out in the following manner.
Sterile 4-ounce screw cap bottles were completely filled with mine
drainage water at the outfall  site  and tightly capped.  One ml of the

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  140 i—
  130 -
             O  SYRACUSE CULTURE
             9  BECK CULTURE
              8
12
36
Figure
                16   20    24   28   32
                   HOURS  INCUBATION
CARBON CONTENT OF  GROWING  IRON BACTERIAL
CULTURES
40

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    8000
    7000
  T 6000
  o>
    5OOO
  V)

  O 4000
  cc
  a:
  ui
    3000
    2000
    1000
                    X	X
                                     FERROUS IRON
                           CARBON
                          I
             24    48     72    , 96

                       HOUR INCUBATION
120
144
                                                   10
             20
               o
               UJ
                                                   30
                                                   40
               O
               CD
               K
               5
               u.
               o
             50
               en

             60 o
               o:
               o
             70




             80



             90

             95
Figure 2  CARBON PRODUCTION AND FERROUS IRON

          OXIDATION BY SOUTHWEST-1 CULTURE

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  MEDIUM
  RESERVOIR
  20 LITERS
     AIR
                   PUMP
        FIRST STAGE VESSEL

           6 LITERS
                                                        ASPIRATOR
SECOND STAGE VESSEL

     4  LITERS
 WASTE
20 LITERS
Figure 3   SCHEMATIC OF CONTINUOUS  FEED BIOOXIDATION SYSTEM
                               16

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water was added to a screw cap culture tube containing 10 ml of sterile
API sulfate agar (Difco) at ^-5°C.  The tube was inverted three times to
distribute the sample into the medium and then allowed to solidify at
room temperature.  Replicate cultures of each of the waters were incu-
bated at 35°C.  After characteristic black (due to the production of
iron sulfide) colonies had developed within the agar, the tubes were
entered and a sterile loop was used to transfer isolates to tubes of
sterile API sulfate broth containing 0.1 percent agar.  After these
tubes blackened, one ml of culture was withdrawn and used to inoculate
a new tube of the 'same medium.  Stock cultures were maintained in this
manner until needed.
                                   17

-------
                               SECTIOI V

                             EXPERIMENTAL

Two-day old aliquots of three iron bacterial field cultures (Marchand,
Arden and Southwest-l) and pure cultures of Ferrobacillus (Syracuse)
and Thiobacillus (Beck) were used to inoculate one-liter quantities of
sterile 9K medium  in two-liter erlenmeyer flasks.  The flasks were then
incubated at 200 rpm at 25 °C on a gyrotory shaker.  Although all the
cultures had been  transferred in 9K medium at weekly intervals for s,ix
weeks, the two pure cultures displayed shorter lag periods and more
rapid rates of iron oxidation than did the three field cultures (Figure 4).
In all cases the cell populations reached densities of 10T to 108 bacteria
per ml within 24 hours (determined by tube serial dilution).  Additional
oxygen supplied to the cultures by air sparging or by increased shaker
speed did not detectably increase growth or iron oxidation rates.
Excessive aeration was found to significantly retard rates of growth and
iron oxidation.

                       Nutritional Requirements

In order to achieve maximal iron bacterial activity in mine discharge
water, it is obviously necessary that adequate concentrations of required
nutrients be present.  Oxygen, carbon dioxide, nitrogen, phosphorus,
ferrous iron and sulfate are nutrients that must be present in significant
amounts.  Chemical compositions of several acid mine drainage waters are
shown in Table J.

Nitrogen

Ammonium sulfate,  sodium nitrate and sodium nitrite were quantitatively
evaluated as nitrogen sources for the Southwest-l culture.

Media containing 0, 1.0, 10 and 100 mg/1 ammonium ion as the sole known
source of available nitrogen were prepared by adding appropriate amounts
of reagent grade (NH4)2S04 to modified 9K medium (modified by leaving
out the amount of  (NH4T2S04 normally present).  Normal 9K medium, con-
taining 819 mg/1 ammonium ion, served as a control along with modified
medium to which no ammonium salt was added.  Following sterilization,
the media were inoculated with equal quantities of washed Southwest-l
culture recovered  from ^00 ml of a 48-hour old culture grown in 9K
medium.  Incubation was at 25 °C in 2-liter flasks on a gyrotory shaker.
Resultant iron oxidation profiles are shown in Figure 5-  In order to
examine the possibility that available nitrogen was introduced in the
test systems by inoculum carry-over in spite of the cell washings, two
successive subcultures were made from these flasks into fresh sets of
media and rates of iron oxidation followed.  No iron oxidation or growth
occurred in the subculture flask which contained no added (NH4)2S04
while iron oxidation and cell growth were severely limited in both
subcultures containing only 1.0 mg/1 ammonium ion.  Assuming that the
other reagent grade constituents of 9K medium did not supply available
                                  19

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                                          LEGEND
                                         O MARCHAND CULTURE
                                         X ARDEN CULTURE
                                         O SOUTHWEST-1 CULTURE
                                         A SYRACUSE CULTURE
                                         • BECK CULTURE
                                         • UNINOCULATED CONTROL
         48  12  16  20  24 28  32  36  40 44  48  52  56
                        HOURS INCUBATION

Figure 4   Fe*OXIDATION  RATES IN  9K  MEDIUM
          SHAKE FLASKS

                        10 % INOCULUM
                        20

-------
                               TABLE 3

        CHEMICAL COMPOSITIONS OF SEVERAL MIME DRAINAGE WATERS
                                   Mine Drainage Water -
                              Component Concentration in mg/1
pH

H2S
Cl
S04
C03
EC03
Na
Mg
Ca
Ba
Iron (Total)
     (Dissolved)
Al
Mn
P
N (Total)
  (NH4)

Total Salts
Suspended Solids
Specific Gravity
Rw-73°F Ohm Meters
Core
3-1
0
532
9400
0
0
3360
294
4i6
0
630
540
99
11


5
4-5
i46oo
78
1.013
0.778
Bolby
3-
0
241
3000
0
0
962
93
344
0
550
4oo
50
7
_ y» f"^-(-
HOT
5
5
4920
> 150
l.
2.
Mill Fetty
6 '5.2
0
319
6200
0
34
2290
93
440
0
430
268
0
4


6
5
9810
> 150
005 1.009
49 1.06
Arden
6.2
0
142
1250
0
198
441
59
184
0
^9
0
0
3


2
1-5
2320
> 150
1.002
3.63
                                 21

-------
   8000
   7OOO
~  60OO
  .
 £
 — 50OO
 2
 O
 a:

 o
 a:
 a:
 LU
   4000
   30OO
   2000
   1000
     0
 mg/liter AMMONIUM ION


X 819 (UN INOCULATED CONTROL)

D 819 ( 9K)

•   0

•   I

O   10

A 100
             I   I   I   I    I   I   I   I   I
      0   4   8   12  16  20  24  28 32 36 4O  44  48  52  56
                      HOURS INCUBATION

Figure 5  Fe* OXIDATION AT VARIOUS  AMMONIUM  ION

          CONCENTRATIONS

       SOUTHWEST-1 CULTURE -10% WASHED CELL INOCULUM
                       22

-------
nitrogen to the organisms, it appears that one to ten mg/1 of M4 ion
is sufficient to support growth of this iron bacterial culture and
accompanying ferrous iron oxidation in 9K medium.

Qualitative studies were carried out to examine the ability of the
Southwest-1 culture to utilize nitrate or nitrite as a nitrogen source.
Nitrate was examined at 100 and 1,000 mg/1 concentrations, and nitrite
was tested at 100 mg/1.  When 1,000 mg/1 of nitrite was added to 9K
medium minus ammonium sulfate, a voluminous precipitate occurred;
therefore, this concentration of nitrite was not examined.  Iron oxidation
at the nitrate and nitrite levels tested are shown in Figure 6.  Sub-
sequent subculture responses were identical.  While these nitrogen sources
can be utilized by the test organism, the ammonium salt is more
readily used (compare Figures 5 and 6).

Phosphorus

Since most microorganisms utilize phosphate ion as a source of phosphorus,
phosphorus requirements were studied using a phosphate salt, dibasic
potassium phosphate (K2HP04).  Both the Southwest-1 culture and a pure
culture of T. ferrooxidans (Beck) were used as subjects for the phosphate
requirement investigation.  The base medium utilized was 9K medium modi-
fied by omission of the phosphate constituent, K2HP04.  K^KP04 was then
added to this altered 9K medium to produce media containing 0.55; 5-5
and 55 nig/1 phosphate.  Standard 9K medium containing 275 mg/1 phosphate
ion and the modified 9K base containing no added phosphate served as
controls.  Following inoculation with washed cells grown on 9K medium,
the test flasks were incubated at room temperature on a gyrotory shaker.
Subsequent iron oxidation patterns are shown in Figures 7 and 8.  Results
of successive subcultures carried out with the pure culture are shown  in
Figure 9«  Although not shown in Figure 9, no ferrous iron oxidation or
iron bacterial growth occurred in subcultures containing no added phosphate.
Assuming no phosphate present in other medium constituents, the data
reveals that phosphate becomes rate limiting for iron bacteria in 9K
medium at a concentration between 0.55 and 5-5 mg/l (the Southwest-1
culture was not tested at 0.55 mg/1 phosphate).  Oxidation activity in
the flask containing no added phosphate was apparently due to carry-over
in spite of careful cell washing.

Ferrous Iron

The effect of initial ferrous iron concentration on rate of ferrous iron
oxidation in batch culture was examined at concentrations of 10, 100,
500, 1,000 and 9,QQQ mg/1 of ferrous iron.  At the first four concentrations,
oxidations were followed for only about six hours on the assumption that
the microbial population would not significantly increase in this short
period of time after inoculation.  A more complete picture of the 9,000
mg/1 system was obtained.  Cultures used for this study were the Southwest-1,
F. ferrooxidans (Syracuse) and T. ferrooxidans (Beck).  Incubation was on
a gyrotory shaker at 25°C.  Equal volumes of ^8-hour old cultures were
utilized as inoculum in attempting to produce approximately equal initial

-------
  9000
  8000 —
  700O —
  6000 —
  5000 —
o
i
UJ
                                           A	
              o   I 00 mg/liter N03

              D  1000 mg/liter NO,

              •   100 mg/liter
              A  UNINOCULATED CONTROL
  4000 —
   3000 —
  2000 —
   1000 —
Figure 6
            12  18
                  24 30  36  42 48 54
                    HOURS INCUBATION
60 66 72 78
           Fe* OX I DAT ION USING NOe OR NOjAS
           NITROGEN  SOURCE IN 9K MEDIUM
           SHAKE FLASKS.
          SOUTHWEST-1 CULTURE -10% WASHED
          CELLS INOCULUM

-------
     8000
     7000
     60OO
  z  5OOO
  (C
  CO
  ce  4000
  CC
     3000
     200O
     1000
mg/liter PHOSPHATE ION
•  275
•   55
n    5.5
a    0
X  275 (UNINOCULATED
       CONTROL)
            I   I   I    I   I   I    I
                             I   I   I    I   I
        0   10  20  30 40 50  6O  70 8O  90  IOO 110 120 130 140
                          HOURS INCUBATION
Figure  7 Fe*OXIDATION AT VARIOUS POf CONCENTRATIONS
         SOUTHWEST-1 CULTURE-10% WASHED CELL INOCULUM

-------
   8000
  700O
  6000
  5000
o>
E
o

- 4000


§
o:
o:
lil

"• 3000
   20OO
   1000
      0
          mg/liter PHOSPHATE ION
n

x

6

A
275  (UN INOCULATED

    CONTROL)


275


 55

  5.5

  0.55


  0
(INITIAL EXPOSURE-WASHED

 CELL INOCULUM)
                                I
  12
      24     36     48

      HOURS INCUBATION
60
  Figure 8  EFFECT OF PHOSPHATE CONCENTRATION

           ON FERROUS IRON OXIDATION BY

           THIOBACILLUS FERROOXIDANS (BECK)
                   26

-------
                            X
                            A
                            O
 mq/liter P04
-  55.0
    5. 5
    0.55
             SUBCULTURE- I
 o>
 E
 O
 cc
        O   12  2*  36  48  60
          HOURS INCUBATION
O
cc
         12   24  36  48  60  72  84  96
             HOURS  INCUBATION
                                               SUBCULTURE-4
            12  24  36  48  60  72
             HOURS INCUBATION
           12  24  36 48  60  72  84
              HOURS  INCUBATION
           INOCULATED WITH 10% V/V  OF SPENT 9K MEDIUM FROM PRIMARY OXIDATION FLASK
           INOCULATED WITH 10 % V/V  OF SPENT MEDIUM FROM PREVIOUS SUBCULTURE
Figure 9 EFFECT OF PHOSPHATE CONCENTRATION ON  FERROUS
          IRON OXIDATION BY JJ_FERROOXIDANS_(BECK)- SUB-
          CULTURES
                               27

-------
concentrations of cells in each flask.  The oxidation of ferrous iron
during the first six hours of incubation for all five concentrations of
iron are shown in Figure 10 as a function of time and formation of ferric
iron.  Due to the similar behaviors of the three cultures under these
conditions, their values were averaged to produce the functions shown in
Figure 10.

Iron oxidation patterns for the three cultures in the 9,000 mg/1 ferrous
iron system are shown in Figure 11.  These data were noted to be very
similar to that for a similar system reported by Lacey and Lawson (iS).
This comparison is shown in Figure 12.

                      Other Environmental Factors

Temperature

The effect of temperature on ferrous iron oxidation was examined for
four acidophilic iron bacterial cultures:  Southwest-1, F. ferrooxidans
(Syracuse), F. ferrooxidans (Dugan) and T. ferrooxidans iBeck) .  Tempera-
tures studied were 10°C, 20°C, 25°C, 30°C, 35°C and 40°C.  In each case
the temperature was maintained at ± 0.5"C.  Cultures were temperature
acclimated by three successive transfers at each of the selected tempera-
tures prior to inoculation.  The test medium consisted of 1-liter volumes
of 9K medium modified to contain approximately 1,000 mg/1 of ferrous
iron.  The inocula were adjusted so that all test flasks contained 106
bacterial cells per ml at the time of inoculation.  Incubation at the
various temperatures was carried out using a New Brunswick PsychroTherm
gyrotory shaking apparatus.  Ferrous iron oxidation patterns and popula-
tion developments at the selected temperatures by the four cultures are
shown in Figures 13, 1^, 15 and l6.  Surprisingly, temperatures from 20°C
to JO°C did not appear to significantly influence iron oxidation patterns
exhibited by any of the cultures, and only in the case of the Southwest-1
culture did 35 °C effect iron oxidation (in the latter instance oxidation
was impaired).  The T. ferrooxidans cultures (Figure 1^) were unaffected
by ^0°C, but the F. ferrooxidans culture examined also at 40°C was quite
adversely affected by this temperature (Figure 15)•  None of the cultures
functioned well at. 10°C.

Profiles of batch microbial ferrous iron oxidation at 10°C, 20°C and 35°C
were then examined as a function of initial ferrous iron concentration.
Ferrous iron levels selected for study were 10, 100, 500 and 1,000 mg/1.
Results of these investigations are shown in Figures 17 through 22 and
in Table U.  As may be seen, the same effect of initial ferrous iron on
rate of ferrous iron oxidation that was observed at 25°C was found to
hold at higher and lower temperatures.  The pure culture of T. ferrooxidans
(Beck)  was more effective than the field culture at 25°C and 35°C while
the field culture was slightly more effective at 10°C.
                                  28

-------
 500r—
                                            INITIAL Fe++
                                            CONCENTRATION
                                             (mg / I iter)
                                                     9OOO
                                                     1000
                                                     500
                                                     100
                         TIME - HOURS

Figure 10  EFFECT OF  INITIAL FERROUS IRON  CONCENTRATION
          ON EARLY IRON OXIDATION IN  BATCH CULTURE
                       29

-------
                                    CULTURE
   10,000
   5,000 -
 o>
 6
 z
 O
 CO
 3
 O
 cc
 ir
 UJ
 u.
    1,000 —
     500 —
     100
                  12
                                  • T. FERROOXIDANS ( BECK )

                                  O F  FERROOXIDANS (SYRACUSE)

                                  A SOUTHWEST-I
24         36

   TIME- HOURS
48
60
Figure  II  FERROUS IRON OXIDATION AT AN INITIAL  IRON

          CONCENTRATION OF  9,000  mg / liter

-------
  4OOO»—
  1,000
   500
o>
E
Z
o
or
o
cr.
cc
   100
   50
   20
                               4000
            FROM  Figure
                          FROM LACEY 8.LAWSON ( 18)
               1,000 —
                500 —
              z
              o
              

              o
              cc
              <£
              Ul
              U-
           I
1
I
     44    46    48   50

           TIME , HOURS
          52
                  6     8    10

                    TIME , HOURS
12
14
Figure  12   IRON  OXIDATION  AT AN INITIAL  FERROUS  IRON

           CONCENTRATION  OF ABOUT 10,000 mg / liter

-------
                                     POPULATION
  8000
          12
              24
36   48   60   72   84
   HOURS INCUBATION
96
108
Figure 13  EFFECT OF TEMPERATURE ON FERROUS IRON
          OX I DATION BY SOUTHWEST- I CULTURE
120

-------
                                       POPULATION
    800O,
            16  24
          36
 48   60  72   84
HOURS INCUBATION
96
108  120
Figure 14
EFFECT OF TEMPERATURE ON FERROUS IRON
OXIDATION BY THIOBACI LLUS FERROOXIDANS
(BECK)
                        33

-------
                                      POPULATION
          12
     24
36
 48   60   72   84
HOURS INCUBATION
                                       96
108
120
Figure 15
EFFECT OF TEMPERATURE ON FERROUS IRON
OXIDATION BY FERROBACILLUS
FERROOX I DANS  I SYRACUSE )

-------
                               POPULATION
                             48 HOURS MAXIMUM
                2O°C
                25 °C
                30°C
                35°C
              I07
              I09
              I09
              I07
                                     10
                                       8
                                     I09
                                     I09
                                     I08
     0
12
36   48
 HOURS
  60   72
NCUBATION
Figure 16  EFFECT OF TEMPERATURE  ON FERROUS
         IRON OXIDATION BY FERROBACILLUS
         FERROOXIDANS  (DUGAN)

-------
                  X = AUTOOXIDATION
 lOOOg 	
r eoo
o>

^ 600
i
en
§400
OL
OC.
£
  200
    -\
                                            100
                                    rso
                                    0>
                                           £60
                                           z
                                           o
I   I   I  I   I   I
                          1   1   1   1   1  \°~ 1
       4  8  12  16  20 24 28 32  36 4O 44 48 52  56

                              HOURS INCUBATION
                                                 i   i   i   i
                                                 3  6  9 12 15  18 21
 500

                                           ]
          8  12 16  20  24 28 32 36 4O 44 48 52 56
                              HOURS  INCUBATION

Figure  17  FERROUS IRON OXIDATION  AT VARIED INITIAL  Fe
          CONCENTRATIONS  BY BECK CULTURE-SHAKE FLASKS  IO°C

-------
                   X = AUTOOXIDATION
   1000
    800
 3 600 -
  O
  cc
  o
    400 -
    2OO -
                 8  10 12  14  16 18 20 22 24


                             HOURS  INCUBATION
   2468

400
k_
0>
™
\ 300
o>

z 200
o
0)
3 IfV^
O IUO
u.
o
X
-\
o
: \
V
w- O
k
\
\
_ X
\
i i i i i 1^1
         2  4  6  8  10 12  14
246
                            HOURS INCUBATION
                                                          •H-
Figure  18  FERROUS IRON  OXIDATION  AT VARIED INITIAL Fe

           CONCENTRATIONS BY BECK CULTURE-SHAKE FLASKS 20°C

-------
                              X = AUTO-OXIDATION
                                         5OO&-            '
          24   6  8  10  12 14
           HOURS INCUBATION
    2  4  6  8  10
  HOURS  INCUBATION
                                                   —X
          246
      HRS. INCUBATION
    246
HRS. INCUBATION
Figure 19  FERROUS IRON OXIDATION  AT VARIED INITIAL Fe+*
          CONCENTRATIONS BY BECK CULTURE-SHAKE FLASKS 35°C.
                             38

-------
                           X = AUTOOXIDATION
                                                         —X
             8  12
                                             100
             16 20 24 28  32 36 40 44 48

                      HOURS  INCUBATION
                                                     8  12  16
     500
   ~ 400
   o>
   O
   IT
   CO

   O
   ac
   IE
   IU
     300
20O
100
     I   J   I   I  I   I   I
O PRIMARY OXIDATION
D SUBCULTURE-I

A SUBCULTURE-2
                                 I  I   I   I
        04  8  12162024283236404448

                            HOURS INCUBATION
                                                0  4  8  12
Figure 20   FERROUS  IRON OXIDATION AT  VARIED  INITIAL  Fe
           CONCENTRATIONS  BY SOUTHWEST-I  CULTURE -
                       SHAKE FLASKS  IO°C
                              39

-------
                           	. x500X	
    0
        2  46  8 10 12 14  16 18  20
           HOURS INCUBATION
2 4  6  8  10 12  14  16  18 20
      HOURS INCUBATlpN
                            X = AUTO-OXIDATION
                 	X
        100
              246  8  10
            HRS.  INCUBATION
      246
   HRS. INCUBATION
Figure 21  FERROUS  IRON OXIDATION AT VARIED INITIAL Fe+4
          CONCENTRATIONS BY  SOUTH WEST-I  CULTURE -
          SHAKE FLASKS  20 °C

-------
                                     J
          2  4  6 8  IO  12  14 16  18 20 22
              HOURS INCUBATION
                                              100
                                                         	X
                                            0)
                                            i  80
                                            x.
                                            en
                                            E
                                            —  SO
                                            o
                                            
-------
Beck Culture
                               TABLE k


         FERROUS IRON OXIDATION RATES AT VARIED INITIAL Fe++
     CONCENTRATIONS AND INCUBATION TEMPERATURES IN SHAKE FLASKS
                Average Fe++ Oxidation Rate
             	mg/l/hr	
 Initial
mg/1 Fe++    10°C  20°C  25"C  35"C  Controls

    10        1.5   3.it   2.2   3.it      0

   100        9.3  13.4  13.0  18.8      o

   500       11.0  35.5  32.7  53-3      o

  1000       17.0  1+0.5  4o.it  80.it      o


    10        1.7   4.5   1.3   k.i      o

   100        9.3  12.8   8.0  12.it      0

   500       16.0  2it.8  32.7  2k. 0      0

  1000       20.6  46.1  4o.it  47.2      o
Southwest-1 Culture
                                 it2

-------
Initial pH

It has been established in the literature that acidophilic iron bacteria
grow and oxidize ferrous iron poorly at pH values above 4  (l9)«
However, air oxidation of ferrous iron occurs more rapidly as the pH is
raised from 4, and acidity is produced when ferrous iron is oxidized by
air or by microbial catalysis.  Thus, if some oxidation of ferrous iron
takes place, regardless of the mechanism, the pH of the solution decreases
unless highly buffered.  In view of these factors, it seemed reasonable
to assume that microbial oxidation of ferrous iron might take place
reasonably well even in systems having an initial or influent pH of 4 or
above.

Using three different iron bacterial cultures, Southwest-1, T. ferro-
oxidans (Beck) and F. ferrooxidans (Syracuse), the effect of initial pH
on microbial oxidation of ferrous iron was examined.  The studies were
carried out batchwise in shake flasks at 25 °C using 9K medium modified
to contain about ^00 mg/1 ferrous iron.  The cultures were used to inocu-
late sets of flasks in which the medium had been adjusted to pH 3, 4, 5
or 6 with potassium hydroxide.  Iron oxidation rates were monitored.
Uninoculated flasks at the four different pH values were used to deter-
mine the extent of auto-oxidation under these conditions as well as
accompanying pH changes.  Results are presented in Table 5 and in
Figure 23.

As may be seen, no ferrous iron oxidation or pH shift took place at
pH 3 in "the absence of microbial growth.  Rapid ferrous iron oxidation
with no lag period was displayed at pH 3 by all three cultures.  There
was a small amount of ferrous iron oxidation by air at pH k with a
concomitant decrease in pH of about 0.5 unit.  The T. ferrooxidans
culture appeared to be inhibited until this pH shift occurred and then
displayed a rather long lag period before rapidly oxidizing the ferrous
iron present.  The other two cultures appeared to be essentially uneffected
by pH 4.

At pH 5 there was a significant degree of ferrous iron oxidation by air,
and it occurred in the first few hours of incubation.  While all three
cultures appeared to be inactive until the pH was dropped by air oxida-
tion of the iron, only the T. ferrooxidans culture displayed a relatively
long lag period once the pH had dropped.

As might be expected, more air oxidation of iron was found at pH 6; and
a more pronounced effect on microbial oxidation of iron was noted at
this pH.  All of the cultures displayed a significant lag period at this
pH with again the Thiobacillus culture being effected most severely.

Folyelectrolyte Catalyst

The polyelectrolyte, sodium polyvinylsulfpnate, has been reported to be
a very effective catalyst for some oxidation reactions at a concentration
of 1 gram of polyelectrolyte per liter (20).  When this polymer was added

-------
                         TABLE 5

        AIR OXIDATION OF FERROUS IRON AT SEVERAL
                INITIAL pH VALUES AT 25 °C

    Initial pH adjusted with KOH., uninoculated flasks
        incubated on a gyrotory shaker at 200 rpm
                     	Hours Incubated	

                      0          4          24         48

Ferrous Iron*        429        425        ^25        425
pH                   3.0        3.0        3.0        3.0

Ferrous Iron*        429        420        420        422
pH                   4.0        3.8        3.6        3.5

Ferrous Iron*        429        385        384        384
PH                   5-0        3.7        3.7        3.7

Ferrous Iron*        429        340        337        330
pH>                   6.0        4.2        4.1        2.8


     * mg Fe++/l
                           44

-------
                          •  pH 3
                          A  pH 4
                          n  pH 5
                          o  pH6
  300 I-  THIOBACILLUS FERROOXIDANS
SOUTHWEST-I
           I— FEftROBACILLUS FERROOXIDANS
       I I  I  I I  I I\J  I t9 I  I
                        36 0      12     24  0
                          HOURS INCUBATION
Figure 23   EFFECT OF I N I T I A L pH  ON  FERROUS
            OXIDATION IN  SHAKE FLASKS - 25°C

-------
to 9K medium containing 500 mg/1 of ferrous iron and inoculated with.
Southwest-1 culture, a poorer rate of iron oxidation was experienced
than in a control system void of the polymer (see Table 6).  Subsequent
investigation showed that the polymer also inhibited air oxidation of
ferrous iron.

Redwood Leachate

It became important to consider potential effects of water soluble
components of redwood on iron bacterial oxidation of ferrous iron when
tanks constructed of redwood were being considered for pilot plant oxida-
tion vessels.  Redwood shavings were added to 9K medium which was then
inoculated with the Southwest-1 iron bacterial culture.  Resultant ferrous
iron oxidation data (Figure 2k) indicated that the presence of redwood
did not significantly effect the iron oxidation pattern.

                      Continuous Culture Studies

With few exceptions waste treatment' is most economically accomplished
using continuous flow systems.  While batch data is useful in suggesting
continuous culture conditions, it is usually wise to actually carry out
continuous culture studies prior to the design and construction of larger
systems.  Therefore, a small bench-scale continuous feed system was
assembled and used to check out factors suggested by the previous batch
work.

To ascertain the effect of air oxidation of ferrous iron in the system,
9K medium was fed at rates to give 87- or 12-hour average residence
periods in the oxidation reservoir.  Each residence period was examined
for two-week periods of operation, and 1/3 WM of aeration was utilized.
Wo microbial inoculation was made, and no ferrous iron oxidation was
observed under either condition.

To establish an active microbial population in the system, the first
oxidation vessel was filled to the six-liter mark with nonsterile 9K
medium and inoculated with the Southwest-1 culture.  The medium was
aerated at 1/3 WM and incubated at room temperature (controlled at 25 °c) •
After iron oxidation began to occur, ferrous iron (Fe2S04) was added on
a batch basis until oxidation was found to be occurring at a rapid rate.
Continuous feed of 9K medium and withdrawal was then initiated at rates
calculated to give an average residence time of 87 hours in the oxidation
vessel.  Steady-state operation was maintained for 7 days, and then the
flow rate was increased to give an average residence time of 77 hours.
Following this pattern of establishing steady state then increasing flow
rate, it was found that the residence time could be decreased to li hours
without upset.  At a 12-hour residence period, steady-state operation  could
not be maintained.  Data from these studies are shown in Table 7-

From the results shown in Table 7, it may be seen that, at a feed of
9,000 mg/1 ferrous iron, the minimum residence time that can be maintained

-------
                           TABLE 6

         EFFECT OF SODIUM POLYVINYLSULFONATE ON THE
        BIO-OXIDATION OF FERROUS IRON IN SHAKE FLASKS
                        Residual Ferrous Iron (mg/l)
Hours
Incubation
0
16
24.75
40.75
64.25
90
Inoculated
Medium*
510
478.5
409.2
33-5
0
0
Inoculated
Medium + SPVS**
478.5
431.5
427-1
424.8
380.1
58.1
Un inoculated
Medium + SPVS**
458.4
458.4

458.4
458.4
458.4
Medium = 9K medium containing 500 mg/l ferrous iron

SPVS = sodium polyvinylsulfonate - Hoechst Chemical Company
       molecular weight 2= 20,000

-------
    9000
    8000 —
CONTROL -NO REDWOOD

25 cm2 REDWOOD
SURFACE/liter

IOO cm2 REDWOOD
SURFACE/I iter
             12     24    36    48    60    72

                      HOURS INCUBATION AT 25°C
            84
96
Figure 24  EFFECT OF UNLEACH ED REDWOOD ON FERROUS
          OXIDATION
             SOUTHWEST -I  CULTURE, SHAKE FLASK STUDY

-------
                   TABLE 7

OXIDATION OF 9K MEDIUM BY CONTINUOUS CULTURE

       FERROUS IRON FEED - 9,000 mg/1
       CULTURE - Southwest-1 culture
Average Residence Time
(hours)
8?
77
43
29
25
16
12 ( 1st Day)
12 ( 4th Day)
12 ( 8th Day)
12 (10th Day)
12 (12th Day)
32
20
14
12 ( 1st Day)
12 ( 4th Day)
12 ( 5th. Day)
Effluent Fe++ Concentration
(mg/l)
81
131
163
191
217
295
I0it9
2053
961
2500
5289
165
307
474
629
1975
5624
Average Oxidation Rate
(mg/l/hr)
102
115
205
304
351
531
662
579
695
5^2
310
276
1+34
609
710
598
294
Population
(per ml)
10s
10s
10s
10s
10s
10s
108
10s
10s
10s
10s
10s
10s
10s
108
10s
10s

-------
is about 14 hours.  As expected, the rate of iron oxidation increased
as residence time was decreased.  At the 14-hour residence point, ferrous
iron oxidation was occurring at about 600 mg/l/hr.

Increased aeration and/or addition of more ammonium and phosphate salts
did not influence operation; therefore, it appears that iron oxidation'was
being limited by the metabolic capabilities of the bacterial cells present.

Operation at Lower Ferrous Iron Feed Concentrations

Using the same technique described for the 9K medium operation, the
continuous feed system was used to examine 9K media modified to contain
1,000 mg/1 or 500 mg/1 ferrous iron.  Results of these investigations
are shown in Table 8.  As may be seen, operation at lower ferrous iron
influent concentrations allowed the concomitant use of lower residence
times.  As might be anticipated, lower rates of iron oxidation were
experienced at these lower iron concentrations.

Aeration Rate

To insure that aeration rate was not limiting rate of iron oxidation,
a rather extensive check was made during operation of the continuous feed
system being fed ferrous iron at ^00 mg/1-  Aeration rates were decreased
at two residence times:  two hours and six hours.  The resultant data is
shown in Table 9-  As may be seen, aeration at 1/3 or 1/4 WM should not
come close to limiting the rate of iron oxidation at iron concentrations
substantially higher than any utilized in studies reported in this
communication.

Carbon Dioxide Enrichment

In order to determine if insufficient carbon dioxide might be limiting the
rate of iron bacterial growth, and thereby iron oxidation, the partial
pressure of C02 in the sparger gas was increased.  Because of the very
low concentration of C02 relative to oxygen in air, it was possible to
make large increases in the C02 content of air without significantly
altering the percentage of oxygen present.

As may be seen in Table 10, the addition of about 1 1/2 order of magnitude
more C02 to the microbial oxidation system had no significant effect on
iron oxidation.

Multistaging of Oxidation Vessels

Effect on total efficiency of iron oxidation was examined by the addition
of one or two oxidation vessels in series downstream of the original
continuous feed oxidation tank.  Although neither of these added vessels
was quite as large as the first stage, it was still possible to develop
a picture of the economics of such a setup.  The results of this study
are presented in Table 11.

-------
H
                                                       TABLE 8

                       OXIDATION OP 1000 mg/i ^ 500 Ja&/1 FERROUS
1000
1000
1000
1000
1000
1000
operation
500
500
500
500
500
500
500
\ ilUUi'tJ/
8.0
5-9
4.2
2.1
1.6
0-95
at 0.95 hr ret
8.0
6.0
4.0
3.0
2.0
1.8
1.0
                                                               48
                                                               56
                                  119
                                  160
                                  222
                                  420
              200                 500
              400                 624
residence time became progressively poorer)
                                                   (operation
                                                              78
                                                             102
                                                             105
                                                             109
                                                             120
             57
             70
            100
            135
            195
            210
progressively poorer)
                                                                                               (per ml)
                             10"
                             10s
                             10s
                             10s
                             10s
                             10s
                                                  10s
                                                  10s
                                                  10s
                                                  10s
                                                  10s
                                                  10s

-------
                           TABLE 9

        EFFECT OF AERATION ON THE MICROBIAL OXIDATION
                       OF FERROUS IRON

         Steady State Values over Four-Day Intervals
      Influent Ferrous Iron Concentrations of 5°° mg/1
                       Aeration Rate      Fe"*"+ Oxidation Rate
                                               (mg/l/hr)
Average Residence
   of 2 Hours              1/4                    185
                           1/3                    202
                           1/6                    215
                           1/12                   200
                           1/15                   192
                           1/20                   192

Average Residence
   of 6 Hours              1/4                     75
                           1/25                    80
                           1/30                    71
                           1/40                    75
                           1/80                    73
                           1/100                   69
                           1/200                   40

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             TABLE 10

EFFECT OF CARBON DIOXIDE CONTENT OF
   SPARGER GAS ON IRON OXIDATION
% C02 in
Sparger Gas
0.03
0-7
3-2
6.2
16.7
% 02
Sparger
21
21
20
19
17
in
Gas





                             Fe++ Oxidation
                             Rate (mg/l/hr)

                                   48

                                   42

                                   47

                                   44

                                   40

-------
                              TABLE 11

          MULTISTAGE CONTINUOUS OXIDATION OF FERROUS IRON

Medium:                  9K Medium Modified to Contain 1000 or 500
                         mg/1 Fe++
Aeration Rate:           1/3 WM
Oxidation Vessel Sizes:   6000 ml, 4000 ml, 4000 ml
                      Retention Time (hours)
Fe++ Feed
(mg/l)
1000
1000
1000
1000
1000
1000
500
500
500
500
500
500

Stage I
8
4.8
3-4
5
3
2.1
6
3.6
2.6
4
2.4
1.8

Stage II
0
3-2
2-3
0
2
1.4
0
2.4
1.7
0
1.6
1.2

Stage III
0
0
2-3
0
0
1.4
0
0
1-7
0
0
1.2

Total
8
8
8
5
5
4.9
6
6
6
4
4
4.2
Effluent
Fe++ (mg/l)
48
34
29
65
60
42
78
46
38
102
58
38

-------
Although the effects were not large, it may be observed that multistaging
at equivalent total residence times does provide better quality effluents
in terms of ferrous iron concentration.  Apparently this effect is due to
the presence of higher bacterial cell concentrations in Stages II and III
than in Stage I presumably due to washout from Stage I.  It was observed
that the cell populations in the stages downstream of Stage I were about
one order of magnitude larger than in Stage I.

Natural Mine Drainage Water as Substrate

Once the characteristics of microbial oxidation of ferrous iron were
established in defined media, it became necessary to determine if the
same patterns would extend to characteristic field waters.  For these
studies water from two coal mine discharges, Lynch Shaft and Core, were
obtained and transported to the laboratory using appropriate precautions
to minimize changes in the water during transit.  Important chemical
characteristics of these waters are given in Table 12.  The data indicate
that appropriate types and concentrations of nitrogen and phosphorus
were present in the waters to adequately support iron bacterial growth.
Initial operating conditions of the continuous feed oxidation system were
selected that would most likely provide rapid biological activity and
consisted of a residence time of six hours in Stage I, aeration at
1/5 WM and a temperature of 25°C.  The iron oxidation data thus obtained
are presented in Table 13.  By comparing oxidation rates in Table 13 with
those in Table 8 using synthetic medium, it may be seen that the Core
water was oxidized much more rapidly than the synthetic medium, while the
Lynch Shaft water was oxidized at about the same rate as the synthetic
medium.

Effect of Interrupted Aeration

The possibility of a mechanical failure always exists in a plant or
process system operation.  In order to simulate just what effect such an
occurrence might produce, aeration to the continuous feed system was
suddenly stopped while operating at a six-hour residence time in Stage I
and a four-hour residence time in Stage II.  The dissolved oxygen content
dropped from ^-.8 mg/1 to 2.2 mg/1 after 60 minutes, and the ferrous iron
concentration in the oxidation vessels more than doubled within 100
minutes (Table 14).  Without aeration, the system degenerated quite
rapidly.

Limestone Neutralization

The necessity of converting ferrous iron to the ferric state prior to use
of limestone to neutralize acidity and precipitate ferric hydroxide from
mine drainage water has been discussed in an earlier portion of this
report.  However, it is questionable whether bio-oxidation of the iron
needs to be carried to completion or if some significant quantity of
ferrous iron could remain and limestone still be efficiently utilized to
produce an acceptable treated water.  This final treatment must provide

-------
                               TABLE 12




               CHEMICAL ANALYSIS OF MIME DRAINAGE WATERS
Core MDW*
**
Lynch Shaft*
**
Acidity
pH ( mg/l)
3-3 3430
3-3
2.45 3260
2.8
Fe Total Fe
(mg/l) (mg/l)
1535 1550
1506.6
735 1130
660.7
(mg/l)
20.0
1.1
47-0
(mg/l)
15.0
2.4
13.0
*  Analysis of MDW when shipped November 26, 1968




** Analysis of MDW when received December 2., 1968
                                  56

-------
                                                     TABLE 13

                                 CONTINUOUS FEED STUDIES WITH NATURAL MINE WATERS


       	Stage I* - 6 Hours Retention	    	Stage II* - 4 Hours Retention	

                     Fe++ in         Fe++                     Fe++       Fe++ in         Fe++
       Fe++ Feed    Effluent    Oxidation Rate    P.P.**    Influent    Effluent    Oxidation Rate    P.P.**
       "(mg/l)(mg/l)        (mg/l/hr)(mg/l)     (mg/l)      (mg/l)         (mg/l/hr)(mg/l)

                                                   Core MDW Feed
-q
        1454.5        38.7           235.9          6.9       38.7        10.1            7.2           7.7

                                               Lynch Shaft MDW Feed

         465.0        26.4            73-1          7-0       26.4        11.1            3.9           7.8


       *  Aeration rate 1/5 WM - 1200 cc/min. Stage I, 800 cc/min. Stage II

       **D.O. - Dissolved oxygen

-------
CO
                                                     TABLE 14




                        EFFECT OF NON-AERATION ON FERROUS OXIDATION DURING CONTINUOUS FEED






                        Stage 1-6 Hours Residence	        Stage II - 4 Hours Residence
Non-Aeration
Time (Min.)
0
20
60
100
Feed
(Wl Fe++)
437-5

435-3
432.8
Effluent
(mg/1 Fe++)
49.1

78.1
104.9
D.O.
(Wl)
4.8
3.6
2.2
0.4
Feed
(Wl Fe++)
49.1

78.1
104.9
Effluent D.O.
(mg/1 Fe++) (mg/l)
8.9 6.8
6.4
8.9 6.2
15.6 5.4

-------
effluent water containing not more than 7 mg  total  iron per liter, having
a pH In the range of 6 to 9, and no un-neutralized  acidity.

Effluent samples from Stage II of the  continuous feed system were used
for limestone neutralization studies in an attempt  to clarify this uncer-
tainty.  Limestone rock dust with a calcium carbonate content of approxi-
mately eighty percent was used to neutralize  partially oxidized effluents.

Stoichiometric quantities of rock dust required were calculated from the
titratable acidity of each individual  sample.  These amounts of limestone
were added to one-liter quantities of  each effluent and the resultant
slurries mixed for ten minutes with a  magnetic stirrer.  The neutralized
liquors were then allowed to settle, and samples of the supernatants
taken at subsequent times were analyzed for total iron contents.  The
data obtained from these experiments are presented  in Table 15 and
Figure 25-  Significantly, it was found that  waters containing as much as
89 mg ferrous iron per liter could be  limestone neutralized to produce
a water containing less than 7 mg/1 total iron and  meet the other criteria
as well.

Effect of Added Solids during Lmestone Neutralization

The previous experiments indicated that neutralized solids settled more
rapidly in samples containing higher concentrations of total iron.
Therefore, the possibility of decreasing settling time by the addition of
iron solids prior to limestone treatment was  investigated.  Some precipi-
tation of ferric iron always occurred  in the  first  stage of the bio-oxidation
system during normal operation.  At low aeration rates there was insuf-
ficient agitation to carry all of these solids out  of the oxidation vessels.

Solids from 50° ml of Stage I slurry were collected on Whatman No. 1
filter paper by vacuum filtration.  The moisture content of the solids
collected in this manner was determined to be approximately 65 weight
percent.  Solids were weighed and added to one liter of Stage II oxi-
dized effluent.  Analyses for total iron, ferrous iron, acidity and pH
were performed and rock dust neutralization carried out.  The data for
two settling rate studies are presented in Table l6 and Figure 26.  Also
shown in Figure 26 is a control to which no iron solids were added.  The
effect of solids addition was quite significant in  that the required
settling time was decreased from l6 hours to  5 hours.  Solids removal from
the oxidation vessel in this manner with return of  the filtrate to the
oxidation vessel did not adversely effect bio-oxidation of ferrous iron
in the oxidation vessel.

                       Sulfate-Reducing Bacteria

Effect of pH on Growth

Of the cultures listed in Table 2, none were  found  capable of growing or
producing hydrogen sulfide at pH values of 5  or lower.  Incubation was at
25°C and 35°C in Difco Sulfate API Broth adjusted to pH 2, J, k, 5, 6, 7
                                  59

-------
cr\
o
      Total Residence

          (hours)
5-8








6.7




7-5




7-5
                                                      TABLE 15



                             EFFECT OF RESIDUAL FERROUS IRON ON LIMESTONE NEUTRALIZATION
                                  Stage  II Oxidized Liquor
                                                               Limestone Neutralized Liquor

Total Fe
(mg/l)




435



487

453

469







Fe++
(mg/l)
85



85



49

65

89







Acidity
pH (mg/l CaCOn)
2.4 2100



2.4 1900



2.5 1770

2.4 1750

2.4 i860







Settling Time
pH (hours)
6.25 2
4
6
22.5
6.2 2
4
6
23
6.3 2
22
6.5 1
17
6.5 0-75
2.0
3-0
4.0
5-0
10.75
21.0
Total Iron in
Supernatant
(mg/l)
35-7
29.0
24.6
2.2
42.4
27-2
26.8
6.7
20.1
3-3
15.6
6.7
46.9
40.2
37-9
33-5
33-5
15.6
2.2

-------
   0)
   O>
   E
   OC
   tu
   Q.
   Z>
   (O
48



44



40



36


32



28



24



20


16



12



 8


4


OJ
                    6   8   10   12   14  16
                      SETTLING TIME-HOURS
                                   18  20   22  24
Figure  25  IRON  PRECIPITATION  FOLLOWING LIMESTONE
           NEUTRALIZATION OF PARTIALLY  OXIDIZED
           MEDIUM.

-------
       Total
       Iron
Fe
  ++
        453
 65
                  60
cr\
ro
        469
       2745
 67
                                                     TABLE 16

                          NEUTRALIZATION AND SETTLING STUDIES OF STAGE II OXIDIZED LIQUOR

                                          Total Retention Time 7«5 Hours
 pH

2.4


2.4
         2.4
Acidity
(mg/1
CaCOa)
1750

2020

i860






2920






Settling
Neutralized Tame
pH (Hours)
6.5 1
IT
6.1 l
17
6.5 0.75
2.0
3.0
4.0
5.0
10.75
21.0
6.15 0.75
2.0
3.0
4.o
5.0
10.75
21.0
Supernatant
(mg/1
Total Iron)
15-6
6.7
6.7
2.2
46.9
40.2
37-9
33-5
33-5
15.6
2.2
15.6
8.9
8.9
8.9
6.7
2.2
2.2
      Study A

Oxidized Liquor

Oxidized Liquor
plus Stage I Solids
(15.4 gms/l)*

      Study B

Oxidized Liquor
                                                             Oxidized Liquor
                                                             plus Stage I Solids
                                                             (23.5 gms/l)*
        Wet weight of solids

-------
o
(E
<
O
o»
E
h-
I

-------
or 8 with KOH or HC1.  The media were deaerated by autoclaving prior to
inoculation with 28-hour old cultures growing at pH 7-  Bottles were
held for JO days before discarding.  All cultures grew and produced H2S
at pH 6, 7 and 8.  Attempts to acclimate several of the field cultures to
low pH by stepwise decrease of pH at half-unit intervals were unsuccessful.

Growth in Mine Drainage Water

Sodium lactate at 0.1 weight percent was added to four acid mine drainage
waters and the resultant media inoculated with sulfate-reducer cultures
isolated from these corresponding waters in the field.  In no instance
did growth or H2S production occur.  The API strain also refused to grow
in these media.

Growth in Synthetic Media

As in the previous system, sodium lactate (0.1 weight percent) was used
as the carbon source in 9K medium modified to contain ^00 mg/1 ferrous
iron.  pH adjustments were also made using KOH or HC1.  Of six cultures
tested, all grew at pH 6, but none grew or produced H2S at lower pH values.
Acclimation did result in growth at pH 5-5 "but not at pH 5-0.

Using a platinum - calomel electrode system an effluent sample from the
Stage II oxidation vessel of the continuous feed oxidation system was
found to possess an Eh (oxidation-reduction potential) of +40 mv.  An
aliquot of this material was purged with H2S until the Eh reached -115 nw-
Following pH adjustments from 2.5 to 6.5 at 0-5-unit intervals, flasks
of the reduced media were inoculated with the Southwest sulfate-reducer
culture.  As before, growth occurred no lower than pH 5-5-

                          Pilot Plant Studies

In order to field test the iron bacterial oxidation of ferrous iron in
acid mine drainage water, a pilot plant was designed and constructed by
the Consolidation Coal Company near Enterprise, ¥est Virginia.  The plant
was constructed and housed in a small building near an acid mine water
source that normally contained about ^00 mg/1 ferrous iron and very
little ferric iron.

Physically the pilot plant consisted of two, 1,000-gallon tanks in
series, a 50~gallon inoculum tank and facilities to aerate the tanks,
heat the two, 1,000-gallon oxidation tanks and add phosphoric acid and/or
ammonia to the acid mine water influent if necessary.  A flow diagram of
the plant is shown in Figure 27-

Temperature of liquor in the two oxidation vessels was controlled by four,
22-kilowatt immersion heaters located in each stage.  Air was supplied by
a centrifugal blower rated at 100 CFM and 6 PSIG.  Aeration rates to the
oxidation tanks could be read and controlled by means of flowmeters
upstream of each tank.

-------
NUTRIENT FEED
MINE DRAINAGE
  WATER
                MINE
               DRAINAGE
               WATER
           AIR
                         AIR
                         HEATER
                 INOCULUM
                   TANK
           HEATERS

                                              - OXIDIZED
                                               EFFLUENT


                                                AIR
HEATERS
                      STAGE 1     STAGE E
Figure 27  BIO-OXIDATION  PI LOT PLANT

-------
The heated, aerated inoculum vessel was used to propagate an active iron
bacterial culture for start-up as well as to maintain a constant supply
of active culture in the event it might be needed during plant operation.

Mine drainage water was pumped from a mine to the surface and into a
10,000-gallon surge tank for minimizing flow and composition fluctuations.
This supply water was pumped continuously to the bio-pilot plant at a
rate of 10 to 12 GPM.  A side stream was taken from this continuous flow
as feed to the pilot plant.  Post-oxidation treatment of the water was
not piloted since this aspect of treatment has been relatively well
covered in the literature and was somewhat outside the purpose of the
overall investigation.

Start-Up

The inoculum tank was filled with acid mine water and inoculated with
five gallons of laboratory culture grown in 9K medium.  This mixed
culture was composed of the cultures listed in Table 2 plus iron bacteria
cultivated from the water to be used as feed to the pilot plant.  Ferrous
sulfate (l g/l), anhydrous ammonia (20 mg/l) and phosphoric acid (20 mg/l)
were added to the inoculum tank to insure rapid growth.  The temperature
was controlled at approximately 25"C, and the tank was aerated at 1/5 WM.

When the iron bacterial population reached 108/ml in the inoculum tank,
the 1,000-gallon bio-oxidation vessels were filled with mine drainage water
containing about 500 mg/l ferrous iron, aeration begun at 1/25 WM, the
temperature controlled at 26.5°C and the system inoculated.  One day after
inoculation the ferrous iron concentration had decreased to approximately
200 mg/l.  At this point sufficient ferrous sulfate to raise the ferrous
iron content back to 'yOO mg/l, 10 mg/l of ammonia and 20 mg/l phosphoric
acid were added to the two oxidation vessels.  This medium supplementation
was subsequently done on a daily basis until the fourth day after inoculation
when the bacterial population in the oxidation vessels reached 10 /ml.

Continuous Feed Operation

Based on previous laboratory experience, a 12-hour residence time, 1/25
WM aeration and 26.5°C were selected as appropriate for good initial
continuous feed, operation.

Since the acid mine drainage water to be studied contained an average of
12 mg/l phosphate and 13 mg/l of ammonia nitrogen, these nutrients were
not supplied.  As might be expected of a field water, the iron content
varied from time to time, but the variation was usually less than
± 100 mg/l from a 500 mg/l average ferrous iron level.  Very little ferric
iron was ever detected in the water-  When continuous flow operation
was initiated, temperature was checked at all extreme spots as well as
central locations, in the two oxidation tanks.  Temperature uniformity
at all locations indicated adequate tank mixing by the aeration system.

-------
After  steady-state  iron oxidation was  established,  ferrous  iron concentra-
tions  at  these  same locations  in the tanks were  found to  be equally uniform
verifying that  the  tanks were  completely mixed.

Continuous feed oxidation  at the 12-hour average residence  time is  shown
in the table below:

                                TABLE 1?

          EFFECT OF FERROUS FEED VARIATION ON OXIDATION RATE

                   Average Residence Time:   12 hours
                    Operating Temperature:    26.5°C
                   Aeration Rate:            1/25 WM
                             Effluent
         MD¥ Feed       Fe++ Concentration       Oxidation Rate
         .mg  Fe++/l             (mg/l)             mg Fe++/l/hr
                                                    Stage I
                                 310                   Ik

             560                  347                   18

From these data  it  can be  seen  that  a variation  of about 80 mg/l in the
influent produced only a minor  change in  oxidation rate.  More signi-
ficantly, oxidation rates  at  this  influent ferrous iron concentration and
residence time were disappointingly  low.

When the aeration rate to  the oxidation tanks was increased from 1/25 WM
to 1/10 WM, the average oxidation rate at the 12-hour retention time
rose from l6 mg/l/hr to 38 mg/l/hr.  Although dissolved oxygen values in
the oxidation tanks were around 8  mg/l at the 1/25 WM aeration rate and
previous laboratory data indicated that 1/25 WM aeration was adequate,
the increased aeration produced a  striking increase  in rate of ferrous
iron oxidation.

Using the 1/10 WM  aeration rate,  retention time in  the oxidation tanks
was decreased to 8  hours and  steady-state operation  established.  Subse-
quently the  residence time was  dropped to 7 hours.   Shorter residence times
at 26.5°C were not  investigated in order  to be able  to examine certain
other pilot  plant operational characteristics within the project time
allocation.

If the data  in Table 18 are compared to the laboratory data in Table 8,
it is apparent that the pilot plant  was operating about 15 percent poorer
than the laboratory findings  would predict.

Attempts to  improve  oxidation by further  increasing  the aeration rate,
addition of  phosphate and  addition of ammonia proved fruitless.  The mine


                                   67

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                                                             TABLE 18

                                        EFFECT OF RESIDENCE  TIME  OH FEEEOUS OXIDATION


                                                                   	Stage I	  	Stage II

          P»air1(=snn(=      Apr>a "hi nn   IVITlW "Pi=(=>r1   Ttrrmp"-r&-f-.nrv=  RpHlrhin"
8
Residence
Time - Hours
12
8.0
7.0
Aeration
Rate - WM
1/10
1/10
1/10
MOW Feed
mg Fe+"yl
517
530
492
Temperature
Degree C
26.5
26.5
26.5
mg Fe++/l
60
147
119
Oxidation
mg Fe++/l/hr
39
48
53
Re s idual
mg Fe++/l
21
9
8
Oxidation
mg Fe++/l/hr
3
17
16

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drainage feed was examined for the presence of organic  carbon and heavy
metals that might have deterred microbial activity, but none of these
agents were detectable in the water.

The temperature of the oxidation tanks was then dropped to 20 °C where
oxidation was characterized.  Further data were obtained at 11°C which
represented the average temperature of the influent mine drainage water
during the time of the year at which this study was conducted (January to
February).  Results of these studies are shown in Table 19.

Rates of oxidation at 20"C were lower than the rate attained at 26.5°C,
and doubling the aeration rate only increased oxidation by 5 mg Fe++/l/hour.
The increase in oxidation rate at four hours over that  at eight hours is
almost directly proportional to the ferrous iron concentration in the mine
drainage water feed.

Bench-Scale Studies Using Pilot Plant Feed Mine Drainage Water

In view of the somewhat poorer showing of the pilot plant operation in
comparison to previously demonstrated microbial oxidation capabilities
in the laboratory, the continuous feed system used in the laboratory was
transported to the pilot plant site for studies on the  fresh pilot plant
feed water.  This system was inoculated in the same manner as the pilot
plant and operated on 9K medium modified to contain ^00 mg/1 of ferrous
iron until rapid iron oxidation was taking place.

The medium was then altered by substituting pilot plant feed water for
the distilled water in 9K medium and leaving the ferrous iron concentra-
tion at approximately 500 mg/1.  This step was taken to positively deter-
mine if the field mine drainage water contained any components that might
adversely effect iron oxidation.  Steady-state operating conditions
following substitution of this medium for the 9K medium containing
500 mg/1 ferrous iron remained constant for 4 days indicating there were
no -inhibitory problems inherent to the pilot plant feed water.   System
operation utilizing this feed is shown in Table 20.

At this point the medium being fed to the unit was altered to the pilot
plant feed water to which was added sufficient ammonium sulfate and
dibasic potassium phosphate to equal concentrations of  these agents in
9K medium.  Again, no impairment in iron oxidation rates was detectable.

Finally, the influent to the bench-scale unit was altered to the unmodi-
fied pilot plant feed water.  Resultant operation data  are presented in
Table 20.  As .may be observed from the table, the system oxidized ferrous
iron at rates predictable from the earlier laboratory data.

From the data available, the only apparent difference between the bench-
scale system and the pilot plant system might have been the surface-to-
volume ratios.  Although microscopic examination of samples taken from
oxidation vessel surfaces as well as from central locations within the
vessels indicated no detectable difference in microbial cell densities,


                                   69

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o
                                                     TABLE 19




                                    EFFECT OF TEMPERATURE ON FERROUS OXIDATION
MDW
Feed
mg Fe /I
550
550
575
550
700
725
Average
Residence
Time - Hours
8
8
8
8
4
4
Aeration
Rate - WM
1/10
1/10
1/10
1/5
1/10
1/10
Temperature
Degrees C
26.5
20
11
20
20
11
Oxidation Rate
mg Fe++/l/hr
Stage
40
25
20
30
31
26
I Stage II
17
22
19
22
30
23
mg Fe+Yl
Final Effluent
94
224
263
134
456
529

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                                    TABLE 20

                     EFFECT OF FEED WATER ON OXIDATION RATE
   Residence          Aeration     Feed Water    Temperature       Oxidation Rate
 Time - Hours        Rate - WM     mg Fe++/l     Degrees  C        mg Fe++/l/hr
Stage I
Stage II



Stage I
Stage II
Modified MDW Feed
7.8
6.0
5.0
4.3
11.7
9.0
7-5
6.5
1/10
1/10
1/10
1/10
515
355
438
429
25
25
25
22
57
52
77
81
5
4
7
11
                               Unmodified MDW Feed

4.3         6.5         1/10           421           22            80          11

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surface-to-volume ratios of the pilot plant and bench-scale units were
calculated.  The resultant values (Table 2l)  perhaps offer an explanation
for the wide discrepancies in efficiencies of the two systems.

                               TABLE 21

          PILOT PLANT AND BENCH UNIT SURFACE-TO-VOLUME RATIOS


            Oxidation Vessel        Surface-to-Volume Ratio

               Pilot Plant                  0.13:1

               Bench Unit                   0.82:1
                                  72

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                             SECTION VI

                             DISCUSSION

Iron Bacterial Treatment of Acid Mine Drainage Waters

Although, the literature reveals a  significant amount of knowledge about
the kinetics of acidophilic iron bacterial oxidation of ferrous iron in
synthetic media, there have been few real efforts to utilize the capabili-
ties of these organisms in the treatment of acid ferrous mine discharge
waters.  In this study attention was focused on determining if these iron
bacteria would function well in field mine drainage waters and on charac-
terizing the nutritional and physical factors that would have Important
economic bearing on  the iron bacterial oxidation of ferrous iron in mine
drainage waters.

Bench-scale investigations revealed that acidophilic iron bacteria can
perform equally well in synthetic  media and field mine drainage waters.
Based on nitrogen and phosphorus studies, these elements are present in
-appropriate forms and quantities in many acid mine discharges so that
supplementation for  maximal bacterial activity would not be necessary.
Although aeration of the water to  supply oxygen and carbon dioxide is
essential for rapid  microbial activity, it appears that aeration rates of
around 1/100 WM do  not limit iron bacterial activity in waters containing
up to 500 mg/1 ferrous iron if the aeration technique results in efficient
gas transfer to the  water.  In terms of aerobic microbial conversion
operations, this aeration requirement is very low.

Since non-ferrous nutritional factors do not appear to be economically
important in the microbial oxidation of ferrous iron in mine drainage
waters, the most crucial economic  factors are residence time and tempera-
ture.  Based on the  literature and the data presented in this communica-
tion, microbial oxidation of iron  at very low pH values can be accomplished
using an equal or lower average residence time than can air oxidation of
lime-neutralized equivalent waters.  Therefore, capital investment and
operating cost requirements for detention and aeration should be similar
for these two treatment techniques.

At mine drainage water temperatures below 20°C, microbial oxidation of
ferrous iron is impaired.  At 10°C efficiency appears to be about JO to
lj-0 percent of that at 20 °C.  Although it might be possible to find or
develop a strain of  iron bacteria  that would function somewhat better at
10°C than the cultures that have been studied, it is not likely that such
an effort would be very successful.  At present it appears that an iron
bacterial oxidation  operation receiving water at 10°C must bear the burden
of an increased residence time or  the cost of heat to elevate the water
temperature to about 20°C.  The economic significance of this problem
will depend greatly  upon the specific location where water treatment is
necessary and obviously upon the influent raw water temperature.  Glover
(21, 22) has reported that high aeration rates and solids recycle signi-
ficantly aid microbial oxidation of iron in acid mine waters.  While the

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necessity of high aeration rates is not clear, solids recycle may be an
important consideration.  Although not reported in the previous sections
of this report, indications of higher concentrations of microorganisms on
or adjacent to solids in oxidation vessels were noted.  This situation
needs to be clarified for it might indicate that other types of microbial  -
mine drainage water contact systems should be investigated.  Many new
waste treatment techniques utilize physical configurations that provide
large surface areas for microbial activity to take place.

Finally, the scale-up problems encountered in this study when attempts were
made to evaluate bench data with data obtained in a 1,000-gallon pilot plant
need to be thoroughly investigated.  There is little doubt that the problems,
once clarified., could be solved by simple pilot plant modifications.

Microbial Production of Hydrogen Sulfide in Mine Drainage Waters

Although sulfate-reducing bacteria do not appear capable of growing or pro-
ducing hydrogen sulfide at pH values below 5.5, this factor should not rule
out their potential application to the treatment of acid mine drainage
waters.  Since these organisms will grow in such waters at higher pH values,
it might be feasible to utilize their hydrogen sulfide generating capability
downstream of the neutralization step.  It would, however, be essential that
the oxygen content and oxidation-reduction potential of the water be kept
very low.

A further complication is that the sulfate-reducing bacteria are not auto-
trophic and, therefore, require organic carbon for growth.  Obviously, the
provision of organic carbon to a mine drainage water would entail some
expense; therefore, only a very cheap carbon source would be suitable.  To
supply such a carbon source at mine drainage treatment locations may prove
difficult.

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                          SECTION VII
                        ACKNOWLEDGEMENTS
Acknowledgement  is  given  the  Consolidation Coal Company for the pilot
plant  facility and  to Dr.  G.L. Barthauer of Consol for important advice
throughout  this  study.  Also  acknowledged are the helpful suggestions
made by Mr. Z.V. Kosowski  and Mr. J. Lonbardo of Consol.  The dedicated
efforts of  Mr. F.J. Beafore in operating the pilot plant made such data
available for the project.

Thanks are  given to Drs.  J.V. Beck, Brigham Young University; D.G. Lundgren,
Syracuse University; W.W.  Leathen, Gulf Research; and P.R. Dugan, Ohio
State  University in supplying pure cultures of iron bacteria for the
project study.
A  significant  objective  of  this project was to investigate the use of
microbiological  treatment to  remove iron from mine drainage waters.  Such
research projects,  intended to assist in the prevention of pollution of
water by industry,  are conducted under Section 6b of the Water Pollution
Control Act, as  amended.  This project of EPA was conducted under the
•direction  of the Pollution  Control Analysis Section, Ernst P. Hall,
Chief, Dr. James M.  Shackelford, Project Manager, and Dr. James E. Moyer,
Project Officer.
                                75

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                              SECTION VIII

                               REFERENCES

 1.  Martin, E. J., and Hill, R. D., "Mine Drainage Research Program of
     the Federal Water Pollution Control Administration," Second Symposium
     on Coal Mine Drainage Research, Mellon Institute, pp 46-63 (1968).

 2.  Tybout, R. A., "A Cost-Benefit Analysis of Mine Drainage," Second
     Symposium on Coal Mine Drainage Research, Mellon Institute, pp 33^-371
     (1968).

 3-  Conrad, J. W., "Solving the Problem of Mine Acid Water Pollution,"
     The Analyzer, Beckman Instruments Incorporated, 2_, pp 16-17 (1967)-

 *K  Rose, J. L., "Treatment of Acid Mine Drainage by Ion Exchange Processes,"
     Third Symposium on Goal Mine Drainage Research, Mellon Institute,
     pp 267-278 (1970).

 5-  Mason, D. G., "Treatment of Acid Mine Drainage by Reverse Osmosis,"
     Third Symposium on Coal Mine Drainage Research, Mellon Institute,
     pp 227-2^0 (1970).

 6.  Mihok, E. A., "Applied Advance Technology to Eliminate Aeration in
     Mine Water Treatment," Third Symposium on Coal Mine Drainage Research,
     Mellon Institute, pp l8l-l87 (1970).

 7-  Bikerman, J. J., Hanson, P. J., and Rose, S. H., "Treatment of Acid
     Mine Drainage by Foam Separation," Water Pollution Control Research
     Series, 1^010 DEE 12/70.

 8.  Applied Science Laboratories, Inc., "Purification of Mine Water by
     Freezing," Report FWQA Grant No. 1^010 DRZ (1970).

 9-  Bituminous Coal Research, Inc., "Sulfide Treatment of Acid Mine
     Drainage," Report FWPCA Grant No. 1^010 DLC (1969)-

10.  Dugan, P. R. "Removal of Mine Water Ions by Microbial Polymers,"
     Third Symposium on Coal Mine Drainage Research, Mellon Institute,
     pp 279-283 (1970J.

11.  Wilmoth, R. C., and Hill, R. D., "Neutralization of High Ferric Iron
     Acid Mine Drainage," Report FWQA Program 1^010 ET7 (1970)•

12.  Dean, R. B., "Disposal of Chemical Sludges and Brines," Third Symposium
     on Coal Mine Drainage Research, Mellon Institute, pp 367-375 (1970).

13.  Holland, C. T., Berkshire, R. C., and D. F. Golden, "An Experimental
     Investigation of the Treatment of Acid Mine Water Containing High
     Concentrations of Ferrous Iron with Limestone," Third Symposium on
     Coal Mine Drainage Research, Mellon Institute, pp 52-65 (1970).
                                   77

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1^.  Harvard University, "Oxygenation of Ferrous Iron," Report FWQA Contract
     PH 36-66-107 (1970).

15-  Wakeman, S. A., and Joffe, J. S., "Microorganisms Concerned in the
     Oxidation of Sulfur in the Soil/1 Journal of Bacteriology, 1, pp 239-256
     (1922).

16.  Hamilton, L. F., and Simpson, S. G., Qualitative Chemical Analysis, 12,
     The Macmillan Company, New York (19647^

17-  Silverman, M. P., and Lundgren, D. G., "Studies on the Chemotrophic
     Iron Bacterium Ferrobacillus ferrooxidans," Journal of Bacteriology, 77>
     pp 642-647 (1959T

18.  Lacey, D. T., and Lawson, F=, "Kinetics of the Liquid-Phase Oxidation
     of Acid Ferrous Sulfate by the Bacterium Thiobacillus ferrooxidans,"
     Biotechnology and Bioengineering, 12, pp 29~5° (1970)•

19-  Duncan,  D. ¥• Walden, C. C-, and Trussell, P. C.-, "Biological Leaching
     of Mill Products," Canadian Mining and Metallurgical Bulletin, 59;
     pp 653 (1966).

20.  Morawetz, H., and Vogel, Bo, "Catalysis of Ionic Reactions by Poly-
     electrolytes," Journal of the American Chemical Society, 9~L, pp 563
     (1969)•

21.  Glover,  H. G., "The Control of Acid Mine Drainage Pollution by
     Biochemical Oxidation and Limestone Neutralization Treatment,"
     22nd Industrial Waste Conference, Purdue University, May (1967).

22.  Glover,  H. G., Hunt, J., and Kenyon, W. G., "Process for the Bacteri-
     ological Oxidation of Ferrous Salts in Acid Solution," U. S. Patent
     3,218,252, November, 1965.
                                   78

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    Accession Number
  w
                            Subject Field & Group
                                           SELECTED WATER RESOURCES ABSTRACTS
                                                  INPUT TRANSACTION  FORM
     Organization
               Continental Oil  Company,  Ponca City, Oklahoma
               Research and Development Department
     Title
               Microbiological  Treatment of Acid Mine Drainage Waters
 10
Authors)

 Whitesell,  Louis B., Jr.
 Huddleston, Robert L.
 Allred,  Ray C=
16
    Project Designation
                                            EPA, WQO Contract No. lij-010 EKW
                                     21
                                        Note
 22
     Citation
 23
Descriptors (Starred First)

 *Acid Mine Water,  *Iron Bacteria, Sulfate-Reducing Bacteria,
 Ferrous Iron Oxidation
 25
     Identifiers (Starred First)
      *Acid Mine Water Treatment,  Microbial Oxidation of Mine Water
 27
     Abstract
            Laboratory  studies  demonstrated that both pure cultures and fresh field  cul-
 tures of acidophilic  iron bacteria could readily oxidize ferrous iron in both synthetic
 and natural acid mine drainage  waters.   In the presence of adequate forms and amounts  of
 oxygen, carbon dioxide, nitrogen and phosphorus, ferrous iron was oxidized at rates up to
 600 mg/l/hr.  Average oxidation rate was found to vary with the ferrous iron concentration
 in the water; i.e., the higher  the ferrous iron content, the greater the oxidation rate.
 Approximate requirements of  oxygen,  carbon dioxide, nitrogen and phosphorus by the iron
 bacteria were established.
     Multistaging of oxidation vessels in series was found to produce a more effective
 microbial oxidation system than use  of  a single oxidation reservoir.
     Limestone neutralizations of partially oxidized acid mine waters showed that  such
 waters containing up  to 90 mg/1 ferrous iron could be successfully neutralized and result
 in discharge waters containing  < 1 mg/1 total iron.
     For reasons as yet undetermined,  attempts to duplicate laboratory findings with a
 2,000-gallon pilot plant were not completely successful.
     Although sulfate-reducing bacteria  were isolated from all of nine acid mine discharges
 examined,  attempts to-grow the  cultures or produce hydrogen sulfide at pH values  below 5.5
 were unsuccessful.
Abstractor
         Authors
                               Institution
                                   Continental Oil  Company
 WR:102 (REV. JULY 1969)
 WRSI C
                        SEND, WITH COPY OF DOCUMENT, TO; WATER RESOURCES SCIENTIFIC INFORMATION CENTER
                                                  U.S. DEPARTMENT OF THE INTERIOR
                                                  WASHINGTON, D. C. 20240
                                                                               * GPO: 1 970-389-930

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