United States
              Environmental Protection
              Agency
               Environmental Monitoring
               and Support Laboratory
               P.O. Box 15027
               Las Vegas NV 89114
EPA-600 3-79-048
April 1979
              Research and Development
&EPA
Possible Use of
Alcaligenes paradoxus
as a  Biological  Monitor

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                  RESEARCH REPORTING SERIES

 Research reports of  the Office of Research and Development, U.S. Environmental
 Protection Agency, have been grouped into nine series. These nine broad categories
 were established to facilitate further development and application of environmental
 technology.   Elimination of traditional  grouping was consciously planned to foster
 technology transfer and a maximim interface in related fields.  The nine sereies are:


       1.  Environmental Health Effects Research
       2.  Environmental Protection Technology
       3.  Ecological Research
       4.  Environmental Monitoring
       5.  Socioeconomic Environmental Studies
       6.  Scientific and Technical Assessment Reports (STAR)
       7.  Interagency Energy—Environment Research and Development
       8.  "Special" Reports
       9.  Miscellaneous Reports
 This report has been assigned to the ECOLOGICAL RESEARCH series.  This series
 describes research on the effects of pollution on humans.plant and animal species, and
 materials.  Problems are assessed for their long-and short-term influences. Investiga-
 tions include  formations,  transport, and pathway studies  to determine the fate of
 pollutants and their effects. This work provided the technical basis for setting standards
 to minimize undesirable changes in living organisms in the aquatic, terrestrial, and
 atmospheric environments.
This document is available to the public through the National Technical Information
Service, Springfield, Virginia  22161

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                                                    EPA-600/3-79-048
                                                    April 1979
              POSSIBLE USE OF ALCALIGENES PARADOXUS
                    AS A BIOLOGICAL MONITOR
                               By
Donald V. Bradley, Jr., Robert D. Rogers, and James C. McFarlane
       Monitoring Systems Research and Development Division
         Environmental Monitoring and Support Laboratory
                    Las Vegas, Nevada  89114
         ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
               OFFICE OF RESEARCH AND DEVELOPMENT
              U.S. ENVIRONMENTAL PROTECTION AGENCY
                    LAS VEGAS, NEVADA  89114

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                                 DISCLAIMER
     This report has been reviewed by the Environmental Monitoring and
Support Laboratory-Las Vegas, U.S. Environmental Protection Agency, and
approved for publication.  Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.

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                                  FOREWORD
      Protection  of  the  environment  requires effective regulatory actions
 that  are based on sound technical and scientific  information.   This infor-
 mation must  include the quantitative  description  and linking of pollutant
 sources, transport  mechanisms,  interactions,  and  resulting effects on man
 r«.nd his environment.  Because of the  complexities involved,  assessment
 of specific  pollutants  in the environment  requires a total systems approach
 that  transcends  the media of air, water, and land.   The Environmental
 Monitoring and Support  Laboratory-Las Vegas contributes to the formation
 and enhancement  of  a sound monitoring data base for exposure assessment
 through programs designed to:

           •  develop and optimize systems and strategies for moni-
             toring  pollutants and their impact  on the environment

           •  demonstrate new monitoring systems  and technologies by
             applying them to fulfill  special monitoring needs  of
             the  Agency's operating  programs

      This r,eport is concerned with  the development of a method to detect
 the bioavailable levels of pollutants in environmental samples.   In this
 study the possible  use  of the bacterium, Aloal'igeT'ies paradoxus3  to rapidly
 and cost-effectively detect low levels of  mercury,  cadmium,  and lead is
 examined.  The results  from this preliminary research should be useful
 in the further development of this  and other biological monitors and the
 assessment of the actual biological hazard posed  by different  toxic sub-
'stances.  This report should be valuable to all persons involved in the
 research and regulation of toxic substances whether in government,  industry,
 or university programs,

                                           V
                                           George  B. Morgan
                                              Director
                           Environmental Monitoring and Support Laboratory
                                              Las Vegas
                                      111

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                                   PREFACE
     The existence of the so-called "hydrogen bacteria" has been known for
many years, and a considerable number of investigations into their proper-
ties have been conducted.  These bacteria represent a group of facultative
chemolithotrophs which obtain their energy from the oxidation of hydrogen
and use this energy for the assimilation of carbon dioxide and other metabol-
ic processes.

     Their heterotrophic growth can occur at the expense of a wide variety
of organic compounds.  These bacteria differ from heterotrophic hydrogen-
oxidizing microorganisms by virtue of their ability to use hydrogen as a
sole source of energy for autotrophic growth while the others require added
organics and thus are obligate heterotrophs.

     The studies described herein came about as a result of the metabolic
activity of some of these organisms.  In studies on the oxidation of elemen-
tal tritium (3H2> by plants, it was discovered that the reaction was rapidly
occurring in soil without plants.  Tritium is the isotope of hydrogen with
two added neutrons and is radioactive due to its spontaneous decay  (t^, =
12.3 years) and emission of a weak beta particle.  This oxidation of elemen-
tal tritium was found not to occur in autoclaved soils, and subsequent work
led to the isolation of a bacterium capable of carrying out the reaction in
pure culture.  This bacterium was later determined to belong to the pre-
viously mentioned group of "hydrogen bacteria."

     Subsequent research into the properties of this organism was done and
fell into  four main areas into which the body of this report has been divided:

     1.  Taxonomic Studies
     2.  Growth  Studies
     3.  Resting Cell Studies
     4.  Heavy Metal Effect Studies

     Initially,  interest was directed towards determining which organism was
capable of rapidly oxidizing the tritium; but as the properties of the bac-
terium were defined, it was realized that this organism had the potential
for use as a biological indicator of selected pollutants.  The growth and
resting cell studies resulted from a need for baseline data prior to initia-
tion and understanding of those with the heavy metals.
                                     IV

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                                   SUMMARY

     A tritium  (  Ha)-oxidizing  soil  isolate  was  identified as AlcaUgenes
paradoxuSj  a  gram-negative,  rod-shaped bacterium.   This  organism belongs to
a  group of  facultative  autotrophs  referred to  as the  "hydrogen bacteria" due
to their unique ability to utilize hydrogen  as a sole source  of energy for
chemolithotrophic growth.

     Growth studies  showed that A. paradoxus had autotrophic  doubling times
from 5.0 to 6.0 hours when grown in  a mineral  salts medium plus an  atmosphere
of 70  percent HZ,  20 percent 02, and 10 percent  CO2.   The  optimal pH for
growth was  found  to  be  near  7.0 although growth  occurred over the range from
5.0 to 9.0.   No growth  occurred at pH 4.0 or 10.0.  The  addition of glucose,
fructose, leucine, or trypticase soy broth  (TSB) to the  autotrophic medium
 (pH 7.0) stimulated  growth  (except for leucine in one instance)  and shortened
the doubling  times to 4.57.  3.07,  4.36, and  1.77 hours,  respectively,  when
added  at the  time of inoculation with the cells.  When these  same organics
were added  after  the cells had  been  allowed  to undergo two autotrophic divi-
sions,  glucose, fructose, and TSB  produced doubling times  of  5.38,  4.12,  and
2.40 hours  after  little or no lag  period.
     Experiments  with washed cells of A. paradoxus  suspended  in 0.025M potas-
sium phosphate buffer  (resting  cells) showed that the range of pH's over
vtiich hydrogen (tritium)  oxidation occurred was  from  4.0 to  9.0.   The optimal
rate and extent of the  reaction occurred at  pH 7.0.
     Further  studies with resting  cells showed that 1.0  ppm mercury [as
Hg(NO3)2] caused  a 95 percent reduction in hydrogen oxidation,  whereas,  con-
centration  of 0.1 ppm and lower showed no inhibitory  effects.   When suspen-
sions  of A. paradoxus were added to  sterile  soil and  then  amended with mercury
at concentrations of 1.0, 10.0, and  100.0 ppm, hydrogen  oxidation was  reduced
80 percent, 85 percent,  and  95  percent,, respectively,  compared to soil con-
trols  with  no mercury added.  This showed that soil afforded,  at least tempo-
rarily, some  protection from toxic mercury effects.
     When cadmium or lead was used in solution,  it  was found  that up to
100.0  ppm of  these metals produced no detectable inhibition in the  rate or
extent of the hydrogen  oxidation reaction at either pH 7.0 or 5.0.   However,
pretreatment  of A. paradoxus cells with a combination of ethylenediaminetetra-
acetate (EDTA) and tris-(hydroxymethyl)-aminomethane  (Tris),  at a pH of 8.0,
resulted in a reduction in oxidation in solutions containing  10.0 ppm of
cadmium or  lead,  with the greater  reduction  being due to cadmium.   These
results suggested that  although the  cells may  initially  be insensitive to
certain compounds, their sensitivity may be  enhanced  thus  making them more
suitable for  potential  future development as bioindicators of pollutants.

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                            TABLE OF CONTENTS

                                                                        Page
FOREWORD                                                                 iii
PREFACE                                                                   iv
SUMMARY                                                                    v
LIST OF FIGURES                                                         viii
LIST OF TABLES                                                             x
ACKNOWLEDGMENTS                                                           xi
INTRODUCTION                                                               1
CONCLUSIONS AND RECOMMENDATIONS                                            1
     SECTION I.  TAXONOMIC STUDIES                                         2
                      INTRODUCTION                                         2
                      MATERIALS AND METHODS                                3
                      RESULTS AND DISCUSSION                               5
     SECTION 2.  GROWTH STUDIES                                            5
                      INTRODUCTION                                         5
                      MATERIALS AND METHODS                                7
                      RESULTS AND DISCUSSION                               9
     SECTION 3.  RESTING CELL STUDIES                                     16
                      INTRODUCTION                                        16
                      MATERIALS AND METHODS                               16
                        pH Study                                          16
                        Organic Effect Study                              17
                      RESULTS AND DISCUSSION                              18
     SECTION 4.  HEAVY METAL EFFECT STUDIES                               20
                     INTRODUCTION                                         20
                     MATERIALS AND METHODS                                20
                       Mercury, Cadmium, and Lead in Solution
                       Experiments                                        20
                       Mercury-in-Soil Experiments                        21
                       Cadmium-in-Soil Experiments                        21
                       Cadmium and Lead Effect on Cells in a
                       Solution of EDTA and Tris                          22
                     RESULTS AND DISCUSSION                               22
                       Mercury Studies                                    22
                       Cadmium and Lead                                   24
LITERATURE CITED                                                          28
                                    vii

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                               LIST OF FIGURES

 Number                                                                Page

 1,  The autotrophic growth rate, k (generations/hour) , of              11
     Aloaligenes paradoxus as a function of the pH

 2.  The growth rates of Aloaligenes paradoxus in the auto-             11
     trophic medium before and after the addition of
     glucose

 3.  The growth rates of Alcaligenes paradoxus in the auto-             13
     trophic medium before and after the addition of
     fructose

 4.  The growth rates of Aloaligenes paradoxus in the auto-             13
     trophic medium before and after the addition of leucine

 5.  The growth rates of Aloaligenes paradoxus in the auto-             14
     trophic medium before and after the addition of TSB

 6.  The growth rate of Aloaligenes paradoxus in the auto-              14
     trophic medium at pH 7.0

 7.  Tritium oxidation by Aloaligenes paradoxus in 0.025tl               23
     potassium phosphate solution, pH 7.2, and amended
     with either 0.01, 0.1, or 1.0 ppm mercury Cas
 8.  Tritium oxidation by Alcaligenes paradoxus in sterilized           23
     clay loam soil, and amended with either 1.0, 10.0, or
     100.0 ppm mercury [as Hg(NOa)2]

 9.  Tritium oxidation by Aloaligenes paradoxus in 0.025M               25
     potassium phosphate solution, pH 7.2, and amended with
     100.0 ppm lead  (as PbCla) or cadmium  (as CdCl2)

10.  Tritium oxidation by Aloaligenes paradoxus after 40                25
     minutes pretreatment in a solution of 30 mM Tris, pH
     8.0, and 10~2tl EDTA followed by washing and resuspen-
     sion in distilled water amended with 0.0 or 10.0 ppm
     lead (as PbCla) or cadmium as
                                    viii

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                          LIST OF FIGURES Cont'd

Number                                                                Page

11.  Tritium oxidation by Alcaligenes paradoxus after 48                27
     hours pretreatment in a solution of 30 mM Tris, pH
     8.0, and 10~2M^ EDTA followed by washing and resuspen-
     sion in distilled water amended with either 0.0 or
     10.0 ppm lead (as PbCla) or cadmium (as CdCl2)

12.  Tritium oxidation by AloaLigenes paradoxus after                   27
     inoculation into otherwise sterile soils that
     were amended with no cadmium (soil 1), or 20 ppm
     cadmium (as CdCla) (soil 4) in 1973 and now have
     levels of 0.18 and 0.28 ppm cadmium respectively
                                    IX

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                              LIST OF TABLES

Number

1.  A Comparison of Some Characteristics of other Gram-
    negative, Rod-shaped, Yellow-pigmented Hydrogen
    Bacteria with the Tritium-oxidizing Isolate

2.  A Comparison of the Autotrophic Growth Rates of                     10
    Aloal-igenes popadoxus at Different pH's

3.  The Growth Rate of Alcaligenes paradoxus  in the                     12
    Autotrophic Medium at pH 7.0  (control) and with
    Supplements of Either Glucose, Fructose,  Leucine,
    or TSB

4.  Autotrophic Doubling Times of Aloaligenes paradoxus                 15
     (hours)

5.  The Rate and Extent of  Tritium Oxidation  by Resting                 19
    Cells of Alcal-Lgenes paradoxus at Different pH's

6.  The Rate and Extent of  Tritium Oxidation  by Resting                 19
    Cells of Alodl-Lgenes paradoxus in the Presence of
    Different Organics

7.  The Concentration of Cadmium  (as CdCl2) Measured in                 21
    the Field Soils Amended with  0  (Soil 1),  3  (Soil 2),
    11  (Soil 3), and 20  (Soil 4) ppm Cadmium  in 1973

8.  The Rate and Extent of  Tritium Oxidation  by Resting                 24
    Cells of Aleal'igenes paradoxus in a Solution of
    0.025M  Potassium Phosphate, pH 7.2, Amended with
    Lead  (as PbCla) or Cadmium  (as CdCl2)
                                     x

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                              ACKNOWLEDGMENTS
     Thanks are due to Mr. Rick Thiriot for his hard work and attention to
detail during these studies.  The statistical assistance from Dr. Robert
Kinnison and Mr. Alan Crockett is also gratefully acknowledged.
                                     xx

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                                INTRODUCTION
     The increased awareness in recent years of the hazards presented by the
deposition of toxic substances in the environment has stimulated research
into methods for detecting their presence and the biological threat these
pollutants pose.  As a result we can now measure low levels of many toxic
compounds in environmental samples, and much progress has been made in
identifying the biological effects these substances cause.  However, prob-
lems still exist in the determination of the actual bioavailable levels of
many pollutants.  Also rapid assessment of environmental samples for their
biological toxicity is presently not possible.  Plant or animal studies are
generally slow and expensive.

     Described herein are preliminary studies of a method to determine rapid-
ly and cost-effectively the bioavailability of certain inorganic or organic
pollutants by observing their inhibitory effect on the easily monitored
enzymatic oxidation of hydrogen gas [using tritium (3Ha) as a tracer] by the
bacterium, Aloaligenes paradoxus.

     Since very little information was available on this microorganism,
it was necessary to do a number of background studies prior to those using
pollutants.  Therefore, this report has been divided into four sections
starting with studies to determine if the microorganism would suffice for
the wide variety typically encountered in environmental samples and finish-
ing with toxic heavy metal effects.

     The first section deals with the identification of the bacterium after
it was isolated from a soil sample.  The second is concerned with the rate
of growth both autotrophically and heterotrophically of the microorganism.
In the third section, the ability of A, parodoxus to carry out the enzymatic
conversion of tritium to tritiated water under a variety of conditions is
examined.  In section four the effect on the microorganism of the previously
mentioned pollutants, mercury, cadmium, and lead, is studied.
                       CONCLUSIONS AND RECOMMENDATIONS


     The results of studies with the tritium-oxidizing bacterium, Alealigenes
       uSj indicate that it may be useful as a biological monitor for avail-
able levels of certain toxic substances.  This research has demonstrated the
A. paradoxus sensitivity to mercury, lead, and cadmium.

     The ability of A. paradoxus to rapidly convert elemental tritium (hydro-
gen) to tritiated water and the ease with which the reaction can be followed

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present a simple, effective method for assessing the concentration and effect
of certain pollutants on a living organism.  The autotrophic mode of existence
of A. paradoxus obviates the need for organic supplements and the reaction
with tritium will occur rapidly with cells suspended in buffer or soil.
Additionally, although the presence of certain organics can stimulate the
rate of growth of A. paradsxus under autotrophic conditions, these compounds
do not appear to have a significant effect on the ability of A. paradoxus to
carry out the oxidation reaction.

     The wide range of pH's that can support both autotrophic growth and the
oxidation reaction further increases the versatility of this microorganism.
Thus, it should be possible to monitor the rate of tritium oxidation by A.
paradoxus in samples as diverse as beach sand to peat bog soil and distilled
water to untreated sewage.

     The ability of 1.0 ppm mercury to inhibit almost completely the oxida-
tion reaction and the development of a method for increasing its sensitivity
to lead and cadmium at the 10.0 ppm level as shown in preliminary experiments,
strongly suggests that future  studies into the feasibility of utilizing A.
paradoxus and/or similar organisms for the detection of biologically avail-
able levels of  selected pollutants be given careful consideration.

     Future standardization of technique and research into contributing
factors can almost certainly make such organisms as A. paradoxus extremely
reliable, inexpensive, and rapid indicators of the bioavailable levels of
toxic substances in the environment.
                        SECTION I.   TAXONOMIC STUDIES
 INTRODUCTION

      The  hydrogen bacteria have been found to comprise  a diverse  group  of
 microorganisms  spanning a number of genera.   At one  time they  were  all
 relegated to the  genus  Hydrogenomonas due to their ability to  utilize
 molecular hydrogen as a source of energy for chemolithotrophic growth
 (Bergey's manual, 1957).   However,  this was later found to be  a poor basis
 of  classification since many of these bacteria had so few other characters
 in  common (Davis, 1969).   In addition,  the defining  character  of  the genus,
 namely  the ability to grow autotrophically at the expense of hydrogen could
 even  be lost from some  of these organisms after repeated cultivation with
 organic substrates (Kluyver and Manten, 1942;  Packer and Vishniac,  1955;
 Wilson  et al.,  1953).

      As a result  of these observations the various hydrogenomonads  were
 relegated to other genera more appropriately suited  to  them based on their
 other properties.  These genera included Pseudornonas3 Alcaligenes*  and
 Paraaocaus.   Subsequent work has resulted in the isolation of  hydrogen
 bacteria  belonging to still more genera including Noeardia (Aggag and Schlegel,
 1973);  Corynebaeterium  (Andressen and Schlegel, 1974);  Myoobacterium  (Park and
 DeCicco,  1974); and Streptomyoes (Takamiya and Tubaki,  1956).

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     The  isolate  used  in  tEis  study  was  isolated  by enrichment from soil and
 identified by means of the  taxonomic criteria established for the identifi-
 cation of many of the  hydrogen bacteria  (Davis et al.,  1970).

     The  following will detail the numerous morphological, biochemical,  and
 physiological tests used  in establishing the  identity of  the  tritium-oxidizing
 isolate.

 MATERIALS AND METHODS

     The  soil isolate  was obtained in the following manner  (Rogers  et  al.,
 1978).  Mineral salts  plates were prepared with:

                            KH2POi»         0.8  grams (g)
                                          0.8  g
                               7H20       0.2  g
                            CaCl2          0.002 g
                       FeSOi, •  7H20       0.001 g
                            NaNO3          4.0  g
                       Distilled water     1000 milliliters  (ml)

     The media were solidified by the addition of  1.5 percent Noble  agar
 (Difco) and sterilized by steam autoclaving.  These plates were then inocu-
 lated with various dilutions of a silty  clay loam soil and put in a BBL
 (Baltimore Biological  Labs)  gas pack that generated an atmosphere of hydro-
 gen and carbon dioxide.  The packs are designed for the cultivation of
 anaerobes, but by removing  the oxygen scavenger disc it was possible to
 obtain an atmosphere with oxygen and the  aforementioned gases.

     A variety of colonies  appeared on the plates after approximately 10 days
 incubation at room temperature.  Representative colonial types were selected
 and pure cultures obtained.   Not all microorganisms that appeared were capa-
 ble of hydrogen oxidation but, as determined by later experiments,  one yellow
 colony (several strains of which were isolated) and an apparent streptomycete
were the only active tritium-oxidizers isolated from the plates.

     The yellow bacterial isolates were  initially chosen for further investi-
 gation and, due to the success obtained with them, the streptomycete was not
 examined further.

     After tests with other autotrophic media, it was found that the use of
 Repaske's media (1966)  produced the best growth; and it was used thereafter
 for maintenance of all autotrophic cultures.  It was also found that the use
 of an atmosphere of 70 percent hydrogen, 20 percent oxygen, and 10  percent
 carbon dioxide (Repaske et  al., 1971) provided satisfactory gaseous condi-
 tions.   After the initial isolation, subsequent cultures were maintained in
dessicator jars which were  evacuated, then refilled with the above  gas mix-
 ture.

     Determinations of the  ability of the organism to utilize organic com-
pounds as a sole source of  carbon and energy were conducted according to

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the procedures described by Stanier et al.   (1966).  All chemicals used were
of reagent grade quality.

     To examine isolates for the presence of cytochrome oxidase, a 1-percent
solution of NjN.N^N1 tetramethyl-p-phenylenediamine dihydrochloride was pre-
pared in distilled water.  A small strip of filter paper was impregnated with
this solution and then a colony of the isolate was picked with a Pasteur
pipette and rubbed on the filter paper.  Development of a dark color within
20 seconds was considered a positive reaction.

     The test for the presence of catalase was performed by dripping a 3-per-
cent solution of hydrogen peroxide on colonies on nutrient agar.  Bubbling
was a positive response.

     Motility and sulfide and/or indole production were determined using
SIM media  (Difco).

     Nitrate reduction was determined by removing to separate wells small
aliquots from a culture grown in a nitrate broth then adding a drop of
sulfanilic acid solution [sulfanilic acid, 0.8 g, acetic acid  (5N), 100 ml],
followed by a drop of dimethyl-^-naphthylamine [N,N-dimethyl-l-naphthylamine,
0.5 g, acetic acid  (5N), 100 ml].  The development of a red color indicated
the presence of nitrite.

     Denitrification was tested for using nitrate broth which was inoculated
with the test organism and allowed to incubate for over 1 week.  Gas forma-
tion within Durham tubes was considered positive for denitrification.

     The ability to hydrolyze starch was checked by streaking organisms on
nutrient agar plates supplemented with 0.2 percent weight/volume soluble
starch then flooding the plates with Gram's  iodine solution after good growth
had occurred.  Hydrolysis would be represented by clear areas around colonies.

     Flagellation was determined from young  cultures grown on trypticase soy
agar  (TSA) slants  (BBL) .  Slides used for the test v:erc* soaked 1 week in
freshly prepared dichromic acid.  The stain  was freshly prepared from BBL
flagella stain.  The procedure consisted of  adding a 1- to 3-ml amount of
distilled water to the  slants thus obtaining a suspension of the cells in the
water.  Some of this water was then gently removed with a Pasteur pipette and
1 drop allowed to run down the length of several dry glass slides.  When the
solution had dried, the slides were stained  as per the instructions supplied
by BBL.

     The test for the presence of indole was done using trypticase soy broth
cultures in test tubes that were layered over with 1.0 ml of Kovac's reagent
[ (p-dimethylaminobenzaldehyde, 5 g; amyl alcohol, 75 ml; hydrochloric acid
(cone.), 25 ml)], shaken gently, then observed for the development of a red
color in the reagent which rises to the top.

     Dry weight determinations were conducted by first growing the bacterial
isolate under autotrophic conditions in the medium described by Repaske  (1966)
to an optical density of 0,35 as determined with a Bausch & Lomb Spectronic 20

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colorimeter set at 540 nanometers  (nm).  Aliquots of  10.0 ml were  then remov-
ed and centrifuged for 20 minutes  at 8,000 x g.  The  supernate was discarded
and the cells were resuspended in  a small volume of distilled water,  then
recentrifuged as before.  The water was decanted and  the packed  cells were
carefully washed out of the centrifuge tubes into preweighed tins  and dried
at 65° C for 24 hours before being reweighed.  Dilution plates were prepared
to determine cell numbers in the culture media at the time the samples were
removed.  The dilutions were plated out on TSA since  it had been determined
by earlier tests that dilutions of autotrophic cell cultures actually yield-
ed higher cell counts on the TSA than on autotrophic  plates.
RESULTS AND DISCUSSION

     Because the bacterial isolates all gave identical reactions, they were
considered to be the same organism.  The organism was found to be a gram-
negative, yellow-pigmented rod capable of tenacious, slimy growth under
autotrophic conditions.

     Table 1, taken largely from Davis et al.   (1970) compares the charac-
teristics of a number of other similar hydrogen bacteria with the isolate.
As can be seen, the isolate appears to be identical to Alodti-genes paradoxus.

     In addition, the isolate possessed "degenerately peritrichous" flagella,
generally one or two, which originated subpolarly and extended several times
the length of the cell, and a characteristically unpleasant odor when grown
autotrophically.  Both of these characters also agreed well with the descrip-
tion of A. paradoxus.

     The properties for which there is no comparable information regarding
A. paradoxus  are: negative for indole formation, nitrate is reduced to
nitrite, sulfide is not produced, a heavy pellicle is formed in SIM medium,
and no growth occurs with L-(+)-cysteine.

     Growth under autotrophic conditions resulted in such a tenacious slime
that it was extremely difficult to obtain individual colonies by ordinary
streaking.  On the other hand, growth on TSA plates resulted in soft colonies
that were easy to pick and restreak, resulting in good colonial separation.

     Triplicated dry weight determinations from autotrophically grown cells
yielded 1.67 x 1Q-13 g/cell with a standard deviation of 7.51 x 10~15 g/cell.
                        SECTION 2.  GROWTH STUDIES

INTRODUCTION

     Due to their unique dual modes of growth, the hydrogen bacteria have
been the subject of numerous growth studies involving either their auto-
trophic, heterotrophic, or mixotrophic (autotrophic and heterotrophic pro-
cesses occurring simultaneously) metabolism.  For example, studies have been
done on the possible use of certain hydrogenotnonads in closed cycle systems

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TABLE 1.  A COMPARISON OF SOME CHARACTERISTICS OF OTHER GRAM-NEGATIVE,
          ROD-SHAPED, YELLOW-PIGMENTED HYDROGEN BACTERIA WITH THE
          TRITIUM-OXIDIZING ISOLATE  (TAKEN LARGELY FROM DAVIS ET AL.,
          1970)

Yellow Pigmentation
Oxidase Reaction
Catalase Reaction
Motility
Tolerance to 20% 02 with H2
Hydrolysis of Starch
Organotrophic Denitrification
Utilization of:
H2
glucose
D-xylose
L-arabinose
D-mannose
D-galactose
sucrose
mannitol
pora-hydroxybenzoate
L-leucine
L-tryptophan
histidine
Pseudomonas
flava
+
+
+
+
-
-
-
+
+
-
+
+
+
+
+
-
-
-
-
Strain
450
+
+
+
+
+
-
-
+
+
(+)d
+
+
+
+
+
-
+
-
+
Pseudomonas
palleron-Li
+
Strain
363
+
+ +
+ +
+
** ; +
_
-
+
+
-
-
-
T*
~
-
+
+
+
-
-

+
+
-
-
-
-
-
T-
-
+
T-
-
Aloal-Lgenes
paradoxus
+
+
+
+
<+)a
i-
r-
+
+
+
+
+
+
-
+
+
+
*d
±c
Unknown
Isolate
+
+
+
+
+
r-
r-
+
+
+
+
+
+
r*
+
+
+
+
+
 Parentheses  indicate that mutation and selection may be required before
 the  indicated reaction occurs.

 DOne  of two strains is positive,  the other negative for this character.

 :Ten  of eleven strains are known  to be positive for this character.
  Seven  of eleven strains are known to be positive for this character.

-------
for waste recycling and protein production in spacecraft  (Foster and
Litchfield, 1964; 1969) and other continuous culture systems  (Amman et al. ,
1968; Bongers, 1970).  Many studies have also been done determining the nutri-
tional and gaseous requirements for autotrophic growth  (Schatz and Bovell,
1952; Repaske, 1962; Bartha and Ordal, 1965; Amman and Reed, 1967; Repaske
et al., 1971; Repaske and Repaske, 1976; Repaske and Mayer, 1976).  All these
investigations were concerned with autotrophic growth and the rates of
several species were established.

     In a similar manner growth studies involving the metabolism  of various
organic compounds including amino acids (Fraser-Smith et al., 1968; Brown
and Clark, 1974; DeCicco and Stukus, 1968) and carbohydrates and/or organic
acids (Rittenberg and Goodman, 1969; Kluyver and Manten, 1942; Wilson et al.,
1953; Crouch and Ramsey, 1962; Cook et al., 1967; Marino and Clifton, 1955;
Stukus and DeCicco, 1970)  have been done.   Most of these studies on hetero-
trophic growth were also concerned with mixotrophic growth, and from these it
was generally found that addition of an organic compound to autotrophically
growing cells resulted in increased gas uptake, rate of growth, and cell
yield.  Frequently, there was a lag period between the time of addition of
the organic substrate and the subsequent increased metabolic activity.  This
lag apparently represented the time required to synthesize the enzymes neces-
sary for the metabolism of the added substrate.

     The object of this study was to determine the growth rate of A. paradoxus
in a suitable mineral salts medium under an appropriate gas mixture of hydro-
gen, oxygen, and carbon dioxide.  This was done at different pH's to esta-
blish the range of tolerance under autotrophic conditions.  Next, after an
optimal pH had been established, various organic nutrients were added to the
media to evaluate their effect on the growth rate.
MATERIALS AND METHODS

     Initially, A- pcyadoxus was grown in the autotrophic media used for the
dry weight determinations; however, pH control was very difficult due to the
use of NH4C1.  Therefore, Repaske' s media were modified by replacing the NH4C1
with 10~2M (NH4)2HP04.  This also eliminated the need for a separate phosphate
addition.  Thus, the modified growth media contained the following reagents
in the listed final concentrations:

                        (NH4)2HPO4              1,0 x 10"2M
                     MgS04 ' 7H20              5.0 x 10"4M

                     CaCl2 • 2H20              7,0 x 1CT5M
                     NaHC03                    1.2 x 10"%

              Fe(NH4)2(S04)2 • 6H20            6.0 x 10"5M

     One percent of the trace metals stock solution described by Repaske (1962)
was also added.  The -iron was sterilized by filtration through .0.22-micrometer
     Millex filters (Millipore) and added after the other ingredient had been

-------
autoclaved.  Distilled water was always used and the pH was adjusted with
HC1 or NaOH,  The pH was checked after autoclaving and several times there-
after during the growth rate determinations.

     Cultures were grown in 1-liter volumetric flasks with sidearms suitable
for insertion into a Bausch & Lomb Spectronic 20 colorimeter.  Each flask
contained 250 ml of media which was then inoculated with 1 ml of a fully
grown fresh culture of A. paradoxus.  Since a fully grown culture of A.
paradoxus contains about 2 to 5 x 109 cells/ml, this inoculum resulted in an
initial cell concentration of approximately 10  cells/ml.  Growth of the
culture and thus estimation of the cell concentration was possible using the
colorimeter.  This facilitated finding the proper dilution necessary for
accurately determining the cell numbers at the sample times.  Dichromate
blanks with the same optical density as the uninoculated media were prepared
for each flask.  Thereafter, the appropriate blank was used to zero the
colorimeter before a reading was taken.  A wavelength of 540 run was used
(DeCicco and Stukus, 1968) for all readings.  In this manner it was found
that the correct dilution factor could consistently be selected.

     A gas  line was connected to each flask via a glass tube through each
flask's neoprene stopper.  The gas pressure was kept near 1 atmosphere by
using two  10-liter carboys with a water displacement system as described in
Repaske  (1962).  The flasks were shaken in a water bath maintained at 30° C.

     Samples were removed from each flask through a septum on the end of a
short length of glass tubing that penetrated the stopper.  Pre-sterilized,
disposable  l*-cubic centimeter  (cm^) syringes were used.  It was found that
somewhat more variation  than was desired occurred while attempting to remove
exactly 1.0 ml of the culture for dilution purposes.  Therefore, in the
first dilution only 0.1  ml of the culture was used since it was possible
to obtain much better accuracy when going from mark-to-mark on the syringe
as opposed  to expelling  the entire contents.

     Dilutions were done in blanks filled with tap water.  Since accurate
knowledge of the dilution factors was of prime importance, the dry dilution
bottles and their caps were individually weighed, water was added, and then
the bottles were steam sterilized.  After sterilization, they were allowed
to cool, all external moisture was allowed to evaporate, and they were then
reweighed.  The difference in weights  (in grams) was considered to be
exactly equal to the number of milliliters of water in each blank.  Using
this technique, consistently accurate cell counts were obtained.

     Cell  counts were done using spread plates of TSA layered with from
0.1 to 0.4  ml of diluent.  Four plates were prepared for each sample.

     For growth studies  involving the addition of organics to the auto»-
trophic media, three of  the organics used, dextrose, fructose, and leucine,
were added  by filtration to the autoclaved mineral salts media.  These organ'
ic additions were brought up to a final concentration of 10"2M.  The fourth
organic used was trypticase soy broth  (TSB)  (BBL) which was too viscous to
be filtered and so was separately autoclaved and then added to a final con-
centration  of one-half its normal strength, i.e., 3.25 g/250 ml.

                                     8

-------
     The heterotrophic/autotrophic studies with the organics were run  in  two
different ways.  In the first, the organics were added to the mineral  salts
medium at the same time that the cells were added.  In the second, the inoc-
ulum was added and the cells were allowed to undergo one or two divisions
autotrophically before the addition of the organics.

     Aseptic technique was always observed when working with sterile materials,
and samples were withdrawn from flasks as quickly as possible to avoid exces-
sive interruption of growth.  Also, in experiments where organics were added
to the growth media, the gas mixture in the carboys was frequently changed
even though more than half of it had not yet been utilized.  This was  done
because of possible radical changes in the ratios of the gases due to  simul-
taneous growth under both autotrophic and heterotrophic conditions.
RESULTS AND DISCUSSION

     There were two ways of determining the growth rate of A. paradoxus .
The exponential growth rate constant, k, could have been determined by
the following equation (Stanier et al. , 1966) :

                                  log2Nt " 10g2N0
where

     N  = cell concentration at time t
     N  = cell concentration at time zero
      t = time in hours.

Thus, k represents the number of generations per hour.  The reciprocal of
this, 1/k, could also be computed.  This represents the mean doubling time
and is in hours per generation.

     The other way of determining the growth rate, if during exponential
growth the cell concentration at more than two times is known, is to plot
the Iog2 of the cell concentration versus the time in hours and determine
the slope of the regression line that best fits the points.  This can be done
because during exponential growth such a semi logarithmic plot should yield a
straight line (Stainer et al, 1966) .  The slope thus determined is equal to
k.  Also, every unit increase on the log_ scale represents one doubling of
the cells.

     This latter method was used for the determination of both k and 1/k in
all growth experiments shown except for the autotrophic growth of A. paradoxus
at pH 5.0 (Table 2) in which usable data points were obtained for only two
times.  In all other cases data points at three or more times were obtained
and so the regression line was used.  In these cases the points were statisti-
cally examined to determine the validity and usefulness of the data.  A lack
of fit test was run to" determine if there was a significant lack of fit of the
points to a straight line at either the 95 percent or 99 percent confidence

-------
levels.  A regression test was also run  on  each  set  of  points  to determine,
at the same confidence levels, if there  was a  significant regression.

     The results of the autotrophic growth  experiment determining the  range
of pH tolerance are shown in Figure 1  and Table  2.   No  growth  occurred at
pH 4.0 or 10.0.

TABLE 2.  A COMPARISON OF THE AUTOTROPHIC GROWTH RATES  OF Alcaligenes
          paradoxus AT DIFFERENT pH's

PH
5.0
6.0
7.0
8.0
9.0
k
(generations/h)
0.146
0.165
0.156
0.105
0.086
1A
(h/generation)
6.480
6.059
6.402
9.485
11.662

     For the slow rate of autotrophic growth it  is more meaningful to refer
to the mean doubling time when comparing the rates at different pH's.  This
way it can be seen that the shortest doubling times occurred at pH 6.0 follow-
ed closely by pH 7.0 and 5.0.  The others, pH 8.0 and 9.0, were considerably
slower.  This range for autotrophic growth is not surprising based on the
findings of Lascelles and Still  (1946) who determined the pH optimum for E.
ooli was 5.9 to 6.4, Schatz and Bovell  (1952) who found the highest rate of
oxidation for another hydrogenomonad, Hydrogenomonas fac-ili,s3  to be at pH
6.0, and Repaske  (1962) who obtained the highest rate of growth of Hydrogen-
omonas eutvopha (Aloal-igenes eutrophus) at pH 6.6.

     For growth involving organic additions, two sets of experiments were
run.  In the first, the cells were added to the  flask at the same time as
the various organics.  In the second, the cells  were added first and the
organics added after one or more autotrophic divisions had occurred.  In
this way the lag period between strictly autotrophic growth and heterotrophic/
autotrophic growth could be better determined.   Both sets of experiments had
a control flask consisting of the autotrophic medium at pH 7.0.

     The results of the first run are shown in Table 3.  It can be seen that
TSB gave the shortest doubling times followed by fructose, leucine, glucose,
and the pH 7.0 control, in that order.  Statistically, none showed a signi-
ficant lack of fit to a straight line and the regression was significant at
the 99 percent confidence level.

     In the second run, the organics were added  after the cultures had been
maintained for 16 hours under autotrophic conditions.  It was  found that
with the glucose and fructose additions a statistically significant increase
in slope (growth rate) occurred within 2 hours or less (Figures 2 and 3) .

                                    10

-------
                                                
-------
In addition, the lines described by both the 0-, 11-, and  15-hour points ana
the 18-, 22-, and 29-hour points each had a significant regression at the
99 percent confidence level and showed no significant lack of linearity.
TABLE 3.  THE GROWTH RATE OF ALCALIGENES PARADOXVS IN THE AUTOTROPHIC MEDIUM
          AT pH 7.0 (CONTROL) AND WITH SUPPLEMENTS OF EITHER GLUCOSE,
          FRUCTOSE, LEUCINE, OR TSB
       Supplement to                  k                        1/k
     autotrophic media           (generations/h)            (h/generation)
None (control)
Glucose
Fructose
Leucine
TSB
0.174
0.219
0.326
0.229
0.565
5.738
4.570
3.066
4.364
1.770

     For some reason the addition of leucine, Figure 4, actually decreased
the growth rate even though it had been used previously with success both
in liquid and on solid media.  No explanation is available for this result.
Further tests would be needed to help clarify this response.

     As for the TSB, it had four separate rates of growth, as can be seen
in Figure 5.  The rate of growth between 0 and 11 hours is the true auto-
trophic growth rate since the decrease between 11 and 18 hours can be attrib-
uted to the half hour during  that  time that  the  flask was not being shaken
or connected to its gas supply while some problems in adding the TSB to the
flask were resolved.  Therefore, the doubling time of 5.030 hours was con-
sidered correct.  After the addition of the TSB, the growth rate greatly
increased between 18 and 22 hours, then decreased somewhat between 22 and
29 hours.  The decrease was probably due to depletion of some nutrient or
overcrowding since at 22 hours the cell concentration had already exceeded
109 cells/ml which is very near the maximum generally obtained.  Thus, the
doubling time after the addition of TSB was considered to be 2.398 hours.

     The control flask, Figure 6, showed no variation in slope during the
entire experiment, and a statistically significant regression and fit of a
single straight line through  all data points could be obtained.

     In Table 4 the different doubling times obtained during the period of
autotrophic growth in this second set of organic additions experiments are
compared.  The range was from about 5 to 6 hours.  Doubling times found by
other investigators for other hydrogen bacteria are: 3.2 hours for Hydro-
genomonas H 20  (Schlegel, 1966), 1.7 hours with Eydrogencmonas eutropha
(Bongers, 1970), 3.5 to 4.0 hours with Hydrogenomcnas eutvopha  (Repaske, 1962)


                                    12

-------
                                = 0.243, _ = 4.11 hrs.
                                    = 0.181.—= 5.537 hrs.
                                           k
                                                    I 95% Level
                                                    D Mean Value
            ^""™r   i   i   i    i   i   i    i   i    i    i
        25
              2   4  6   8  10  12 14  16  18 20 22 24 26  28 30
                               Time (hours)
Figure 3.   The growth rates of Aloaligenes paradoxus in the autotrophic
           medium before  and after the addition of fructose.
        !5i
                                    k = 0.121,—= 8.28 hrs.
                                             k
                                 -k = 0.166,L= 6.025 hrs.
                                            k
                                                    I 95% Level
                                                    D Mean Value
               2  4  6   8  10 12 14  16 18 20  22 24  26 28 30
                               Time (hours)
Figure 4.   The growth rates of Alcaligenes paradoxus in the autotrophic
           medium before  and after the .addition of leucine.
                                  13

-------
                        k = 0.417,T-= 2.399 hrs.
                                 k
                          :	k = 0.199.— = 5.030 hrs.
                                                 I 95% Level
                                                 D Mean Value
            V

            0   2  4   6   8  10 12  14  16 18  20 22 24  26 28  30
                                Time (hours)
Figure 5.  The growth  rates of Alodl-igenes paradoxus in the  autotrophic
           medium before  and after the addition of TSB.
                                    	k = 0.190.-^-= 5.257 hrs.
                                                 I 95% Level
                                                 D Mean Value
         25
            0   2  4  6   8  10 12  14 16 18  20 22 24  26 28 30
                                Time (hours)
Figure 6.  The growth rate of Alaaligenes paradoxus in the autotrophic
           medium at pH 7.0.
                                   14

-------
and 2 hours with Aloaligenes eutrophus  (Hydrogenomonas eutropha)  (Repaske,
1966) .   No growth experiments using A. paPCtdoxus have yet been published.

TABLE 4.  AUTOTROPHIC DOUBLING TIMES  OF Aloaligenes poradoxus  (HOURS)

pH 7.0 control
Leucine flask
Glucose flask
Fructose flask
TSB flask


(before
(before
(before
(before


organics
organics
organics
organics


added ;
added ;
added ;
added ;


0
0
0
0


to
to
to
to

standard

15
15
15
11


h)
h)
h)
h)
x =
deviation =
5.
6.
5.
5.
5.
5.
0.
257
025
914
537
030
553
423

     The autotrophic doubling times obtained in these experiments are  some-
what longer due to the following critical factors that have been shown to
be important  (Repaske, 1962, 1966; Schlegel, 1966):

     1)  good aeration
     2)  supplements of nutrients as needed
     3)  pH control
     4)  proper gas mixture

     Of these, the first is the only factor which really affected these
results.  For the period of time growth was followed, there was no nutrient
depletion or pH fluctuation, and in addition the gas supply was frequently
changed.  However, adequate aeration was a problem since the shaking water
bath used was not capable of high speeds and the flasks used were smooth-
walled.  Baffles on the inside walls would have greatly increased aeration,
however, such flasks were not available in time to be used.  Also, some of
the doubling times of other investigators, namely the 1.7- and 2-hour  times,
were obtained using continuous culture apparatus with high impeller speeds
and electrodes to constantly monitor the dissolved gas concentrations  and pH.
In this manner they were able to maintain exponential growth almost indefi-
nitely.

     In the experiments involving the organic additions, a question arises
regarding the mechanisms responsible for the increase in growth after  the
addition of the organics (the second run with leucine excepted).  There
exist two possibilities:

     1)  Heterotrophic growth occurred after the addition of the
organics and autotrophic growth ceased;

     2)  Both autotrophic and heterotrophic growth occurred simul-
taneously (mixotroffhic growth).
                                     15

-------
     Mixotrophic growth of the closely related Aloal-igenes eutrophus has been
shown by DeCicco and Stukus  (1968), Rittenberg and Goodman  (1969), Stukus
and^DeCicco (1970), and Brown and Clark  (1974).  Hydrogenomonas  (Pseudomonas)
faaiHs  has also been shown to exhibit mixotrophic growth  (Wilson et al., '
1953; Brown and Clark, 1974; DeCicco and Stukus, 1968).  Thus, it would not
be surprising if A. paradoxus was also capable of it.

     Although determination with certainty of mixotrophic growth would be
useful, it was not the intention here.  Rather, this study has shown the
growth rate under autotrophic and autotrophic/heterotrophic conditions.
                     SECTION  3.  RESTING CELL STUDIES

INTRODUCTION

     In many  instances  an  experiment may require  that  the number of bacterial
cells be constant while measuring  some parameter.   Bacteria can be prepared
in this way by  suspending  a known  number of cells in some medium that will
not greatly affect  their reactivity or viability  and will also not allow
cell division to occur.  Such suspensions  are frequently known as "resting
cells" although they are certainly not "resting"  except from  cell division.

     Many  investigators have  used  this technique  to observe the uptake of
gases by hydrogenomonads  (Kluyver  and Manten, 1942; Marino and Clifton,
1955; Schatz  and Bovell, 1952;  Schatz, 1952; Lascelles and Still, 1946;
Packer and Vishniac, 1955;  Atkinson, 1955;  Lindsay and Syrett, 1958; Kanai
et al., 1960) under autotrophic, heterotrophic  and mixotrophic conditions.

     In this  study  suspensions of  the soil isolate, A.paradoxus3 were pre-
pared in buffers of varying pH to  determine the range  of their tritium-
oxidizing  ability.   The effect of  organics on this reaction is also examined.
MATERIALS AND METHODS

      Cultures of A.  paradoxus were prepared under strict autotrophic condi-
tions as described in  the preceding chapter.  These cells were then centri-
fuged down  at 4,000 to 5,000 x g for at least 20 minutes.  The supernate was
decanted, and the cells were rinsed twice with distilled water,  then re-
suspended in  a  small volume of the same.

pH  Study

      One-liter,  round-bottomed flasks were used.  A series of 8  or 9 flasks
was used for  each pH examined.  Each of the 8 or 9 flasks had 15.0 ml of
0.025M potassium phosphate buffer at the desired pH and the flasks were
then  positioned on a rotary shaker.  Next, approximately 0.5 ml  of the
washed cell suspension was added to each flask using an automatic pipette.
The pipette had previously been checked for accuracy and was found to con-
sistently deliver the  same volume within 0.001 ml.  Thus, each flask could
be  expected to  start with the same cell concentration.   This was verified

                                      16

-------
by cell counts on TSA spread plates using appropriate dilutions.  During  the
entire inoculation time the flasks remained unstoppered.

     When this was complete, each flask was stoppered and injected with
5 cm3 of nitrogen gas containing 930 nanocuries  (nCi) of elemental tritium
(3^) .  The stoppering and injecting took place together and the exact time
of injection of each individual flask was recorded.

     When all the flasks were injected  (which usually took only 10 to 15
minutes) , the shaker was started.  Flasks were then removed at the desired
times and flushed with air which removed any remaining tritium, thus stopping
the reaction, and the contents were poured into a small vial and capped.

     After all the samples had been collected, they were filtered through
0.045-ym filters which removed the cells and any other debris.  The filtrate
could then be counted by liquid scintillation as described by Lieberman and
Moghissi (1970) .  Appropriate quenching standards were prepared to correct
for the presence of the buffer.  In this manner, the precise amount of gas-
eous tritium converted to tritiated water could be determined for each sam-
ple.

     This produced a sequence of points which yielded a curve described by
a regression function known as the exponential growth model:
                         Y = P [1-EXP(-P t)] + E
where
              Y = tritium level
             P  = the asymptotic tritium concentration

             P  = the reaction rate parameter
              t = the time in hours
              E = the error function, assumed to be Gaussian

     The derivative of formula (1) with respect to time gives the velocity
of the reaction.
The velocity is maximal and equals PI? 2 at time zero.  In this manner the
initial velocity (calculated at time zero) and asymptote were determined
for each pH examined.
Organic Effect Study

     The cells were.prepared as before.  However, all the flasks except those
using TSB had 15.0 ml of the buffer at pH 7.0 with a 10~2fl concentration of
either glucose, fructose, leucine, or histidine.  The series with TSB as the


                                    17

-------
organic source used only the broth prepared at its normal strength  (7.5 g/
250 ml).  Replicate series of pH 7.0 were also run as a standard against
which to compare the rate of oxidation in the presence of the organics.

     Due to the presence of the organics, it was decided to autoclave all
the flasks and materials that came into contact with either the cells or
media.  Cell counts were done as previously described.  The injection of
tritium was performed just as before and the shaker started.

     Collection of the sample after the specified time was done somewhat
differently.  The reaction was stopped as before by opening the flask and
flushing it with a stream of air, but then the water was extracted  in a
benzene azeotrope  (Moghissi et al., 1973).  As before, appropriate  standards
were prepared and the tritium content was determined by liquid scintillation.
Mathematical computations of the initial velocity and the asymptote of the
reactions were done as in the pH study.
RESULTS AND DISCUSSION

     The results obtained from the resting cell experiments provided the
desired preliminary data.  Before pursuing further serious investigation
into the potential of this organism  for use as a biological monitor, it was
necessary to determine what effect two important environmental factors,
pH and organics, would have on the ability of A. paradoxus to oxidize
tritium.  No attempt was made to accurately determine the variation from
pH-to-pH or organic-to-organic.  Rather,  it was important only to know if
the oxidation reaction occurred at all or was very severely inhibited or
enhanced under the various conditions examined.  The results obtained
answered these questions.

     No conversion of tritium to tritiated water was found to occur at pH
3.0 or 10.0.  The rates and limits of the reactions from pH 4.0 to 9.0
are shown in Table 5.  As can be seen, the initial velocities and asymptotes
for the oxidation reaction at these  pH's  were quite similar.  This would
seem to indicate that tritium oxidation occurs at a near optimal rate over
a wide range of hydrogen ion concentrations.  This may prove to be quite
important since, as will become evident in the next section, the intent was
to develop this organism as a biological  monitor; and in environmental
samples, whether they be soil or water, a wide range of  pH's can be ex-
pected.

     The effect of the addition of organics can be seen  in Table 6.  It
should be noticed that the initial velocities for the reactions at pH 7.0
are all much slower here than those  obtained in the pH study.  This can be
attributed to batch-to-batch variation in the hydrogen dehydrogenase activity
of cell dultures.  However, within any one batch of cells the activity was
constant.

     The addition of organics was found to cause no shutdown of the tritium-
oxidizing activity.  It appears from Table 6 that, if anything, some of the
organics actually caused a slight increase in the reaction rate.  However,

                                     18

-------
no significance can be  given  to  that possibility  since  this  experiment was
run only once and one would want to run  at  least  triplicates to  be fairly
sure of the variation around  each data point.
TABLE 5.  THE RATE AND EXTENT OF TRITIUM OXIDATION BY  RESTING  CELLS  OF
          Alodl-igen.es paradoxus AT DIFFERENT pH's

PH
4.0
5.0
6.0
7.0
8.0
9.0
initial velocity
(nCi/h)
175
141
151
139
172
123
asymptote
(nCi)
706
800
785
857
788
767

TABLE 6.  THE RATE AND EXTENT OF TRITIUM OXIDATION BY RESTING CELLS OF
          Alodligenes paradoxus IN THE PRESENCE OF DIFFERENT ORGANICS

sample
pH 7.0 #1
pH 7.0 #2
Glucose
Fructose
Leucine
Histidine
TSB
initial velocity
(nCi/h)
46
42
47
53
55
62
41
asymptote
(nCi)
628
736
821
706
668
638
671

     These data clearly show that the addition of A. paradoxus to an environ-
mental sample with high organic levels will not result in inactivation of the
oxidation reaction.  While it is certainly true that not all possible organics
one could expect to find in nature were examined, the TSB series showed good
activity; and TSB contains a number of protein digests and yeast extract, and
so has a fairly high concentration and variety of organic compounds.  And
certainly, a 10  £1 concentration of the other organics tested is far higher
than one would expect in  almost any environmental sample.
                                     19

-------
                  SECTION 4.  HEAVY METAT EFFECT STUDIES
INTRODUCTION
     Numerous studies have been done on the toxicity of mercury to animals,
plants, man, and microorganisms.  Similar investigations have been conduct-
ed into the toxicity of cadmium and lead.  A number of excellent reviews on
mercury (Friberg and Vostal, 1972), cadmium  (Friberg et al., 1971), and lead
(U.S. Public Health Service, 1966) are available in addition to many less
comprehensive publications on the toxicity, availability, removal of these
metals, and the general reactions they can undergo.

     A common problem encountered by these investigators is the determination
of the actual biologically available levels of these compounds in the sample
being examined.  The absolute concentration can be determined by a number of
methods, but these concentrations may not necessarily correspond to the levels
free to react with a biological system.  In general, it has been found that
organic compounds in particular will strongly bind with mercury, lead, and
cadmium in this order of affinity:

                            Hg2+ > Pb2+ » Cd2+                      (3)

(Ramamoorthy and Kushner, 1975).  Also, clay soils have been shown to reduce
the toxicity of cadmium  (Babich and Stortzky, 1977b) and mercury  (van Fraassen,
1973) compared to sand soils.  The pH effect on cadmium toxicity has also
been examined with regard to various bacteria, fungi, and actinomycetes
(Babich and Stortzky, 1977a).

     The object of this study was to examine the effect of mercury, lead,
and cadmium on the rate of oxidation of tritium  (hydrogen) by the soil
isolate of A, paradoxus previously described.  The hope was to use the
techniques acquired from the resting cell studies to analyze rapidly samples
for the bioavailable concentration of the particular metal of interest as
opposed to current methodology which can analyze only for levels chemically
available.  This study also examines the effect in soil of these metals on
the rate of tritium oxidation.
MATERIALS  AND METHODS

     Cells of A, paradoxus  were cultured,  washed and resuspended  as describ-
ed  in  the  previous  Section.

Mercury, Cadmium, and  Lead  in SolutionExperiments

     One-liter,  round-bottomed flasks were prepared with 0.025M potassium
phosphate  buffer, pH 5.0, 7.0, or 7.2 as detailed in the previous Section.
These  contents were then amended with either Hg(N03)2»  CdCl2»  or  PbCl2  to
yield  the  desired final concentrations.  Controls received a volume of  dis-
tilled water equal  to  the volume of the metal solution  addition.   Cells were
then added to the flasks which were stoppered, injected, and shaken as

                                      20

-------
described before.  The final volume of liquid within each  flask was  15.0 ml.
Collection of the water by benzene distillation and the analysis  for its
tritium content were also done as before.

Mercury-in-Soil Experiments

     Twenty grams of steam-sterilized clay loam were put into each one-liter,
round-bottomed flask.  Sterilization was verified by plating soil dilutions
on TSA.  After this, 14.0 ml of the A. paradoxus cell suspension  and 1.0 ml
of the proper dilution of Hg(N03)2 were added to yield a final concentration
of 1.0, 10.0, or 100.0 ppm.  Controls received 1.0 ml of distilled water
instead of the mercury solution.  The flasks were then stoppered  and in-
jected and the mixture was well shaken to create a "slurry" covering the
entire inner surface of the flask.  The flasks were then incubated without
shaking in a room at 30° C.  Samples were removed and the water distilled
and analyzed as before.

Cadmium-in-Soil Experiments

     Field soils were obtained which had been amended with cadmium (as
CdCl2) in 1973.  In the years since then the levels of residual cadmium
have steadily decreased as shown in Table 7  (A. G. Wollum, personal  commu-
nication) .  The 1977 data were obtained for this study by extraction of  the
soils using the methods of Follett and Lindsay (1971) followed by atomic
absorption analysis.

     At the time of this study the levels of cadmium in all four  soils
were near background concentrations.  The pH's of the soils were  also very
similar being 5.1, 4.8, 5.2, and 4.8, respectively.

     In the experiments, 20 g of the soil was put into each flask followed
by the addition of a sufficient volume of the cell suspension to obtain  a
good slurry of the mixture on the inside of the flasks.  The injection,
stoppering, and analysis were done as before.
TABLE 7.  THE CONCENTRATION (IN ppm) OF CADMIUM (AS CdCl2) MEASURED IN THE
          FIELD SOILS AMENDED WITH 0 (SOIL 1), 3 (SOIL 2), 11 (SOIL 3), AND
          20 (SOIL 4) ppm CADMIUM IN 1973
     Year        Soil 1          Soil 2          Soil 3          Soil 4
1974
1975
1977
0.20
0.19
0.18
2.16
0.86
0.24
5.58
2.35
0.26
12.37
4.03
0.28
                                    21

-------
Cadmium and Lead Effect on Cells in a Solution of EDTA and Tris

     Each flask was prepared as in the lead and cadmium solution experiments.
The buffer was always pH 7.0; however, the washed cells, diluted to a con-
centration of approximately 5 X 104 g/ml were suspended in 30 mM tris-
(hydroxymethyl)-aminomethane  (Tris), pH 8.0, and 10~2M disodium ethylene-
diaminetetraacetic acid  (EDTA)  (Repaske, 1958) for either 40 minutes at
room temperature or 48 hours at 7° C, and then recentrifuged and washed
twice more in distilled water to remove the Tris and EDTA.  The cells were
next pipetted into the test flasks which were then stoppered, injected with
the tritium, and shaken for the desired time.  Removal, extraction of the
water, and tritium analysis were done by benzene distillation and liquid
scintillation as previously described.
RESULTS AND DISCUSSION

Mercury Studies

     The results of a variety of mercury  concentrations  in  solution, pH 7.2,
on the ability of A. paradoxus to oxidize tritium  are  shown in Figure 7.
It is evident that A. pcafadoxus is very sensitive  to somewhere between 0.1
and 1.0 ppm mercury in an uncomplexed  solution.  In the  sterilized  soil,
Figure 8, amended with A. papadoxus and mercury, 1.0 ppm was sufficient to
completely inhibit the oxidation reaction, but only after approximately
20 percent conversion of the tritium to tritiated  water  had taken place.  For
the same concentration of mercury in solution, only about 5 percent conversion
took place.  It is noteworthy that 100.0  ppm mercury in  the soil produced the
same effect as 1.0 ppm in solution.  This suggests that  the clay loam soil
offered some degree of protection not  available  in solution.  This  is consist-
ent with the results obtained^ by Babich and Stortzky  (1976b)  who found that
cadmium toxicity was reduced by clay minerals.   They determined that the degree
of protection was directly  related to  the cation exchange capacity  of the clays
used.  Work by van Faassen  (1973) seems to indicate that this is true for mer-
cury also since he found that in sandy soils the toxicity of mercury was great-
er than in clay soils.  Thus, since the soil used  here was  a clay loam, it
should not be surprising that some protection from mercury  was afforded to
the cells.  The negatively  charged clay particles  could  be  binding  the Hg2+
ions, thus preventing their interaction with the cells.  Exchange of the
Hg2+ ions with other cations in the solution probably  accounted for the
eventual toxic effect on the bacteria.

     A concern in determining the usefulness of  A. paradoxus for future
biological monitoring was that it might be resistant to  low levels  of
heavy metals.  For mercury  this appears not to be  the  case.  Although this
study is far from conclusive, it clearly  shows that A. popodoxus is sensi-
tive to low levels of mercury both in  solution and soil. Future work could
fill in the gaps and cover  a larger range of mercury concentrations and con-
ditions of- exposure.  It seems that A. paradoxus has the potential, given
sufficient research emphasis, of serving  as a sensitive  bioindicator of
environmental mercury.
                                     22

-------

(0
         100-
          90-
          80^
          70-
          60-
          50-
          40-
          30-
          20-
          10-
                                             „,-"—— *
                                    ^.^^as^	'	"•
                                            Control
                                            0.1 ppm Hg2+
                                         -. 1.0 ppm Hg2+
                                           • 0.01 ppm Hg2+
                      8   10  12  14 16
                          Time (hours)
                                                18  20  22  24
 Figure 7.
     Tritium oxidation by Alcal'igenes paradoxus  in  0.025M
     potassium phosphate solution, pH 7.2,  and amended with
     either 0.01, 0.1,  or 1.0 ppm mercury [as Hg(N03)2]-
100-

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                                                Control
                                              	1.0 ppm Hg2+
                                                 0.0 ppm Hg2+
                                      •—'—^-—-- 100 ppm Hg2+
                                  ,#m ———--- — _ — --- — — ---•*
Figure 8.
                  6   8  10  12  14  16  18  20  22  24
                        Time (hours)
    Tritium oxidation by Aloaligenes paradoxus in  sterilized
    clay loam soil,  and amended with either 1.0, 10.0, or
    100.0 ppm mercury [as Hg(N03)2]-
                               23

-------
Cadmium and Lead

     The results obtained from the incubation of A. pafadoxus with low
levels of cadmium and lead at pH 7.2 in a phosphate buffered solution are
shown in Table 8.  They reveal a different pattern than found for mercury
since here even 10.0 ppm lead or cadmium had no discernible effect on the
ability of the cells to oxidize the tritium to tritiated water.  Therefore,
a similar test, this time using 100.0 ppm lead and cadmium was run; however,
once again no inhibitory effect was noted  (Figure 9).

     Since to use even higher concentrations to establish the upper limit of
sensitivity would have been pointless in light of the planned use for A.
paradoxus as a monitor of low environmental levels of the metals, it was
decided to attempt to increase the sensitivity of A. paradoxus to the lead
and cadmium.  Thus, the cells were again incubated with 1.0 and 10.0 ppm of
lead and cadmium in phosphate buffered solution, however, this time the pH
was 5.0.  The results were identical to those before, namely no reduction in
the rate or extent of the oxidation reaction.  At this pH all the lead and
cadmium could certainly be expected to be in solution  (as Pb2+ and Cd^+ ions),
so it appeared that A. paradoxus was fairly resistant to these two metals.


TABLE 8.  THE RATE AND EXTENT OF TRITIUM OXIDATION BY RESTING CELLS OF
          Aloaligenes paradoxus IN A SOLUTION OF 0.025M POTASSIUM
          PHOSPHATE, pH 7.2, AMENDED WITH LEAD  (AS PbCl ) OR CADMIUM
           (AS CdCl2)


                                     Initial Velocity       Asymptote
Concentration  (ppm) and Element       (% conversion/h)      (% conversion)
Control
2+
0.1 Cd
1.0 Cd2+
10.0 Cd2+
0.1 Pb2+
1.0 Pb2+
10.0 Pb2+
15

13
14
13
17
14
13
95

99
92
98
91
97
90
      It was  then found that the effect of certain antibiotics could be
enhanced,  presumably by increasing bacterial permeability to them (Weiser
et  al.,  1969;  Brown and Richards,  1965;  Heppel,  1967).   These techniques
all required pretreatment of the bacterial cells with the disodium salt of
EDTA followed  by the antibiotic exposure.
                                      24

-------
Figure 9.
» 70-
CB
« 60-
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£
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• i 1 1 1 	
12345
                Time (hours)
Tritium oxidation by Alcal-igenes paPodoxus in
0.025M potassium phosphate  solution, pH 7.2, and
amended with 100.0 ppm lead (as PbC^) or cadmium
(as CdCl2).

     50-
     45-
   k.
 "o ^ 40-

 .2 * 35'

 1 | 3°-
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 2 | 15-
 *% ID-
      S'

                                                    Control
                                                    10 ppm Pb2+
                                                    10 ppm Cd2+
                                  2
                                                          I
Figure 10.
        01234
                      Time (hours)
 Tritium oxidation by Alcdl'igenes paPadoxus after
 40 minutes pretreatment in solution of 30 mM^Tris,
 pH 8.0, and 10~^M EDTA followed by washing and re-
 suspension in distilled water amended with 0.0 or
 10.0 ppm lead  (as PbCl2)  or cadmium as (CdCl2)-
                              25

-------
     Other investigators found that EDTA produces structural changes in the
outer membrane of Esoheriohia coli  (Bayer and Leive, 1977) and a variety of
other gram-negative bacteria  (Gray and Wilkinson, 1965b).  EDTA has also
been found, especially when used in conjunction with Tris buffer, to stimu-
late the rate of lysis of gram-negative organisms by lysozyme  (Repaske,
1956; Repaske, 1958; Goldschmidt and Wyss, 1967; Voss, 1964).  Gray and
Wilkinson  (1965a), also showed that EDTA alone has direct bacteriocidal
action against Pseudomonas aeruginosa and Aloali-genes faeoalis.  Thus, it
was decided to use a combination of some of these techniques on A. paradoxus
with the hope of increasing its sensitivity to lead and  cadmium.

     The results of the EDTA-Tris treatment of A. paradoxus cells can be
seen in Figures 10 and 11.  Both the 40-minute and the 2-day treatment
produced a definite reduction in the extent of tritium oxidation while
using only 10.0 ppm cadmium and lead.  In both cases also, the effect of
lead was less than that of cadmium by an apparently similar percentage.
This was a considerable difference  from the earlier results and showed
that it is definitely possible to modify the activity of A. paradoxus to
make it more suitable, at least in  this case, for detecting the desired
low levels of certain compounds.

     This  shows that preliminary studies indicate it is  feasible to chemi-
cally alter the response of A. paradoxus to show desired  effects, in this
instance by increasing its sensitivity to two heavy metals.  Considerable
work still needs to be done to clearly define the types  of conditions that
can cause  the effects observed and  to standardize procedures, but the poten-
tial for sensitizing A. paradoxus to these and perhaps lower levels of lead
and cadmium exists.

     In the course of this work it  was found that there  were soils available
that had been amended with cadmium  in 1973.  As shown in Table 5, the levels
of cadmium in the soils at the time of these studies were near background
levels, and in view of the just-described results with cadmium, no response
from A. paradoxus to such low levels was expected.  However, as can be seen
in Figure  12, there was a distinct  difference in the oxidizing ability of
A. paradoxus in  soil 1 compared to  soil 4.  Soils 2 and  3, although not
shown, were also below soil 1 in the extent and rate of  their reaction.
They are not shown because the data did not allow lines  to be drawn through
their points with sufficient  confidence to justify adding them to the graph.
This was not the case with the data presented.  The fit  of the lines to the
points was good.

     The question now arises  as to  what was causing the  effect.  The cadmium
levels are so low and similar that  it is doubtful that the cause was due to
cadmium.   More likely it seems that there was some residual effect on the
soils from the cadmium additions.   The only difference in the soils was
supposedly the amounts of cadmium added in 1973, so the  possibility of
other differences inherent in the soils can be excluded. Plant studies run
in 1975 also showed poorer growth on soil 4 compared to  soil 1  (A. G. Wollum,
personal communication), although admittedly the cadmium levels were higher
then.
                                     26

-------
              7-
          X  5-
          09
          +*
          i  «J
          (0

          I   n
                                                    Control
                                                   ••' 10 ppm Cd2+
                                                  . 10 ppm
                                          I
                                          3
               01        2345
                                  Time (hours)
Figure 11.  Tritium oxidation by Alcal'Lgenes paradoxus  after 48  hours
            pretreatment in a solution  of  30 mM  Tris, pH 8.0,  and 10~2M
            EDTA followed by washing and resuspension in distilled
            water amended with either 0.0  or 10.0 ppm lead  (as PbCl2)
            or cadmium  (as CdCl_).
                               £1

°i
'ercent Conversior
tium to Tritiated V\
£

100-
90-
80-
70-
60-
50-
40-
30-
20-
10-


^^
AM <0
/ jf*
/•*
^ ....... ..Soil 4
/
r
Illlllllllll
               0
                                 Time (hours)

Figure 12.  Tritium oxidation by Aloal-Lgenes paradoxus  after inoculatior
            into otherwise sterile soils that were  amended with no cad-
            mium (.soil 1) , or 20 ppm  cadmium  (as CdC^)  (soil 4)  in 197
            and now have levels of 0.18 and 0.28 ppm cadmium respective
                                  27,

-------
     Whatever caused the effect may or may not be  found.  However,  the
differences in the rate and extent of the tritium  oxidation between the
two soils is very interesting and would certainly  seem worthy of  further
investigation, especially in light of the somewhat similar research findings
of McFarlane et al.  (1978).  They found that  identical soils fumigated with
varying concentrations of sulfur dioxide  (SC>2) for 1 and  2 years  showed
differences in their ability to carry out the oxidation of tritium  by their
indigenous microbial populations.  These differences in the ability to carry
out the oxidation reaction were found to be a function of the degree of SC>2
fumigation even though no residual SO2 was present in the soils at  the time
of the tests.

     These findings, even though done with unidentified hydrogen-oxidizing
microorganisms, indicate once again that even though virtually no detectable
chemical differences can be found between soils, biological effects, viz.,
the ability to oxidize tritium by microorganisms,  can be  observed.  What
these results mean and why they occur are intriguing questions awaiting
future research.
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2.  Amman, E. C. B., and  L.  L.  Reed.   1967.  Metabolism of nitrogen com-
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3.  Amman, E. C. B., L. L. Reed,  and J.  E.  Durichek, Jr.  1968.  Gas
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4.  Andressen,  M., and H. G.  Schlegel.   1974.   A new coryneform hydrogen
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7.  Babich,  H., and G. Stortzky.  1977b.  Reductions in the toxicity of
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8.  Bartha,  R., and E. J. Ordal.  1965.  Nickel-dependent chemolithotrophic
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                                     28

-------
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                                     29

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22.  Fraser-Smith, E. C. B., M. A. Austin,  and  L.  L.  Reed.   1969.   Utiliza-
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                                     30

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47.  Rittenbuerg, S. C., and N. S. Goodman.  1969.  Mixotrophic growth of
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                                     31

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                                      32

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
 EPA-600/3-79-048
                             2.
                                                           3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
 POSSIBLE USE OF ALCALIGENES PARADOXUS AS  A
 BIOLOGICAL MONITOR
                                                           5. REPORT DATE
                                  April  1979
                               6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
 Donald V.  Bradley,  Jr., Robert D. Rogers,  and
 James C. McFarlane
                                                           8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
 Environmental  Monitoring and Support Laboratory
 Office of Research and Development
 U.S. Environmental Protection Agency
 Las Vegas, Nevada  89114
                               10. PROGRAM ELEMENT NO.
                                  IHE 775
                               11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
 U.S. Environmental Protection Agency-Las Vegas, NV
 Office of Research and Development
 Environmental Monitoring and Support Laboratory
 Las Vegas, Nevada   89114
                               13. TYPE OF REPORT AND PERIOD COVERED
                                 Final          	  	
                               14. SPONSORING AGENCY CODE

                                 EPA/600/07
15. SUPPLEMENTARY NOTES
16. ABSTRACT
      A tritium  ( H2)-oxidizing soil isolate was identified as Aloaligenes paradoxus.,
 a gram-negative, rod-shaped bacterium.  This organism belongs to a group of facultative
 autotrophs referred  to as the "hydrogen bacteria" due to  their unique ability to
 utilize hydrogen as  a sole source of energy for chemolithotrophic growth.
      Experiments with washed cells of A. paradoxus  suspended in 0.025M potassium
 phosphate buffer  (resting cells)  showed that 1.0 ppm mercury [as Hg(No"3)2] caused
 a 95 percent reduction in hydrogen oxidation, whereas,  concentration of 0.1 ppm and
 lower showed no inhibitory effects.  When suspensions of  A.  paradoxus were added to
 sterile soil and then amended with mercury at concentrations of 1.0, 10.0, and 100.0
 ppm, hydrogen oxidation was reduced 80 percent, 85  percent,  and 95 percent, respective-
 ly, compared to soil  controls with no mercury added.  This showed that soil afforded,
 at least temporarily,  some protection from toxic mercury  effects.
      When cadmium or  lead was used in solution, it  was  found that up to 100.0 ppm of
 these metals produced no detectable inhibition in the rate or extent of the hydrogen
 oxidation reaction at either pH 7.0 or 5.0.  However, pretreatment of A.  paradoxus
 cells with a combination of ethylenediaminetetraacetate (EDTA)  and tris-(hydroxymethyl)
 aminomethane (Tris),  at a pH of 8.0, resulted in a  reduction in oxidation in solutions
 containing 10.0 ppm of cadmium or lead, with the greater  reduction being due to cadmium.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b.lDENTIFIERS/OPEN ENDED TERMS
                                            c.  COS AT I Field/Group
 Tritium
 Hydrogen-oxidizing
 Hydrogen bacteria
 Soils
 Resting cell studies
 Mercury
 Cadmium
Lead
Resting cell studies
06M
07B
18B,H
18. DISTRIBUTION STATEMENT
 RELEASE TO PUBLIC
                                              19. SECURITY CLASS (This Report)
                                                UNCLASSIFIED
                                            21. NO. OF PAGES
                                              48
                 20. SECURITY CLASS (Thispage)
                    UNCLASSIFIED
                          22. PRICE
                            A03
EPA Form 2220-1 (Rev. 4-77)   PREVIOUS EDITION is OBSOLETE

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